(EAAP Scientific Series) Lapierre, Hélène - Skomiał, Jacek - Energy and Protein Metabolism and Nutrition 2016-Wageningen Academic Publishers (2016)

Energy and protein
metabolism and nutrition
EAAP publication No. 137
edited by:
Jacek Skomiał
Hélène Lapierre
Energy and protein metabolism and nutrition
EAAP – European Federation of Animal Science
The Kielanowski Institute of Animal Physiology and Nutrition

of the Polish Academy of Sciences
The Committee of Animal Sciences and Aquaculture

of the Polish Academy of Sciences
University of Agriculture in Krakow
Warsaw University of Life Sciences – SGGW
The European Association for Animal Production wishes to express its appreciation to the
Ministero per le Politiche Agricole e Forestali and the Associazione Italiana Allevatori for their
valuable support of its activities
Energy and protein
5th EAAP International Symposium on Energy and Protein Metabolism

and Nutrition
Krakow, Poland
12-15 September 2016
EAAP publication No. 137
edited by:
Jacek Skomiał
Hélène Lapierre
Wageningen Academic
P u b l i s h e r s
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The 5th EAAP International Symposium on Energy and Protein
Metabolism and Nutrition was organised in Krakow, Poland on 12-15 September 2016.
The symposium was chaired by:

• Jacek Skomiał
The scientific programme of the symposium was co-chaired by:
• Hélène Lapierre
In consultation with the International Scientific Committee:
• Johan de Boever (Belgium)
• Mario Luiz Chizotti (Brazil)
• Gert van Duinkerken (President, the Netherlands)
• Michael Kreuzer (Secretary, Switzerland)
• Hélène Lapierre (Canada)
• Toyomizu Masaaki (Japan)
• James Oltjen (USA)
• Jacek Skomiał (Poland)
• Jürgen Zentek (Germany)
and liasing with the National Organizing and Scientific Committees:
• Marcin Taciak
• Ewa Sawosz-Chwalibog
• Zygmunt M. Kowalski
• Marcin Barszcz
• Anna Tuśnio
• Piotr Zakrzewski
Energy and protein metabolism and nutrition 5

Table of contents
Preface 21
Keynotes
Timing of nutrient delivery impacts muscle protein synthesis and lean growth 25
T.A. Davis, S.W. El-Kadi, C. Boutry, A. Suryawan and M.L. Fiorotto
Interaction between immunological system, microflora of the gastrointestinal tract and

metabolism 27
M. Bailey
Pork production with maximal nitrogen efficiency 35

S. Millet, M. Aluwé, J. De Boever and S. De Campeneere
Feeding strategies to manipulate in vivo protein turnover and post mortem proteolysis in meat 39
M. Therkildsen and N. Oksbjerg
Hepatic metabolism of glucose in the adaptation to the transition period in the dairy cow 41
H.M. Hammon, C.T. Schäff, J. Gruse and C. Weber
Proteomic applications to study livestock growth efficiency and meat quality 53

S.M. Lonergan, E. Huff-Lonergan, J.K. Grubbs, S.M. Cruzen and K.B. Carlson
Fasting heat production and metabolic body weight in non-ruminant growing animals 55
E. Labussière, S. Dubois, J. van Milgen and J. Noblet
Part 1.
Physiological aspects of protein and energy metabolism and nutrition: ruminants
Oral presentations
Energy from fat increases milk lactose yield from dairy cows to the same extent as energy
from protein 67
K. Nichols, H. van Laar, A. Bannink and J. Dijkstra
Rate of protein growth and energy for maintenance parameter changes in the Davis
Growth Model 69
J.W. Oltjen, R.D. Sainz, L.G. Barioni and S.R. Medeiros
Evaluation of the 2001 Dairy NRC and derivation of new equations 71

R.R. White, Y. Roman-Garcia, J.L. Firkins, M.H. VandeHaar, L.E. Armentano, T. McGill,
R. Garnett and M.D. Hanigan
Age effects on energy balance of dairy cows subjected to different diet types since first calving 73
F. Grandl, M. Kreuzer, J.O. Zeitz and A. Schwarm
Predicting variation in feed efficiency in veal calves by early life characterization 75

M.S. Gilbert, J.J.G.C. van den Borne, C.G. van Reenen and W.J.J. Gerrits

Isotopic N fractionation as a biomarker of nitrogen use efficiency by ruminants:
a meta-analysis 77
G. Cantalapiedra-Hijar, R.J. Dewhurst, L. Cheng, A.R.J. Cabrita, A.J.M. Fonseca,
H. Fouillet, P. Nozière and I. Ortigues-Marty
Effect of canola meal heat treatment and glycerol inclusion in calf starter on GIT development 81
K. Burakowska, P. Górka, Z.M. Kowalski, B. Laarveld and G.B. Penner
Associations between body weight change, hepatic and intestinal oxygen consumption in
pregnant cows 83
A.B.P. Fontoura, F.E. Keomanivong, L.D. Prezotto, L.E. Camacho, Y.R. Montanholi,
K.A. Vonnahme and K.C. Swanson
Overfeeding alters hepatic lipidomic and gene expression profiles in the periparturient
dairy cows 85
N. Qin, T. Kokkonen, S. Salin, S. Selim, T. Seppänen-Laakso, J. Taponen, A. Vanhatalo
and K. Elo
Ruminal metabolism of rapeseed meal insoluble nitrogen 87

T. Stefański, P. Huhtanen, A. Vanhatalo, K.J. Shingfield and S. Ahvenjärvi
Effects of solid feed intake on nutrient utilisation from milk replacer in veal calves 89
J.J.G.C. van den Borne, S.J.J. Alferink, M.J.W. Heetkamp and W.J.J. Gerrits
Bovine utero-placental glucose and cationic amino acid transporters during early gestation 91
M.S. Crouse, K.J. McLean, M.S. Crosswhite, L.P. Reynolds, C.R. Dahlen, B.W. Neville,
P.P. Borowicz and J.S. Caton
Heat production in ruminants: from experimental data to feed unit systems through
meta-analysis 93
D. Sauvant, S. Giger-Reverdin, P. Nozière and I. Ortigues-Marty
Meta-analysis of the efficiency of metabolisable protein utilisation in dairy cows and goats 95
D. Sauvant, J.B. Daniel, G. Cantalapiedra-Hijar and P. Nozière
Determinants of feed preferences and intake in calves 97

H. Berends, W.J.J. Gerrits, L.E. Webb, E.A.M. Bokkers and C.G. van Reenen
Heat stress involves activation of pAMPK and FOXO3 regulating glycolysis and
proteolysis in the skeletal muscle of dairy cows 99
F. Koch, O. Lamp and B. Kuhla
Performance and body composition in high and low RFI beef cattle 101
R.D. Sainz, K.C. Dykier, F.M. Mitloehner and J.W. Oltjen
Energy metabolism in high and low RFI beef cattle 103

R.D. Sainz, K.C. Dykier, F.M. Mitloehner and J.W. Oltjen
Relation of leukocyte activation and proliferation to feed efficiency in peripartal cows 105
S. Meese, S.E. Ulbrich, H. Bollwein, R. Bruckmaier, O. Wellnitz, M. Kreuzer, M. Röntgen,
U. Gimsa and A. Schwarm
8 Energy and protein metabolism and nutrition

Assessing intestinal absorption of amino acids 107
M.D. Hanigan, K. Estes and J. Castro
Production factors have a larger impact than genetic selection for lamb growth 109
K.R. Kelman, C. Alston, D.W. Pethick and G.E. Gardner
Increasing growth breeding values in Merino lambs decreases the non-esterified fatty acid
response during feed deprivation 111
S.M. Stewart, D.W. Pethick, G.E. Gardner and P. McGilchrist
Energy and protein restriction in the goat mammary gland: proteomics and metabolomics
profiling 113
M. Palma, L.E. Hernandez-Castellano, A.M. Ferreira, P. Nanni, J. Grossmann,
A. Arguello, N. Castro, J. Capote, M Matzapetakis and A.M. Almeida
Estimation of endogenous urinary N excretion in lactating dairy cattle 115

H. Lapierre, J.W. Spek, C.E. Galindo and D.R. Ouellet
Representation of essential amino acid use by the portal drained viscera and liver in cattle 117
A.J. Myers, M.D. Hanigan, J. Castro Marquez, R.R. White, H. Lapierre, R. Martineau and
J. France
Modelling homeorhetic trajectories of milk component yields, body composition and dry
matter intake in dairy cows: influence of parity, phenotypic potential and breed 121
J.B. Daniel, N.C. Friggens, H. Van Laar, K.L. Ingvartsen and D. Sauvant
Posters
Milk metabolites as biomarkers of energy balance in goats during lactation 123

P. Criscioni, T. Larsen and C. Fernández
Effect of grain increase in the diet on brush border enzymes activity in cattle 125
P. Górka, A. Błońska, B.L. Schurmann, M.E. Walpole, S. Li, J.C. Plaizier, Z.M. Kowalski
and G.B. Penner
Effects of rumen thermodynamics on volatile fatty acid production and interconversion in

dairy cattle 127
L.B. Harthan, R.R. White and M.D. Hanigan
Mammary and whole body energy metabolism in lactating cows fed high-roughage diets 129
K. Higuchi, F. Ohtani, Y. Kobayashi, I. Nonaka, O. Enishi, M. Sutoh and K. Yayou
Estimation of duodenal endogenous protein flow in dairy cattle: a regression approach 131
H. Lapierre, M.D. Hanigan and D.R. Ouellet
Evaluation of the INRA Systali digestive model through measured net portal appearance
of nutrients in ruminants 133
P. Nozière, J. Vernet, F. Raulhac, P. Chapoutot, H. Lapierre, D. Sauvant and
I. Ortigues-Marty
Are circulating blood metabolite concentrations related to changes in absorption in ruminants? 135
I. Ortigues-Marty, J. Vernet, S. Ashaba, H. Lapierre and P. Nozière

Proteomics of total and phosphorylated proteins in skeletal muscle of Angus and Nellore
cattle 137
R.T.S. Rodrigues, M.L. Chizzotti, C.E. Vital, M.T. Baracat-Pereira, E. Barros, R.A. Gomes,
K.C. Busato and M.M. Ladeira
Diet-dependent energy metabolism of neonatal calves determined by indirect calorimetric

measurements 139
C.T. Schäff, J. Gruse, M. Derno and H.M. Hammon
Amino acid composition of rumen microorganisms in cattle 141

M. Sok, D.R. Ouellet, J. Firkins, D. Pellerin and H. Lapierre
Ruminal absorption kinetics of D and L-lactate in lactating dairy cows under washed
rumen conditions 143
A.C. Storm, T. Larsen and M. Larsen
Exchanging fat for lactose in milk replacer stimulates de novo lipogenesis in calves 145
J.J.G.C. van den Borne, E. Labussière, M. Mielenz, H. Sauerwein and W.J.J. Gerrits
Effect of plasma factors on in vitro activation and proliferation of leukocytes from

peripartal cows 147
S. Wang, S. Meese, S.E. Ulbrich, M. Röntgen, M. Kreuzer and A. Schwarm
Energy requirements for maintenance and weight gain during pregnancy in dairy goats 149
I.A.M.A. Teixeira, C.J. Härter, L.D. Lima, H.G.O. Silva, D.S. Castagnino, A.R. Rivera and
K.T. Resende
Part 2.
Physiological aspects of protein and energy metabolism and nutrition: monogastrics
Oral presentations
Changes in muscle metabolism in Iberian and Landrace × Large-White pigs fed lysine
deficient diets 153
P. Palma-Granados, A. Haro, I. Seiquer, L. Lara, J.F. Aguilera and R. Nieto
Effect of dietary live yeast supplementation on thermal heat acclimatization in finishing

male pigs 155
E. Labussière, S. Dubois, M. Castex and D. Renaudeau
Citrulline and arginine synthesis in perinatal and young pigs 157

J.C. Marini, U. Agarwal, J. Robinson, Y. Yuan, I.C. Didelija, B. Stoll and D.G. Burrin
Dietary citrulline supplementation is an efficient strategy to increase arginine availability 159

J.C. Marini, U. Agarwal and I.C. Didelija
Lowering the dietary protein content in piglets: how far can we go? 161
A.J.M. Jansman, H. van Diepen, M. Rovers and E. Corrent

Does a reduction in dietary protein affect immune status of pigs kept under different
sanitary conditions? 163
Y. van der Meer, W.J.J. Gerrits, A.J.M. Jansman, B. Kemp and A. Lammers
Dietary ammonia appearance in portal blood of pigs fed diets deficient in non-essential
amino acid nitrogen is incomplete 165
W.D. Mansilla, A.K. Agyekum, C.L. Zhu, J.K. Htoo, C.M. Nyachoti and C.F.M. de Lange
The effect of the rate of starch digestion on energy efficiency in low and high performing
piglets 167
R.J.J. van Erp, H.M.J. van Hees, R.T. Zijlstra and W.J.J. Gerrits
Influence of deoxynivalenol (DON) on the piglet’s immune system with due regard to
sodium sulfite decontaminated feed: in vivo results 169
A.T. Tran, M. Paulick, A. Berk, J. Kluess, J. Frahm, D. Schatzmayr and S. Dänicke
Impact of dietary crude protein and amino acid restriction on the amino acid deposition
rate and profile in the empty body of modern Swiss Large White pigs 173
I. Ruiz-Ascacibar, P. Stoll and G. Bee
Effect of dietary proteins on immunity and metabolism in mice 175

S.K. Kar, A.J.M. Jansman, L. Kruijt, N. Benis and M.A. Smits
Methionine, leucine, isoleucine or threonine effects on mammary cell signalling and pup
growth in lactating mice 177
G.M. Liu, M.D. Hanigan, X.Y. Lin, K. Zhao and Z.H. Wang
Effects of supplemental dietary leucine and immune system stimulation on whole body
protein turnover in starter pigs 179
M. Rudar, C.L. Zhu and C.F.M. de Lange
Roles of corticosterone and superoxide in the ubiquitin proteasome system in heat-

stressed chickens 181
K. Furukawa, M. Kikusato and M. Toyomizu
A comparison of reactive oxygen species regulation in skeletal muscle with different

muscle fibre compositions from heat-stressed birds 183
M. Kikusato and M. Toyomizu
Breast muscle protein turnover in broiler breeder parent stock 185

K. Vignale, J.V. Caldas, J.A. England, N. Boonsinchai, P. Sodsee, M. Putsakum,
E.D. Pollock, S. Dridi, C.M. Owens and C.N. Coon
De novo lipogenesis in broiler breeder hens 187

N. Boonsinchai, K. Hilton, G. Mullenix, J.V. Caldas, A. Magnuson, J.A. England and
C.N. Coon
Application of compartmental analysis to study nitrogen kinetics in broilers 191

A.R. Troni, M.H. Green, J.L. Ford, N.K. Sakomura, R.M. Suzuki, D.M.B. Campos,
N.J. Peruzzi and L.G. Pacheco

Incorporation of N from l-threonine in plasma of broilers 193
R.M. Suzuki N.K. Sakomura, J.A. Bendassolli J.C. Denadai A.R. Troni D.M.B. Campos
L.G. Pacheco and P.M. Júnior
Biomarkers of optimum dietary branched chain amino acids for the best growth
performance in pigs 195
E.A. Soumeh, M.S. Hedemann, H.D. Poulsen, E. Corrent, J. van Milgen and J.V. Nørgaard
Posters
Potential contribution of net portal absorption of volatile fatty acids to energy expenditure
in Iberian gilts fed acorn 197
L. González-Valero, M. Lachica, J.M. Rodríguez-López, L. Lara and I. Fernández-Fígares
Effect of dietary net energy and digestible lysine levels on growth performance of
growing pigs 199
J.K. Htoo and J. Morales
Prediction of true and apparent ileal digestibility of amino acids of wheat for broiler chickens 201
O. Lasek, R. Augustyn and J. Barteczko
Replacing dietary non-essential amino acids with ammonia nitrogen does not alter amino
acid profile of retained body protein in growing pigs fed a diet deficient in non-essential
amino acid nitrogen 203
W.D. Mansilla, J.K. Htoo and C.F.M. de Lange
The protein requirement before and after implantation in mink 205

C.F. Matthiesen, C. Larsson, P. Junghans and A.-H. Tauson
Transgenic flax overexpressing polyphenols and inflammation: anti-inflammatory

mechanisms 209
M. Matusiewicz, I. Kosieradzka, M. Zuk, T. Niemiec, A. Łozicki, G. Halik, M. Makarski
and J. Szopa
Rice diet containing high fat and rice hull affects the growth performance of heat-exposed
broiler chickens 211
F. Nanto, M. Kikusato, S. Ohwada and M. Toyomizu
Lysine deficiency and genotype affect amino acid composition of carcass protein of
growing pigs 213
P. Palma-Granados, N. Hidalgo-Checa, L. Lara, J.F. Aguilera and R. Nieto
Effect of copper nanoparticles and copper sulphate on metabolic rate and growth of broiler
embryos 215
A. Scott, K.P. Vadalasetty and A. Chwalibog
The effect of the rate of starch digestion on diurnal heat production and RQ in low and
high performing piglets 217
R.J.J. van Erp, H.M.J. van Hees, R.T. Zijlstra and W.J.J. Gerrits

Interactions among leucine and threonine on growth and amino acid metabolism in
weaned piglets 219
A.G. Wessels, H. Kluge, F. Hirche, J. Bartelt, E. Corrent and G.I. Stangl
The optimal lysine requirement of modern genotype piglets 221

S. Millet, M. Aluwé, E. Le Gall, E. Corrent, J. De Sutter and S. De Campeneere
Part 3.
Animal product quality and health in the light of protein and energy metabolism and
nutrition
Oral presentations
Lamb intramuscular fat percentage is correlated between muscles, with whole body
fatness, and with muscle oxidative capacity in the loin 225
G.E. Gardner, D.W. Pethick and F. Anderson
Alicar: a database for carcass characteristics, diet composition and intake in ruminants 229
J. Vernet, M. Reichstadt, M. Al-Jammas and I. Ortigues-Marty
Effects of diet composition on carcass fat in beef cattle: a meta-analysis 233

M. Al-Jammas, J. Agabriel, J. Vernet and I. Ortigues-Marty
Effect of dietary starch content on prediction of feed intake by six different models for
dairy cows 235
P. Nørgaard and L.M. Jensen
Posters
The effect of silica-calcite sedimentary rock (opoka) in the diet on texture parameter of
selected muscule in broiler 239
M. Makarski, T. Niemiec, A. Łozicki, I. Kosieradzka, D. Pietrzak, L. Adamczak,
M. Matusiewicz and P. Koczoń
Muscle anserine content is associated with pork meat quality and carnosine synthase gene
expression 241
M.F. Palin, J. D’Astous-Pagé, R. Blouin, S. Cliche, F. Fortin, B. Sullivan and C. Gariépy
Effect of white striping myopathy on breast muscle protein turnover and gene expression
in broilers 243
K. Vignale, J.V. Caldas, J.A. England, N. Boonsinchai, A. Magnuson, E.D. Pollock,
S. Dridi, C.M. Owens and C.N. Coon
Performance and fatty acid status in dairy cows fed a diet with reduced essential fatty acid
content 245
C. Weber, A. Tröscher, H. Kienberger, M. Rychlik and H.M. Hammon

Part 4. Environmental and animal welfare aspects of protein and energy nutrition
Oral presentations
Agriculture without animals? The environmental and economic role of livestock in food
production 249
R.R. White and M.B. Hall
Soybean meal supplementation in automatic milking systems 251

M.R. Weisbjerg, M. Johansen, M. Larsen and P. Lund
Update of protein requirements for Zebu beef cattle 253

L.F. Costa e Silva, S.C. Valadares Filho, P.P. Rotta, M.I. Marcondes, M.L. Chizzotti and
A.C.B. Menezes
Intravenous lipid infusion affects methane production apart from reducing dry matter
intake in late lactation German Holstein cows 255
O. Lamp, M. Derno, G. Nürnberg, C.C. Metges and B. Kuhla
Effects of dietary carbohydrate source on milk production and environmental impact of

lactating dairy cows 257
E.H. Cabezas-Garcia, S.J. Krizsan, K.J. Shingfield and P. Huhtanen
Delineation of relationships between feed composition, methane emission and milk fatty
acids in cows – prerequisite for the development of an indirect methane indicator 259
S.W. Engelke, G. Daş, M. Derno, B. Kuhla and C.C. Metges
Posters
Temperature effect on fast heat production of Anglo Nubian and Saanen goats 261
M.H.M.R. Fernandes, A.R.C. Lima, C.I.S Oporto, K.T. Resende, B. Biagioli and
I.A.M.A. Teixeira
Long term implications of feeding low protein diets to first lactation dairy cows 263
C.K. Reynolds, L.A. Crompton, D.J. Humphries and A.K. Jones
Influence of rutin and rutin-containing buckwheat seeds on methane emission in lactating

dairy cows 265
A.-K. Stoldt, M. Derno, G. Daş, J.M. Weitzel, S. Wolffram and C.C. Metges
Effect of genetic group and feeding level on methane production in lactating crossbred
and Zebu cows in Brazil 267
A.L.C.C. Borges, P.A.D. Vivenza, R.R. Silva, E.O.S. Saliba, P.H.A. Carvalho, H.F. Lage,
I. Borges, A.U. Carvalho and J.R.M. Ruas

Part 5.
Feed sources and feed processing related to energy and protein digestion and metabolism
Oral presentations
Rumen degradation kinetics of protein rich feedstuffs in dairy cows and intensively fed
beef cattle 271
A. Navarro-Villa, H. van Laar, J. Doorenbos and J. Martín-Tereso
Effect of starch content of milk replacer on energy metabolism in growing-finishing veal

calves 273
E. Labussière, J.N. Thibault, T. Lefèbvre and C. Martineau
Effects of dietary fatty acid chain length on performance of early lactation dairy cows 275
D.E. Rico, P.Y. Chouinard, C. Cohou, J.E. Parales, M. Plante-Dubé and R. Gervais
Altering the starch and fat content of the peri-conception diet has no effect on fertility in
breeding ewe lambs 277
D.W. Miller, E.J. Bowen and C.L. Jacobson
Effect of toasting time on proteolysis of soluble and insoluble protein fractions of

rapeseed meal 279
S. Salazar-Villanea, E.M.A.M. Bruininx, P. Carré, A. Quinsac and A.F.B. van der Poel
Rapeseed meal, faba beans and microalga (Spirulina platensis) as protein supplements for
dairy cows on grass silage based diets 281
A. Halmemies-Beauchet-Filleau, M. Lamminen, T. Kokkonen, A. Vanhatalo and
S. Jaakkola
Microalgae as a substitute for soya bean meal in the grass silage based dairy cow diets 285
M. Lamminen, A. Halmemies-Beauchet-Filleau, T. Kokkonen, S. Jaakkola and
A. Vanhatalo
Impact of grain source and distillers fat level on ruminal enzymes, pH and methane
production 289
F. Keomanivong, M. Rodenhuis, M. Ruch, M. Crouse, J. Kirsch, M. Bauer, M. Borhan,
S. Rahman and K. Swanson
Oilseed meal processing affects whole body amino acid retention and composition in
growing pigs 291
T.G. Hulshof, A.F.B. van der Poel and P. Bikker
Oilseed meal processing affects protein digestion kinetics and metabolic organ load of
growing pigs 293
T.G. Hulshof, A.F.B. van der Poel and P. Bikker
Increasing level of full-fat rapeseeds in broiler chicken diets changes the plasma
nontargeted metabolic profile 295
E. Ivarsson, K. Hanhineva and H. Wall
Protein digestion kinetics of different protein sources in broilers 297

H. Chen, S. de Vries, J. de los Mozos and A.J.M. Jansman

Maintenance energy requirements in modern broilers fed exogenous enzymes 299
J.V. Caldas, K.M. Hilton, N. Boonsinchai, G. Mullenix, J.A. England and C.N. Coon
Laying performance of layer vs dual purpose genotypes under low methionine supply 301
S. Mueller, R.E. Messikommer, M. Kreuzer and I.D.M. Gangnat
Possible mechanism of down-regulation of atrogin-1 mRNA level in butoxybutyl

alcohol-fed chicken 303
T. Kamizono, M. Kikusato, K. Hayashi and M. Toyomizu
Effects of increased diet density through increased dietary fat level on energy balance
characteristics of broilers during the first week of life 305
D.M. Lamot, D. Sapkota, P.J.A. Wijtten, I. van den Anker, M.J.W. Heetkamp, B. Kemp and
H. van den Brand
Posters
Effects of metabolisable energy and crude protein levels in balanced digestible essential
amino acids on body weight gain and carcass composition of Brown laying hens in the
late phase of production 307
N. Chauychuwong and K. Soisuwan
Protein digestion kinetics of different protein sources in pigs 309

H. Chen, P.A Wierenga and A.J.M. Jansman
Impact of silages with bioactive compounds on ruminal fermentation and microbial

protein synthesis 311
A. Grosse Brinkhaus, G. Bee, F. Dohme-Meier, M. Kreuzer and J.O. Zeitz
Postprandial net portal and liver fluxes of essential amino acids in dairy cows fed rumen
escape protein 313
M. Larsen and A.C. Storm
Effect of the type of dietary fibre in the feed on digestibility and fermentation parameters
in dogs 315
O. Lasek
Digestion pattern in horses of fibre in grass haylage cut at different stages of maturity 317
J.E. Lindberg and S. Ragnarsson
Effect of the site of starch infusion on urea kinetics in dairy cows 319
D.R. Ouellet, F. Hassanat and H. Lapierre
Effect of pelleted feed use for Addax nasomaculatus on feed intake and nutrient digestibility 321
M. Przybyło, P. Tyl, J. Kański, A. Kloska and P. Górka
Lactobacillus buchneri on sugarcane silage fermentation and performance of cattle in

Brazil: a meta-analysis 323
C.H.S. Rabelo, C.J. Härter and R.A. Reis

Lactobacillus buchneri applied in corn silage or directly into the rumen of wethers: effect
on apparent digestibility and ruminal fermentation 325
C.H.S. Rabelo, L.G.O. Jorge, F.C. Basso, E.C. Lara, C.J. Härter, L.M. Delevatti and
R.A. Reis
Effects of the level of soybean oil on the kinetics of fibre digestion in dairy cows fed
sugarcane based diets 327
J.P.P. Rodrigues, R.M. de Paula, L.N. Rennó, M.M.S. Fontes, P.P. Rotta, S.C. Valadares
Filho, P. Huhtanen and M.I. Marcondes
Effects of metabolisable energy and crude protein levels in balanced digestible essential
amino acids on production performances and egg quality of Brown laying hens in the late
phase of production 329
K. Soisuwan and N. Chauychuwong
Effect of level and micronizing of lupin seeds on microbial activity in the large intestine
of pigs 331
A. Tuśnio, M. Barszcz, E. Święch, J. Skomiał and M. Taciak
Influence of silver nanoparticles on growth and health of broiler chickens challenged with
Campylobacter jejuni 333
K.P. Vadalasetty, C. Lauridsen, R.G. Engberg, E. Sawosz and A. Chwalibog
The effects of dried leaves of Manihot esculenta and Artemisia annua on coccidiosis in
organically reared pullets in Brazil 335
G.F.D. Almeida, S.M. Thamsborg, D.M.B. Campos, K. Horsted, P.M. de Magalhães,
J.F.S. Ferreira and J.E. Hermansen
Effects of rich in docosahexaenoic acid algae supplementation on performance of calves 337

J. Flaga, Ł. Korytkowski, P. Górka and Z.M. Kowalski
Determining the optimal essential amino acid ratios in juvenile of Nile tilapia 339
F.H.F. Rodrigues, J.C.P. Dorigam, N.K. Sakomura, T.M.T. Nascimento, C.F.M. Mansano,
E.P. Silva and J.B.K. Fernandes
Part 6.
Methodological aspects of research on protein and energy metabolism and nutrition
Oral presentations
A new modelling system to estimate lactation requirements and the efficiency of utilising
metabolisable protein to synthesise milk protein 343
L.E. Moraes, E. Kebreab, L. Doepel and H. Lapierre
Incorporating Theil-Sen regression method into Bartlett’s 3-group Type II unknown

variances model to improve its robustness 345
M.S. Dhanoa and R. Sanderson

Use of the pig to determine digestible indispensable amino acid scores (DIAAS) in human
foods 347
H.H. Stein and J.K. Mathai
Blood biomarkers in short-term studies on amino acid requirement in pigs 349

J.V. Nørgaard, E.A. Soumeh, M. Curtasu, H.D. Poulsen, E. Corrent, J. van Milgen and
M.S. Hedemann
Variation in protein content and efficiency of lysine utilisation in growing-finishing pigs 351
S. Ghimire, C. Pomar and A. Remus
Evaluation of compound-specific N isotope analysis in key amino acids to predict feed

efficiency in growing lambs 353
G. Cantalapiedra-Hijar, S. Prache, I. Téa, S. Faure, C. Chantelauze and
I. Ortigues-Marty
Using computed tomography to assess fat distribution in lamb carcasses 355

F. Anderson, A. Williams, L. Pannier, D.W. Pethick and G.E. Gardner
Dynamics of nutrient utilisation, heat production and body composition in broiler breeder
hens during egg production 357
J.V. Caldas, K. Hilton, M. Schlumbohm, N. Boonsinchai, J.A. England and C.N. Coon
Use of artificial neural networks to improve estimation of energy requirements of cattle 359
M.P. Gionbelli, D.D. Ferreira, L.K. Ferreira, M.L. Chizzotti and S.C. Valadares Filho
Posters
Mature weight of male and female Saanen goats 361

A.K. Almeida, K.T. Resende, L.O. Tedeschi, M.H.M.R. Fernandes and I.A.M.A. Teixeira
Dual NMR and LC-MS-ToF analysis highlights new markers of nitrogen use in dairy cows 363
H. Boudra, M. Lagrée, M. Doreau, P. Nozière and D.P. Morgavi
Early life nutritional programming of long-term weight gain and feed intake in the porcine
model 365
C. Clouard, W.J.J. Gerrits, B. Kemp, D. Val-Laillet and J.E. Bolhuis
Net energy requirements for maintenance of F1 – Holstein × Gyr crossbred bulls

determined by the calorimetry and comparative slaughter technique 367
A.L. Ferreira, A.L.C.C. Borges, R.R. Silva, A.S. Souza, A.C.A. Duque, J.S. Silva,
J.R.M. Ruas, L.C. Gonçalves and E.O.S. Saliba
Energy requirements during pregnancy in dairy goats 369

C.J. Härter, L.D. Lima, H.G.O. Silva, D.S. Castagnino, A.R. Rivera, J.L. Ellis, J. France
and I.A.M.A. Teixeira
Intake and digestibility of diets containing crude glycerin to steers determined with markers 371
C.R.M. Silva, E.O.S. Saliba, F.A. Silva, G.S.S.C. Barbosa, G.M. Rocha and H. Lopes

Estimate of the dry matter intake for grazing horses 373
R.H.P. Silva, A.S.C. Rezende, E.O.S. Saliba, D.F.S. Inácio, S. Maruch, J.N.S.M. Queiroz
and K.M.C. Barcelos
Use of stable isotopes as a tracer for broiler chickens 375

A.R. Troni, D.M.B. Campos, R.M. Suzuki, H.S. Doreto, N.K. Sakomura and
J.A. Bendassolli
Author index 379

Preface
In 2003, the previously independent Energy or Protein Symposia merged with the International
Symposium on Energy and Protein Metabolism and Nutrition (ISEP) in Rostock-Warnemünde,
Germany, associated with the European Federation for Animal Science (EAAP), formerly known
as the European Association for Animal Production. The tradition then followed every three years:
in Vichy, France (2007), in Parma, Italy (2010) and in Sacramento, California, USA (2013). The
5th ISEP was held on 12-15 September 2016 at the Holiday Inn Hotel in Krakow, Poland, and was
organized by the Kielanowski Institute of Animal Physiology and Nutrition Polish Academy of
Sciences in Jabłonna, in cooperation with University of Agriculture in Krakow and Warsaw University
of Life Sciences – SGGW.
Development in agricultural sciences, particularly in farm animal sciences, resulted in the increased
productivity to meet the demand for high quality and relatively cheap protein sources for human
nutrition. In parallel, this increased productivity challenges the adequate supply of nutrients, including
protein and energy, needed to cover not only high performances, but also insure health and animal
welfare, reproduction and quality of products in a sustainable environment. We are convinced that the
precise understanding of animal biology is crucial for animal health and welfare, sustainable animal
production, and health of animal product consumers. Continuing the strategy of the previous EAAP
symposia, the 5th EAAP ISEP focused on combining basic and applied research and its practical
applications. To achieve these goals, many topics have been presented and discussed in detail.
Physiological aspects of protein and energy metabolism and nutrition, including: metabolic and
neurohormonal regulations and role of microbiota in the gastrointestinal tract and genetic regulation,
animal health and welfare metabolic related issues, effect of feeds and feed processing on energy
and protein digestion and metabolism, methodological aspects of research on protein and energy
metabolism, environment protection and enhancement of the quality and health-promoting features
of animal products were the most important issues discussed during the meeting.
Interesting lectures given by famous scientists, as well as oral and poster presentations by the
participants, gave the opportunity to share the results of the newest research performed all over
the world. Over 150 participants from 21 countries presented lectures and contributions, in which
participated more than 700 authors/co-authors. Apart from the perfectly presented speeches, the
strength of the ISEP lies in its workshop spirit, and in the interaction and collaboration among
scientists from all these countries with a true desire to exchange their expertise and knowledge.
To show some Polish highlights and to familiarize the participants with Polish hospitality and delicious
cuisine, interesting excursions and events were organized within Krakow and its neighbourhoods
(Wieliczka Salt Mine, Arabian horses’ Stud in Michałów, Niepołomice Royal Castle).
We thank all those who participated in the organisation of this symposium, particularly the International
Scientific Committee, Committee of Animal Sciences of the Polish Academy of Sciences, the local
organisers and the sponsors.
My special gratitude goes to the speakers who accepted our invitation and all the symposium
participants. Your presence created the opportunity to discuss metabolic and nutritional issues with
a look into the future research trends.
Jacek Skomiał

Keynotes
Timing of nutrient delivery impacts muscle protein synthesis and lean
growth
T.A. Davis1*, S.W. El-Kadi2, C. Boutry1, A. Suryawan1 and M.L. Fiorotto1
1USDA/ARS Children’s Nutrition Research Center, Department of Pediatrics, Baylor College
of Medicine, Houston, TX, USA; 2Department of Animal and Poultry Sciences, Virginia Tech,
Blacksburg, VA, USA; tdavis@bcm.edu
Abstract
The ingestion of food stimulates the synthesis of protein in skeletal muscle and it is especially
marked before maturity is attained. This feeding-induced stimulation of protein synthesis is crucial
to support the rapid muscle growth during early postnatal life and the maintenance of body protein
in adulthood. Although total daily protein intake impacts the rate of protein synthesis, the timing
of the nutrient delivery also influences the overall rate of muscle protein synthesis and ultimately,
muscle mass. Our studies in young pigs have shown that intermittent bolus feeding, similar to meal
feeding, enhances muscle protein synthesis compared to continuous delivery of the same nutrient
load. The increase in muscle protein synthesis with intermittent bolus feeding is enabled by the
rapid and profound increases in insulin and amino acids that occur following a bolus meal. This
pulsatile pattern of insulin and amino acids activates the insulin and amino acid signalling pathways
that lead to translation initiation. By contrast, the low and constant hormone and substrate pattern
elicited by continuous feeding attenuates translation initiation signalling and resulting in a blunted
rate of muscle protein synthesis. Protein degradation appears unaffected by the pattern of nutrient
delivery, suggesting that the higher rate of protein deposition with intermittent bolus feeding is
mainly due to a higher rate of protein synthesis. The higher muscle protein synthesis rates achieved
by the intermittent bolus pattern of nutrient delivery can be sustained long term to promote protein
deposition and increase lean body mass and growth.
Keywords: amino acids, insulin, growth, translation initiation, protein metabolism
Introduction
In this brief synopsis, we address our current understanding of how the timing with which nutrients
are delivered modulates the rate of protein synthesis in skeletal muscle and, ultimately impacts
protein deposition and lean growth. We have utilised the young pig as an animal model in these
studies because its anatomy, developmental physiology, and metabolism is similar to the human and
its heightened responsiveness of muscle protein synthesis to variations in nutrient intake enables
rapid growth rates to be attained. These studies have demonstrated that feeding stimulates protein
synthesis and this response is more profound in skeletal muscle than in other tissues and organs
and this response decreases with development (Davis and Fiorotto, 2009). Although it is known
that total daily protein intake impacts the rates of skeletal muscle protein synthesis and accretion,
we have shown that the timing with which nutrients are delivered also impacts these processes. In
studies of young pigs we demonstrated that the intermittent bolus feeding pattern enhances muscle
protein synthesis more than continuous feeding and that this effect is due to the pulsatile pattern of
circulating insulin and amino acids, which activates the insulin and amino acid signalling pathways
leading to an increase in translation initiation.
Insulin and amino acids mediate the feeding-induced stimulation of

muscle protein synthesis
Circulating insulin and amino acids rise after a meal and each independently mediate the postprandial
stimulation of protein synthesis in skeletal muscle (Davis and Fiorotto, 2009). The rise in insulin

after a meal activates the insulin receptor and downstream insulin signalling cascade that leads
to the activation of mammalian target of rapamycin (mTOR). Amino acids also signal to mTOR
although the signalling pathway has not been as well characterized. Together, stimulation of mTOR
by these anabolic agents modulates the phosphorylation of downstream targets, 4EBP1, S6K1, and
formation of the eIF4E-eIF4G complex that regulates translation initiation. We have shown that the
time course of the changes in protein synthesis after a meal parallels the time course of the changes in
the activation of these mTOR signalling proteins and that these time-dependent changes correspond
with the pulsatile pattern of circulating insulin and amino acids (Wilson et al., 2009).
Effects of intermittent bolus and continuous delivery of nutrients on

protein synthesis
To test whether the surge in insulin and amino acids enables a more efficient utilisation of nutrients
for protein synthesis, pigs were fed the same amount of a complete formula by orogastric tube
either as an intermittent bolus feed every 4 hours or as a continuous infusion. The results show that
circulating insulin and amino acids are low and constant in continuously fed pigs, whereas intermittent
bolus feeds elicit a pulsatile insulin and amino acid pattern (Gazzaneo et al., 2011). This pulsatile
hormone and substrate pattern enhances muscle protein synthesis in association with the activation
of the mTOR signalling pathway. Utilisation of a multi-catheterized piglet model showed that the
increase in protein synthesis leads to an increase in protein deposition, whereas protein degradation
is insensitive to the timing of nutrient delivery (El-Kadi et al., 2012). Recent studies indicate that
this increase in protein deposition, when sustained chronically, translates into an increase in skeletal
muscle weight, lean body mass accretion, and whole body growth.
References
Davis, T. and M. Fiorotto, 2009. Regulation of muscle growth in neonates. Current Opinion in Clinical Nutrition and
Metabolic Care 12: 78-85.
El-Kadi, S, A. Suryawan, M. Gazzaneo, N. Srivastava, R. Orellana, H. Nguyen, G. Lobley and T. Davis, 2012. Anabolic
signaling and protein deposition are enhanced by intermittent as compared with continuous feeding in skeletal
muscle of neonates. American Journal of Physiology 302: E674-E686.
Gazzaneo, M., A. Suryawan, R. Orellana, R. Torraza, S. El-Kadi, F. Wilson, S. Kimball, N. Srivastava, H. Nguyen,
M. Fiorotto and T. Davis, 2011. Intermittent bolus feeding has a greater stimulatory effect on protein synthesis in
skeletal muscle than continuous feeding in neonatal pig. Journal of Nutrition 141: 2152-2158.
Wilson, F., A. Suryawan, R. Orellana, S. Kimball, M. Gazzaneo, H. Nguyen, M. Fiorotto and T. Davis, 2009. Feeding
rapidly stimulates protein synthesis in skeletal muscle of the neonatal pig by enhancing translation initiation.
Journal of Nutrition 139: 1873-1880.

Interaction between immunological system, microflora
of the gastrointestinal tract and metabolism
M. Bailey
Comparative Immunology, School of Veterinary Science, Bristol University, Langford House,
Langford, Bristol, United Kingdom; mick.bailey@bristol.ac.uk
Abstract
‘Enteric health’ is a poorly-defined goal of increasing interest to the livestock industries. Empirically, it
can be defined by optimum growth over input, but issues such as welfare and freedom from disease are
important modifiers. Current products largely rely on empirical evidence but an understanding of the
mechanisms by which enteric health is obtained is essential to improve existing products and develop
new approaches. One important component determining enteric health is the ability of the mucosal
immune system to respond appropriately to pathogens and to harmless antigens including food and
commensal bacteria: failure to respond actively to pathogen or vaccines results in susceptibility to
infectious diseases, while responses to food or commensals results in gut inflammation and reduced
feed utilisation. Experimental studies in mice and pigs, and epidemiological studies in humans,
suggest that this ability to respond appropriately is driven by early life colonisation with normal
commensal microbiota. Further, relatively subtle distinctions between the types of microbiota, such
as between indoor and outdoor pigs, or even between different farms, may affect the way the immune
system develops. In humans, this process of early colonisation with microbiota has also been linked
to differences in metabolism such as energy capture, and predispositions to obesity and diabetes.
Thus, it is increasingly apparent that the process of early life colonisation by intestinal bacteria
can affect susceptibility to immunological and metabolic dysfunction in a range of target species
including pigs. In future, interventions need to be targeted at rational manipulation of this process.
Keywords: pig, microbiota, immune development, early life, metabolism
What is gut health?

Prior to 2000, the terms ‘enteric health’ or ‘gut health’ were rarely used. Since then, there has been
an exponential increase in the numbers of research papers referring to it as a concept, and an even
more striking increase in references to those articles, indicating the level of interest. Despite this,
the term ‘enteric health’ is relatively poorly defined. As such, it is often not easy to identify what
a particular author is referring to by the term. The simple fact that we refer to ‘enteric health’ at
all, when we tend not to use the terms ‘musculoskeletal health’ or ‘renal health’ implies that there
are specific considerations around optimal gut function. Specifically, the intestine provides a host
surface which, unlike skin, has to allow a level of access to the external environment in order to
absorb nutrients. In doing so, it concentrates those nutrients over a partially permeable surface and
provides a substrate for massive colonisation by environmental microbiota (bacteria, yeasts, viruses,
some multicellular parasites).
These factors (the size of the enteric surface, the permeability of the gut and the presence of
microbiota) need to be managed in order to maintain the health of the animal. Estimates from humans
are that the unfolded surface of the intestine is about 250 m2 (ten-fold greater than skin), while
estimates of the number of bacterial cells are about 1014 (for comparison, the number of human cells
is only about 1012: that is, humans and pigs contain fewer human or pig cells than they do bacteria).
The optimal situation is one where the pig has maximal availability of nutrients absorbed across the
intestine, with minimal exposure to any toxic or otherwise dangerous molecules. Changes in the area
available for absorption of nutrients can affect the amount of nutrients available to the animal, while

changes in the permeability of the intestine can affect the types of molecules which cross. Both of
these will impact on the metabolic and immune systems of the pig.
For the purposes of this paper, I will use the term ‘enteric health’ to mean optimal functioning of
three specific components of the intestine: intestinal barrier function; the intestinal immune system;
and the metabolic system of the pig.
Intestinal barrier function
Through most of the intestine, mucus provides an initial barrier to particles (like bacteria) and large
molecules. Recent work has clarified the roles of mucus in exclusion of bacteria from the host tissues
(Johansson et al., 2013). In the colon, the mucus forms two layers: (1) an outer, loose layer, which
contains bacteria and is used by them as a substrate; and (2) an inner, dense layer which is normally
impermeable to these bacteria and is the primary mechanism which prevents their direct access to
the epithelium. In the small intestine, the inner, dense layer is almost non-existent, while the outer,
loose layer fills the spaces between adjacent villi, leaving some villus tips free above the surface. In
the small intestine, protection against luminal bacteria is provided predominantly by the presence
of anti-microbial peptides secreted by a subset of the intestinal epithelial cells.
Much of the function of this mucus defence layer is a response to microbial colonisation. In germ-free
animals, relatively little colonic or bacterial mucus is produced and, where it is, there are defects in the
way in which it is released from the epithelium. Colonisation of germ-free mice with a conventional
microbiota results in upregulation of mucus production and, in the colon, the appearance of a dense
mucus layer capable of excluding luminal bacteria. However, this process is relatively slow: in rodent
models, it can take 4 weeks after colonisation before full exclusion is attained.
The second, much better understood barrier is that provided by enterocytes themselves. This involves
complex, tight cell junctions close to the apical surfaces of intestinal enterocytes which provide
molecular continuity between cell membranes of adjacent cells. These junctions are formed by a
series of proteins including claudins, occludins and the zona occludens protein 1. Deeper to these
are the other, less complete junctions, including desmosomes. Integrity of, in particular, the tight
junctions is essential for controlling macromolecular transfer between the lumen and the internal
tissues of the pig.
Immunity
Classically, the function of the immune system is to recognise and to mount responses to non-self
molecules. However, it is now apparent that this is an over-simplification. Although the vast majority
of macromolecules are degraded to small, non-antigenic molecules before absorption, small but
significant amounts of macromolecules are absorbed intact (Bailey, 2009). These absorbed molecules
undergo relatively little structural modification and can be recognised by the immune system. We and
others have demonstrated that weaned piglets absorb significant amounts of soya or egg proteins from
the weaner diet into blood, and that this triggers active immune responses characterised by antibody
and cellular reactions. Expression of immune responses at mucosal surfaces is usually associated
with inflammation and loss of function, and this response to food proteins has been linked to the
occurrence of postweaning diarrhoea.
Continued feeding of novel proteins at weaning results in a peak in antibody response at around two
weeks after weaning, and the response subsequently wanes. Importantly, when animals which have
mounted these responses are challenged by injection of the same protein systemically, they fail to
mount a normal response: that is, they have become specifically immunologically tolerant to the
antigens with which they have been fed. This process of so-called ‘oral tolerance’ has been seen as

a mechanism whereby the immune system prevents expression of potentially damaging responses
to harmless, non-self molecules (in this case food) but the same process is likely to apply to antigens
derived from gut bacteria as well.
However, although the mucosal immune system must be able to switch off inappropriate responses
to harmless antigens, it must retain the ability to express effective, defensive responses to potentially
harmful pathogens. Thus, one of the most crucial aspects of the intestinal immune system is the
ability to discriminate between these different classes of antigen and to respond appropriately.
Identifying the mechanism(s) by which this discrimination is made represents the ‘holy grail’ of
mucosal immunology, since it would enable the development of rational approaches to control
allergic diseases in humans and animals, and to engineer non-replicating vaccines effective when
given orally. Despite decades of research in rodents, humans and pigs, we still do not understand
these mechanisms. However, it is becoming clear that the presence of the commensal intestinal
microbiota contributes to the development of an immune system capable of making this distinction
Host-microbial co-metabolism
Conventional approaches to metabolic profiling have involved submitting blood or urine samples
for analysis of levels of specific, known metabolites and comparison with ‘normal’ and ‘disease’
ranges. However, the availability of high-throughput, next generation approaches to characterisation
of small molecules in biological fluids and tissues has identified an enormous range of metabolites,
many of which are not components of well characterised metabolic pathways. It is now possible
to submit a sample for simultaneous quantitative analysis of over 500 characterised metabolites.
Experimental approaches allow even more detailed comparison of metabolic profiles including the
unknown metabolites. Although the full pathways in which these molecules are involved is often not
known, they can be used as empirical ‘biomarkers’ for health and disease (Nicholson et al., 2012).
Part of the difficulty in identifying pathways associated with many of these molecules arises from
the fact that they are often products of microbial metabolism rather than host, and that they are
present in blood or urine as a consequence of absorption across a normal or damaged intestine. Their
presence, therefore, may be indicative of colonisation by particular classes of bacteria engaged in
specific pathways of metabolic activity. Even greater difficulty arises from the fact that in many cases,
these molecules are host-microbial co-metabolites: that is, they are initially synthesised by intestinal
bacteria then absorbed and subsequently modified by host metabolism or vice-versa. While, at the
moment, this raises difficulties in formally identifying the pathways by which these molecules are
produced, it does mean that many of them are indicative of the relationship between the mammalian
host (human or pig) and the intestinal microbiota.
Experimental studies of enteric health

One thing which is clear from the previous section is the extent to which the intestinal microbiota
is involved in generating and maintaining ‘enteric health’, although the exact interactions are only
beginning to emerge. Most of the work has been carried out in laboratory rodents, some in humans
and a small amount in pigs.
Experimental models in laboratory rodents
Laboratory mice are, clearly, monogastric omnivores and experimental studies are likely to be at
least relevant to pigs. They are highly genetically manipulable, and very well characterised in terms
of reagents. This characterisation is important for genetic and epigenetic studies, but it is worth
noting that it is less important for high-throughput approaches to phenotypic characterisation such
as proteomics and metabolomics. One of the major difficulties in interpretation of the rodent studies

is that they have been subjected to a long history of laboratory breeding. This process has involved
repeated re-derivations by Caesarean section and rearing in germ-free and gnotobiotic environments,
to the point where the extent to which the kind of microbiota present in the intestine of a laboratory
mouse is representative of normal, wild mice is not clear. It has become apparent, for example,
that at least some studies identifying effects of particular genes on microbiota were confounded by
the fact that a knock-out strain and the wild type strain were kept in separate colonies, and that the
microbiotas in the two colonies have diverged for stochastic reasons rather than as a consequence
of the gene knock-out.
One further problem with rodents is that the young are altricial and difficult to rear away from the
mother. As a consequence, studies of the interaction between microbiota and host are usually carried
out by colonising adult, weaned, germ-free mice with a defined or a conventional microbiota. Whether
such studies are representative of colonisation of humans or piglets with commensal microbiota in
the neonatal period is also not clear.
Given the caveats above, mouse studies have demonstrated a clear, causal link between microbial
colonisation of the intestine and development of a normal, functional immune system (Hooper et
al., 2012). In truly germ-free mice, both the systemic and the mucosal immune system are poorly
developed. Importantly, several independent studies have demonstrated that germ-free mice are not
able to generate normal oral tolerance to fed proteins. Although the interpretation of these studies is
difficult, they do clearly demonstrate the importance of microbiota. Colonisation with a conventional
microbiota or a defined, restricted microbiota can restore relatively normal immunological function.
In fact, some studies have suggested that colonisation with a single bacterial species or even a single
capsular polysaccharide can restore normal function in laboratory rodents.
Similar studies have implicated microbiota in the development of metabolic disease in mouse models.
In diabetes-prone mice, modification of the intestinal microbiota either directly by administration
of specific strains, or indirectly by administration of prebiotics, can influence the development of
disease. The microbiota of obese mice differs from that of normal mice, and transfer of the ‘obese’
microbiota can transfer a predisposition to obesity. Effects of microbiota on metabolism, energy
balance and fat deposition are clearly relevant to pigs as production animals, but the mechanisms
are not sufficiently clear to allow direct translation. In addition, the effectiveness of single strains
of bacteria, or the association between disease and single strains, is probably an artefact of the
laboratory rodent as a model.
Epidemiological studies in humans
Although limited intervention experiments are possible in humans, the majority of studies are
epidemiological. These studies have become possible as a consequence of developments in DNA
sequencing technology which allow direct sequencing of specific genes in the full microbial
community in the human intestine (Voreades et al., 2014). As a consequence, it has become apparent
that the normal microbiota is much more diverse than originally thought, containing one to two
thousand species or strains from an identified pool of around five thousand. Many of these species
or strains are known only from their sequence, since around 60% of the gut microbiome turns out
to be unculturable by current, conventional techniques.
Studies using these approaches in human infants have demonstrated that the initial microbiome
appearing immediately after birth has relatively limited diversity, but that this diversity increases over
time. Importantly, however, it seems that the microbiota of infants is not a subset of the microbiota
of adults: the composition of the microbiota changes with age in a process similar to that seen in
the succession of plant species seen after a major woodland fire. This analogy is important: studies
in humans suggest that the gut microbiota needs to be considered as an ecosystem. In this model,

complex interactions between species and the host determine the abundance of different species
and strains, and environmental influences around birth, weaning or antibiotic treatment can have
either transient or long-lasting effects, depending on the robustness or resilience of the ecosystem
A large body of epidemiological literature now supports the relationship between microbiota and
immunological disease in humans. While genetic predispositions are clear, early life environment
has a strong influence on later susceptibility to allergic disease and to inflammatory bowel disease.
Specific factors known to be associated are a history of pathogen infection, urban or farm-living,
exposure to animals. Similarly, differences in early and later microbiota have been linked to the
development of diabetes and obesity in humans and, in some cases, causal links have been suggested
by transfer of human microbiota into mice.
Intervention studies in pigs
Experimental studies in pigs are clearly of value for understanding the role of the microbiota in
determining health and productivity of pigs as agricultural species. However, they are also of value
as models for human infants. Large litter size and the fact that piglets are precocial means that
individual piglets can be removed from the sow and reared in a range of different conditions. Studies
on true germ-free piglets have confirmed the observations from mouse models that colonisation
drives development of the mucosal immune system and the ability to respond to defined antigens.
However, the husbandry difference between germ-free and conventional pigs is dramatic and far
exceeds the kind of differences seen between conventional piglet environments. We have further
demonstrated that farm-to-farm differences also influence both development of the microbiota and
the immune system. Importantly, some of these differences are laid down early and appear to persist
for significant periods of time. In our studies, we have not maintained piglets beyond 8 weeks but
it is still possible, at this point, to identify differences between piglets born on different farms and
moved to the same environment after the first 24 hours only. Studies on the appearance and resilience
of the microbiota in young piglets confirm that it may be determined early in life and be relatively
difficult to change. Using inbred piglets to minimise genetic effects, litters were mixed after weaning
and the microbiota sampled up to four weeks later. Not surprisingly, weaning resulted in a marked
shift in the microbiota. However, four weeks after weaning, litter was a still much more important
determinant of microbiota than was postweaning pen, indicating that the microbiota acquired before
weaning was a strong determinant of the later composition.
Finally, we have attempted to manipulate the developing microbiota, the immune system and the
metabolism of young piglets by administering a human probiotic as a model organism (Lewis et
al., 2013). Probiotic administration had clear effects on all three components, although the effects
were not entirely as expected. The probiotic resulted in clear upregulation of proteins associated
with epithelial tight cell junctions, indicating increased barrier function in supplemented animals.
In contrast, the effects on synthesis of immunoglobulins in mucosal tissues were anomalous. The
European Food Standards Agency accepts increased presence of IgA (the immunoglobulin class
specialised for mucosal defence) as evidence of probiotic activity. However, in our studies, IgA was
decreased in the majority of mucosal tissues in probiotic supplemented animals, and we interpret this
as further evidence for increased barrier function resulting in reduced uptake of antigenic material
and, consequently, reduced synthesis of specific antibody. Importantly, the only tissue in which IgA
was increased was colon, and this might explain the increases seen in other trials where IgA was
measured in faeces. The study we carried out included two feeding arms, where either soya or egg
formed the major protein source. Not surprisingly, weaning diet had effects on metabolism, but also
interacted with probiotic supplementation in their effects on metabolism and immunity, indicating
that the effects of intervention are likely to be diet-dependent (Merrifield et al., 2013).

Future studies: what do we need to know?
Despite the amount of work in humans, rodents and pigs, there are important questions which
remain. The answers to these questions are likely to come from work in humans, since investment
in studies of the relationship between microbiota, metabolism, immunity and ‘health’ is massively
greater than that available for pigs or rodents.
Firstly, is the normal, adult, intestinal microbiota stable or fluctuating? Studies to date indicate that
it is relatively stable in any one individual, changing slowly over time and probably containing a
core group of organisms. However, the studies demonstrating this have largely used sequencing of
a single gene, encoding the 16S subunit of the ribosomal RNA, and assumed that similar sequences
indicate the same species, strain or operational taxonomic unit. This is a relatively coarse approach
and the new techniques of metagenomics, in which whole genomes are sequenced, are likely to
identify variation previously undetected by 16S rRNA sequencing. While the microbiome is likely
to be stable at the coarse level of 16S rRNA sequencing, it remains to be seen how stable it is at a
more detailed level. Importantly, we will then need to determine how biologically important such
fine variation is.
Secondly what actually constitutes a ‘healthy’ microbiota? There have been, and continue to be,
a number of simple algorithms to achieve this. For example, the diversity of the microbiome is
frequently cited as a measure of enteric health. One possibility is that diversity is negatively associated
with resilience: it may be that a reduction in diversity makes the microbiota less stable and more likely
to allow ingress of pathogens. While there are precedents for this in ecology, the evidence for it in
‘enteric health’ is largely empirical: microbiotas associated with some disease states do show reduced
diversity but there is no direct evidence that it is the diversity which actually causes the problem,
rather than the composition. Further, we need to be careful in interpretation, since current techniques
are actually measuring a genotype, rather than a phenotype. From the point of view of the pig, it is
unlikely that the genetic composition of the microbiota is the key determinant of ‘health’. It seems
more likely that it is the function of the ecosystem which determines ‘health’ or ‘disease’. Future
approaches to understanding microbial ecosystems in the gut are likely to include metagenomics,
metatranscriptomics, proteomics and metabolomics: however, the bioinformatics and biostatistics
tools necessary to link these different data domains also need to be developed.
However, the questions we need to ask can probably be formulated now. For simplicity, we can
think of a multi-dimensional ‘space’ of possible microbiotas or microbial ecosystems. Within the
total space of all possible variations, there will be areas which will be lethal, corresponding to major
pathogen infections, areas which are survivable, and areas which will be ‘healthy’. The questions
relate to whether there is a single space corresponding to optimal ‘enteric health’ or multiple separate
spaces; whether these spaces are actually optimally healthy under different rearing conditions;
whether microbiotas move within and between these spaces slowly or in jumps; and, importantly,
how can we drive microbiotas towards specific, optimally healthy areas.
References
Bailey, M., 2009. The mucosal immune system: recent developments and future directions in the pig. Developmental
and Comparative Immunology 33(3): 375-383.
Hooper, L.V., D.R. Littman and A.J. Macpherson, 2012. Interactions between the microbiota and the immune system.
Science 336(6086): 1268-1273.
Johansson, M.E.V., H. Sjovall and G.C. Hansson, 2013. The gastrointestinal mucus system in health and disease. Nature
Reviews Gastroenterology & Hepatology 10(6): 352-361.

Lewis, M.C., D.V. Patel, J. Fowler, S. Duncker, A.W. Zuercher, A. Mercenier and M. Bailey, 2013. Dietary
supplementation with Bifidobacterium lactis NCC2818 from weaning reduces local immunoglobulin production
in lymphoid-associated tissues but increases systemic antibodies in healthy neonates. British Journal of Nutrition
110(7): 1243-1252.
Merrifield, C.A., M.C. Lewis, S.P. Claus, J.T.M. Pearce, O. Cloarec, S. Duncker, S.S. Heinzmann, M.E. Dumas, S.
Kochhar, S. Rezzi, A. Mercenier, J.K. Nicholson, M. Bailey and E. Holmes, 2013. Weaning diet induces sustained
metabolic phenotype shift in the pig and influences host response to Bifidobacterium lactis NCC2818. Gut 62(6):
842-851.
Nicholson, J.K., E. Holmes, J. Kinross, R. Burcelin, G. Gibson, W. Jia, and S. Pettersson, 2012. Host-gut microbiota
metabolic interactions. Science 336(6086): 1262-1267.
Voreades, N., A. Kozil and T.L. Weir, 2014. Diet and the development of the human intestinal microbiome. Frontiers
in Microbiology 5: 494.

Pork production with maximal nitrogen efficiency
S. Millet*, M. Aluwé, J. De Boever and S. De Campeneere
ILVO (Institute for Agricultural and Fisheries Research), Scheldeweg 68, 9090 Melle, Belgium;
sam.millet@ilvo.vlaanderen.be
Abstract
By raising pigs, plant protein is converted into animal protein. The major part of ingested protein is
excreted to the manure, with potential losses to the environment. For sustainable pork production,
it is important to maximize the conversion of plant to animal production. The latter can be obtained
through different management and nutritional strategies, including housing conditions, genetic
selection, castration decision, slaughter weight and at last the diet.
Keywords: protein, nitrogen excretion, amino acids, management strategies, nutrition
Introduction
In contemporary pig production, approximately 6.3 kg N is used to raise an 8 kg piglet to a 110 kg
finishing pig (own calculations based on current Belgian feeds and breeds). While approximately
45% of this N is retained in the animal, the other 55% is excreted, mainly through faeces and urine.
The excreted N can in turn be used as nutrient for plant production. However, part of this N is lost
as ammonia (NH3), nitrous oxide (N2O) and N oxides (NOx) emissions to the air and leaching and
runoff of nitrate (NO3) and other N compounds to ground and surface water (Leip et al., 2014).
This abstract describes different management and nutritional strategies aiming to maximize the
conversion of plant to animal protein.
Management strategies
From animal to farm
Group housing is common practice for piglets and fattening pigs on farms. Feed formulation is
adapted according to the different age groups. However, also individual pigs of the same age group
differ in protein deposition capacity and hence, they may differ in amino acid (AA) requirement.
This variation is important when formulating recommendations for feeding pigs in groups and may
explain differences in research results on individual or group level. Recommendations on group
level may be of most practical value. For recommendations on farm level, optimisation should be
done per ‘animal place’ on the farm, taking into account all trade-offs between inputs and outputs.
Genetic selection
Improving feed energy efficiency is a major objective of current animal breeding programs (Shirali et
al., 2012). Without changing the diet, it is clear that pigs with a low feed conversion ratio consume,
and hence excrete less N per kg of gain. Still, in experiments with genetically different pig lines on
feeds that limited growth, the efficiency of protein use did not differ between breeds (Kyriazakis et
al., 1994; Susenbeth et al., 1999). As such, one could expect that more efficient pigs – with mostly
a higher lean meat content – have higher dietary AA needs per kg gain, but not per kg lean gain.
In contrast with previous authors, Moehn et al. (2004) stated that it is possible to select for lysine
catabolism. Also, with a higher proportion of muscle nitrogen (N) in the body, the amount of meat
per kg of N input and the ratio of muscle to maintenance N is probably higher.

Castration decision
Boars have a higher protein deposition than gilts or barrows. Immunocastrates can be considered
boars until the second vaccination. Thereafter, their feed intake increases drastically. Differences in
N efficiency may be mainly visible in diets with sufficient AA levels. In line with the observations
of Moehn et al. (2004), one could expect higher marginal lysine efficiency in boars compared to
barrows. Moreover, the ratio of muscle to total body protein and the ratio of protein for growth
versus maintenance may also be higher in boars versus barrows.
Slaughter weight
Empirical results suggest that the marginal efficiency of using standardised ileal digestible (SID)
lysine intake for protein deposition decreases with increasing bodyweight (BW), from 0.68 at 20 kg
to 0.57 at 120 kg BW (NRC, 2012). Above, maintenance AA requirements increase with increasing
BW. Both imply a higher need of AA per kg of lean gain with increasing BW. On the other hand, a
higher BW involves a lower number of pigs per 1000 kg pork.
Nutritional strategies: precision feeding for maximal nitrogen efficiency

Decreasing the dietary crude protein content with optimal AA concentrations
Decreasing the dietary crude protein (CP) content while maintaining optimal SID AA concentrations
has been proven a successful strategy to reduce N input per kg of lean gain. This can be obtained by
combining highly digestible AA sources and formulation of feeds for the optimal AA composition.
The increasing availability of feed grade AA makes it possible to decrease the CP content in the
diet. However, with decreasing CP levels, it is important to know the requirements of all essential
AA as a deficiency in one AA may lead to suboptimal use of other AA. Although feeding highly
digestible AA sources is an efficient way to decrease faecal N output, one should also consider other
aspects of sustainability. For example, using local protein sources may be less digestible but more
sustainable than better digestible sources. Also, it may be more important to reduce urinary than
faecal N excretion.
Optimal SID lysine:CP ratio
While it is generally accepted that animals need AA rather than protein, the question remains at
which level N per se is limiting. Already in 1993, Henry and Dourmad suggested that the crude
lysine:protein ratio should not exceed 0.065 to 0.068. This ratio was suggested to limit the risk for
deficiencies in non-essential AA or in essential AA that have not been taken into account. With a
better knowledge on the requirement of all essential AA, it is possible that the CP content can be
diminished further until N by itself becomes limiting. Wu (2014) states that minimal levels are also
required for non-essential AA. Functional AA are absorbed in the small intestine. Non-protein N can
also be absorbed in the large intestine and increase efficiency if the ratio of non-essential to essential
AA is too low (Mansilla et al., 2015). Thus, for practical diet formulation, it would be good to give
recommendations for the maximal SID lysine/CP ratio.
Phase feeding and compensatory growth
Phase feeding – adapting the dietary AA content to the physiological needs of an animal – is a
recognized strategy to lower N inputs while maintaining maximal performance. An alternative
approach for reducing overall N input per kg pork is using compensatory growth mechanisms (Fabian
et al., 2004; Millet and Aluwé, 2014). However, results between studies are somewhat conflicting,

making it difficult to give general recommendations for maintaining profitability while minimizing
N excretion through short term dietary protein deficiencies.
References
Fabian, J., L.I. Chiba, L.T. Frobish, W.H. McElhenney, D.L. Kuhlers and K. Nadarajah, 2004. Compensatory growth
and nitrogen balance in grower-finisher pigs. Journal of Animal Science 82(9): 2579-2587.
Kyriazakis, I., D. Dotas and G.C. Emmans, 1994. The effect of breed on the relationship between feed composition
and the efficiency of protein utilization in pigs. British Journal of Nutrition 71(6): 849-860.
Leip, A., F. Weiss, J.P. Lesschen and H. Westhoek, 2014. The nitrogen footprint of food products in the European
Union. The Journal of Agricultural Science 152(S1): 20-33.
Mansilla, W.D., D.A. Columbus, J.K. Htoo and C.F. De Lange, 2015. Nitrogen absorbed from the large intestine
increases whole-body nitrogen retention in pigs fed a diet deficient in dispensable amino acid nitrogen. The Journal
of Nutrition 145(6): 1163-1169.
Millet, S. and M. Aluwé, 2014. Compensatory growth response and carcass quality after a period of lysine restriction
in lean meat type barrows. Archives of Animal Nutrition 68(1): 16-28.
Moehn, S., R.O. Ball, M.F. Fuller, A.M. Gillis, and C.F. De Lange, 2004. Growth potential, but not body weight or
moderate limitation of lysine intake, affects inevitable lysine catabolism in growing pigs. The Journal of Nutrition
134(9): 2287-2292.
National Research Council (NRC), 2012. Nutrient requirements of swine (11th rev. Ed.). The National Academy Press,
Washington, DC, USA, 420 pp.
Shirali, M., A. Doeschl-Wilson, P.W. Knap, C. Duthie, E. Kanis, J.A.M. van Arendonk and R. Roehe, 2012. Nitrogen
excretion at different stages of growth and its association with production traits in growing pigs. Journal of Animal
Science 90(6): 1756-1765.
Susenbeth, A., T. Dickel, A. Diekenhorst and D. Höhler, 1999. The effect of energy intake, genotype, and body weight
on protein retention in pigs when dietary lysine is the first-limiting factor. Journal of Animal Science, 77(11):
2985-2989.
Wu, G., 2014. Dietary requirements of synthesizable amino acids by animals: a paradigm shift in protein nutrition.
Journal of Animal Science and Biotechnology 5: 34.

Feeding strategies to manipulate in vivo protein turnover
and post mortem proteolysis in meat
M. Therkildsen* and N. Oksbjerg
Department of Food Science, Aarhus University, Blichers Allé 20, 8830 Tjele, Denmark;
margrethe.therkildsen@food.au.dk
Abstract
Muscle protein turnover (synthesis and degradation) in meat animals is a dynamic process with
immediate response to the feeding level. A feeding strategy which leads to high daily gain is often
established on the basis of high muscle protein synthesis but also high muscle protein degradation.
This may be in favour of the meat tenderness post mortem, as it is the same proteolytic enzymes
which is involved in muscle protein degradation in vivo and post mortem, leading to tenderization.
Thus ad libitum feeding versus restricted feeding will lead to increased protein turnover and more
tender meat, but also a compensatory growth strategy can stimulate protein turnover and lead to
more tender meat.
Keywords: compensatory growth, muscle protein degradation, tenderness
Introduction
Muscle protein turnover in meat producing animals is a dynamic process with a continuously
turnover of proteins throughout life, and which is dependent on the environmental conditions of the
animal. Muscle protein turnover consists of two processes: the muscle protein synthesis and muscle
protein degradation, the difference being muscle protein accretion – leading to muscle growth. In
the production of meat an optimised growth rate can be achieved in several ways, by an increased
protein synthesis, decreased protein degradation or an increase in both processes although with a
difference in rate. The interest in muscle protein turnover is relevant when it comes to animal growth,
however the rate of muscle protein degradation at the time of slaughter also influences the rate of
proteolysis post mortem and thereby the final meat quality post-mortem as degradation of muscle
proteins post-mortem is one of the main processes in meat tenderisation as well as flavour generation.
Meat tenderisation
Tenderness of meat is dependent on the species, sex, age, muscle type, as well as factors related to
the slaughter and handling post mortem. However, in a specific muscle, the level of tenderness is
related to level and stability of collagen, the degree of contraction of the muscle fibres and finally on
the level of muscle protein degradation which involves proteolysis by enzymes. The more activity of
proteolytic enzymes post mortem the tenderer is the meat. The proteolytic enzyme system the calpains,
which involves the calcium-activated enzymes µ- and m-calpain and the inhibitor calpastatin, seems
to be the rate limiting enzymes in the protein degradation, initiating the release and fragmentation of
the structural and cytoskeletal proteins leading to increased tenderness. Likewise, the calpain system
also plays an important role in the in vivo protein degradation, and consequently the muscle protein
turnover. Thus it is hypothesised, that increased protein degradation in muscle in vivo establishes
the potential for increased protein degradation post mortem and hence more tender meat.
Feeding level, protein turnover and meat tenderness

This hypothesis has been supported by manipulating with the feeding level offered to meat animals.
When cattle and pigs are on a long time restricted diet relative to ad libitum feeding, both protein
synthesis and degradation is decreased (Chang and Wei, 2005; Jones et al., 1990). In addition to

this, meat from animals fed ad libitum versus a restricted diet has been shown to be more tender
(Aberle et al., 1981; Kristensen et al., 2002).
Compensatory growth, protein turnover and meat tenderness

The feeding strategy compensatory growth has been shown to manipulate with the protein turnover in
the living muscle and thus may also affect the post mortem muscle protein degradation. Compensatory
growth is the phenomenon where an increased growth rate in response to ad libitum feeding follows
a period of feed restriction, which exceeds the growth rate of continuously ad libitum feed animals.
In experiments with cattle and pigs it has been demonstrated that both protein synthesis and
degradation is increased in the period with compensatory growth (Jones et al., 1990; Therkildsen
et al., 2004). Thus, compensatory growth may be a strategy to improve meat tenderness, and this
effect has been confirmed in experiments with pigs and cattle (Skiba et al., 2012; Therkildsen et
al., 2008). Compensatory growth is usually seen in growing animals, however in dairy cows, which
are fed to produce milk, muscle growth is also minimized. A strategy was tested where culled dairy
cows were subsequently exposes to a compensatory growth strategy; 3 weeks of restrictive feeding
followed by 6 weeks ad libitum feeding. The meat from these cows was tenderer relative to cows
slaughtered while in milk production. This suggests that the protein turnover can be stimulated even
in fully grown animals (Therkildsen et al., 2011).
Conclusions
Muscle protein turnover in vivo, post mortem degradation and meat tenderness is connected. It is
possible to stimulate post mortem degradation through a specific feeding strategy allowing for a high
growth rate at time of slaughter. This effect is valuable in production systems where the growth rate
is sub-optimal in periods – like extensive pasture or organic systems and where the meat quality
will benefit from a finishing feeding period.
References
Aberle, E.D., E.S. Reeves, M.D. Judge, R.E. Hunsley and T.W. Perry, 1981. Palatability and muscle characteristics of
cattle with controlled weight gain: time on a high energy diet. Journal of Animal Science 52: 757-763.
Chang, Y.M. and H.W. Wei, 2005. The effects of dietary lysine deficiency on muscle protein turnover in postweanling
pigs. Asian-Australasian Journal of Animal Sciences 18: 1326-1335.
Jones, S.J., D.L. Starkey, C.R. Calkins and J.D. Crouse, 1990. Myofibrillar protein turnover in feed-restricted and
realimented beef cattle. Journal of Animal Science 68: 2707-2715.
Kristensen, L., M. Therkildsen, B. Riis, M.T. Sørensen, N. Oksbjerg, P.P. Purslow and P. Ertbjerg, 2002. Dietary-induced
changes of muscle growth rate in pigs: effect on in vivo and postmortem muscle proteolysis and meat quality.
Journal of Animal Science 80: 2862-2871.
Skiba, G., R. Stanisława, E. Poławska, B. Pastuszewska, G. Elminowska-Wenda, J. Bogucka and D. Knecht, 2012.
Profile of fatty acids, muscle structure and shear force of musculus longissimus dorsi (MLD) in growing pigs
as affected by energy and protein or protein restriction followed by realimentation. Meat Science 91: 339-346.
Therkildsen, M., M.B. Houbak and D.V. Byrne, 2008. Feeding strategy for improving tenderness has opposite effects
in two different muscles. Meat Science 80: 1037-1045.
Therkildsen, M., S. Stolzenbach and D.V. Byrne., 2011. Sensory profiling of textural properties of meat from dairy
cows exposed to a compensatory finishing strategy. Meat Science 87: 73-80.
Therkildsen, M., M. Vestergaard, H. Busk, M.T. Jensen, B. Riis, A.H. Karlsson, L. Kristensen, P. Ertbjerg and N.
Oksbjerg, 2004. Compensatory growth in slaughter pigs – in vitro muscle protein turnover at slaughter, circulating
IGF-I, performance and carcass quality. Livestock Production Science 88: 63-75.

Hepatic metabolism of glucose in the adaptation to the transition period
in the dairy cow
H.M. Hammon*, C.T. Schäff, J. Gruse and C. Weber
Institute of Nutritional Physiology ‘Oskar Kellner’, Leibniz Institute for Farm Animal Biology (FBN),
18196 Dummerstorf, Germany; hammon@fbn-dummerstorf.de
Abstract
Ruminants such as dairy cows absorb very low amounts of glucose from the intestine. To meet
the huge glucose requirement for milk production with the onset of lactation, high-yielding dairy
cows produce enormous amounts of glucose predominantly in the liver. Therefore, the liver has to
adapt quickly to this elevated glucose demand by stimulation of endogenous glucose production,
i.e. glycogenolysis and gluconeogenesis. Marked changes in glucogenic enzyme syntheses and
activities around parturition ensure the high level of hepatic glucose production. The variable
enzymatic changes in liver mirror the different glucogenic precursor supply during first weeks of
lactation. Besides substrate regulation, the endocrine changes around parturition support hepatic
glucose production, enabling a coordinated regulation of precursor supply and glucose synthesis to
cover the glucose demand for lactation.
Keywords: endogenous glucose production, gluconeogenesis, glucogenic enzymes, endocrine and

substrate regulation
Introduction
High-yielding dairy cows nowadays utilise enormous amounts of glucose during lactation. Cows that
produce 40-60 kg milk/day may require more than 3-4 kg glucose/day, primarily for milk lactose
output (Aschenbach et al., 2010; Bell and Bauman, 1997; Reynolds, 2005). Glucose concentrations
in blood plasma decrease shortly after parturition and usually recover during the first month in
lactation (Gross et al., 2011; Hammon et al., 2009; Weber et al., 2013b). The decrease of plasma
glucose concentrations after parturition in high-yielding dairy cows mirrors the high priority of
the mammary gland for glucose utilisation (Bauman, 2000; Drackley et al., 2001). The amount of
available glucose is a precondition for achieving the possible genetically determinate milk production
because lactose is the major osmoregulator for mammary water uptake (Linzell, 1972; Rigout
et al., 2002). A close relationship between whole-body glucose flux and milk volume has been
already proposed (Aschenbach et al., 2010; Danfær, 1994). Some glucose is also needed for milk
fat synthesis, whereas glucose utilisation in extra-mammary tissue and for maintenance decreases
to minimal levels during early lactation (Bauman et al., 1970; Reynolds, 2005; Stangassinger and
Sallmann, 2004). In late gestation, significant amounts of glucose are also required for foetal growth
and development, but glucose requirements before calving are still much lower than during early
lactation (Bell, 1995; Drackley et al., 2001; Reynolds et al., 2003).
Ruminants barely absorb glucose in the intestine after feeding as most carbohydrates are fermented
in the forestomach. Thus, endogenous glucose production provides more than 90% of the glucose
(Aschenbach et al., 2010; Bergman, 1973, 1990; Young, 1977). The distinct increase of hepatic blood
flow and hepatic glucose output during the transition from pregnancy to lactation clearly demonstrates
the importance of hepatic glucose production to meet glucose demands of the mammary gland
(Danfær, 1994; Reynolds et al., 2003). An increased hepatic glucose production is accompanied
by elevated metabolisable energy intake after parturition (Reynolds, 2005). As stated above, the
level of endogenous glucose production is strongly related to milk performance (Aschenbach et al.,
2010; Hammon et al., 2010; Reynolds, 2005). However, digestive infusion of casein and propionate
stimulates whole-body rate of glucose appearance in dairy cows, but only casein infusion increases

mammary lactose output, indicating no direct link between whole-body glucose rate of appearance
and milk lactose output (Lemosquet et al., 2009b).
The contribution of intestinally absorbed glucose to whole-body rate of glucose appearance in

ruminants is still under discussion, because diets rich in ruminally undegradable starch stimulate
intestinal starch digestion and glucose absorption, and the true contribution of glucose from portal-
drained viscera (PDV) to whole-body rate of glucose appearance adds up to 20% (Breves and
Wolffram, 2006; Galindo et al., 2011; Loncke et al., 2009). Providing additional starch, glucose or
propionate by adapting the diet composition or by abomasal infusion does not necessarily result in
an elevated milk production, but changes milk composition and may improve the metabolic status
of the cows during early lactation (Reynolds, 2005; Rigout et al., 2003). On the other hand, the
contribution of renal glucose production to total glucose supply seems to be of minor relevance in
lactating cows (Galindo et al., 2011).
Hepatic glucose production in the transition period

As indicated above, hepatic glucose production, i.e. glycogenolysis and gluconeogenesis, is the
primary metabolic pathway to ensure glucose supply in ruminants (Aschenbach et al., 2010; Bergman,
1973; Young, 1977). With the onset of lactation, liver glycogen concentration rapidly decreases,
making glucose immediately available to cover demands for milk production (Drackley and Andersen,
2006; Duske et al., 2009; Weber et al., 2013b). At the same time, hepatic gluconeogenesis markedly
increases in dairy cows and the hepatic glucose output doubles after parturition (Aschenbach et
al., 2010; Bauman, 2000; Drackley et al., 2001; Reynolds et al., 2003). On the other hand, there
is no activity of glucokinase and no net utilisation of glucose in the liver, indicating that glucose
plays a minor role in hepatic energy production by glycolysis, although some postpartum changes
in glycolytic enzymes are described in literature (Aschenbach et al., 2010; Brockman, 2005; Loor,
2010; Rawson et al., 2012; Reynolds et al., 2003; Schäff et al., 2012). In addition, hepatic glycolysis
may be important to provide substrates for amino acid and lipid metabolism in the liver.
Substrates used for hepatic gluconeogenesis and changes during the

transition period
Regarding gluconeogenesis in liver, propionate originating from ruminal fermentation is the main
precursor, followed by lactate, amino acids, glycerol and others that are of minor importance
(Aschenbach et al., 2010; Bergman, 1973; Reynolds, 2005). During the transition from pregnancy to
lactation and in the early lactation, hepatic glucose release from propionate increases and propionate
is still the dominant glucogenic precursor in dairy cows. However, its relative contribution to hepatic
gluconeogenesis decreases immediately after parturition and other precursors, mainly lactate,
become more important (Aschenbach et al., 2010; Drackley et al., 2001; Larsen and Kristensen,
2013; Reynolds et al., 2003).
The main reason for the use of different glucogenic precursors is probably that the increase of feed
intake and propionate synthesis in the rumen deviate from the increase in hepatic glucose production
which does not meet glucose requirements for milk production (Aschenbach et al., 2010; Drackley
et al., 2001; Larsen and Kristensen, 2013; Reynolds et al., 2003). Gluconeogenesis from propionate
depends on feed (and metabolisable energy) intake and decreases during times of reduced feed intake
or starvation (Aschenbach et al., 2010; Bergman, 1973; Young, 1977). In early lactation, when
feed intake does not meet energy requirements for milk production and hepatic glucose production
enlarges at the same time, the demand of using other glucogenic precursors than propionate for
hepatic gluconeogenesis becomes obvious (Larsen and Kristensen, 2013). Especially hepatic lactate
removal increases during the transition period in dairy cows and the fraction of lactate utilisation for
gluconeogenesis rises (Aschenbach et al., 2010; Doepel et al., 2009; Drackley et al., 2001; Reynolds

et al., 2003). The available lactate results from increased PDV release, but enhanced endogenous
lactate production by Cori cycling also contributes to hepatic glucose production (Larsen and
Kristensen, 2013; Lomax and Baird, 1983; Reynolds, 2005; Reynolds et al., 2003; Stangassinger,
1997; Stangassinger and Sallmann, 2004). In fact, metabolic enzymes in skeletal muscle that
favour glycolysis and lactate formation increase, whereas enzymes involved in Krebs cycling and
glycogenesis as well as glycogen content in muscle decrease during the transition period in dairy
cows (Kuhla et al., 2011; Schäff et al., 2013).
Although lactate becomes more important as glucogenic precursor during the transition period,
this seems to be not the case for glucogenic amino acids (Aschenbach et al., 2010; Larsen and
Kristensen, 2013; Reynolds, 2005). Generally, most of the amino acids are able to serve as glucogenic
precursor in ruminants, but during the transition period cows may use amino acids principally for
milk protein synthesis (Bauman, 2000; Bergman, 1973; Larsen and Kristensen, 2013). Supporting
this assessment, abomasal amino acid infusion did not enhance hepatic glucose output in dairy
cows during early lactation (Galindo et al., 2015), albeit, an increase of hepatic glucose output was
seen after amino acid infusion in mid-lactation (Galindo et al., 2011). These ambiguous findings
on amino acid metabolism in cows at different stages of lactation suggest that metabolic priority is
changing with ongoing lactation and utilisation of amino acids for hepatic gluconeogenesis is less
important in early lactation. Among all amino acids alanine contributes to glucose re-cycling by
the alanine-glucose cycle and alanine is the preferential amino acid used as glucogenic precursor
in the liver (Aschenbach et al., 2010; Bergman, 1973; Larsen and Kristensen, 2013). However, the
contribution of alanine to hepatic glucose output after parturition does not exceed 5.6% (Larsen and
Kristensen, 2013; Reynolds et al., 2003).
The contribution of glycerol to hepatic glucose release is even less than the one of alanine (Larsen
and Kristensen, 2013; Reynolds et al., 2003), although glycerol is released during the transition
period from adipose tissue due to intensive lipolysis (Bauman, 2000; Drackley et al., 2001). Hepatic
glycerol removal increases immediately after parturition, but hepatic gluconeogenesis from glycerol
does not exceed 5% to total hepatic glucose production (Larsen and Kristensen, 2013; Reynolds et al.,
2003). Further substrates from ruminal fermentation like microbial D-lactate, ribose and pyrimidine
may additionally contribute to hepatic gluconeogenesis, but their ruminal synthesis depends on feed
intake (as this is the case for propionate, isobutyrate and valerate). Thus, the elevated dry matter
intake and incremental microbial activity may magnify their contribution to hepatic glucose output
with ongoing lactation (Bergman, 1990; Larsen and Kristensen, 2013).
Interestingly, Larsen and Kristensen (2013) summarised that 40% of liver glucose release is from
recycling of glucose carbon (lactate, alanine, glycerol) at day 4 of lactation. This calculation
implies the importance of endogenous sources for net hepatic glucose production in early lactation,
when feed intake does not meet energy requirements for milk production, supporting the previous
assumption on endogenous lactate metabolism (Stangassinger and Sallmann, 2004). At the same
time, irreversible loss of glucose decreases in non-mammary tissues, indicating a shift in energy
utilisation in these tissues using free fatty acids and ketone bodies instead of glucose (Bauman, 2000;
Larsen and Kristensen, 2013; Stangassinger and Sallmann, 2004). Besides an elevated hepatic glucose
production, the reduction of glucose utilisation in non-mammary tissue is an important mechanism
to guarantee glucose supply to the mammary gland.
Gluconeogenic enzymes involved in hepatic glucose production

The enzymes pyruvate carboxylase (PC), phosphoenolpyruvate carboxykinase (PEPCK), fructose-
1,6-bisphosphatase (FBPase) and glucose-6-phosphatase (G6Pase) catalyse the rate-limiting steps
of gluconeogenesis in the liver (Figure 1). PC catalyses the formation of oxaloacetate from pyruvate
in the mitochondria, and PEPCK the conversion of oxaloacetate to phosphoenolpyruvate both in

Figure 1. Simplified scheme of hepatic glucose metabolism in dairy cows after parturition.
Rate-limiting enzymes of endogenous glucose production (gluconeogenesis, glycogenolysis) are
bold black, enzymes of glucose consuming pathways (glycolysis, glycogen synthesis) are italic
grey. Abbreviations: FBPase = fructose-1,6-bisphosphatase (EC 3.1.3.11); G6Pase = glucose
6-phosphatase (EC 3.1.3.9); GYS = glycogen synthase (EC 2.4.1.11); PC = pyruvate carboxylase
(EC 6.4.1.1); PEPCK = phosphoenolpyruvate carboxykinase, (c = soluble; m = mitochondrial; EC
4.1.1.32); PFK = phosphofructokinase (EC 2.7.1.11); PK = pyruvate kinase (EC 2.7.1.40); PYG =
glycogen phosphorylase (EC 2.4.1.1). Day postpartum (d pp) close to PC, PEPCKm, PEPCKc and
G6Pase refers to the time point when gene expression starts to increase after parturition (Graber
et al., 2010; Greenfield et al., 2000; Hammon et al., 2009; Kinoshita et al., 2016; Ostrowska et al.,
2013; Van Dorland et al., 2009; Weber et al., 2013a).
the mitochondria and in the cytosol. FBPase facilitates the conversion of fructose-1,6-bisphosphate
to fructose-6-phosphate in the cytosol, and G6Pase, a membrane-bound enzyme complex in the
endoplasmic reticulum, catalyses the release of free glucose that is released into the blood (Bergman,
1973; Danfær, 1994; Donkin, 1999; Hanson and Reshef, 1997; Jitrapakdee and Wallace, 1999;
Kraus-Friedmann, 1984; Pilkis and Granner, 1992; Rognstad, 1979; Van Schaftingen and Gerin,
2002; Young, 1977). These enzymes are subjected to both nutritional and hormonal control in dairy
cows (Aschenbach et al., 2010; Baird et al., 1980; Bergman, 1973; Danfær, 1994; Donkin, 1999;
Young, 1977).
Hepatic gene expression of PC, cytosolic (PCK1) and mitochondrial form of PEPCK (PCK2) and
G6Pase (G6PC) increases during the transition from pregnancy to lactation, but time changes differ
among enzymes. Gene expression of PC, PCK2 and G6PC increases immediately at parturition,
whereas PCK1 mRNA abundance increases 2 weeks after parturition (Greenfield et al., 2000;
Hammon et al., 2009; Van Dorland et al., 2009; Weber et al., 2013a, 2015; Figure 1). It is quite
evident that the regulation of hepatic gene expression is different among gluconeogenic enzymes in
dairy cows and obviously related to the metabolic changes with the onset of lactation. As discussed

above, due to insufficient propionate supply at parturition, a shift in substrate availability occurs for
hepatic gluconeogenesis resulting in elevated fractional lactate utilisation and decreased fractional
propionate utilisation for glucose production (Aschenbach et al., 2010; Lomax and Baird, 1983;
Reynolds et al., 2003). These changes in substrate availability might favour gene expression of PC
and PCK2 instead of PCK1 immediately after parturition (Brockman, 2005; Kinoshita et al., 2016;
Loor, 2010; Ostrowska et al., 2013; Weber et al., 2013a) and partly fit to changes in gluconeogenic
enzyme expression seen after feed restriction in dairy cows (Velez and Donkin, 2005), indicating
comparable mRNA expression patterns during the early transition period and feed restriction.
The increased PC gene and protein expression coincides with the time point of lowest feed intake
and highest plasma concentrations of non-esterified fatty acids (NEFA), pointing at greatest degree
of body fat mobilisation around parturition (Drackley et al., 2001; Greenfield et al., 2000; Ingvartsen
and Andersen, 2000; Karcher et al., 2007; Loor et al., 2006; Ostrowska et al., 2013; Sejersen et al.,
2012; Weber et al., 2013a). The increasing NEFA release around parturition may stimulate hepatic PC
gene expression by activating PC promoter 1 (White et al., 2011), but elevated doses of NEFA inhibit
PC mRNA abundance and enzyme activity in bovine hepatocytes (Li et al., 2012). The stimulation
of oxaloacetate synthesis by PC results in elevated substrate utilisation for glucose production,
but at the same time augments the tricarboxylic acid cycle and enhances fatty acid oxidation.
Therefore, elevated PC activity around parturition mirrors both elevated gluconeogenesis and fatty
acid oxidation, and fatty acid oxidation provides the energy necessary for gluconeogenesis (Danfær,
1994; Loor, 2010; Nelson and Cox, 2001; Rawson et al., 2012). This is supported by the fact that
acetyl-CoA is a direct allosteric activator of PC (Aschenbach et al., 2010; Nelson and Cox, 2001).
Therefore, oxaloacetate is a key-substrate in hepatic energy metabolism and a deficit of oxaloacetate
in the tricarboxylic acid cycle shifts hepatic energy metabolism to ketogenesis (Aschenbach et al.,
2010; Bergman, 1973; Drackley et al., 2001; Young, 1977).
On the other hand, the increase of PCK1 gene expression after parturition is delayed and is different
from the time change of PCK2 gene expression, which occurs immediately after parturition and is
associated with the rise in PC gene expression (Graber et al., 2010; Greenfield et al., 2000; Hammon
et al., 2009; Karcher et al., 2007; Ostrowska et al., 2013; Van Dorland et al., 2009; Weber et al.,
2013a). The different time pattern of PCK1 and PCK2 gene expression may result from different
substrate availability of glucogenic precursors for gluconeogenesis at parturition. Time changes of
PCK1 are associated with the rise of postpartum feed intake and the gradual increase of propionate
supply (Bergman, 1973; Brockman, 2005; Young, 1977). Obviously, propionate can directly affect
the promotor region of the PCK1 gene and establish a feed-forward mechanism of substrate control
(Aschenbach et al., 2010; Koser et al., 2008; White et al., 2016; Zhang et al., 2015), but in vitro
studies indicate a stimulation of PC and PCK2 mRNA abundance in hepatocytes of neonatal calves
as well (Zhang et al., 2016). The immediate rise of PCK2 gene expression at parturition may mirror
the enhanced fractional utilisation of lactate for gluconeogenesis, as described above (Aschenbach
et al., 2010; Doepel et al., 2009; Greenfield et al., 2000; Reynolds et al., 2003; Weber et al., 2013a).
Lactate is the preferred precursor when phosphoenolpyruvate is synthesized from oxaloacetate by
mitochondrial PEPCK. The conversion of lactate to pyruvate in the cytosol provides NADH that is
needed in the gluconeogenic pathway as well as in other cytosolic pathways associated with amino
acid and lipid metabolism (Aschenbach et al., 2010; Hanson and Reshef, 1997; Nelson and Cox,
2001). Supporting this suggestion, lactate dehydrogenase mRNA abundance increases strongly at
parturition (Ostrowska et al., 2013).
As propionate is a significant substrate for gluconeogenesis in ruminants this implies additional activity
of enzymes related to hepatic propionate entry into the gluconeogenic pathway (Aschenbach et al.,
2010; Bergman, 1973, 1990; Pilkis and Granner, 1992). Propionate is converted by mitochondrial
propionyl-CoA carboxylase (PCC), methylmalonyl-CoA mutase and in the tricarboxylic acid cycle
to oxaloacetate (Aschenbach et al., 2010; Bergman, 1973). Although PCC is affected by feed intake

(Baird and Young, 1975), the post-calving increase of gene expression and activity of PCC and its
association with elevated feed intake in early lactation is weak, but the time pattern of the postpartum
increase is consistent with the time pattern of PCK1 (Aschenbach et al., 2010; Kinoshita et al., 2016;
Murondoti et al., 2004; Weber et al., 2013a). Changes of the FBPase activity are barely seen during
the transition period in dairy cows (Murondoti et al., 2004; Rukkwamsuk et al., 1999). Studies on
the gene expression of FBPase indicate less regulation of this enzyme at the transcriptional level
(Aschenbach et al., 2010; Ostrowska et al., 2013; She et al., 1999). Activity and gene expression of
G6Pase increase after parturition in dairy cows (Murondoti et al., 2004; Rukkwamsuk et al., 1999;
Weber et al., 2013a). Time changes relative to parturition for G6PC gene expression differ from
changes of other enzymes, because mRNA abundance of G6PC increases immediately after birth,
as seen for PC and PCK2, but remains on a high level during first weeks after parturition (Cedeño
et al., 2008; Graber et al., 2010; Kinoshita et al., 2016b; Weber et al., 2013a). Therefore, the time
changes of G6PC after parturition may best fit to elevated hepatic glucose production, comprising
hepatic glycogenolysis and gluconeogenesis. The great enzyme activity of G6Pase in dairy cows
mirrors the importance of G6Pase for hepatic glucose output (Aschenbach et al., 2010).
Plasma glucose concentrations were not associated with protein concentrations of gluconeogenic
enzymes in the liver of dairy cows (Sejersen et al., 2012) and challenges of the glucose status in
dairy cows indicated only minor changes in the gene expression of gluconeogenic enzymes, implying
less regulation of gluconeogenic enzyme gene expression by the glucose in dairy cows (Al-Trad
et al., 2010). On the other hand, a negative relationship was observed between hepatic glycogen
concentration and particularly PC mRNA abundance during lactation, pointing to a feedback
mechanism of stored hepatic glycogen on gluconeogenesis (Weber et al., 2013a).
Endocrine regulation of hepatic glucose production in the transition

period
Besides substrate regulation, hepatic gene expression of gluconeogenic enzymes is under endocrine
control and insulin, glucagon, glucocorticoids, catecholamines and thyroid hormones are the primary
regulators of glucose metabolism in cattle (Aschenbach et al., 2010; Bauman, 2000; Bell and Bauman,
1997; Brockman and Laarveld, 1986; Donkin, 1999; Loor, 2010; McDowell, 1983; Vernon, 2005;
Weekes, 1991). During the transition from pregnancy to lactation plasma concentration of insulin
decreases and that of glucagon increases, resulting in a reduction of the insulin to glucagon ratio at the
beginning of lactation (Drackley et al., 2001; Hammon et al., 2009; Weber et al., 2013b). In addition,
plasma concentrations of cortisol and catecholamines are elevated at the time of calving (Weber et
al., 2013b). These endocrine changes favor hepatic glucose production by inducing gluconeogenesis
and glycogenolysis as the inhibitory action of insulin on both metabolic pathways is reduced. In
contrast, glucagon, cortisol and adrenaline stimulate hepatic glucose production by affecting gene
expression and enzyme activity of the gluconeogenic enzymes (Brockman and Laarveld, 1986;
Kraus-Friedmann, 1984; McDowell, 1983; Pilkis and Granner, 1992; Weekes, 1991).
Insulin inhibits gene expression of gluconeogenic enzymes during the transition period when applied
in supraphysiological doses during hyperinsulinaemic-euglycaemic clamp studies (Hammon et al.,
2012), but not in hypoglycaemic clamp studies (Kreipe et al., 2011). Especially gluconeogenesis
relying on propionate is less suppressed by insulin in adult ruminants (Donkin and Armentano,
1995; Smith et al., 2008). Furthermore, insulin impedes the propionate-induced stimulation of PC,
PCK1 and PCK2 mRNA abundance in bovine hepatocytes of neonatal calves (Zhang et al., 2016).
However, this effect might be reduced in the transition cow because of the low plasma insulin
concentration and the proposed insulin-resistant state of these cows (Bauman, 2000; De Koster
and Opsomer, 2013; Kautzsch et al., 2012; Vernon, 2005). Supporting this assumption, cortisol is
known to cause peripheral insulin resistance and to reduce glucose uptake in peripheral tissues of
dairy cows, finally leading to elevated plasma glucose concentrations. Hepatic glucose production

is less affected by cortisol (Kusenda et al., 2013; Starke et al., 2009). These cortisol effects are also
observed in neonatal calves (Scheuer et al., 2006). Reduced insulin sensitivity is further reflected
by decreasing plasma concentrations of adiponectin during the transition period in cows (Mielenz
et al., 2013) as adiponectin is an adipokine supporting insulin sensitivity (Yadav et al., 2013). In
ruminants, insulin does not completely depress gluconeogenesis as it is the case in monogastric
species, indicating a different regulation of gluconeogenesis (Kautzsch et al., 2012; Weekes, 1991).
This impaired depression of gluconeogenesis by insulin is also seen in neonatal and veal calves
(Hugi et al., 1998; Scheuer et al., 2006).
Glucagon is the main antagonist of insulin action with regard to glucose metabolism. It increases
plasma glucose concentrations and is therefore needed for glucose homeostasis (Brockman and
Laarveld, 1986; Kraus-Friedmann, 1984; McDowell, 1983; Pilkis and Granner, 1992; Weekes,
1991). Glucagon secretion is stimulated by reduced plasma glucose or by an elevated insulin status,
but stimulation of glucagon release by insulin in dairy cows depends on the level of plasma glucose
(Zarrin et al., 2015). In vitro studies with hepatocyte monolayers from neonatal calves indicate the
regulation of gluconeogenesis by glucagon (Donkin and Armentano, 1995) and glucagon application
affects gluconeogenic enzyme gene expression in dairy cows (She et al., 1999). So, elevated plasma
glucagon and the reduced insulin to glucagon ratio during the transition period contribute to the
stimulation of hepatic PC mRNA abundance in dairy cows (Hammon et al., 2009; Hanson and
Reshef, 1997; Jitrapakdee and Wallace, 1999; Weber et al., 2013a).
As mentioned above, the gluconeogenic effects of glucocorticoids have been questioned in the liver
of dairy cows, although major genes (PC and PCK1) contain responsive elements for glucocorticoids
in their promoters. Glucocorticoids are known to stimulate enzyme activities and gluconeogenesis
in other species (Aschenbach et al., 2010; Bergman, 1973; Drackley et al., 2001; Hanson and
Reshef, 1997; Kraus-Friedmann, 1984; Pilkis and Granner, 1992). Their main effect in dairy cows is
probably the provision of substrates for gluconeogenesis, e. g., amino acids, by stimulation of protein
degradation (Aschenbach et al., 2010; Bergman, 1973; Brockman and Laarveld, 1986; McDowell,
1983). Albeit glucocorticoids do not stimulate hepatic glucose output, they act anti-ketonic in dairy
cows thus justifying the treatment of ketosis with glucocorticoid agents (Baird and Heitzman, 1971;
Starke et al., 2009). Catecholamines stimulate PCK1 gene expression by enhancing intracellular
cAMP, as does glucagon. Catecholamines promote hepatic gluconeogenesis and glycogenolysis
by binding to hepatic α1- and β2-adrenergic receptors and by providing glycerol and lactate as
precursors for glucose synthesis (Bergman, 1973; Brockman, 1991; Brockman and Laarveld, 1986;
Kraus-Friedmann, 1984; McDowell, 1983; Pilkis and Granner, 1992). Thyroid hormones may also
be involved in the regulation of the hepatic glucose output, but probably more indirectly by affecting
the metabolic rate and with permissive action on other hormones. However, studies on glucose
metabolism in cattle are rare (Hanson and Reshef, 1997; Jitrapakdee and Wallace, 1999; McDowell,
1983; Kraus-Friedmann, 1984; Pilkis and Granner, 1992; Yen, 2001). Growth hormone may affect
hepatic glucose output both by inhibition of hepatic insulin action and by stimulation of the glucose
irreversible loss rate though enhanced lactose output, but it may not directly affect gene expression
or activity of gluconeogenic enzymes (Bergman, 1973; Brockman and Laarveld, 1986; Drackley et
al., 2001; Etherton, and Bauman, 1998; Kraus-Friedmann, 1984; McDowell, 1983).
Conclusions
Hepatic glucose metabolism adapts to the increased glucose demand for milk production by stimulation
of the hepatic glucose output, originating from elevated glycogenolysis and gluconeogenesis. The
increase of hepatic glucose production seems to be well balanced to provide sufficient glucose for
adequate milk production. Hence, the administration of extra glucose often fails to enhance milk
production (Al-Trad et al., 2009; Lemosquet et al., 2009a,b) and glucose supply seems not to be a
limiting factor for milk production in high-yielding, healthy cows (Reynolds et al., 2003). However,

in sick cows the challenged immune system may affect insulin-dependent glucose metabolism and
thus impairs hepatic glucose production (Garcia et al., 2015; Moyes, 2015; Moyes et al., 2013;
Vernay et al., 2012). An elevated body fat mobilisation and hepatic fat content may not impair hepatic
glucose output per se in healthy cows, but may affect hepatic propionate metabolism (Kautzsch et
al., 2012; McCarthy et al., 2015; Weber et al., 2013a,b).
Variable regulation of hepatic glucose production results from distinct changes of glucogenic
precursor availability. As the fractional propionate utilisation is reduced and propionate supply does
not increase adequately to the needs of the enhanced hepatic glucose production in early lactation,
lactate becomes more important as a glucogenic precursor (Larsen and Kristensen, 2013; Reynolds
et al., 2003). Therefore, hepatic conversion of lactate to glucose increases during early lactation
in dairy cows. The changes in glucogenic enzyme gene expression around parturition mirror these
changes in fractional substrate supply for gluconeogenesis and are specified in this review.
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Proteomic applications to study livestock growth efficiency
and meat quality
S.M. Lonergan1*, E. Huff-Lonergan1, J.K. Grubbs1, S.M. Cruzen2 and K.B. Carlson1
1Department of Animal Science, Iowa State University, Ames, IA, USA; 2School of Animal Sciences,
Louisiana State University, Baton Rouge, LA, USA; slonerga@iastate.edu
Abstract
Two major goals of animal agriculture are to improve the efficiency of meat production by optimizing
the growth performance of livestock and to achieve consistent production of high quality products
for the consumer market. Our long-term goal is to define the contribution of muscle metabolism
and function to both of these goals. A primary contributor to muscle phenotype and meat quality
characteristics is the muscle proteome. We have used protein chemistry and proteomic approaches
to define proteins and protein modifications that are linked to both muscle metabolism and meat
quality. Improvement in muscle growth efficiency has been achieved with genetic selection. The
cellular mechanisms at play appear to be both energy metabolism and protein turnover. Improved
muscle growth in swine has been linked to less electron leakage from the mitochondria. A parallel
observation is that muscle from more efficient pigs had a greater capacity to decrease protein
degradation and conserve muscle mass. Oxidative conditions interrupt the inhibition of calpain-1 by
calpastatin, perhaps linking oxidative stress to increased initiation of myofibrillar disassembly. Very
different proteomic results are noticed when pigs are exposed to acute heat stress. Muscle from heat
stressed pigs shows an increase in abundance of proteins involved with coping mechanisms to avoid
structural damage and protein oxidation. The combined proteomic evidence demonstrates abundance
and modification of structural, metabolic, stress response, and antioxidant proteins govern muscle
growth and maintenance. These conditions set the stage for variation in response to the conversion
of muscle to meat and therefore the ultimate quality of fresh meat. Protein oxidation, modification
of stress proteins, and protein degradation are all considerations in the balance between livestock
growth efficiency and meat quality.
Keywords: muscle, meat, proteomics
Introduction
Efficient production of food is a primary goal in agriculture. Production of food with fewer inputs
of valuable resources such as feedstuffs, water, and space continues to be a primary goal in a world
facing increasing population and fewer land resources to produce food. In livestock production, feed
efficiency is defined as the growth achieved by a unit of feedstuff. Muscle growth and protein accretion
are known to be affected by selection for improved growth efficiency or by outside stressors like
heat stress. Changes in efficiency are accompanied by changes in protein expression and abundance.
These changes set the venue for the muscle response to conversion to meat and therefore they have
the capacity to impact fresh meat quality. The objective of this presentation is to define the influence
of muscle metabolism on meat quality with the use of proteomic and protein chemistry techniques.
Improved muscle growth

One component of improvement in efficiency of growth is cellular metabolism, and there is
evidence that mitochondria function – or dysfunction – can have a direct effect on the efficiency
of the conversion of dietary energy to cellular fuel. As a consequence of normal electron transport,
electrons can escape from the electron transport chain to induce the formation of superoxide anions.
Mitochondrial efficiency could be evaluated by the rate and extent of the production of these products.
Investigations in our laboratory have demonstrated that muscle mitochondria from efficient pigs

exhibit less electron leakage from the electron transport chain (Grubbs et al., 2013a). Mitochondria
from muscle of the more efficient pigs had a greater abundance of heat shock protein 70, heat shock
protein 60, malate dehydrogenase, and ERO1 alpha. It is important to note that post-translational
modification of proteins plays a central role in mitochondria function. Numerous mitochondrial
proteins modified by phosphorylation (Grubbs et al., 2013b, 2014).
Heat stress
Acute heat stress is documented to change the muscle growth, even when accounting for an expected
change in feed intake (Cruzen et al., 2015). Heat stress generally results in an increase in enzymes in
the glycolytic pathway. Muscles that are more predominantly glycolytic in nature are prone to exhibit
a more disorganized microtubule structure, and an apparent capacity to strengthen the microfilament
structure. Finally it is noted that acute heat stress stimulates a response in muscle to utilise and
express antioxidant proteins such as superoxide dismutase, peroxiredoxin-2, and peroxiredoxin-6.
Connection of muscle metabolism to meat quality

The potential for protein oxidation tends to be greater in muscle of less efficient pigs (Grubbs et
al., 2013b), and the calpain:calpastatin ratio is lower in that muscle (Cruzen et al., 2013). It is
reasonable to suggest that a small increase in mitochondrial produced ROS and calcium could
result in small changes in protein turnover and accretion. Furthermore, a slower rate of postmortem
desmin degradation has been noted in muscle from low RFI pigs (Arkfeld et al., 2015). Less protein
degradation is frequently linked to poorer ultimate meat quality (Huff-Lonergan et al., 2010).
References
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Fasting heat production and metabolic body weight in non-ruminant
growing animals
E. Labussière*, S. Dubois, J. van Milgen and J. Noblet
INRA, UMR 1348 PEGASE, Domaine de la Prise, 35590 Saint-Gilles, France; Agrocampus Ouest,
Domaine de la Prise, UMR 1348 PEGASE, 35000 Rennes, France; etienne.labussiere@rennes.inra.fr
Abstract
Fasting heat production (FHP) of growing animals is indicative of their basal metabolic rate and it is
proportional to the metabolic body weight (MBW), calculated as BW raised to a certain exponent. It
can be estimated from the analysis of the decreasing kinetic of total heat production during a rather
short period of feed deprivation (about one day), as the horizontal asymptotic value corrected for zero
physical activity. Specific exponents should be used to calculate MBW in growing animals over the
growing period; a compilation of our data suggest 0.60 in pigs, 0.70 in broilers, 0.75 in turkeys and
0.85 in calves. Therefore, they may differ from the classical 0.75 exponent more adapted to adults.
From measurements conducted at different feeding levels, it appears that FHP varies by 0.13, 0.14
and 0.22 kJ per kJ variation in metabolisable energy intake prior to the fasting period, in turkeys, pigs
and calves, respectively. The size of the visceral organs and its evolution during growth would explain
the difference between species and the effect of feeding level on basal metabolic rate. Within species,
differences in FHP between breeds can be attributed to differences in visceral and protein mass,
whereas differences between sexes were only significant when animals approach sexual maturity.
Finally, high ambient temperature is associated with decreased FHP that can be mainly explained
by the anorexic effect of heat stress. To conclude, variations in FHP are indicative of variations in
maintenance energy requirements that should be taken into account in nutritional recommendations.
Keywords: fasting heat production, mathematical modelling, maintenance energy
Introduction
Fasting heat production (FHP) of animals is defined as the minimum energy expenditure of resting,
healthy, non-reproductive, fasting and adult animals that are in a thermoneutral environment during
the inactive circadian phase (McNab, 1997). It is indicative of their basal metabolic rate (Baker et
al., 1991). It is used to determine their maintenance energy requirements and to calculate net energy
value of diets and feedstuffs. In growing and producing animals, it is not possible to estimate directly
the FHP since total heat production (HP) has to be partitioned between what is due to maintenance
(FHP) and what is due to productive functions. The difference between total HP in a fed state and
FHP corresponds to heat increment of the feed which includes HP due to physical activity (AHP)
and HP related to the metabolic use of nutrients provided by the feed (TEF). Several methods have
been proposed to estimate FHP either based on measured HP during a rather long period of feed
deprivation, or from extrapolation to zero energy intake of the relationship between HP and energy
intake using linear or nonlinear models. Nevertheless, it has been demonstrated that the time to
measure the plateau HP during starvation may vary between species and at least 3 to 4 days of feed
deprivation should be considered to reach such a plateau (Chwalibog et al., 2005; Close and Mount,
1975). In addition, the value of FHP as estimated after this long duration of feed deprivation is
questionable and may not be representative of the ‘true’ value of FHP. Additionally, feed deprivation
may favour the occurrence of behavioural disturbances and associated HP that can deeply bias
and overestimate FHP values (Gerrits et al., 2015). Alternatively, the relationship between energy
intake and HP has been used, considering that the extrapolation of this relationship at zero energy
intake represents an estimate of FHP. This approach requires the implementation of different levels
of energy intake. Nevertheless, it considers that the intercept of the relationship between HP and
metabolisable energy (ME) intake does not depend on energy intake, that is the adaptation of energy

metabolism to low levels of energy intake is not considered, despite experimental evidences in pigs
and ruminants (Ferrell et al., 1986; Kim et al., 2015; Koong et al., 1985).
The concept of metabolic body weight (MBW) is closely linked with FHP. MBW has been proposed
to define a unit for metabolic measurements so that they are constant over a rather large BW range
when they are expressed relative to MBW. In mature animals, the exponent 0.75 was adopted for
interspecies comparisons (Kleiber, 1965), but the validity of this exponent for intraspecies studies
in growing animals has been questioned (Da Silva et al., 2006; Thonney et al., 1976; White and
Seymour, 2005). In growing animals, FHP instead of total HP should be used to define MBW as it
is indicative of the minimal energy expenditure and may not be affected by the level of BW gain, its
composition and associated heat increment. The objectives of the paper are to describe a methodology
to estimate FHP and MBW in growing animals and to highlight the main variation factors of FHP
in growing pigs, calves, broilers and turkeys.
Methodologies to estimate fasting heat production

Conceptually, total HP is partitioned between what is due to basal metabolism without physical
activity (FHP), to physical activity (AHP) and to the thermic effect of feeding (TEF) (Figure 1). To
partition HP in its components, several methods have been proposed that are based on the statistical
analysis of the daily dynamic patterns of HP in fed animals (Even et al., 1991; Van Klinken et al.,
2012). However, insufficient delay between successive meals does not allow a sufficient decrease
in instantaneous HP that lead to an erroneous estimate of the basal HP. Consequently, the value of
basal HP as estimated by these methods may include a contribution from TEF (Chwalibog et al.,
2004). To limit the contribution of TEF to the estimate of FHP, animals can be fasted, considering that
feed deprivation reduces glucose utilisation and stops protein deposition (MacDonald and Webber,
1995). Indeed, HP during a short period of feed deprivation (about one day) can be approached
as the sum of a constant FHP, AHP (which can be estimated from a quantitative measurement of
physical activity) and the adaptive HP from the fed state to fasting. The latter was modelled as a
first-order decline between the fed state and fasting, which resulted in an estimate of FHP that does
80
70
60
Heat production (MJ/day)
50
40 AHP
30
20
TEF
10
FHP
0
8:30 11:30 14:30 17:30 20:30 23:30 2:30 5:30
Time
Figure 1. Daily kinetics of heat production in a growing pig and its partitioning between components
due to basal metabolic rate (fasting heat production; FHP), thermic effect of feeding (TEF) and
physical activity (AHP).

not account for any contribution of remnant TEF and physical activity (Van Milgen et al., 1997). The
modelling approach was implemented on measurements of O2 consumption and CO2 production per
10 s intervals obtained with animals housed in open-circuit respiration chambers. The cage where
the animals were housed was mounted on force sensors that give an ‘instantaneous’ and quantitative
estimate of physical activity that contributes to whole body HP.
In order to consider the effects of previous feeding and housing conditions on the estimate of FHP,
several experiments with growing pigs, calves, broilers and turkeys were conducted in open-circuit
respiration chambers regulated in air temperature and relative humidity. Animals used to spend
at least 5 days in a fed state in the respiration chamber, either individually housed (calves, pigs)
or group-housed (pigs, broilers and turkeys), while nitrogen and energy balances were measured.
The animals were then fasted during one day. The size (1.7 or 12 m3) and the ventilation rate
(from 1 to 15 m3/h) of the chambers varied according to the weight and production level of the
animals. Gas concentrations in the outgoing air of each chamber were measured continuously
using paramagnetic differential (for O2) and non-dispersive infra-red (for CO2) analysers. Details
regarding the mathematical model and procedures to partition HP can be found in previous papers
(Labussière et al., 2013; Van Milgen et al., 1997). Results presented in this paper were obtained
using this latter methodology when animals were either housed at thermoneutrality and under heat
stress to determine the effects of this latter condition on FHP.
Intraspecies determination of metabolic body weight

In order to determine the best exponent for calculating MBW, experiments should be conducted over
a large range of BW, and ideally at least over a BW range that corresponds to the production period
of each species. Animals should not suffer from cold stress, which indicates that ambient temperature
in the respiration chamber should be higher than the lower critical temperature so that the lowest HP
is reached. The exponent for calculating MBW was then estimated through the relationship between
FHP and MBW and ME intake during the previous days to account for the effect of feeding level
on basal metabolic rate (Labussière et al., 2008b). Additionally, logarithmic transformation of the
relationship corrected the heteroscedasticity of the data, aiming at giving the same influence in the
relationship of data obtained at low or at high BW. The best exponent to calculate MBW during the
growing period differed from 0.75 in growing pigs, broilers and calves and was 0.60, 0.70, 0.75
and 0.85 for pigs, broilers, turkeys and veal calves, respectively (Labussière et al., 2008b; Noblet
et al., 1999, 2015; Rivera-Torres et al., 2010b). These values were estimated over the BW range
that corresponds to the production stages (25-100 kg in pigs; 60-265 kg in veal calves; 0.4-24.0
kg in turkeys, and 0.6-2.8 kg in broilers). The exponents did not differ between sexes and breeds
within the same species (Noblet et al., 2015; Rivera-Torres et al., 2010a; Van Milgen et al., 1998).
Because the visceral mass contributes to a great extent to whole body FHP (more than 35% of FHP
due to liver, heart and kidney; Ferrell et al., 1976), the classification of the exponents between
species follows the same order as the classification of allometric growth of visceral organs relative
to the whole body (from 0.7 in pigs to more than 1.0 in calves; Noblet et al., 1999; Rivera-Torres
et al., 2011; Robelin, 1986). When the basal metabolic rate was estimated at a particular BW and
then extrapolated over the whole growing period to estimate the maintenance energy requirements,
the utilisation of 0.75 exponent instead of species-specific exponents results in underestimating the
basal metabolic rate for BW lower than the BW used during measurements and overestimating it for
BW higher than the BW used during measurements for pigs, turkeys and broilers and the opposite
for veal calves. When a complete dataset with FHP measured at different BW is used to determine
a general estimate of maintenance energy requirements, the utilisation of the 0.75 exponent results
in systematically underestimating maintenance requirements (Noblet et al., 1999).

Effects of energy intake on fasting heat production
In growing pigs, calves, broilers and turkeys, FHP at zero activity has been estimated at different
stages of growth and the average values are presented in Table 1. Except for veal calves, FHP
contributes to 50% of total HP. The high contribution of FHP to total HP in veal calves (75%) has
to be related to the great size of the visceral tract in this species and to the high efficiency of using
energy from milk replacers (Labussière et al., 2009b). In growing pigs fed ad libitum, FHP averaged
0.77 MJ/kg BW0.60/d, which is in the range of values previously estimated with animals feed-
deprived for more than one day (0.57 to 0.94 MJ/kg BW0.60/d; Close and Mount, 1975; Koong et al.,
1983; Tess et al., 1984). In veal calves, the FHP averaged 0.30 MJ/kg BW0.85/d, in agreement with
previous estimates (0.30 to 0.35 MJ/kg BW0.85/d; Holmes and Davey, 1976; Webster et al., 1974).
These values were nevertheless twice than those that can be estimated from the extrapolation at zero
energy intake of the relationship between HP and ME intake (0.38 MJ/kg BW0.60/d and 0.14 MJ/kg
BW0.85/d in pigs and calves, respectively; Labussière et al., 2011). In growing broilers and turkeys,
FHP averaged 0.43 MJ/kg BW0.70/d and 0.41 MJ/kg BW0.75/d, respectively. These values are in
the range of values previously reported (Johnson and Farrell, 1985) or slightly lower (MacLeod et
al., 1979). In all species, it is rather difficult to compare our values with values reported previously,
because in most cases, these latter values were calculated from direct measurement of HP during
starvation and they included the contribution of variable levels of physical activity. Depending on
the intensity of feed deprivation and associated behavioural disturbances that have opposite effects
on the estimate of FHP, it is possible to reach similar values of FHP, but that do not include the
same components of metabolism. It may nevertheless be pointed out that behavioural disturbances
as stimulated by feed deprivation can deeply bias the estimate of basal HP, leading to erroneous
values of the maintenance components of energy requirements and energy value of diets. Differences
in housing conditions (mainly ambient temperature) can also affect the estimate of FHP (see later).
Because of the high contribution of visceral mass to whole body energy expenditure (Noblet et
al., 1999; Ortigues et al., 1995), any factor that affects the size of visceral organs would induce a
modification in FHP (Koong et al., 1985). Among them, the effects of variations in energy intake
are the most important. Preliminary studies have shown that previous feeding level modifies the
estimate of FHP (Koong et al., 1982; Vermorel et al., 1980). Using more than three levels of energy
Table 1. Interspecies comparison of losses of metabolisable energy (ME) intake as heat production
(HP) and its component fasting HP (FHP) in growing pigs, calves, broilers and turkeys fed ad
libitum a standard diet.1,2
Pigs3 Calves4 Broilers5 Turkeys6
MBW BW0.60 BW0.85 BW0.70 BW0.75

BW (kg) 72 (26) 156 (67) 1.47 (0.06) 9.3 (7.8)
ME intake (MJ/kg MBW per day) 2.59 (0.25) 0.62 (0.07) 1.63 (0.13) 1.25 (0.28)
FHP (MJ/kg MBW per day) 0.77 (0.07) 0.30 (0.02) 0.43 (0.02) 0.41 (0.05)
Utilisation of ME intake (% of ME)
As FHP 29.7 (1.2) 48.9 (2.9) 26.7 (1.5) 34.0 (5.4)
As HP 55.0 (2.1) 65.3 (3.5) 54.1 (6.5) 60.6 (4.8)
1 Values are means and their standard deviations in brackets

2 BW = body weight; MBW = metabolic body weight.
3 Le Bellego et al., 2001; Le Goff et al., 2002; De Lange et al., 2006; Barea et al., 2010; Labussière et al., 2013.
4 Labussière et al., 2008a, 2009a,b.
5 Noblet et al., 2007, 2009, 2015.
6 Rivera-Torres et al., 2010a,b.

intake, it is possible to calculate the contribution of previous feeding level on the estimate of FHP
(Figure 2). In growing turkeys, pigs and calves, the marginal increase of FHP equals 0.13, 0.14 and
0.22 kJ per supplementary kJ ME intake, respectively (Labussière et al., 2011). It is rather difficult
to compare the slopes calculated for the different species but it may be considered that the difference
in size of the visceral tract (with calves higher than pigs and poultry) may be responsible for the
differences between species. It may also be argued that calves were fed liquid milk that generates
rapid and massive absorption of nutrients postprandially. The increased feed intake is therefore
associated with large modification of energy and glucose metabolism, forcing the animal to cope
with a strong hyperglycemia postprandially.
Effects of sex and genotype on fasting heat production

Differences between breeds have been mainly evaluated in growing pigs and studies revealed that FHP
highly depends on BW composition and particularly on the size of the visceral and muscle masses
(Van Milgen et al., 1998). Nevertheless, all the variations between breeds cannot be explained by these
factors, whereas differences in composition of the visceral mass can lead to differences in FHP (Noblet
et al., 1999). The contribution of fat mass to FHP and the effect of genetically-induced differences in
fat content of BW are more controversial (no modification or reduction down to 30%; Baker et al.,
1991; Koong et al., 1983; Van Milgen et al., 1998). It seems that high body fat is associated with a
decreased FHP, but it is not known to what extent one is the cause or the consequence of the other
because decreased FHP can let energy available for fat deposition.
Within breeds, selection of animals for improved feed efficiency can also generate differences in FHP.
As an example, the selection for lower residual feed intake resulted in decreased feed intake (-7%)
and a lower FHP (-9%; Barea et al., 2010). The direct decrease in FHP induced by the decreased
feed intake (see above) only explains 30% of the difference in FHP between the two genetic lines.
A B
400
FHP (kJ/kg MBW/d)
800
750 350
700
300
650
600 250
550
500 200
1,200 1,700 2,200 2,700 400 600 800
C
500
400
300
200
700 900 1,100 1,300 1,500 1,700
ME intake (kJ/kg MBW/d)
Figure 2. Effects of metabolisable energy (ME) intake on fasting heat production (FHP) in growing
(A) pigs (De Lange et al., 2006), (B) calves (Labussière et al., 2009b), and (C) turkeys (Rivera-Torres
et al., 2010a). MBW = metabolic body weight calculated as BW0.60, BW0.85 and BW0.75 for pigs,
calves, and turkeys, respectively.

The difference in catabolic pathways activity involved in the Cori cycle may substantially explain
the decreased FHP in the most efficient animals (Le Naou et al., 2012).
Finally, the effects of sex on FHP were limited in pigs (Noblet et al., 1999). It may nevertheless be
pointed out that entire male pigs exhibited higher FHP relative to castrated pigs (0.86 vs 0.74 MJ/
kg BW0.60/d), only when they approach maturity (Labussière et al., 2013; Van Milgen et al., 1998).
This observation is consistent with what was observed between female and male turkeys (Rivera-
Torres et al., 2010a).
Effect of ambient temperature on fasting heat production

Apart from animal-based variations of FHP, environmental factors may also influence FHP estimates
in growing animals. One that has been widely studied is the effect of ambient temperature, when
it increased above the thermoneutral zone. In growing pigs, thermal heat stress induces metabolic
and behavioural adaptations that are mainly characterized by a severe decrease in feed intake and a
subsequent 20% decrease of FHP (Figure 3). However, approximately half of the difference may be
explained by the decreased ME intake directly induced by the anorexic effect of ambient temperature
(Campos et al., 2014; Collin et al., 2001a,b). Only the remnant decrease in FHP can be attributed
to decreased metabolic activity, independently of feed intake variations.
Conclusions and perspectives

The utilisation of modelling techniques to determine FHP in growing animals is useful to distinguish
the contributions of basal metabolic rate, heat increment, physical activity, (possible thermoregulation)
to total HP. The estimation of FHP at zero physical activity and immediately after a feeding period
limits the bias induced by behavioural disturbances generated by feed deprivation and adaptation
of metabolism. Using this methodology, it appears that specific coefficients should be used for
calculating MBW in growing farm animals. These coefficients differ from the classical 0.75 exponent
0.85
Fasting heat production
0.80
(MJ/kg BW0.60/d)
0.75 1
2
0.70
3
0.65
0.60
20 25 30 35
Ambient temperature (°C)
Figure 3. Effect of ambient temperature on fasting heat production in growing pigs. 1: Collin et al.,
2001a; individually housed 25 kg piglets; ME intake decreased from 2.52 to 1.95 MJ/kg BW0.60/d
between thermoneutrality (23 °C) and heat stress (33 °C); 2: Collin et al., 2001b; group-housed 30
kg piglets; ME intake decreased from 2.98 to 2.20 MJ/kg BW0.60/d between thermoneutrality (23 °C)
and heat stress (33 °C); 3: Campos et al., 2014; group-housed 70 kg pigs; ME intake decreased
from 1.42 to 1.21 MJ/kg BW0.60/d between thermoneutrality (24 °C) and heat stress (30 °C). Open
symbols are corrected fasting heat production (FHP) values calculated as measured FHP plus a
variation in FHP caused by the variation in ME intake between thermoneutrality and heat stress
(δME) and using a correction factor of 0.14 kJ/kJ δME (see above).

in pigs, broilers and calves. The utilisation of these coefficients when calculating energy requirements
during a prolonged period is fundamental to adequately describe the variations in maintenance
requirements during growth. Using this methodology, it is possible to determine the effects of
feeding level on basal metabolic rate. These effects should be taken into account when defining
energy requirements in growing animals.
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Part 1.
Physiological aspects of protein and energy metabolism and
nutrition: ruminants
Energy from fat increases milk lactose yield from dairy cows to the same
extent as energy from protein
K. Nichols1,3*, H. van Laar2, A. Bannink3 and J. Dijkstra1
1Animal Nutrition Group, Wageningen University, P.O. Box 338, 6700 AH, Wageningen, the
Netherlands; 2Trouw Nutrition R&D, P.O. Box 220, 5830 AE, Boxmeer, the Netherlands; 3Animal
Nutrition, Wageningen UR Livestock Research, P.O. Box 338, 6700 AH, Wageningen, the Netherlands;
kelly.nichols@wur.nl
Abstract
Fifty-six cows were used to test the effect of supplemental energy from protein (PT), fat (FT) or
both. Four total mixed rations were fed (4 weeks) at 95% of individual intakes and supplemented
such that additional energy intake consisted entirely of PT or FT. PT increased milk, lactose and
protein yield. FT increased milk, lactose, protein and fat yield. Arterial essential amino acid (EAA)
concentration and mammary EAA uptake were increased on PT, unaffected by FT, and a PT × FT
interaction occurred for EAA concentration. Glucose uptake was unaffected, but long-chain fatty
acid uptake decreased on PT and increased on FT suggesting differences in mammary metabolism
exist when different energy substrates are used for synthesis of milk components.
Keywords: dietary protein, dietary fat, mammary gland, amino acid
Introduction
A relationship exists between protein supply and lactose yield in dairy cows independent of glucose
supply, where amino acid (AA) infusions increase protein yield but also lactose yield (Nichols et
al., 2016). Mammary glands preferentially use glucose for protein and lactose synthesis; however,
fat is often added to dairy rations to increase energy density. Lactose yield increases from cows
fed lipogenic diets compared with glucogenic diets (Hammon et al., 2008). Thus, intra-mammary
metabolism must be flexible to derive substrates for lactose when supplied with aminogenic or
lipogenic precursors. Our objective was to determine the effects of supplemental energy from protein
or fat on lactation performance and mammary kinetics.
Material and methods

Fifty-six Holstein cows (167±87 days in milk; 2.8±1.9 lactations) were used in a randomized complete
block design where energy from protein (PT) or fat (FT) was tested. Cows were adapted (4 weeks)
to a total mixed rations (TMR) meeting net energy for lactation (NEL) and metabolisable protein
(MP) requirements, then blocked and randomly assigned to 1 of 4 experimental TMRs for a further
4 weeks: (1) low protein, low fat (LP/LF; 95% MP, 95% NEL); (2) high protein, low fat (HP/LF;
131% MP, 107% NEL); (3) high fat, low protein (LP/HF; 95% MP, 107% NEL); or (4) high protein
and high fat (HP/HF; 131% MP, 119% NEL). Individual cows were restricted to 95% of their ad
libitum intake during adaptation. Rumen-protected products (soybean + rapeseed, hydrogenated
palm fatty acids) were supplemented and entirely covered additional energy intake. Cows were fed
from intake control boxes and milked 2× daily with milk sampled during the final 7 d of each period.
Arterial and venous mammary blood samples (n=4) collected on the final day of each period were
analysed for plasma AA and metabolites and mammary uptake was calculated according to Nichols
et al. (2016). Data was subject to ANOVA with PT and FT and their interaction as fixed effects and
values from the final 7 d of adaptation as covariates.

Results and discussion
PT increased milk, lactose and protein yield and FT increased milk, lactose, protein and fat yield
(Table 1). Arterial concentration and mammary uptake of essential amino acid (EAA) increased on
PT, were unaffected by FT, but a PT × FT interaction for arterial concentration suggests that EAA
concentration increased more when HP was fed in a LF diet than in a HF diet. Arterial long-chain
fatty acid (LCFA) increased on FT and glucose was unaffected. Glucose uptake was also unaffected,
whereas uptake of LCFA was increased by FT and decreased by PT. Increased lactose yield with PT
and FT agrees with the observations of Nichols et al. (2016) and Hammon et al. (2008), respectively.
Neither arterial glucose nor glucose uptake was increased by PT or FT, suggesting intra-mammary
glucose partitioning was altered. Decreased de novo fatty acid synthesis on HF diets, suggested by
increased uptake of LCFA, reduces glucose oxidation and may have partitioned glucose towards
lactose synthesis. In conclusion, these results highlight differences between the effect of energy
supplied through protein or fat on milk component synthesis.
Acknowledgements
The work is part of the Feed4Foodure program supported by the Vereniging Diervoederonderzoek
Nederland and the Dutch Ministry of Economic Affairs.
Table 1. Effect of protein (PT) or fat (FT) on milk yield, arterial plasma concentrations and mammary
uptake of essential amino acids (EAA) and metabolites.1
Treatment P-value
LP/LF HP/LF LP/HF HP/HF SEM PT FT PT × FT
Milk (kg/d) 26.6b 28.7ab 28.4ab 30.0a 0.70 0.01 0.03 0.71
Protein (g/d) 915b 1,013a 974ab 1,041a 24.1 <0.01 0.05 0.50
Fat (g/d) 1,199c 1,254bc 1,325ab 1,375a 33.0 0.10 <0.01 0.93
Lactose (g/d) 1,205b 1,307ab 1,301ab 1,375a 30.8 <0.01 0.01 0.66
Arterial concentration
EAA2 (µM) 838b 1,111a 881b 1,018a 31.2 <0.01 0.44 0.04
Glucose (mM) 2.62 2.66 2.61 2.68 0.060 0.39 0.96 0.76
LCFA3 (µM) 254b 246b 309a 295a 9.4 0.20 <0.01 0.77
Plasma flow (l/h) 703 608 694 675 37.6 0.09 0.39 0.26
Uptake (mmol/h)
EAA 168a 196b 185b 198b 9.6 0.02 0.28 0.40
Glucose 548 519 572 563 40.4 0.64 0.41 0.80
TAG 15.6b 13.7b 24.0a 20.0ab 1.60 0.08 <0.01 0.52
LCFA 34.9ab 26.0b 54.7a 39.3ab 5.24 0.03 <0.01 0.54
1 LP = low protein; LF = low fat; HP = high protein; HF = high fat; TAG = triacylglycerol.
2 EAA = Arg, His, Ile, Leu, Lys, Met, Phe, Thr, Trp, Val.
3 LCFA = long-chain fatty acids; calculated on a molar basis as 3 × triacyglycerol + non-esterified fatty acids.
References
Hammon, H.M., C.C. Metges, P. Junghans, F. Becker, O. Bellmann, F. Schneider, G. Nürnberg, P. Dubreuil and H.
Lapierre, 2008. Metabolic changes and net portal flux in dairy cows fed a ration containing rumen-protected fat
as compared to a control diet. Journal of Dairy Science 91: 208-217.
Nichols, K., J.J.M. Kim, M. Carson, J.A. Metcalf, J.P. Cant and J. Doelman, 2016. Glucose supplementation stimulates
peripheral branched-chain amino acid catabolism in lactating dairy cows during essential amino acid infusions.
Journal of Dairy Science 99: 1145-1160.

Rate of protein growth and energy for maintenance parameter changes
in the Davis Growth Model
J.W. Oltjen1*, R.D. Sainz1, L.G. Barioni2 and S.R. Medeiros3
1University of California, Davis, CA 95616, USA; 2EMBRAPA Agricultural Informatics, Campinas,
SP, Brazil; 3EMBRAPA Beef Cattle, Campo Grande, MS, Brazil; jwoltjen@ucdavis.edu
Abstract
The Davis Growth Model simulates protein and fat growth of the empty body of beef cattle. We
have improved both model structure and parameter estimates to reflect trends in protein and fat
accretion for today’s more productive cattle. Initial DNA is now estimated by an equation which
requires both body protein and previous rates of protein accretion and energy intake. Because the
newer DNA estimates differ from the original ones, other parameters in the model were re-estimated
using the same data with which the model was originally parameterized. Later data has been used
to reparametrize the model for a number of different studies. Protein accretion and maintenance
energy parameters have increased.
Keywords: beef cattle, growth, body composition, energy requirements
Introduction
The Davis Growth Model (Oltjen et al., 1986) simulates protein and fat growth of the empty body
of beef cattle. It is based on the net energy system but includes mechanistic representation of protein
growth by simulating both DNA accretion and protein turnover. Experience with its use has revealed
strengths and weaknesses, and improvements have been made both within the model structure and
to parameter estimates. These changes reflect trends in protein and fat accretion due to selection
pressure for more productive cattle.
Model structure
Originally initial DNA was an interpolation based on body fatness between DNA of a well-fed animal
and an animal of similar protein content fed near maintenance. However, DNA estimates diverge
from the model’s simulated DNA at heavier weights. In subsequent implementation, initial DNA is
estimated by the following equation which requires both body protein (PROT) and previous rates
of protein accretion and energy intake (NUT2):
DNA = ( K3×PROT0.73 +
NUT2×K2
dt
)
dPROT 1/0.73
Where K2 (protein synthesis rate constant), K3 (protein degradation rate constant) and NUT2 are
defined as in the original model. This equation provides estimates of initial DNA that are within
1 g of simulated DNA across the entire growth path for both implanted and non-implanted steers.
Because the newer DNA estimates differ from the original ones, other parameters in the model
were re-estimated using the same data with which the model was originally parameterized (Oltjen
et al., 1986; original parameter values in parenthesis): K1=0.00493 (0.00429), K2=0.0444 (0.0461),
K3=0.143 (0.143 fixed due to unidentifiability), Alpha=0.0841 (0.0858). Also, the increase in protein
synthesis due to anabolic implant became 3.9% instead of the original 4.2%.

Model parameter changes over time
Data for original parameterization spanned 1960-1980. Later data was used to reparametrize the
model (Table 1). Protein growth and maintenance parameters have increased, similar to increases
in apparent maintenance requirements, efficiency of lactation (kL), and efficiency of growth (kG) in
dairy cattle (Moraes et al., 2013). An exception is for Nellore bulls with reduced K1, K3 and alpha
in these slower growing Bos indicus cattle (Sainz et al., 2006). Also, our recent work (unpublished
data) has shown decreased K3 with use of beta-agonists.
Table 1. Estimates of growth and maintenance parameters in the Davis Growth Model.
Description K1 K2 K3 Alpha Reference
Original estimates 0.00429 0.0461 0.143 0.0858 Oltjen et al., 1986

Revised estimates (above) 0.00493 0.0444 0.143 0.0841 Oltjen et al., 2014
Angus-Hereford Steers 0.053 0.0961 Garcia et al., 2008
Charolais Bulls 0.058 0.1372
Salers Heifers 0.056 0.0901
British Breed Steers 0.047 0.0983 McPhee et al., 2009
Nellore Bulls 0.00416 0.13 0.0768 Sainz et al., 2006
Angus Steers Sainz and Oltjen, 2014
Low RFI (residual feed 0.1375 0.062
intake)
Medium RFI 0.1436 0.0737
High RFI 0.1477 0.086
References
Garcia, F., R.D. Sainz, J. Agabriel, L.G. Barioni and J.W. Oltjen, 2008. Comparative analysis of two dynamic mechanistic
models of beef cattle growth. Animal Feed Science and Technology 143: 220-241.
McPhee, M.J., J.W. Oltjen, J.G. Fadel, D.G. Mayer and R.D. Sainz, 2009. Parameter estimation and sensitivity analysis
of fat deposition models in beef steers using acslXtreme. Mathematics and Computers in Simulation 79: 2701-2712.
Moraes, L.E., A.B. Strathe, E. Kebreab, D.P. Casper, J. Dijkstra, F. France and J.G. Fadel, 2013. A structural equation
model to analyze energy utilization in lactating dairy cows. In: J.W. Oltjen, E. Kebreab and J. Lapierre (eds.).
Energy and protein metabolism and nutrition in sustainable animal production. EAAP Scientific Series No. 134,
Wageningen Academic Publishers, Wageningen, the Netherlands, pp. 327-328.
Oltjen, J.W., A.C. Bywater, R.L. Baldwin and W.N. Garrett, 1986. Development of a dynamic model of beef cattle
growth and composition. Journal of Animal Science 62: 86-97.
Oltjen, J.W., R.D. Sainz, L.B. Barioni, D.P. Lanna and T.Z. Albertini, 2014. Evolution of parameter changes for beef
cattle growth in the Davis Growth Model over 40 years. Animal Production Science 54(12): 52.
Sainz, R.D., L.G. Barioni, P.V. Paulino, S.C. Valadares Filho and J.W. Oltjen, 2006. Growth patterns of Nellore vs
British beef cattle breeds assessed using a dynamic, mechanistic model of cattle growth and composition. In: E.
Kebreab, J. Dijkstra, A. Bannink, W.J.J. Gerrits and J. France (eds.). Nutrient digestion and utilization in farm
animals: modelling approaches CAB International, Wallingford, UK, pp. 160-170.
Sainz, R.D. and J.W. Oltjen, 2014. Dynamic mechanistic modelling of feed efficiency in Bos inducus beef cattle. In:
K.J. Harper, D.M. McNeill and A.W. Bell (eds.). Modelling Nutrient Digestion and Utilization in Farm Animals,
September 15-17, 2014, Cairns, Australia, p. 36.

Evaluation of the 2001 Dairy NRC and derivation of new equations
R.R. White1*, Y. Roman-Garcia2, J.L. Firkins2, M.H. VandeHaar3, L.E. Armentano4, T. McGill1, R.
Garnett1 and M.D. Hanigan1
1Department of Dairy Science, Virginia Tech, Blacksburg, 24061 VA, USA; 2Department of Animal
Sciences, The Ohio State University, Columbus, 43210 OH, USA; 3Department of Animal Science,
Michigan State University, East Lansing, 48824 MI, USA; 4Department of Animal Science, University
of Wisconsin, Madison, 53706 WI, USA; rrwhite@vt.edu
Abstract
The objectives of this work were to use a literature dataset of apparent total tract digestibility of
neutral detergent fibre (NDF), fatty acid (FA), crude protein (CP), organic matter (OM) and starch,
post-ruminal N flows, and milk yield to evaluate the equivalent predictions provided by the 2001
NRC dairy model and to derive new equations, when necessary. A dataset of 550 treatment means was
collected. The NRC model predictions were biased; notable slope biases were identified in predictions
of digestible NDF, CP and FA, rumen undegradable protein (RUP), and microbial N. Digestible
CP and FA, RUP, and milk returned mean bias. Deriving new equations helped to improve the root
mean squared prediction error, reduce mean and slope bias, and improve concordance correlation
coefficient for all measurements evaluated. The final model predicted milk yield with only 10% error.
Keywords: 2001 Dairy NRC, model evaluation, nutrient digestibility, microbial N
Introduction
Ration balancing programs such as the nutrient requirement equations proposed by the National
Research Council (NRC) are an essential component of animal nutrition research, education, and
extension throughout the world. To ensure these ration balancing systems meet their objectives, it
is necessary to evaluate them against published data. Although the NRC (2001) dairy model was
quantitatively evaluated prior to publication, the extent of the evaluation was limited and largely
restricted to protein supply and milk yield (NRC, 2001; St-Pierre, 2003). The objectives of this work
were to use a literature dataset of apparent total tract digestibility of neutral detergent fibre (NDF),
fatty acid (FA), crude protein (CP), organic matter (OM) and starch, post-ruminal N flows, and
milk yield to evaluate the 2001 NRC dairy model predictions and to derive new equations, when
necessary. We hypothesized that: (1) NRC predictions would have bias when compared to measured
data; and (2) biases would be reduced by re-deriving parameters used in the current equation forms.

Data were collected from the original set of publications used to evaluate the 2001 NRC dairy model
and updated with work published between the early 2000’s until mid-2015. Studies were included
in the dataset if they measured duodenal or omasal N flows, or apparent total tract digestibility
measurements. The final dataset contained usable data from 550 treatment means from 147 studies.
Most studies reported inclusion rates of the ingredients used in diets; however, few studies reported
ingredient nutrient composition. When ingredient nutrient composition data were available, they
were used to calculate dietary nutrient provision. Study-level residual dietary FA, NDF, acid
detergent fibre (ADF), dry matter (DM), and CP values were calculated and used to adjust feed
library nutrient composition for feeds so that dietary nutrient composition was unbiased within the
study. Model predictions of digestible nutrients (NDF, CP, and FA) were evaluated in comparison
to apparent total tract nutrient digestibility reported in the evaluation dataset and adjusted to a
true basis. The NRC estimates of rumen undegradable protein (RUP) and microbial protein were
evaluated in comparison to duodenal or omasal flows of non-NH3, non-microbial, non-endogenous

N and microbial N, respectively. The NRC estimate of endogenous N was used to adjust measured
non-NH3, non-microbial N. Milk yield estimates and component yields were compared to measured
performance data.

The NRC (2001) estimates of nutrient fluxes were biased (Table 1). Digestible nutrient biases
were notable and contributed to downstream biases. A new digestible NDF equation was derived
that accounted for forage type (corn silage, grass, legume or non-forage), ADF to NDF ratio, dry
matter intake (DMI), and diet starch. A new digestible CP equation responsive to ADF, ADF and
NDF insoluble protein, DMI, feed CP, and diet CP was derived. The FA digestibility equation was
re-derived based on feed types representing different FA profiles. A new protein system calculating
RUP estimates from an estimated bypass rate of B protein, rather than passage and degradation rates
was constructed. Microbial protein was predicted as a continuous response to increasing digestible
nutrient and RDP intake. New variable efficiencies of protein and energy use for milk were derived.
Table 1. Comparison of fit statistics for the NRC model and the newly derived equations.1
Metric Equation RMSE Slope bias Mean bias CCC

(% mean) (% MSE) (% MSE)
Digestible NDF NRC 39 24 1.8 0.35

New 23 0.0 0.0 0.65
Digestible CP NRC 15 18 17 0.79
New 10 0.0 0.0 0.89
Digestible FA NRC 26 65 9.7 0.66
New 10 0.0 0.0 0.95
Duodenal/omasal non-NH3, non- NRC 39 17 24 0.36
microbial, non-endogenous N New 33 0.0 0.0 0.45
Duodenal/omasal microbial N NRC 37 20 0.4 0.39
New 27 0.0 0.0 0.52
Milk NRC 24 0.8 17 0.85
New 10 0.0 0.0 0.90
1 CCC = concordance correlation coefficient; CP = crude protein; FA = fatty acid; NDF = neutral detergent fibre; RMSE
= root mean squared prediction error.
Acknowledgements
This work was supported by the National Animal Nutrition Program, a National Research Support
Project (NRSP-9) of State Agricultural Experiment Stations, that is funded from Hatch funds
administered by the National Institute of Food and Agriculture, U.S. Department of Agriculture,
Washington DC. Additional funding was provided by Agriculture and Food Research Initiative
Competitive Grant no. 2011-68004-30340 and by Papillon Agricultural Company (Easton, MD)
References
National Research Council (NRC), 2001. Nutrient requirements of dairy cattle (7th Ed.). National Academy Press,
Washington, DC, USA.
St-Pierre, N., 2003. Reassessment of biases in predicted nitrogen flows to the duodenum by NRC 2001. J. Dairy Sci.
86(1): 344-350.

Age effects on energy balance of dairy cows subjected to different diet
types since first calving
F. Grandl1,2, M. Kreuzer1*, J.O. Zeitz1,3 and A. Schwarm1
1ETH Zurich, Institute of Agricultural Sciences, Universitätstrasse 2, 8092 Zurich, Switzerland;
2Qualitas AG, Chamerstrasse 56, 6300 Zug, Switzerland; 3Justus-Liebig-University Gießen, Institute
of Animal Nutrition and Nutritional Physiology, Heinrich-Buff-Ring 26-32, 35392 Gießen, Germany;
michael.kreuzer@inw.agrl.ethz.ch
Abstract
It is unclear whether the efficiency of energy utilisation increases or decreases with age. This
knowledge is important when evaluating ongoing strategies for increasing the longevity of dairy
cattle concerning energy efficiency and environmental impacts. The effects of senescence may also
be dependent on the diet type the animals are kept on. In the present study, energy balance data were
obtained from 30 lactating Brown Swiss dairy cows aged between 2 and 10 years. For that purpose,
open circuit respiration chambers were used. The cows had been fed since their first calving either
on a forage-only diet or the same forage diet supplemented with 5 kg/day of concentrate. Age and
diet effects were analysed by regression analysis. Intake of gross energy and metabolisable energy
increased from the first lactation onwards but the slope of increase was getting smaller with higher
age. Energy digestibility did not change with age, but energy loss as heat increased. Methane energy
loss followed a curvilinear relationship with age and was highest after few calvings. The efficiency
of utilisation of metabolisable energy for milk production (kL) was promoted with increasing age.
Age and feeding regime did not interact significantly. As expected, offering concentrate enhanced
digestibility and metabolisability of energy. In conclusion, older cows rather express an improved
than reduced efficiency of dietary energy utilisation.
Keywords: energy balance, energy utilisation, dairy cow, longevity, forage-only
Introduction
Energy balance studies in dairy cows are numerous, but changes with age of the cows have rarely
been investigated. Age is mostly not even specified, and can only be roughly estimated from parity
number when indicated in studies. It is likely that energy intake and absolute or proportionate losses
of energy during digestive and metabolic processes change as a consequence of senescence. This is at
least suggested from some distinct changes found in eating behaviour and digestion pattern (Grandl
et al., 2016). Such changes might either support or counteract current efforts to promote longevity
in dairy cattle. As energy losses also depend on diet type, the occurrence of interactions between
age and diet type effects cannot be excluded. Therefore, in an energy balance experiment, cows of
a large age range, subjected to two feeding regimes, were compared to evaluate changes due to age.

The experiment involved 30 lactating Brown Swiss dairy cows (24 to 30 kg/day energy-corrected
milk) which were between 199 to 3,648 days old. For their entire productive lifetime, one half of
the animals had been subjected to either a high quality forage-only diet (0-CONC), the other half
to the same forage diet supplemented with 5 kg/day of concentrate (CTRL). Housing and all other
management issues were identical. Data and samples were collected in 8-day periods of excreta
collection and 2 days of open-circuit respiration chamber measurements. Records and samplings
were made for faeces and urine, feed intake and milk excretion, the latter two were obtained during
the entire 10 days. No adaptation to diet was necessary as the diet was not changed. Detailed
energy balances of the cows were calculated. Data were analysed by regression analyses using the

statistical software package R with diet and age included as effects, and milk yield and body weight
as covariates.

There was an increase (P<0.05) in gross energy (GE) intake from primiparous to multiparous cows
which was getting less pronounced with age. This increase was also found with intake of metabolisable
energy (ME; P<0.01) and, by tendency, with energy excretion in milk (P=0.11). Energy loss in faeces
did not change with age (P=0.50), whereas those in urine decreased (P<0.05) and those in heat
increased (P=0.05) with age. This resulted in unchanged total energy loss with age. In the case of
methane energy loss, a curvilinear relationship with age was found (P<0.001 and P<0.01 for age as
untransformed and ln-transformed effect, respectively), with cows being in their 2nd to 3rd lactation
having the highest emissions. Relative to GE intake, energy loss in faeces and as heat did not change
with age (P=0.13 and P=0.17, respectively), those of urine decreased (P<0.01) and those of methane
were again curvilinear (P<0.001). Most interestingly, along with metabolisability, the efficiency of
ME utilisation for milk production (kL) increased (P<0.05) with age by maximum one percentage
unit from the youngest to the oldest cows. The changes reported by Grandl et al. (2016) for feeding
behaviour also did not show large age changes. Pérez-Barbería and Gordon (1998) concluded in
their review that improved chewing ability and modified chewing behaviour can compensate for
increasing tooth wear with age. These observations were also considered responsible for the increase
in feed intake with age. There were no significant interactions between age and feeding regime
demonstrating that age developments were not influenced by the long-term feeding strategy. In the
0-CONC cows, compared with the CTRL cows, GE intake was similar (P=0.25), whereas ME intake
(199 vs 225 MJ/day, P<0.01), energy digestibility (62 vs 66%, P<0.01), energy metabolisability (52
vs 57%, P<0.001) and kL (59 vs 60%, P<0.001) were lower. The net energy intakes of the 0-CONC
cows were slightly below calculated requirements (maintenance and energy excretion with milk),
those of the CTRL cows slightly above requirements. This was corroborated by similar differences
between 0-CONC and CTRL cows in body energy retention.
In conclusion, it seems that the older cows from the present study were more efficient in energy
utilisation in metabolism than the younger cows and this at unchanged energy digestibility. This
provides arguments for increasing the longevity of cows.
Acknowledgements
The project was funded by the Mercator Research Program of the ETH Zurich World Food System
Center. Thanks go to the staff of Plantahof, Strickhof and ETH Zurich, Switzerland.
References
Grandl, F., S.P. Luzi, M. Furger, J.O. Zeitz, F. Leiber, S. Ortmann, M. Kreuzer and A. Schwarm, 2016. Biological
implications of longevity in dairy cows: 1. Changes in feed intake, feeding behavior and digestion with age.
Pérez-Barbería, F.J., and I.J. Gordon, 1998. Factors affecting food comminution during chewing in ruminants: a review.
Biological Journal of the Linnean Society 63: 233-256.

Predicting variation in feed efficiency in veal calves by early life
characterization
M.S. Gilbert1*, J.J.G.C. van den Borne1, C.G. van Reenen2 and W.J.J. Gerrits1
Netherlands; 2Livestock Research, Animal Sciences Group, Wageningen University and Research
Centre, P.O. Box 338, 6700 AH, Wageningen, the Netherlands; myrthe.gilbert@wur.nl
Abstract
The objectives of this study were to examine whether variation in growth performance in healthy veal
calves can be explained by characteristics of calves in early life, and to determine whether their ability
to cope with low-lactose milk replacer (MR) can be predicted in early life. Male Holstein-Friesian
calves were characterized in early life using targeted challenges. In later life, 50% of the calves
received a lactose-based control MR and the other calves received a low-lactose MR in which 51%
of the lactose was iso-calorically replaced by glucose, fructose and glycerol (2:1:2 ratio). Average
daily gain (ADG) in later life was for 17% explained by early life measurements. However, this
was mainly related to variation in solid feed refusals. When ADG was adjusted to equal solid feed
intake, only 7% of the variation in standardised ADG (reflecting feed efficiency) could be explained
by early life measurements. Significant relations between ‘fasting plasma glucose concentrations’,
‘faecal pH’, ‘drinking speed’, and ‘natural antibodies at arrival’ in early life and feed efficiency
in later life depended on MR composition. These measurements are, therefore, potential tools for
screening calves in early life on their ability to cope with MRs varying in lactose content.
Keywords: growth performance, feed efficiency, predicting, early life, veal calves
Introduction
High and fluctuating dairy prices provide an economic incentive to substitute lactose from calf
milk replacer (MR) by other energy sources. In addition, high inter-individual variation in growth
performance is reported in veal calves. This also occurs under equal feeding and husbandry
conditions in healthy calves, indicating that inter-individual variation in growth performance is
largely determined by inter-individual variation in feed efficiency. Replacement of lactose further
increases this variation in growth performance, indicating that calves differ in their capacity to deal
with low-lactose MRs. Inter-individual variation in glucose metabolism may contribute to this,
because the coefficient of variation in urinary glucose excretion increased from 42 to 82% when
partly replacing lactose with glucose, fructose or glycerol (Gilbert et al., 2016). The objectives of
this study were to examine whether variation in growth performance in healthy veal calves can be
explained by characteristics of calves in early life, and to determine whether their ability to cope
with low-lactose MR can be predicted in early life.

The experiment consisted of 2 periods. In period 1 (2-11 weeks of age), 180 male Holstein-Friesian
calves (17±0.3 days of age) were fed a lactose-based control MR and solid feed (concentrates,
rapeseed straw and alfalfa; 70:15:15 ratio). Calves were characterized using challenges related
to feeding motivation, digestion, post-absorptive metabolism, behaviour, stress and immunity. In
period 2 (11-27 weeks of age), 130 out of 180 calves were included and equally divided over 2 MR
treatments, i.e. a control MR which contained 50% lactose as only carbohydrate source and a low-
lactose MR in which 51% of the lactose was iso-calorically replaced by glucose, fructose and glycerol
(2:1:2 ratio; GFG). Solid feed was composed of concentrates and rapeseed straw (80:20 ratio). In both
periods, MR and solid feed were provided individually and MR increased progressively from 400

to 3,000 g/d and solid feed from 85 to 1,170 g/d according to a practical feeding scheme. Early life
measurements were first subjected to principal component analysis per category (i.e. measurements
at arrival, feeding motivation, digestion, post-absorptive metabolism, and behaviour and stress)
to reduce and scale the data. Thereafter, relations between early life characteristics (i.e. principal
components; PC) and growth performance in later life were assessed in 117 clinically healthy calves.

Average daily gain (ADG) in period 2 tended to be greater for control (1,292±14 g) than for GFG
calves (1,267±14 g; P=0.085). Variation in ADG in period 2 was for 17% explained by variation
in early life measurements. However, this was mainly related to variation in solid feed refusals.
When ADG was adjusted to equal solid feed intake, only 7% of the variation in standardised ADG
(reflecting feed efficiency) could be explained by early life measurements. More than 90% of the
variation in feed efficiency in later life could not be explained. It is hypothesized that variation in
health status explains substantial variation in feed efficiency in veal calves. Significant relations
between four PCs in early life and feed efficiency in later life depended on MR composition (Table
1). These measurements are, therefore, potential tools for screening calves in early life on their ability
to cope with MRs varying in lactose content.
Table 1. Effects of early life (2-11 weeks of age) measurements, subjected to principal
components analysis (principal component; PC), and their interaction with milk
replacer treatment on adjusted average daily gain in veal calves in later life (11-27
weeks of age) fed a milk replacer containing lactose as the only carbohydrate source
(CON; n=62) or a milk replacer in which 51% of the lactose was replaced by iso-
energetic amounts of glucose, fructose and glycerol (GFG; n=55).
Item ADG (adjusted for solid feed intake) in later life
P β (CON treatment) β (GFG treatment)
Item Interaction Estimate SE P-value Estimate SE P-value
Treatment 0.101
PC ‘fasting glucose’ 0.579 0.035 -18.0 8.9 0.044 10.6 10.0 0.293
PC ‘faecal pH’ 0.825 0.057 14.6 9.0 0.106 -11.6 10.2 0.260
PC ‘drinking speed’ 0.063 0.088 -24.8 8.1 0.016 -1.1 9.3 0.909
PC ‘natural antibodies’ 0.602 0.095 -14.9 8.5 0.083 7.8 10.5 0.457
Acknowledgements
This project was jointly financed by the European Union, European Regional Development Fund and
The Ministry of Economic Affairs, Agriculture and Innovation, Peaks in the Delta, the Municipality
of Groningen, the Provinces of Groningen, Fryslân and Drenthe as well as the Dutch Carbohydrate
Competence Centre (CCC2 WP21). Financial support was also provided by Tereos Syral, VanDrie
Group and Wageningen University.
References
Gilbert, M.S., A.J. Pantophlet, J.J.G.C. van den Borne, W.H. Hendriks, H.A. Schols, and W.J.J. Gerrits, 2016. Effects
of replacing lactose from milk replacer by glucose, fructose, or glycerol on energy partitioning in veal calves.

Isotopic N fractionation as a biomarker of nitrogen use efficiency
by ruminants: a meta-analysis
G. Cantalapiedra-Hijar1*, R.J. Dewhurst2, L. Cheng3, A.R.J. Cabrita4, A.J.M. Fonseca4, H. Fouillet5,
P. Nozière1 and I. Ortigues-Marty1
1INRA, UMR 1213 Herbivores, 63122 St Genès Champanelle, France; 2Scotland’s Rural College,
Edinburgh EH9 3JG, United Kingdom; 3Faculty of Agriculture and Life Sciences, Lincoln University,
New Zealand; 4REQUIMTE, LAQV, Universidade do Porto, 4050-313 Porto, Portugal; 5INRA, UMR
914 PNCA INRA-AgroParisTech, 75005 Paris, France; gonzalo.cantalapiedra@clermont.inra.fr
Abstract
We evaluated the potential to use the naturally occurring isotopic N fractionation between plasma
or milk of ruminants and their diet (Δ15Nanimal-diet) to predict between-animal variation in N use
efficiency (NUE), as well as assessed the main mechanisms by which Δ15Nanimal-diet is related to
NUE under common feeding conditions. Individual values for NUE (n=217) and Δ15Nanimal-diet
(n=274) from 11 published experiments were analysed by meta-analysis. All dietary treatments were
characterized according to the newly-updated French feeding system. The Δ15Nanimal-diet reflects
between-animal variation in NUE and underlying mechanisms by which Δ15Nanimal-diet is related to
NUE across diets are at both the metabolic and rumen levels.
Keywords: feed efficiency, 15N, individual variability
Introduction
Nitrogen use efficiency (NUE; nitrogen gain or milk nitrogen yield/N intake) is an important
component of animal feed efficiency. A promising biomarker for NUE is based on the phenomenon
of N isotopic fractionation (Δ15Nanimal-diet; the difference between animals and their diet in natural
abundance of 15N). This approach may have potential to discriminate individuals fed the same diet,
but with different N partitioning. A number of studies over the last 5 years have evaluated this new
biomarker for NUE in ruminants, but its potential to reflect individual variation has not been yet
assessed. The objectives of this study were to (1) assess the potential of Δ15Nanimal-diet to predict
variation in NUE at the individual level; and (2) disentangle the main mechanisms by which this
biomarker is related to NUE.

Eight publications (Cabrita et al., 2014; Cantalapiedra-Hijar et al., 2015, 2016; Cheng et al., 2011,
2013a,b, 2014, 2015) reporting NUE and Δ15Nanimal-diet values in ruminants were analysed. There
were 11 separate experiments, 42 treatments and individual values for NUE (n=217) and Δ15Nanimal-
diet (nmilk=145 and nplasma=129). Meta-analysis was performed by GLM (Minitab v14), adopting
both between-experiment and within-treatment approaches. Dietary treatments were characterized
using the newly updated INRA feeding system and the main determinants of NUE were analysed
by partial least square (PLS) regression (XLStat v2015.2.02). Independent variables were: rumen
protein balance (g/kg DM), rumen degradable protein (RDP, g/kg DM), efficiency of microbial
protein synthesis according to either available energy (EMPS_E, g/g rumen fermentable organic
matter) or available protein (EMPS_N, g/g RDP), the metabolisable protein (MP) to net energy ratio
(g MP/MJ), the digestive efficiency of N use (g MP/g crude protein), the efficiency of MP utilisation
for either production (EMPU_prod, g protein in milk or gain/g MP) or the sum of production and
non-productive (Sauvant et al., 2015) losses (EMPU_absol, g/g MP).

A between-experiment quadratic relationship (P<0.001; r2=0.78; RSE=0.63) between individual
Δ15N and NUE values was identified (Figure 1), indicating that Δ15Nanimal-diet progressively reaches
maximum values as NUE approaches zero. The within-treatment linear relationship (P<0.001;
Figure 1) between individual Δ15N and NUE values was significant highlights the potential of this
isotopic biomarker to predict between-animal variation in NUE. The proposed PLS model made
good predictions of variation in Δ15N resulting from dietary characteristics (Q2=0.88; RMSE=0.37)
and contributed to explain most of the variability in observed values (R2(Y)=0.91).
The most important variables explaining Δ15N variation were related to the metabolic efficiency
of N use (variable importance in projection, VIP=1.10-1.19) but there were also relationships with
rumen and digestive N use (VIP=0.85-1.00).
5
Δ 15N animal-diet (‰)
Beween-experiment
4
Y = 5.95 – 6.87X – 16.1X2
r2=0.78; RSE=0.63
3 n=274 and nexp=11
2
Within-experiment
Y = 4.47 – 4.39X
1 r2=0.94; RSE=0.36
n=274 and ntrt=42
0
-0.2 -0.1 0.0 0.1 0.2 0.3 0.4
N use efficiency (g/g)
Figure 1. Between-experiment (––) and within-treatment (--) relationships between individual

Δ15Nanimal-diet and N use efficiency.
References
Cabrita, A.R.J., A.J.M. Fonseca and R. J. Dewhurst, 2014. Short communication: relationship between the efficiency
of utilization of feed nitrogen and 15N enrichment in casein from lactating dairy cows. Journal of Dairy Science
97(11): 7225-7229.
Cantalapiedra-Hijar, G., I. Ortigues-Marty, B. Sepchat, J. Agabriel, J.F. Huneau and H. Fouillet, 2015. Diet-animal
fractionation of nitrogen stable isotopes reflects the efficiency of nitrogen assimilation in ruminants. British Journal
of Nutrition 113(7): 1158-1169.
Cantalapiedra-Hijar, G., H. Fouillet, J.F. Huneau, A. Fanchone, M. Doreau, P. Nozière and I. Ortigues-Marty, 2016.
Relationship between efficiency of nitrogen utilization and isotopic nitrogen fractionation in dairy cows: contribution
of digestion v. metabolism? Animal 10(2): 221-229.
Cheng, L., E.J. Kim, R.J. Merry and R.J. Dewhurst, 2011. Nitrogen partitioning and isotopic fractionation in dairy cows
consuming diets based on a range of contrasting forages. Journal of Dairy Science 94(4): 2031-2041.

Cheng, L., A.J. Sheahan, S.J. Gibbs, A.G. Rius, J.K Kay, S. Meier, G.R. Edwards, R.J. Dewhurst and J.R. Roche,
2013a. Technical note: nitrogen isotopic fractionation can be used to predict nitrogen-use efficiency in dairy cows
fed temperate pasture. Journal of Animal Science 91(12): 5785-5788.
Cheng, L., A.M. Nicol, R.J. Dewhurst and G.R. Edwards, 2013b. The effects of dietary nitrogen to water-soluble
carbohydrate ratio on isotopic fractionation and partitioning of nitrogen in non-lactating sheep. Animal 7(8):
1274-1279.
Cheng, L., S.L. Woodward, R.J. Dewhurst, H. Zhou and G.R. Edwards, 2014. Nitrogen partitioning, energy use
efficiency and isotopic fractionation measurements from cows differing in genetic merit fed low-quality pasture
in late lactation. Animal Production Science 54(10): 1651-1656.
Cheng, L., G.R. Edwards, R.J. Dewhurst, A.M. Nicol and D. Pacheco, 2015. The effect of dietary water soluble
carbohydrate to nitrogen ratio on nitrogen partitioning and isotopic fractionation of lactating goats offered a
high-nitrogen diet. Animal 10: 779-785.
Sauvant, D.G. Cantalapiedra-Hijar, L. Delaby, P. Faverdin and P. Nozière, 2015. Updating protein requirements
in ruminants and determination of the responses of lactating females to metabolisable protein supply. INRA
Productions Animales 28(5): 347-367.

Effect of canola meal heat treatment and glycerol inclusion in calf starter
on GIT development
K. Burakowska1, P. Górka2, Z.M. Kowalski2, B. Laarveld1 and G.B. Penner1*
1Department of Animal and Poultry Science, University of Saskatchewan, 51 Campus Dr., Saskatoon,
SK S7N 5A8, Canada; 2Department of Animal Nutrition and Dietetics, University of Agriculture in
Krakow, al. Mickiewicza 24/28, 30-059 Krakow, Poland; greg.penner@usask.ca
Abstract
The objective of this study was to evaluate the effect of heat-treated canola meal (CM) and inclusion
of glycerol (GLY) in a pelleted starter mixture (SM) on gastro-intestinal tract development of Holstein
calves. Twenty-eight bull calves were assigned to one of four treatments and fed a SM containing: (1)
non-heated CM without GLY; (2) non-heated CM with GLY (5% of SM DM); (3) heat-treated CM
(110 °C for 10 min) without GLY; and (4) heat-treated CM with GLY. On the day after weaning (day
51), calves were killed and the gastro-intestinal tract was dissected for morphometric measurements
and ruminal fluid was analysed for pH, and short chain fatty acid (SCFA) and ammonia concentrations.
Inclusion of GLY in the SM increased SM intake while heat-treated CM tended to decrease it. Heat
treatment of CM decreased weights of the reticulorumen tissue, reticulorumen digesta, jejunum tissue,
ileum digesta and cecum digesta, and decreased lengths of total small intestine and colon relative to
non-heated CM. The inclusion of GLY increased jejunum digesta weight and decreased the cecum
digesta weight, as well as decreased rumen pH and increased the total ruminal SCFA concentration.
Results suggest that heat treatment of CM decreases SM intake in calves and in consequence limits
gastro-intestinal tract development whereas GLY inclusion in SM increases SM intake and enhances
rumen and small intestine development in calves.
Keywords: starter mixture, gastro-intestinal tract, protein source, palatability
Introduction
Low palatability and digestibility of canola meal (CM) have limited its use as a protein source in
starter mixtures (SM) for dairy calves. However, the amino acid composition of CM (namely high
glutamine and glutamate content) might have beneficial effects on small intestinal development,
and in consequence solid feed intake and rumen development. This effect could be enhanced by
heat treatment of CM, which increases the undegradable protein fraction in the rumen (McKinnon
et al., 1991). On the other hand, inclusion of sweet glycerol (GLY) can be used to increase starter
palatability. The objective of this study was to evaluate the effect of heat-treated CM and inclusion
of GLY in a pelleted SM on gastro-intestinal tract (GIT) development of Holstein calves.

Twenty-eight bull calves at eight days of age (43.4±4.0 kg) were assigned to one of four treatments
and fed a SM containing: (1) CM without GLY; (2) CM with GLY (5% of SM DM); (3) heat-treated
CM (110 °C for 10 min) without GLY; and (4) heat-treated CM with GLY. Canola meal constituted
34% DM of the starters. Calves were fed milk replacer until 49 days of age. The starter mixture
was fed ad libitum until 51 days of age when calves were killed and the gastro-intestinal tract was
dissected for morphometric measurements and ruminal fluid was used to determine pH, and short
chain fatty acid (SCFA) and ammonia concentrations. Body weight was recorded weekly and milk
replacer and SM intakes were recorded daily. Data were analysed as randomised complete block
design using PROC MIXED of the SAS (version 9.4; SAS Institute Inc., Cary, NC, USA).

Milk replacer intake and body weight were not different between treatments (P≥0.05). However,
heat-treatment of CM tended to decrease (0.57 vs 0.47 kg/d; P=0.07) and GLY inclusion tended to
increase (0.48 vs 0.56 kg/d; P=0.09) calf average daily gain. Inclusion of GLY in the SM increased
SM intake (199 vs 293 g/d; P=0.037) while heat-treated CM tended to decrease SM intake (289
vs 203 g/d; P=0.06). Calves fed heat-treated CM had reduced reticulorumen tissue (1.21 vs 0.96
kg; P=0.011) and digesta (4.10 vs 2.82 kg; P<0.001) weights, jejunum tissue weight (1.44 vs 1.21
kg; P=0.024), ileum digesta weight (59 vs 24 g; P=0.010), cecum digesta weight (258 vs 196 g;
P=0.018), and the length of small intestine (19.9 vs 18.3 m; P=0.035) and colon (2.69 vs 2.47 m;
P=0.010). On the other hand, GLY inclusion increased the jejunum digesta weight (0.77 vs 1.14
kg; P=0.001) but decreased the cecum digesta weight (262 vs 192 g; P=0.009). Furthermore, GLY
inclusion in the SM decreased ruminal pH (5.31 vs 5.05; P=0.038) and increased total ruminal SCFA
concentration (124.7 vs 145.0 mM; P=0.008). A previous study (Hadam et al., 2016) reported that
CM use in a SM as a full replacement of soybean meal negatively affected average daily gain and
feed efficiency in calves; however, SM intake was not affected. Results of this study suggest that
heat treatment of CM decreases SM intake in calves and in consequence limits gastro-intestinal tract
development whereas GLY inclusion in the SM increases SM intake and enhances rumen and small
intestine development in calves. Even though heat-treatment of CM may have negative effects on
GIT development, the use of CM in starter mixtures, in combination with GLY, might be considered
as an alternative to soybean meal; however, future research is needed to compare canola meal and
soybean meal inclusion in the starters and their effects on gut development.
Acknowledgements
Funding for this project was provided from the Alberta Crop Industry Development Fund, Western
Grains Research Foundation, Saskatchewan Canola Development Commission, and the Saskatchewan
Agriculture Development Fund.
References
Hadam, D., J. Kański, K. Burakowska, G.B. Penner, Z.M. Kowalski and P. Górka, 2016. Short communication: effect
of canola meal use as a protein source in a starter mixture on feeding behaviour and performance of calves during
the weaning transition. Journal of Dairy Science 99: 1247-1252.
McKinnon, J.J., J.A. Olubobokun, D.A. Christensen and R.D.H. Cohen, 1991. The influence of heat and chemical
treatment on ruminal disappearance of canola meal. Canadian Journal of Animal Science 71: 773-780.

Associations between body weight change, hepatic and intestinal oxygen
consumption in pregnant cows
A.B.P. Fontoura1*, F.E. Keomanivong1, L.D. Prezotto2, L.E. Camacho3, Y.R. Montanholi4, K.A.
Vonnahme1 and K.C. Swanson1
1North Dakota State University, 1300 Albrecht Blvd, Fargo, ND 58102, USA; 2Montana State
University, 3710 Assinniboine Rd, Havre, MT 59501, USA; 3University of Arizona, 4101 N.
Campbell, Tucson, AZ 85719, USA; 4Dalhousie University, 58 River Road, B2N 5E3 Truro, Canada;
ananda.fontoura@ndsu.edu
Abstract
Bovine pregnancy leads to increased metabolic rates which may be reflected in the O2 utilisation
by foetal and maternal tissues. This study evaluated the associations between maternal and foetal
hepatic and intestinal O2 consumption with changes in body weight (BW) during gestation. Forty-
six crossbred multiparous gestating beef cows (621±11.3 kg) were fed to meet 100% or 60% of
dietary requirements. Body weights were taken weekly. Cows were ranked and grouped according
to average daily weight change (ADWC; low, medium, high). At slaughter, liver and small intestine
were collected from the cows and foetuses at different stages of pregnancy (day 85, 140, and 254) and
in vitro O2 consumption was determined. Maternal liver O2 consumption (μmol/min/mg) was lower
(P<0.01) in the ADWChigh, and foetal liver mass increased. Foetal BW was negatively correlated
(P≤0.05) with ADWC. Our results suggest that mature cows undergoing a larger BW loss during
gestation partition a greater proportion of energy towards foetal liver development.
Keywords: beef cattle, functional workload, liver metabolism, pregnancy, small intestine metabolism
Introduction
Hepatic and intestinal tissues can account for up to 50% of energy expenditure in ruminants (Ferrell,
1988). A better understanding of energy use during weight fluctuations over gestation might aid us in
further understanding overall efficiency of energy use in mature beef cows. The objectives for this
study were to evaluate the associations between maternal and foetal hepatic and intestinal energy
use as measured using in vitro O2 consumption with changes in BW weight during gestational.

Crossbred multiparous gestating and non-lactating beef cows (n=46; initial BW of 621±11.3 kg)
were individually fed differing amounts of grass hay to either meet (100%) or be deficient (60%)
according to NRC recommendations, as described in detail by Camacho et al. (2014). Body weights
were taken weekly during the experiment. At slaughter, liver and small intestine were collected from
the cows and foetuses at different stages of pregnancy (day 85, 140 and 254). Maternal and foetal liver
and small intestinal samples were analysed for in vitro O2 consumption. Data were normalized and
differences in least square means for the three ADWC groups were tested using Scheffé’s method.
The ADWC of cows was calculated using regression analysis. Cows were ranked according to BW
change and placed in low, medium and high groups (ADWClow, ADWCmedium, ADWChigh). The
relationships between O2 consumption and ADWC in dams and foetuses were measured through
partial correlations, adjusted for dietary treatment and day of gestation, using the MANOVA/PRINTE
statement. For all analysis, results were considered significant when P≤0.05 and trends were noted
when 0.05 ≥ P ≤ 0.10.

The means for ADWC were different (P<0.01) among groups (mean (confidence limits) kg/d;
ADWClow=-0.12 (-0.32 to 0.09), ADWCmedium=-0.40 (-0.50 to -0.30), ADWChigh=-1.05 (-1.21
to -0.98)). Cows in the ADWChigh group tended to have a lighter BW than the ADWClow and
ADWCmedium (526 (478 to 573), 597 (537 to 657), and 589 (560 to 618) kg). Maternal in vitro liver
O2 consumption was lower (P<0.01) in the ADWChigh group compared to others (0.017 (0.01 to
0.02) vs 0.024 (0.02 to 0.03) μmol/min/mg), whereas foetal liver mass relative to BW was greater
(P=0.05). Maternal jejunal O2 consumption was not different between ADWC groups. Associations
between ADWC, hepatic O2 consumption (mol/min per BW) and foetal BW may suggest that livers
from cows in the ADWClow group tend to consume more O2 and have lighter foetuses (Table 1). In
addition, foetal hepatic mass relative to BW was negatively correlated with ADWC. Similarly, jejunal
O2 consumption (μmol/min per mg) tended to be higher in cows with lighter foetuses. This might
be an indication that pregnant cows may metabolise energy reserves and alter their metabolism in
order to meet the energetic demand of the growing foetus (Wood et al., 2013).
In conclusion, average daily weight change may have a stronger association with maternal and
foetal hepatic energy utilisation than with small intestinal energy utilisation. Our results indicate
that cows from the ADWChigh group had lower BW and lower hepatic O2 consumption, along with
foetuses that had heavier livers. Therefore, mature cows undergoing a larger BW loss may partition
a greater amount of energy to maintain foetal hepatic development. These results may indicate the
importance of hepatic tissue development in overall energy use in pregnant mature beef cows and
may aid in understanding and developing strategies for optimizing efficiency of feed utilisation.
Table 1. Partial correlation coefficients between BW measurements with maternal and foetal liver
and jejunal O2 consumption.1
O2 consumption Maternal Foetal
MBW FBW ADWC MBW FBW ADWC
Liver O2 consumption
μmol/min per mg liver 0.01 -0.16 0.24 -0.09 -0.29† -0.07
mol/min per BW -0.14 -0.23 0.27† -0.11 -0.18 -0.17
Liver g/kg BW -0.29† -0.18 0.16 -0.09 0.58* -0.33*
Jejunum O2 consumption
μmol/min per mg jejunum 0.22 -0.30† 0.02 -0.02 -0.33* 0.02
mol/min per BW -0.07 -0.20 -0.09 0.07 -0.30† -0.04
Jejunum g/kg BW 0.36* -0.27† 0.11 0.08 0.06 -0.24
1 ADWC = average daily weight change; BW = body weight; FBW = foetal body weight; MBW = maternal body weight.
*P≤0.05; †0.10≥P>0.05.
References
Camacho, L.E., C.O. Lemley, M.L. van Emon, J.S. Caton, K.C. Swanson and K.A. Vonnahme, 2014. Effects of maternal
nutrient restriction followed by realimentation during early and midgestation on beef cows. I. Maternal performance
and organ weights at different stages of gestation. Journal of Animal Science 92: 520-529.
Ferrell, C.L., 1988. Contribution of visceral organs to animal energy expenditures. Journal of Animal Science 66: 23-34.
Wood, K.M., B.J. Awda, C.J. Fitzsimmons, S.P. Miller, B.W. McBride and K.C. Swanson, 2013. Effect of moderate
dietary restriction on visceral organ weight, hepatic oxygen consumption, and metabolic proteins associated with
energy balance in mature pregnant beef cows. Journal of Animal Science 91: 4245-4255.

Overfeeding alters hepatic lipidomic and gene expression profiles
in the periparturient dairy cows
N. Qin1, T. Kokkonen1, S. Salin1, S. Selim1, T. Seppänen-Laakso2, J. Taponen3, A. Vanhatalo1 and
K. Elo1*
1Department of Agricultural Sciences, P.O. Box 28, 00014 University of Helsinki, Finland; 2Industrial
Biotechnology, VTT Technical Research Centre of Finland, Tietotie 2, P.O. Box 1000, 02044 VTT,
Finland; 3Department of Production Animal Medicine, University of Helsinki, Paroninkuja 20,
04920, Saarentaus, Finland; kari.elo@helsinki.fi
Abstract
Periparturient insulin resistance in dairy cows is associated with the change in hepatic lipid profiles
and gene expression. The aim of the present study was to investigate the effects of prepartal
overfeeding, as a possible factor to promote insulin resistance, on the lipidomic and gene expression
profiles in the liver of dairy cows. Sixteen multiparous cows were distributed to a controlled-energy
(CON) and a high-energy feeding group (HIGH). Liver samples were collected at 10 d before
predicted parturition and at 1 d and 9 d postpartum by biopsy. Prepartal overfeeding elevated the
concentration of sphingomyelin and various phospholipid subgroups in the HIGH group during the
periparturient period. Differentially expressed genes between the two groups were discovered in
the networks including lipid metabolism, small molecule metabolism, hepatic system disease, and
inflammatory responses.
Keywords: periparturient period, lipidomic profile, hepatic metabolism, sphingomyelin
Introduction
Dairy cows undergo physiological adaptions from late pregnancy to early lactation in response
to the alteration of energy balance, characterized by gradual increase of lipid mobilization from
adipose tissue and changes in hepatic metabolism. Prepartal overfeeding may alter the lipid profiles
in the peripheral tissues and subsequently influence hepatic gene expression associated with energy
metabolism via the regulation of insulin resistance. The results from human and mice studies have
highlighted the significant roles of sphingolipids in the development of insulin resistance (Larsen
and Tennagels, 2014). The aim of the present study was to investigate the effects of prepartal
overfeeding on the lipidomic and gene expression profiles in the liver of dairy cows during the
periparturient period.

The experimental animals consisted of 16 multiparous cows, distributed to a controlled-energy (100%
of energy requirement of pregnant cows, CON) and a high-energy feeding group (142% of energy
requirement of pregnant cows, HIGH). The liver samples were collected by biopsy at 10 d before the
predicted parturition, and 1 day and 9 days postpartum. The lipids in the liver samples collected at 10
d prepartum and 9 d postpartum were identified into classes including ceramide, sphingomyelin (SM),
triglyceride, diacylglycerol, lysophophos-phatidylcholine (LysoPC), lysophosphatidylethanolamine
(LysoPE) phosphatidylcholines (PC), phosphatidylethanolamine (PE), and phosphatidylinositol (PI)
and quantified using liquid chromatography-mass spectrometry based lipidomics. The statistical
analyses were conducted using a mixed model involving the random effect of block and fixed
effects of treatment, sampling day and their interaction. The differentially expressed genes between
groups at three timepoints were identified using Affymetrix Gene Chip Bovine Array, followed by
the function and network analyses with Ingenuity Pathway Analysis software.

HIGH group had higher SM concentration (P<0.01) in the liver compared to CON group during the
periparturient period (Table 1). The further sub-classification of SM based on the carbon numbers of
fatty acids showed that SM species with C16, C18, C20, C21, and C23 fatty acids were significantly
increased (P<0.05) in HIGH group. In contrast, SM species with C14, C16, and C24 fatty acids
were not influenced. In addition, HIGH group displayed significantly higher total concentration of
saturated SM (P<0.05) but unaltered unsaturated SM concentration compared to CON group (data
not shown). Prepartal high-energy feeding also resulted in significant elevation or the tendency
of elevation in PC (P<0.10), PE (P<0.05), PI (P<0.05), LysoPC (P<0.01), and LysoPE (P<0.05).
Table.1 Concentration of different lipid classes in the liver (μmol/l) during the periparturient period.1
Lipids Day Mean SEM P-value
CON HIGH Diet Day Diet×Day
Cer 10 d prepartum 0.3547 0.5013 0.0542 0.1940 0.9226 0.5036

9 d postpartum 1.3380 1.3324
SM 10 d prepartum 0.6523 0.8011** 0.0304 0.0054 0.0999 0.4435
9 d postpartum 0.5902 0.6640°
LysoPC 10 d prepartum 0.0353 0.0505** 0.0048 0.0037 0.2873 0.6192
9 d postpartum 0.0394 0.0527**
LysoPE 10 d prepartum 0.0045 0.0046 0.0002 0.0422 0.6982 0.5316
9 d postpartum 0.0042 0.0047
PC 10 d prepartum 17.3552 22.4105* 1.0014 0.0955 0.2546 0.2707
9 d postpartum 17.0571 18.9208
PE 10 d prepartum 1.6043 2.0835° 0.1146 0.0259 0.2096 0.3668
9 d postpartum 2.0008 2.3266*
PI 10 d prepartum 0.0035 0.0051* 0.0004 0.0296 0.0177 0.4262
9 d postpartum 0.0052 0.0064
DG 10 d prepartum 0.0055 0.0052 0.0009 0.9325 0.0005 0.6949
9 d postpartum 0.0119 0.0120
TG 10 d prepartum 3.8605 3.3903 3.5773 0.9139 <0.0001 0.7332
9 d postpartum 24.0189 25.2515
1 Statistical effects of diet in the same row: ° P<0.10; * P<0.05; ** P<0.01.
Pathway analyses suggested that the gene networks associated with lipid metabolism, small molecule
metabolism, and hepatic system disease were differentially expressed between the two groups at
10 d before predicted parturition. Specifically at this timepoint, the acute phase response signalling
pathway was found to be inhibited in HIGH group. After parturition, the most significant gene
networks regulated by overfeeding were associated with lipid metabolism, molecular transport, and
small molecule biochemistry at 1 d postpartum, and small molecule biochemistry and inflammatory
responses at 9 d postpartum.
References
Larsen, P. and N. Tennagels, 2014. On ceramides, other sphingolipids and impaired glucose homeostasis. Molecular
Metabolism 3: 252-260.

Ruminal metabolism of rapeseed meal insoluble nitrogen
T. Stefański1*, P. Huhtanen2, A. Vanhatalo3, K.J. Shingfield4 and S. Ahvenjärvi1
1Green Technology, Nutritional Physiology, Natural Resources Institute (Luke) Finland, 31600,
Jokioinen, Finland; 2Department of Agriculture Sweden, Swedish University of Agricultural Sciences,
90183 Umeå, Sweden; 3University of Helsinki, Department of Agricultural Sciences, P.O. Box 28,
00014 University of Helsinki, Finland; 4Institute of Biological, Environmental and Rural Sciences,
Aberystwyth University, Aberystwyth, SY23 3EB, United Kingdom; tomasz.stefanski@luke.fi
Abstract
The present study investigated the metabolism of 15N labelled insoluble rapeseed meal protein (ISP)
in the rumen. Four lactating cows in mid lactation and fitted with rumen cannula were used. Cows
were offered a total mixed ration based on grass silage and concentrate (forage:concentrate ratio
60:40 on a dry matter (DM) basis) containing 155 g crude protein/kg DM over the course of the
14 d experiment. Each cow received a pulse dose of ISP (696 mg of 15N in excess) administered
by mixing with rumen contents 1 h after morning feeding. Spot samples of rumen digesta were
collected immediately before and up to 82 h after pulse dosing. Total N content and 15N enrichment
was determined for insoluble, soluble non-ammonia (SNAN), ammonia and bacterial N fractions.
Analysis of pool sizes and tracer fluxes indicated that 17% of the 15N label was incorporated into
bacteria within 0.2 h. Rumen outflow and absorption of ammonia-N accounted for 2 and 22% of
the 15N administered, respectively. The majority of 15N label escaped the rumen as bacterial-N,
undegraded ISP and SNAN (recoveries of 44, 20 and 12%, respectively). In conclusion, a substantial
proportion of dietary insoluble nitrogen escapes the rumen as SNAN that represents a source of
amino acids not considered in modern protein evaluation systems.
Keywords: rapeseed, nitrogen, 15N, insoluble protein
Introduction
Microbial protein and rumen undegraded protein (RUP) are the major sources of absorbed amino
acids in ruminants. The potential to increase metabolisable protein (MP) supply from RUP is limited
(Ipharraguerre and Clark, 2005). A recent meta-analysis (Huhtanen and Hristov, 2009) demonstrated
that milk protein yield responses to incremental MP from microbial protein were about 5 times
greater compared with RUP. Evaluation of omasal flow data suggests that current protein evaluation
systems overestimate RUP supply, due to overestimates of the differences in protein degradability
between feed ingredients using the in situ method. Use of 15N labelled feed fractions is a useful
tool to investigate ruminal N metabolism. To better understand the fate of insoluble-N (ISN) in the
rumen an experiment involving pulse dosing of insoluble rapeseed meal protein (ISP) prepared from
15N labelled rapeseed was performed. By measuring 15N enrichment in different N fractions over
an 82 h interval post-dosing it was possible to derive quantitative estimates of the rate and extent
of ruminal insoluble feed protein metabolism in cows fed a diet containing moderate amounts of
crude protein (CP).

Rapeseed was grown under field conditions and fertilised with ammonium sulfate [(NH4)2SO4]
enriched with 10% of 15N/N (Isotec, Miamisburg, OH, USA). Rapeseed meal was prepared by
extracting oil from crushed seeds with diethyl ether. Insoluble N was isolated following removing
of soluble N fractions with bicarbonate-phosphate buffer. Four lactating cows (155±29.6 d in milk
producing 32.9±0.72 kg milk/d and consuming 22.4±0.45 kg dry matter (DM)/d) fitted with rumen
cannula were used. Cows were fed a total mixed ration based on grass silage concentrate (60:40

on DM basis) containing 155 g CP/kg DM for a 14 d period. Each cow received a pulse dose of
ISP (696 mg of 15N in excess) that was mixed with rumen contents 1 h after the morning feeding.
Spot samples of rumen digesta (200 g) were collected 0.1 h before, immediately after and 0.25,
0.50, 0.75, 1.0, 1.25, 1.5, 2, 3, 4, 5, 6, 8, 10, 12, 14, 17, 22, 27, 33, 39, 47, 55, 63, 72 and 82 h after
administration of labelled ISP. Total N was assessed using a Dumas type N analyser (Leco FP-428;
Leco Corporation, ST Joseph, MI) and enrichment of 15N was determined in insoluble, SNAN,
ammonia and bacterial N fractions using a Hydra 20-20 Isotope Ratio Mass Spectrometer and EA
Elemental Analyser Prep. System (Sercon LTD, Crewe, UK). Pool sizes of different N fractions were
determined by repeated (n=3) rumen evacuation and used to calculate rumen pool sizes of 15N for
each N fraction. A model of ruminal N metabolism was developed to estimate the rate of N transfer
between different N pools using the WinSAAM software (available at http://www.winsaam.com).
The model comprised 4 pools: ISN, SNAN, ammonia-N and microbial-N using quantitative estimates
of 10 metabolic fluxes to describe the transactions of N between different fractions. Measurements
of fluid and concentrate particle passage rates were used as fixed parameters in the model. Fluxes of
different N fractions leaving the rumen were estimated with a mechanistic model using initial pool
sizes and predicted rate parameters using the Powersim® 2.5 graphic modelling software.

A large proportion (17±1.3% of 15N label) was recovered in the bacterial N pool within 0.2 h
post-dosing. Recovery of 15N in intracellular pools and adsorption to cell surfaces was higher
(51±3.3%) for the SNAN fraction of rapeseed meal (T. Stefański et al., unpublished). 20±3% of
labelled ISN escaped ruminal degradation as insoluble N and 12±0.65% as SNAN. Incorporation
in microbial N represented the main route for ruminal escape of 15N labelled ISN (44±10.7%)
from the rumen. Furthermore, 12±1.1% of 15N labelled SNAN escaped the rumen as undegraded
feed protein (T. Stefański et al., unpublished data). Earlier reports have shown that dietary N can
escape ruminal degradation as SNAN (Choi et al., 2002). Both earlier and present findings indicate
that the assumptions of the Ørskov and McDonald (1979) model for calculating effective protein
degradability from in situ incubations do not hold true. In conclusion, ruminal metabolism of 15N
labelled insoluble rapeseed meal protein provided clear evidence that a substantial proportion of
dietary insoluble nitrogen can escape the rumen as SNAN which is not accounted for in modern
protein evaluation systems.
References
Choi, C.W., S. Ahvenjärvi, A. Vanhatalo, V. Toivonen and P. Huhtanen, 2002. Quantitation of the flow of soluble non-
ammonia nitrogen entering the omasal canal of dairy cows fed grass silage based diets. Animal Feed Science and
Technology 96: 203-220.
Huhtanen, P. and A.N. Hristov, 2009. A meta-analysis of the effects of dietary protein concentration and degradability
on milk protein yield and milk N efficiency in dairy cows. Journal of Dairy Science 92: 3222-3232.
Ipharraguerre, I.R. and J.H. Clark, 2005. Impacts of source and amount of crude protein on the intestinal supply of
nitrogen fractions and performance of dairy cows. Journal of Dairy Science 88: E22-E37.
Ørskov, E.R. and I. McDonald, 1979. The estimation of protein degradability in the rumen from incubation measurements
weighted according to rate of passage Journal of Agricultural Science Cambridge 92: 499-503.

Effects of solid feed intake on nutrient utilisation from milk
replacer in veal calves
J.J.G.C. van den Borne1, S.J.J. Alferink1, M.J.W. Heetkamp2 and W.J.J. Gerrits1
1Wageningen University, Animal Nutrition Group, P.O. Box 338, 6700 AH Wageningen, the
Netherlands; 2Wageningen University, Adaptation Physiology Group, P.O. Box 338, 6700 AH
Wageningen, the Netherlands; j.vandenborne@schippers.eu
Abstract
This study was designed to assess the effects of solid feed (SF) supplementation on utilisation of
macronutrients derived from milk replacer (MR) in veal calves. Thirty-two male Holstein-Friesian
calves were randomly assigned to pairs, and each pair of calves was assigned to one of two levels
of SF allowance: 9 or 27 g DM SF/kg0.75 per d. The SF consisted of 80% low-protein concentrates,
10% chopped wheat straw and 10% corn silage based on DM. MR was partly exchanged for SF
(as 1:1.7) to achieve similar growth rates across treatments. In four experimental periods, each pair
of calves was measured with or without supplementation of lactose, fat or protein (189 kJ extra
digestible energy per kg0.75 per d). A higher level of SF intake did not affect energy utilisation for
growth, but increased methane production and urinary energy excretion in calves. Utilisation of
digestible nitrogen for growth increased from 53 to 63% with increasing SF level. Supplementation
of protein increased nitrogen retention, but the efficiency of digestible nitrogen utilisation for growth
decreased. Supplementation of lactose increased digestible nitrogen utilisation for growth by 6 (high
SF) to 10% (low SF). The incremental efficiencies of metabolisable energy utilisation for growth
were similar for fat (73%) and lactose (74%), whereas the incremental energetic efficiency for protein
was 39%. In conclusion, the level of SF intake does not affect energy utilisation, but greater intake
of low-protein SF and also lactose supplementation increase the efficiency of protein utilisation for
growth in veal calves.
Keywords: concentrates, energy, lactose, nitrogen, roughage
Introduction
Provision of solid feed (SF) to veal calves has increased substantially during the last decade, and
is associated with a reduction in abnormal oral behaviour in calves without compromising calf
performance and health (Berends, 2014). Nonetheless, the majority of the digestible energy intake
in veal calves still originates from milk replacer (MR). Interactions between MR and SF, or nutrients
derived from these feed sources, may occur during digestion, absorption and metabolism in calves,
possibly impacting feed evaluation. Therefore, the current study was designed to assess the effects of
SF allowance on digestive and metabolic utilisation of macronutrients derived from MR in veal calves.

Thirty-two male Holstein-Friesian calves (77±2 kg) were randomly assigned to pairs, and each pair
of calves was assigned to one of two levels of SF: 9 or 27 g DM SF/kg0.75 per d. The SF consisted
of 80% low-protein concentrates, 10% chopped wheat straw and 10% corn silage based on DM.
For calves at the low SF level, MR was provided at 1.85 × the digestible energy requirements for
maintenance. For calves at the high SF level, MR was exchanged for SF as 0.59 g DM MR per g
DM SF to achieve similar growth rates across SF levels. After six weeks of adaptation to the SF
level, each pair of calves was assigned to each type of macronutrient supplementation (lactose, fat,
protein, or no supplement) in four 2-wk periods in random order. Supplementation was equivalent to
189 kJ digestible energy per kg0.75 per d. Calves were housed in metabolic cages to allow separate
collection of faeces and urine, and measurements occurred during the second week of each period.

In total, 55 energy balances (pair of calves as experimental unit) and 106 nitrogen balances (individual
calf as experimental unit) were successfully completed. Apparent total tract digestibility decreased for
energy (from 90.8 to 86.6%) and protein (from 86.9 to 83.8%) with increasing SF level. Digestibility
of protein and energy increased with protein supplementation, but interactions between macronutrient
supplementation and SF level were absent. An increase in SF intake did not affect energy utilisation
for growth (Table 1), regardless of the type of macronutrient supplemented. Greater SF intake
increased methane production and tended to increase urinary energy excretion in veal calves. Fat
and lactose supplementation with MR inhibited methane production, and these effects were more
pronounced at a high than at a low level of SF intake.
Supplementation of protein increased nitrogen retention, but the efficiency of digestible nitrogen
utilisation for growth decreased by 11% on average. Supplementation of lactose, but not of fat,
increased the utilisation of digestible nitrogen for growth by 6 (high SF) to 10% (low SF). The
incremental efficiencies of metabolisable energy utilisation for growth were unaffected by SF level
and were similar for fat (73%) and lactose (74%), whereas the incremental energetic efficiency for
protein was 39%. It was concluded that the level of SF intake does not affect energy utilisation in
veal calves, but greater intake of low-protein SF as well as lactose supplementation increases the
efficiency of protein utilisation for growth.
Table 1. Effects of level of solid feed (SF; 9 vs 27 g DM/kg0.75 per d) and macronutrient supplementation
(MS; fat, lactose, protein or no supplementation) on energy partitioning in veal calves. Values are
least square means and are expressed as kJ/kg0.75 per d.1
Item SF MS SEM P-value
No Fat Lactose Protein SF MS SF×MS
GE intake Low 1,141 1,327 1,323 1,334 2.7

High 1,037 1,216 1,218 1,231 2.4
DE intake Low 940 1,083 1,107 1,139 10.9 <0.001 <0.001 0.955
High 985 1,124 1,145 1,186 10.9
ME intake Low 887 1,036 1,074 1,074 9.8 <0.001 <0.001 0.246
High 925 1,086 1,085 1,123 9.0
Heat production Low 608 639 643 723 9.1 0.065 <0.001 0.310
High 627 667 669 722 9.5
ER as protein Low 105 109 126 176 3.8 <0.001 <0.001 0.202
High 126 122 128 193 4.3
ER as fat Low 181 287 302 174 12.8 0.646 <0.001 0.454
High 180 289 297 203 14.1
1 GE = gross energy; DE = digestible energy; ME = metabolisable energy; ER = energy retention.
References
Berends, H., 2014. Nutrient utilization, dietary preferences, and gastrointestinal development in veal calves: interactions
between solid feed and milk replacer. PhD thesis, Wageningen University, Wageningen, the Netherlands, 239 pp.

Bovine utero-placental glucose and cationic amino acid transporters
during early gestation
M.S. Crouse1*, K.J. McLean1, M.S. Crosswhite1, L.P. Reynolds1, C.R. Dahlen1, B.W. Neville2, P.P.
Borowicz1 and J.S. Caton1
1Department of Animal Sciences, and Center for Nutrition and Pregnancy, North Dakota State
University, Fargo, ND 58108, USA; 2Central Grasslands Research Extension Center, Streeter, ND
58483, USA; matthew.crouse@ndsu.edu
Abstract
During early gestation, nutrients are transported to the conceptus via transporters in the endometrium
and developing placenta. In the present study, we examined glucose transporters, GLUT1 and
GLUT3, and the cationic amino acid transporters, SLC7A1, SLC7A2, and SLC7A3, in order to test the
hypothesis that relative mRNA expression of transporters would be different due to day of gestation
and utero-placental tissue type. Crossbred Angus heifers (n=46) were ovariohysterectomized on
d16, 22, 28, 34, 40, or 50 of gestation or on d16 of the synchronized estrous cycle (non-pregnant
controls). Uterine and foetal tissues (caruncular (CAR), intercaruncular (ICAR) endometrium, and
foetal membranes) were collected from the uterine horn containing the conceptus. Chorioallantoic,
amniotic, and plasma fluids were collected from heifers on d40 and 50 of gestation to determine
concentrations of glucose and amino acids. Expression of GLUT1 and SLC7A2 showed a tendency
towards being different (P<0.10). Glucose transporter GLUT3 and arginine transporters SLC7A1 and
SLC7A3 showed significant day × tissue interactions (P<0.05). Expression of GLUT3 was greater in
d50 CAR; SLC7A1 was greater on d34 in ICAR; and SLC7A3 was greater in CAR tissue on d34 of
gestation. Glucose concentration showed a trend towards day × fluid interaction (P=0.10). Arginine
concentration showed a day × fluid interaction (P=0.01). This data supports our hypothesis that
transporters for glucose and cationic amino acids differentiate in their level of mRNA expression
due to d of gestation and utero-placental tissue type.
Keywords: arginine, CAT, endometrium, foetal membranes, GLUT
Introduction
Currently, fertilization rates for first service AI are approximately 90% in beef heifers (Bridges
et al., 2013); however, by d 30 of gestation, only 50 to 60% are viable embryos. Moreover, up to
40% of all embryonic loss occurs before d 40 of gestation. The presence of nutrient transporters
and nutrient flow to the growing embryo is crucial for proper development and growth. During this
time, the placenta is developing and the fetus begins to utilise increasing quantities of glucose and
amino acids (Bazer et al., 2014; Groebner et al., 2011). Thus, the expression of glucose and amino
acid transporters in the utero-placenta becomes essential to the viability of the conceptus. The main
utero-placental glucose transporters are GLUT1 and GLUT3 and the main cationic amino acid
transporters are SLC7A1, SLC7A2, and SLC7A3 whose substrates are arginine and lysine, which are
directly linked to angiogenesis and cell proliferation. The aim of this study was to test the hypothesis
that mRNA expression of glucose and cationic amino acid transporters in utero-placental tissues
would be different due to an interaction of day of gestation and specific utero-placental tissue type
during early pregnancy.

Cross-bred Angus heifers (n=46) were ovariohysterectomized on d16, 22, 28, 34, 40, or 50 of gestation
or on d16 of the synchronized estrous cycle (non-pregnant controls; NP). Uterine and foetal tissues
(caruncular (CAR), intercaruncular (ICAR) endometrium, and foetal membranes (FM)) were collected

from the uterine horn containing the conceptus. Chorioallantoic, amniotic, and plasma fluids were
collected from heifers on d40 and 50 of gestation to determine concentrations of glucose and amino
acids. Relative mRNA expression of glucose and cationic amino acid transporters were determined
using qPCR for each tissue type across days of gestation; the endometrium of NP controls provided
baseline values. Arginine and lysine concentration were determined via HPLC method, and glucose
concentration was determined using colorimetric assay. Pairwise comparisons across day of gestation
and utero-placental tissues were conducted when a day × tissue interaction was present (P≤0.05).

Glucose transporter GLUT3 and arginine transporters SLC7A1 and SLC7A3 showed significant
day × tissue interactions (P<0.05). Expression of GLUT3 was greater on d50 CAR compared with
d34 CAR, which was greater than CAR on all other days or ICAR and FM on all days. Glucose
transporter GLUT3 has the highest known affinity of any glucose transporter (1.5 µM) and is
specifically known for its roles as a neuronal glucose transporter (Ganguly et al., 2007). Increased
expression of GLUT3 in caruncular tissue on d50 of gestation may suggest increased foetal energy
requirements which are supplemented by increased transport of glucose at the maternal-foetal
interface. Cationic amino acid transporter SLC7A1 was greater on d34 in ICAR compared with d40
ICAR, which was greater than CAR on all other days or ICAR and FM on all days. Relative mRNA
expression of SLC7A3 was greater in CAR on d16 compared to CAR on all other days or ICAR or
FM on all days. These peaks in cationic amino acid transporter expression coincide with key dates
of gestation; d16 for SLC7A3 with maternal recognition of pregnancy, and SLC7A1 for d34 being a
critical time window for conceptus viability ~d30 of gestation suggesting that cationic amino acid
transporters play influential roles in maintaining conceptus viability by supplying arginine which is
known for its role in angiogenesis and cell proliferation. Arginine concentration showed a day × fluid
interaction (P=0.01), being greater in chorioallantoic fluid on d40 compared with all other fluids and
days. Therefore, the data does support our hypothesis that there is an effect of day of gestation and
utero-placental tissue type on the mRNA expression of glucose and cationic amino acid transporters.
References
Bazer, F.W., G. Wu, G.A. Johnson and X. Wang, 2014. Environmental factors affecting pregnancy: endocrine disrupters,
nutrients and metabolic pathways. Mol. Cell. Endocrinol. 398(1-2): 53-68.
Bridges, G.A., M.L. Day, T.W. Geary and L.H. Cruppe, 2013. Deficiencies in the uterine environment and failure to
support embryonic development. J. Anim. Sci. 91: 3002-3013.
Ganguly, A., R.A McKnight, S. Raychaudhuri, B. Shin, Z. Ma, K. Moley, S.U. Devaskar, 2007. Glucose transporter
isoform-3 mutations cause early pregnancy loss and fetal growth restriction. Am. J. Physiol. Endocrinol. Metab.
292: 1241-1255.
Groebner, A.E., I. Rubio-Aliaga, K. Schulke, J.D. Reichenbach, H. Daniel, E. Wolfe, J.J.D. Meyer and S.E. Ulbrich,
2011. Increase of essential amino acids in the bovine uterine lumen during preimplantation development.
Reproduction 141: 685-695.

Heat production in ruminants: from experimental data to feed unit
systems through meta-analysis
D. Sauvant1*, S. Giger-Reverdin1, P. Nozière2 and I. Ortigues-Marty2
1UMR Modélisation Systémique Appliquée aux Ruminants, INRA, AgroParisTech, Université Paris-
Saclay, 75005, Paris, France;2INRA,UMR 1213 Herbivores, VetagroSup, Theix, 63122 Saint Genès
Champanelle, France; sauvant@agroparistech.fr
Abstract
A meta-analysis of a large database built on calorimetric studies was performed to study the major
factors of variation of heat production (HP) in ruminants. The database comprised 832 treatments
from calorimetry trials in cattle, sheep and goat at maintenance, growing or lactating. It appeared
that the best inter-species coefficient of power of live weight (LW) is not 0.75 as usually assumed,
but 0.95. When data are expressed on LW0.95, generic maintenance requirements are 38 kcal/kg
LW0.95 and the overall conversion efficiency of metabolisable energy (ME) into net energy (NE) is
57.6%. The ratio q = ME/GE (GE = gross energy) significantly explains variations in HP/LW0.95,
and when its effect is included in the prediction of HP, there is no influence of the species and of
the physiological status. The results open the perspective of building a future NE model based on
HP in ruminants.
Keywords: ruminant, heat production, live-weight, meta-analysis
Introduction
Numerous data of heat production (HP) by ruminants have been published for decades and few
researchers have tried to translate them into feed unit systems. The major proposal was that of
Armsby (1917). In France, a feed unit system based on the same principle was used between 1945
and 1978 (Leroy et al., 1950). Otherwise, Emmans (1994) proposed to use HP data to create a
system of ‘effective energy’. Nowadays, all feed unit systems for ruminants are based on NE, which
is calculated as ME multiplied with k, being a coefficient of efficiency of ME utilisation, which
varies according to the type of production (maintenance, growth, milk,...) and diet quality. However,
various arguments suggest to reconsider the possibility of using HP rather than k to calculate NE
and subsequently revise the feed unit systems. Thus, it would be easier to take into account the
influence of external temperature which can be considered as a major factor of variation, when feed
unit systems are used in hot areas. The aim of this study was to interpret by meta-analysis a large
database of calorimetric studies.
Methods
The database ‘Rumener’, including a total of 832 treatments from calorimetry trials in cattle (230
lactating and 281 others), sheep (244) and goat (77), was used for this study. The data were treated
by meta-analysis to split intra- from inter-experiments, or publications, variations according to
Sauvant et al. (2008). To achieve normality of data and residues, live weight (LW) and HP were
transformed into logarithm.

In a first step, we investigated the best power coefficient of LW to relate HP (kcal/d) to LW. The
LW amounted to 554±84, 374±123, 58±26 and 40±11 kg for cows, growing cattle, sheep and goat,
respectively. When considering only animals fed ad libitum (dry matter intake, DMI%LW>0.8),
the global regression is:

logHP = 1.71 + 0.94 logLW (n=783, R2=0.93, RMSE=0.13) (1)
The slope is significantly higher than 0.75 but less than 1 (P=0.09). This regression was improved
by taking into account DMI%LW. The intra-publication equation is:
logHP = 1.54 + 0.95 logLW + 0.54 logDMI%LW (npub = 126, n = 746, RMSE = 0.03) (2)
and therefore:
HP = 33.9 (DMI%LW)0.54 LW0.95 (3)
In a second step after data were expressed on LW0.95, we calculated the average relationship between
HP (48.7±14.9, min. = 14.9, max. = 108.0 kcal/kgLW0.95) and ME intake (MEI) (64.8±32.6, min.
= 12.4, max. = 205.9 kcal/kgLW0.95). The intra-experiment regression was
HP/LW0.95 = 38.0 + 0.424 (MEI/LW0.95 – 38) (nexp=266, n=799, RMSE=2.24) (4)
Thus, the generic maintenance requirement is 38.0 kcal/LW0.95 and the mean conversion efficiency
of ME into NE is 57.6%, close to the rounded value of 60% suggested by Tolkamp (2010).
When the last equation is applied to the dataset, the residuals are significantly influenced by the ratio
q=ME/GE and the sommative equation is:
HP/LW0.95 = 22.3 + 0.439 (MEI/LW0.95 – 17.45 (q-0.6)) (nexp=266, n=801, RMSE=2.12) (5)
Interestingly, there was no influence of species nor of physiological status on the residuals of
Equations 4 and 5, suggesting that Equation 5 can be considered as a generic one and can be used
to predict NE intake (NEI):
NEI/LW0.95 = MEI/LW0.95 – HP/LW0.95.
In conclusion, when HP aspects are considered the optimal power of LW is 0.95 and not 0.75.
Moreover, it is possible to use a generic equation to predict NE intake for all types of ruminants
from ME intake and q.
References
Armsby, H.P., 1917. The nutrition of farm animals. The MacMillan Company, New York, USA.
Emmans, G.C., 1994, Effective energy: a concept of energy utilisation applied across species. British Journal of
Nutrition 71: 801-821.
Leroy, A.M., J. Sentex and J. Delage, 1950. Equivalents fourragers. In: R. Roux (ed.), pp. 20-80.
Sauvant, D., P. Schmidely, J.J. Daudin and N.R. St-Pierre, 2008. Meta-analyses of experimental data in animal nutrition.
Animal 2: 1203-1214.
Tolkamp, B.J., 2010. Efficiency of energy utilisation and voluntary feed intake in ruminants. Animal 4: 1084-1092.

Meta-analysis of the efficiency of metabolisable protein utilisation
in dairy cows and goats
D. Sauvant1*, J.B. Daniel1, G. Cantalapiedra-Hijar2 and P. Nozière2
1UMR Modélisation Systémique Appliquée aux Ruminants (MoSAR), INRA, 16 rue Claude
Bernard, 75231 Paris cedex 05, France; 2INRA, UMR 1213 Herbivores, Theix, 63122 Saint Genès
Champanelle, France; sauvant@agroparistech.fr
Abstract
To update the French PDI (metabolisable protein) system, new relationships were extracted by
meta-analyses and applied to calculate the PDI supplies for a large diversity of diets. Moreover,
a new approach was applied to assess the non-productive protein expenditures: metabolic faecal
protein and urinary N losses, and loss of protein from scurf. Iterative calculation for the efficiency
of PDI utilisation (EffPDI) for milk protein production demonstrated that estimations of EffPDI
were improved when it was assumed to be variable but with a common value for all processes of
protein synthesis and mobilization in the body.
Keywords: lactating ruminants, protein efficiency, response to supply
Introduction
To improve the prediction of protein utilisation in ruminants, INRA updated the way to calculate
metabolisable protein (MP called PDI) supplies (Sauvant and Nozière, 2016) and the estimation of
non-productive protein requirements (Sauvant et al., 2016). Until recently, the efficiency of PDI
into milk protein yield (MPY) was assumed to be constant (67% in NRC, 2001; 64% in INRA), or
variable but only according to the level of PDI supply (Van Duinkerken et al., 2011; Volden, 2011).
The aim of this work was to propose a better assessment of EffPDI by taking into account the major
processes of protein synthesis in the organism.

Two databases of experiments focused on the effect of protein supplies were built from the literature,
one for dairy cows (879 treatments and 296 experiments) and one for dairy goats (114 treatments
and 36 experiments). All equations were fitted using meta-analysis. We calculated the inevitable
nitrogen expenditures which lead to non-productive protein requirements: metabolic faecal protein
and urinary N losses, and loss of protein from scurf. Moreover, the body protein balance was
calculated as a constant ratio of the energy balance. The calculation methods of these expenditures
were described by Sauvant et al. (2016). Then, EffPDI% (= 100 × Σ[produced protein]/Σ[supplied
PDI]) was calculated iteratively assuming 2 hypotheses. The first hypothesis was that EffPDI varies
only for milk protein production as proposed by Van Duinkerken et al. (2011) and Volden (2011).
The second hypothesis was that EffPDI varies for milk protein and for all other protein expenditures
and mobilization processes, except endogenous nitrogen loss which was assumed to have an EffPDI
of 100%.

The best prediction of EffPDI% was achieved when the same value of EffPDI% was applied for all
protein synthesis functions (Hypothesis 2). For dairy cows, EffPDI% decreases non linearly with
increasing dietary PDI content (from 65 to 150 g PDI/kg DM). The intra-experiment equation is:
EffPDI% = 67.0 exp[-0.007 (PDI-100)] (879 tr., 296 exp., RMSE=1.7%)

With Hypothesis 1, this equation is slightly different and has a RMSE of 3.3%. A very similar
equation, a bit less precise, was found for dairy goats:
EffPDI% = 66.0 exp[-0.007 (PDI-100)] (114 tr., 36 exp., RMSE=2.7%)
For both species, at a dietary PDI content of 100 g/kg DM, the EffPDI is equal or not different from
67%, the value proposed as constant by NRC (2001).
Assuming that animals fed at their potential have an EffPDI of 67%, this value is used as a pivot. First,
to assess in a given situation if the PDI supply is in excess or not; second, to calculate the potential
MPY corresponding to this pivot and third, to have a better prediction of N loss in urine. At this
pivot, for dairy cows, it was observed that the marginal MPY response to available PDI supplies is
the same, being 19%, whatever the potential of production (Daniel et al., in press). Thus a common
equation can be used (Daniel et al., in press) to explore the response of MPY to PDI supplies around
the pivot defined as above. This common equation also includes MPY response to net energy supply.
In conclusion, the efficiency of PDI utilisation is variable and equal for all protein syntheses or
mobilization functions of dairy cows and goats. This efficiency can be calculated when the potential
of the animal is not known using a pivot situation corresponding to EffPDI=67%.
References
Daniel, J.-B., N.C. Friggens, P. Chapoutot, H. Van Laar and D. Sauvant, in press. Milk yield and milk composition
responses to change in predicted net energy and metabolizable protein: a meta-analysis. Animal, http://dx.doi.
org/10.1017/S1751731116001245.
National Research Council (NRC), 2001. Nutrient requirements of dairy cattle (7th rev. Ed.). National Academy Press,
Sauvant, D. and P. Nozière, 2016. Quantification of the main digestive processes in ruminants: the equations involved
in the renewed energy and protein feed evaluation systems. Animal 10: 755-770.
Sauvant, D., G. Cantalapiedra-Hijar, L. Delaby, J.-B. Daniel, P. Faverdin and P. Nozière, 2016. Actualisation des besoins
protéiques des ruminants et détermination des réponses des femelles laitières aux apports de protéines digestibles
dans l’intestin. INRA Productions Animales 28: 347-368.
Van Duinkerken, G., M.C. Blok, A. Bannink, J.W. Cone, J. Dijkstra, A.M. Van Vuuren and S. Tamminga, 2011. Update
of the Dutch protein evaluation system for ruminants: the DVE/OEB2010 system. Journal of Agricultural Science
149: 351-367.
Volden, H., 2011. NorFor – the Nordic feed evaluation system. EAAP Scientific Series No. 130, Wageningen Academic
Publishers, Wageningen, the Netherlands, 180 pp.

Determinants of feed preferences and intake in calves
H. Berends1,2*, W.J.J. Gerrits2, L.E. Webb3, E.A.M. Bokkers3 and C.G. van Reenen3,4
1current address: Trouw Nutrition R&D, P.O. Box 220, 5830 AE Boxmeer, the Netherlands; 2Animal
Nutrition, Wageningen University, P.O. Box 338, 6700 AH Wageningen, the Netherlands; 3Animal
Production Systems, Wageningen University, P.O. Box 338, 6700 AH Wageningen, the Netherlands;
4Livestock Research, Wageningen University and Research Centre, P.O. Box 65, 8200 AB Lelystad,
the Netherlands; harma.berends@trouwnutrition.com
Abstract
This study aimed to gain insight in long-term dietary preferences of calves and the determinants of
their preferences and intake in a free-choice setting. Calves were given free access to milk replacer,
water, hay, straw, concentrate and corn silage for 25 weeks. Despite large variation in intake of diet
components, calves choose ration components to achieve a remarkably constant ratio of digestible
protein to digestible energy. Variation in feed preferences could not be explained by diet bulkiness
or energy intake from milk replacer or concentrates.
Keywords: dietary choice, intake regulation, ruminants
Introduction
Satiety leads to inhibition of further eating, decline in hunger, and a determinant of voluntary feed
intake. In ruminants, distension of the reticulorumen is considered as the major regulator of satiety
(Allen, 1996). In addition, the absorption of metabolic fuels, like volatile fatty acids, has been shown
to increase satiety in dairy cows. Although it is known from weaned calves that they show large
variation in feed preferences (Atwood et al., 2001), there is lack of information on diet-related factors
involved in feed intake regulation in young calves provided with milk and solid diet components.
Compared to adult ruminants, effects of the development of the reticulorumen in calves may interfere
with its distension. In addition, feed intake regulation in calves may be driven by the availability
of specific nutrients, and may depend on the choice of ration components available. The aim of the
current study was to gain insight in long-term dietary preferences of calves developing towards
ruminants and the determinants of their preferences and intake in a free-choice setting.

Twenty-four male Holstein-Friesian calves (17±0.5 days of age, 46±0.3 kg BW) were given free
access to milk replacer (via an automated milk dispenser), water, long hay, long barley straw,
pelleted concentrate, and chopped corn silage until 27 weeks of age. Individual intake of each diet
component was recorded for a week at 3 and 6 months of age. Energy and protein digestibility of
feed components was assessed based on literature data. Variation in feed intake related parameters
was expressed by their coefficient of variation (CV), where a low CV was assumed to reflect a
higher similarity of preference between calves for that specific feed intake related parameter. In an
attempt to find cues that drive voluntary feed intake, the importance of bulkiness in the control of
feed intake was evaluated, as a parameter of distension and hypertonicity in the reticulorumen. The
ratio between gross and digestible energy (GE:DE) was chosen as an indicator for the bulkiness
of ration components. DE intake was included as a dependent variable in a mixed model analysis
(PROC MIXED in SAS 9.20), with a fixed effect of age, individual feed intake related parameters
(i.e. bulkiness, DE from milk replacer, DE from concentrates) as a co-variable, and a random effect
of calf to account for repeated observations on the same animal. The interaction between the co-
variable and age was included in all models.

Variation in intake of individual diet components was very large (Table 1), for example shown by
the CV of 25% for milk replacer (6 months of age) and 110% for corn silage (3 months of age).
For total DM intake, the CV was 18% and 16% at 3 and 6 months of age, respectively. Correcting
intake for variation in BW, metabolic BW, as DE intake per metabolic bodyweight (MBW), or as
digestible protein (DCP) intake per MBW further reduced variation. Expressing dietary preferences
as a ratio of DCP:DE intake, mean values of 9.5 and 8.8 at 3 and 6 months of age were found with
a CV of 4.6 and 4.9% at 3 and 6 months of age, respectively. The constant ratio in DCP:DE ratio
is remarkable when considering the large differences in DCP and DE contents and their ratio for
the individual diet components (10.6 for milk replacer, 9.0 for concentrates, 4.3 for hay, 3.4 for
corn silage, 0.9 for straw). Dietary bulkiness was not associated with DE intake at 3 and 6 months
of age. There was no correlation with DE from milk replacer and DE from concentrates either. In
conclusion, variation in feed preferences cannot be explained by diet bulkiness or DE from milk
replacer or concentrates. Instead, results indicate that calves choose ration components to achieve
a remarkably constant ratio of DCP:DE while selecting a diet.
Table 1. Coefficient of variation (CV, %) in feed intake observed in calves with free access to 6
ration components.1
3 months of age 6 months of age
mean±SD CV (%) mean±SD CV (%)
Total dry matter intake (DMI; kg/d) 2.34±0.416 17.8 4.96±0.77 15.5
from milk replacer 1.39±0.367 26.4 1.75±0.44 25.3
from concentrates 0.57±0.435 76.6 2.17±0.70 32.3
from hay 0.31±0.154 49.1 0.57±0.28 49.9
from corn silage 0.03±0.037 110.0 0.43±0.41 94.7
from straw 0.07±0.056 78.9 0.04±0.04 104.4
DMI per MBW (kg/kg0.75/d) 0.07±0.010 14.5 0.08±0.01 12.6
DE per MBW (MJ/kg0.75/d) 1.18±0.147 12.5 1.33±0.16 12.2
Digestible CP per MBW (g CP/kg0.75/d) 11.1±1.39 12.5 11.74±1.51 12.9
Digestible CP:DE 9.46±0.438 4.6 8.82±0.43 4.9
1 DMI = dry matter intake; MBW = metabolic bodyweight; DE = digestible energy; CP = crude protein.
References
Allen, M.S., 1996. Physical constraints on voluntary intake of forages by ruminants. Journal of Animal Science 74:
3063-3075.
Atwood, S.B., F.D. Provenza, R.D. Wiedmeier and R.E. Banner, 2001. Influence of free-choice vs mixed-ration diets
on food intake and performance of fattening calves. Journal of Animal Science 79: 3034-3040.

Heat stress involves activation of pAMPK and FOXO3 regulating
glycolysis and proteolysis in the skeletal muscle of dairy cows
F. Koch1*, O. Lamp1,2 and B. Kuhla1
1Institute of Nutritional Physiology ‘Oskar Kellner’, Leibniz Institute for Farm Animal Biology,
Wilhelm-Stahl-Allee 2, 18196 Dummerstorf, Germany; 2Schleswig Holstein Chamber of
Agriculture, Teaching and Training Center Futterkamp, Gutshof, 24327 Blekendorf, Germany;
koch@fbn-dummerstorf.de
Abstract
Increasing environmental heat during summer periods leads to decreased milk production, reduced
reproduction rate and growth of dairy cows. Particularly, the period of late-gestation and early-
lactation represents an enormous metabolic challenge for the dam, regularly resulting in a negative
energy balance during the early post-partum period. Heat stress aggravates this state especially as
feed intake decreases. The purpose of this study was to determine the adaptation of skeletal muscle
protein and glucose metabolism to heat stress around calving. Thirteen German Holstein cows were
divided into heat-stress or pair-fed groups. Three weeks before and after parturition, both groups were
exposed to 15 °C with ad libitum feeding. After one week, the temperature was gradually increased
to 28 °C for heat stress cows and maintained at thermoneutrality for pair-fed cows. Blood and muscle
biopsies were taken in both periods. Increased temperature had no effect on the abundance of genes
involved in proteolysis. Heat stress reduces glycolysis via the pAMPK pathway in late-gestation
to minimize metabolic heat production in skeletal muscle. Proteolysis is maintained by FOXO3 in
early-lactation after heat stress.
Keywords: environmental heat, dairy cow, glycolysis, protein break down, skeletal muscle
Introduction
The two major stressors adversely affecting the performance of high yielding dairy cows are the
metabolic stress during the transition period from late pregnancy to early lactation, and environmental
heat stress (West, 2003). The combination of these two stressors has severe negative consequences
on feed intake, milk production and metabolic health (Baumgard and Rhoads, 2013; Hahn, 1999).
Muscle tissue plays a primary role in the metabolic adaptation to negative energy balance and heat
stress e.g. by providing substrates for hepatic gluconeogenesis (Rhoads et al., 2013). The adaptation
response of muscle metabolism to heat stress is not yet fully understood. Therefore, the objective
of this study was to evaluate the effect of environmental heat on glucose and protein metabolism at
the protein and mRNA level.

Thirteen German Holstein dairy cows were allocated to heat-stressed (HS; n=7) and pair-fed (PF,
n=6) group. Twenty one days before parturition (ante parturition: ap), both groups were exposed to
the same climate condition (15 °C, RH=63.3%, temperature-humidity-index (THI) = 59) with ad
libitum feeding for one week (period P1). On the following transition day, the air temperature was
continuously increased to permanent 28 °C (RH=52%, THI=76) for HS and maintained at 15 °C
(RH=69%, THI=60) for PF animals. PF cows received the same amount of feed as cows ingested
under heat stress conditions. Cow groups were kept under these conditions for 6 further days (P2).
The experiment was repeated 22 days after parturition (post parturition: pp) with the same animals
in each group. Muscle biopsies (Musculus semitendinosus) were taken in the morning on transition
day (reflecting P1) and on day 6 of P2 after heat-stress exposure or pair-feeding, respectively, both
in the ap and pp period. Blood samples were taken on the last day of period P1 and daily in P2 for

lactate analysis using ABX Pentra 400 (Horiba Medical, Kyoto, Japan). Muscle tissues were used for
Western Blot (pAMPK, AMPK) and mRNA abundance quantified using RT-qPCR (PDK4, PDK2,
FOXO3, LDHA, LDHB, CAPN1, CAPN2).

Activation of AMPK by phosphorylation was increased in HSap cows from P1 to P2 (170%, P<0.03),
whereas phosphorylation of AMPK in PFap remained unaltered. The abundance of PDK4 mRNA
increased from P1 to P2 of HSap (360%, P<0.05) and tended to be higher during PF conditions
(P<0.063), whereas PDK2 mRNA did not differ between P1 and P2. During the post-partum period,
PDK2 but not PDK4 mRNA abundance decreased by 21% in HS from P1 to P2 (P<0.05), but not in
PF cows. A significant reduction of LDHA and LDHB mRNA abundance from P1 to P2 was observed
in HS late-gestation cows (60%, P<0.05 and 47%, P<0.01, respectively), whereas in PF animals
LDHA mRNA abundance tended to be lower in P2 compared to P1 (P<0.063). In PFpp cows, LDHA
was significantly reduced in P2 relative to P1 (66%, P<0.05), but not in HS cows. Furthermore,
HSap cows showed higher plasma lactate concentrations compared to PF cows during the P2 period
(P=0.05). In early-lactation, lactate concentrations did not change in response to HS or PF. The mRNA
abundance of FOXO3, a transcriptional regulator of glycolysis and protein degradation was greater in
P2 compared to P1 in HSpp (130%, P<0.05), but not PFpp cows. CAPN1 mRNA decreased from P1
to P2 in HSap and PFpp cows (21-30%, P<0.05), whereas CAPN2 abundance remained unaltered.
These results indicate that environmental heat activates the pAMPK pathway to control catabolic
pathways in late-gestating dairy cows. Thus, skeletal muscle glycolysis and lactate production are
down-regulated in late-gestation after 6 days of heat exposure and to a lesser extent after pair-feeding,
presumably to spare glucose from circulation and because of exhausted muscle glycogen reserves.
Inhibition of the pyruvate dehydrogenase complex is reduced and anaerobic glycolysis is maintained
under post-partum heat stress. In contrast to increased 1/3-methyl histidine plasma levels of HSap
(Lamp et al., 2015) and increased FOXO3 in HSpp as indicators for muscle proteolysis, increased
proteolysis of muscle tissue could not be detected at mRNA level.
Taken together, these results reflect different metabolic adaptation of bovine skeletal muscle between
thermoneutrality and heat stress. While HS in late pregnancy reduces conversion of pyruvate to acetyl
CoA and lactate but increases nitrogen export presumably to sustain amino acid supply to the fetus,
HS in early lactation maintains protein degradation and lactate metabolism.
References
Baumgard, L.H. and R.P. Rhoads Jr., 2013. Effects of heat stress on postabsorptive metabolism and energetics. Annual
Review Animal Bioscience 1: 311-337.
Hahn, G.L., 1999. Dynamic responses of cattle to thermal heat loads. Journal of Animal Science 77, Suppl. 2: 10-20.
Lamp, O., M. Derno, W. Otten, M. Mielenz, G. Nurnberg and B. Kuhla, 2015. Metabolic heat stress adaption in transition
cows: differences in macronutrient oxidation between late-gestating and early-lactating German Holstein dairy
cows. PLoS One 10: e0125264.
Rhoads, R.P., L.H. Baumgard and J.K. Suagee, 2013. 2011 and 2012 Early Careers Achievement Awards: metabolic
priorities during heat stress with an emphasis on skeletal muscle. Journal of Animal Science 91: 2492-2503.
West, J.W., 2003. Effects of heat-stress on production in dairy cattle. Journal of Dairy Science 86: 2131-2144.

Performance and body composition in high and low RFI beef cattle
R.D. Sainz*, K.C. Dykier, F.M. Mitloehner and J.W. Oltjen
University of California, Davis, CA 95616, USA; rdsainz@ucdavis.edu
Abstract
In order to identify animals with greater or lesser feed efficiency, 98 weaned Angus cross beef calves
(71 steers and 27 heifers) were fed individually for two sequential periods of 56 and 52 days. Feed
offered and refused were measured daily, body weights were taken at 14 day intervals, and ultrasound
measures (longissimus muscle area and subcutaneous fat over the 12th-13th ribs) were taken at the
beginning, middle and end of each trial. Feed was delivered twice a day, on an ad libitum basis for
Trial 1 and at 1.5% of BW for Trial 2. For Trial 1, residual feed intake (RFI) was determined as the
residual of the regression of dry matter intake (DMI) on mid-test BW0.75 and average daily gain
(ADG). For Trial 2, BW0.75 was omitted. High and Low RFI groups were defined as > 0.5 standard
deviation above or below zero, respectively, with intermediate animals classified as medium RFI.
RFI groups in Trial 1 had similar initial and final BW and ADG, and different DMI, gain:feed and
RFI (P<0.001). Differences in DMI were eliminated in Trial 2, and RFI and gain:feed were similar.
Low RFI animals were leaner at the conclusion of both trials (P<0.05). Differences in feed efficiency
in beef cattle reflect mainly differences in appetite and body composition.
Keywords: efficiency, residual feed intake, body composition, beef cattle
Introduction
Residual feed intake (RFI), defined as the residual of the regression of dry matter intake (DMI)
on mid-test BW0.75 and average daily gain (ADG) (Koch et al., 1963), has gained acceptance as
a measure of feed efficiency that is independent of growth rate and mature body size. Factors that
may contribute to variation in RFI include maintenance energy requirement and body composition
(Castro-Bulle et al., 2007; Sainz et al., 2013). This experiment aimed to identify High and Low RFI
beef calves, and determine differences in associated responses, such as variations in body composition,
nutrient balance, behaviour, and physiological parameters.

In Trial 1, 98 weaned calves (steers and heifers, 263±29 d of age) were housed and fed individually
on a high-flaked corn diet 1 for 56 days after adaptation. Animals were weighed and ultrasound-
scanned every 14 d, and feed intakes were monitored daily. Trial 2 began on day 99, under the same
protocol but with feed intake restricted to 1.5% of BW, ending on day 156.
Results
The equation to calculate RFI i.e. ɛ) for Trials 1 and 2 were:
Trial 1: DMI = -2.64 + 0.123 BW0.75 + 0.854 ADG + ɛ; R2 = 0.80, sy.x = 0.53
Trial 2: DMI = 4.03 + 2.34 ADG + ɛ; R2 = 0.46, sy.x = 0.49
As expected, RFI groups in Trial 1 had similar initial and final BW and ADG, and different DMI,
gain:feed and RFI (Table 1). Differences in DMI were eliminated in Trial 2, and RFI and gain:feed
were similar. Low RFI animals were leaner at the conclusion of both trials. Differences in feed
efficiency in beef cattle reflect mainly differences in appetite and body composition.

Table 1. Differences among residual feed intake (RFI) groups in Trial 1 and 2.
Trial Trait High Medium Low SD P-value
1 Initial BW, kg 284.4 281.5 282.6 33.1 0.94

Final BW, kg 394.2 388.4 394.2 46.5 0.83
ADG, kg/d 1.962 1.909 1.992 0.365 0.64
DMI, kg/d 9.30 8.62 8.11 1.11 0.001
Gain:feed 0.211 0.220 0.245 0.026 <0.001
RFI, kg/d 0.554 0.007 -0.650 0.251 <0.001
Ribeye area, cm2 69.10 67.02 68.41 6.86 0.44
12th-13th rib fat, cm 0.91 0.85 0.76 0.18 0.012
2 Initial BW, kg 450.4 454.4 48.8 0.80
Final BW, kg 501.6 505.0 56.7 0.86
ADG, kg/d 0.977 0.965 0.214 0.87
DMI, kg/d 6.28 6.33 0.701 0.82
Gain:feed 0.154 0.152 0.0244 0.78
RFI, kg/d -0.040 0.040 0.489 0.62
Ribeye area, cm2 82.07 82.60 8.26 0.85
12th-13th rib fat, cm 1.25 0.99 0.27 0.005
Conclusions
Low RFI cattle have similar weights and weight gains, but lower intakes and higher feed efficiencies
as high RFI cattle. This may be at least partly due to less fat deposition.
Acknowledgements
Financial support: USDA-NIFA Multistate Project W2010 / CA-D-ASC-2209-RR.
References
Castro-Bulle, F.C.P., P.V. Paulino, A.C. Sanches, and R.D. Sainz, 2007. Growth, carcass quality, protein and energy
metabolism in beef cattle with different growth potentials and residual feed intakes. Journal of Animal Science
85: 928-936.
Koch, R.M., L.A. Swiger, D. Chambers, and K.E. Gregory, 1963. Efficiency of feed use in beef cattle. Journal of
Animal Science 22: 486-494.
Sainz, R.D., G.D. Cruz, E. Mendes, C.U. Magnabosco, Y.B. Farjalla, F.R.C. Araujo, R.C. Gomes and P.R. Leme, 2013.
Performance, efficiency and estimated maintenance energy requirements of Bos taurus and Bos indicus cattle. In:
J.W. Oltjen, E. Kebreab and H. Lapierre (eds.). Energy and protein metabolism and nutrition in sustainable animal
production. EAAP Scientific Series No. 134, Wageningen Academic Publishers, Wageningen, the Netherlands,
pp. 69-70.

Energy metabolism in high and low RFI beef cattle
R.D. Sainz*, K.C. Dykier, F.M. Mitloehner and J.W. Oltjen
University of California, Davis, CA 95616, USA; rdsainz@ucdavis.edu
Abstract
In order to identify animals with greater or lesser feed efficiency, 98 weaned Angus cross beef calves
(71 steers and 27 heifers) were fed individually twice a day for two periods of 56 (Trial 1, ad libitum)
and 52 (Trial 2, 1.5% of BW) days. For Trial 1, residual feed intake (RFI) was determined as the
residual of the regression of dry matter intake on mid-test BW0.75 and average daily gain. For Trial
2, BW0.75 was omitted. High and Low RFI groups were defined as > 0.5 standard deviation above or
below zero, respectively, with intermediate animals classified as medium RFI. At the conclusion of
each trial, animals were placed in group pens and allowed to obtain about 300 g/d of lucerne pellets
from a Greenfeed™ feeder equipped with sampling and in-line measurement of O2, CO2 and CH4.
This was done with normal feeding, and also after 1-3 days of solid feed withdrawal. On day 4 of
fasting, a subset of animals was placed in metabolism crates fitted with a head box with sampling
and measurement of the same gases for 24 hours. Heat production (HE, Mcal/day) was estimated
as O2 uptake (L/day) × 4.825. Both methods yielded similar values for HE, although there was
substantial slope bias between them. Using Greenfeed™ data, RFI groups did not differ (P>0.05)
in HE (0.238 Mcal/kg0.75 for both), but fasting reduced HE (0.298 to 0.179 Mcal/kg0.75, P<0.001).
The methods used were unable to detect differences in HE among RFI groups.
Keywords: efficiency, residual feed intake, heat production, beef cattle
Introduction
Residual feed intake (RFI), defined as the residual of the regression of dry matter intake on mid-
test BW0.75 and average daily gain (Koch et al., 1963), has gained acceptance as a measure of feed
efficiency that is independent of growth rate and mature body size. Factors that may contribute to
variation in RFI include maintenance energy requirement and body composition (Castro-Bulle et
al., 2007; Sainz et al., 2013). This experiment aimed to identify High and Low RFI beef calves, and
determine differences in associated responses, such as heat production in the fed and fasted states,
measured using two different methods (head box and Greenfeed™), and after a period of ad libitum
feeding and after a 30% feed restriction.

In Trial 1, 98 weaned calves (steers and heifers, 263±29 d of age) were housed and fed individually
on a high-flaked corn diet 1 for 56 days after adaptation. Animals were weighed and ultrasound-
scanned every 14 d, and feed intakes were monitored daily. During days 57 to 98, calves ranked as
High or Low RFI were subjected to indirect calorimetry for estimation of heat production in the fed
and fasting states. Trial 2 began on day 99, under the same protocol but with feed intake restricted
to 1.5% of BW, ending on day 156. During days 157 to 190, calves ranked as High or Low RFI
were once again subjected to indirect calorimetry for estimation of heat production in the fed and
fasting states. At each time, animals were placed in group pens and allowed to obtain about 300 g/d
of lucerne pellets from a Greenfeed™ feeder equipped with sampling and in-line measurement of O2,
CO2 and CH4. This was done with normal feeding, and also after 1-3 days of solid feed withdrawal.
On day 4 of fasting, a subset of animals were placed in metabolism crates fitted with a head box
with sampling and measurement of the same gases for 24 hours. Heat production (HE, Mcal/day)
was estimated as O2 uptake (L/day) × 4.825.

Results
Preliminary analyses based only on oxygen uptake and only from the Greenfeed™ system suggest
little or no difference between RFI groups. On the other hand, fasting for 72 hours reduced heat
production by 33% (headboxes) or 27% (Greenfeed). The two measures were correlated, but with
substantial slope bias between them (Figure 1).
Conclusions
Low RFI cattle have similar gains but lower intakes and higher feed efficiencies as high RFI cattle.
This may be due to lower basal metabolism, but the respiratory exchange methods used were unable
to detect differences in HE among RFI groups.
0.400
Y=X
Heat production Mcal∙kg-0.75∙d-1
0.350
0.300
(Greenfeed)
0.250 y = 0.6773x +
0.0753 R² = 0.6765
0.200
0.150 High RFI

Low RFI
0.100
0.100 0.150 0.200 0.250 0.300 0.350 0.400
Heat production Mcal∙kg ∙d (Head boxes)
-0.75 -1
Figure 1. Heat production (Mcal•kg0.75/d) measured using headboxes and Greenfeed™.
Acknowledgements
Financial support: USDA-NIFA Multistate Project W2010/CA-D-ASC-2209-RR.
References
Castro-Bulle, F.C.P., P.V. Paulino, A.C. Sanches, and R.D. Sainz, 2007. Growth, carcass quality, protein and energy
metabolism in beef cattle with different growth potentials and residual feed intakes. Journal of Animal Science
85: 928-936.
Koch, R.M., L.A. Swiger, D. Chambers, and K.E. Gregory, 1963. Efficiency of feed use in beef cattle. Journal of
Sainz, R.D., G.D. Cruz, E. Mendes, C.U. Magnabosco, Y.B. Farjalla, F.R.C. Araujo, R.C. Gomes and P.R. Leme, 2013.
Performance, efficiency and estimated maintenance energy requirements of Bos taurus and Bos indicus cattle. In:
J.W. Oltjen, E. Kebreab and H. Lapierre (eds.). Energy and protein metabolism and nutrition in sustainable animal
production. EAAP Scientific Series No. 134, Wageningen Academic Publishers, Wageningen, the Netherlands,
pp. 69-70.

Relation of leukocyte activation and proliferation to feed efficiency in
peripartal cows
S. Meese1, S.E. Ulbrich1, H. Bollwein2, R. Bruckmaier3, O. Wellnitz3, M. Kreuzer1, M. Röntgen4,
U. Gimsa4 and A. Schwarm1*
1ETH Zurich, Institute of Agricultural Sciences, Universitaetstrasse 2, 8092 Zurich, Switzerland;
2Vetsuisse Faculty Zurich, University of Zurich, Winterthurerstrasse 260, 8057 Zurich, Switzerland;
3Vetsuisse Faculty Berne, University of Berne, Bremgartenstrasse 109a, 3001 Bern, Switzerland;
4Leibniz Institute for Farm Animal Biology, Wilhelm-Stahl-Allee 2, 18196 Dummerstorf, Germany;
angela.schwarm@usys.ethz.ch
Abstract
In the peripartum state, immune function is generally compromised in mammals. In this period,
blood leukocytes displayed decreased DNA synthesis (proliferation) when challenged with mitogens.
Leukocyte activation, the phase of the cell cycle preceding DNA synthetic phase, might already be
impaired by the limited availability of energy and nutrients. We hypothesized that the proliferation
of peripheral blood mononuclear cells (PBMC) is related to their activation and the animal’s feed
intake, feed efficiency and mobilization of body reserves. Eleven pluriparous Holstein cows were
studied at weeks -2, +2 and +2 around calving. Zootechnical parameters as well as in vitro activation
and proliferation of PBMC were assessed by measuring their oxygen consumption rate and MTT
(3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2H-tetrazolium bromide)-reducing activity, respectively.
Cows were grouped by the magnitude of the proliferative response in week 2 postpartum into high-
and low-proliferators. Contrary to the expectations, the observed differences in PBMC proliferation
were not related to the activation and the animal’s feed intake, feed efficiency (milk yield per intake)
and mobilization of body reserves. It can be speculated, that in case of inflammatory and metabolic
diseases, cows with lower immune responses postpartum may have longer recovery rates and thus
exhibit higher levels and durations of milk yield depression.
Keywords: energy partitioning, leukocyte proliferation, dairy cow, early lactation
Introduction
Lactation is an energy intensive process for mammals and might be maintained at the expense of
other processes, such as immune function. Accordingly, in lactating dairy cows, disease susceptibility
is often associated with limitations in energy availability (Mallard et al., 1998). Due to the selection
for very high milk yields, widely exceeding the requirements of the calves, lactating cows have
particularly high energy needs. An impairment of activation (G1 phase of the cell cycle) of
mononuclear leukocytes from the peripheral blood (PBMC: peripheral blood mononuclear cells)
could be a proximate cause of their reduced proliferation. Furthermore, the animal’s feed intake, feed
efficiency and mobilization of body reserves could be the ultimate causation explaining differences
in PBMC proliferation.

An experiment was conducted with 11 Holstein cows (643±66 kg body weight) entering their 2nd to
5th lactation and receiving a total mixed ration ad libitum. Feed intake, milk yield, body condition
score (BCS) and backfat thickness (BFT) were recorded at weeks -2, +2 and +12 relative to
parturition. Blood was collected from jugular vein into vacutainer tubes. The PBMC were isolated
by density gradient centrifugation. Activation and proliferation of PBMC in the presence and in the
absence of the mitogen phytohaemagglutinin (PHA, 4 µg/ml) were assessed by measuring oxygen
consumption rate (OCR) after 24 h (as a measure of ATP production; Schwarm et al., 2013) and

the MTT (3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2H-tetrazolium bromide)-reducing activity
after 72 h of incubation, respectively. The activation was calculated as the difference between OCR
[nmol/min/(107 cells)−1] of PBMC in the presence and in the absence of PHA divided by the OCR
of PBMC in the absence of PHA. The proliferation index (PI) was calculated as the difference
between optical density of PBMC in the presence and in the absence of PHA divided by the optical
density of PBMC in the absence of PHA. Cows were grouped by the magnitude of the proliferative
response in week 2 postpartum into high- (HP; 6 cows; PI=1.5±0.3) and low-proliferators (LP; 5
cows; PI=0.6±0.2). A two-way repeated measurement ANOVA with Holm-Šídák adjustment was
carried out to evaluate the effect of group, week and group × week interaction.

Groups did not differ in daily dry matter intake per metabolic body weight, but intake increased with
time in HP (92, 143 and 180 g/kg0.75 in week -2, +2 and +12, respectively). Feed efficiency was not
affected by group, ranging from 1.4 to 3.1 kg energy-corrected milk/kg dry matter intake. There was
no group effect on BCS and BFT, but both decreased with time. There was a group × week interaction
for PBMC proliferation, with group differences at weeks -2 (PI: HP 0.3 < LP 1.4) and +2 (HP > LP,
values see methods) relative to parturition and an effect of time in HP (PI: 0.3, 1.5 and 0.6 in week
-2, +2 and +12, respectively). Activation of PBMC was not affected by group. Groups did not differ
in plasma concentrations of glucose, non-esterified fatty acids, β-hydroxybutyrate and cortisol. A
time effect only occurred with glucose, reaching a nadir at week +2 (3.4, 2.9 and 3.2 mmol/L in
week -2, +2 and +12, respectively). Contrary to expectations, the PBMC proliferation was not related
to PBMC activation and the animal’s feed intake, feed efficiency and body reserve mobilization.
Interestingly, cows with a low PBMC proliferation in the 2nd week postpartum, displayed a high
PBMC proliferation 2 weeks before calving. It could be speculated that cows with a high PBMC
proliferation preceding parturition were energetically incapable of an adequate response after calving.
Cell cycle analysis by flow cytometry and qRT-PCR are underway. The present results also indicate
that the observed differences in PBMC proliferation as such seem to be unrelated to the amount of
feed energy partitioned towards milk production. However, in case of inflammatory and metabolic
diseases, cows with lower immune responses may have longer recovery rates and thus exhibit higher
levels and durations of milk yield depression.
Acknowledgements
The help of co-workers at author institutions and at Strickhof is greatly acknowledged. The project
was supported by the German Research Foundation (SCHW 1485/3-1).
References
Mallard, B.A., J.C. Dekkers, M.J. Ireland, K.E. Leslie, S. Sharif, C.L. Vankampen, L. Wagter and B.N. Wilkie, 1998.
Alteration in immune responsiveness during the peripartum period and its ramification on dairy cow and calf
health. Journal of Dairy Science 81: 585-595.
Schwarm, A., T. Viergutz, B. Kuhla, H.M. Hammon and M. Schweigel-Röntgen, 2013. Fuel feeds function: energy
balance and bovine peripheral blood mononuclear cell activation. Comparative Biochemistry and Physiology,
Part A 164: 101-110.

Assessing intestinal absorption of amino acids
M.D. Hanigan*, K. Estes and J. Castro
Department of Dairy Science, Virginia Polytechnic Institute and State University, Blacksburg, VA
24060, USA; mhanigan@vt.edu
Abstract
This research utilised a stable isotope based approach to provide an in vivo model to determine AA
bioavailability for ingredients fed in ruminant diets. A trial was conducted with one steer and two
periods where one treatment was tested per period. One treatment acted as a positive control to validate
the method. On the last day of each period, the steer received a 2 hour infusion of a complete mixture
of amino acids (AA) isotopically labeled and blood was collected over a 4 hour period. Plasma was
assessed for isotopic enrichment and bioavailability values were assessed for each AA. Estimated
AA entry rates into circulation presented small errors suggesting this as a promising technique.
Keywords: amino acid, isotope, bioavailability, in vivo, cattle
Introduction
Supplying the proper mix of essential amino acids (EAAs) should result in improved animal
efficiency and reduced release of nitrogen to the environment (Khezri et al., 2011), but this requires
knowledge of microbial protein flows and rumen non-degradable but digestible amino acids (AA)
sources. The Dairy NRC (NRC, 2001) utilised an empirical equation to predict EAA flows, but such
an approach assumes individual AA digestibility to be that of the protein and cannot be used outside
of the range of the data used to derive the equations. The purpose of this research was to test an in
vivo stable isotope based approach to determine absorbed EAA delivered by an abomasal infusion
of raw essential AAs compared to a basal diet. A similar method of assessing absorbed AA using an
isotope bolus method has been tested by Borucki et al. (2008) and found to be valid but requiring
fairly larger isotope dosages which are costly.

This trial was conducted using one steer and two periods where each period was 10 d in length.
By establishing a base diet and assessing the rate of isotope clearance on that diet and then again
after a portion of the diet has been replaced with the ingredient of interest, the difference between
the two reflects the difference in AA supply only. The base diet contained a mix of corn silage,
grass hay, soybean meal, salt, vitamins and minerals. The steer was fed a high protein diet (~24%
CP) to ensure tissue protein synthesis was not affected by the ingredient being tested. Treatments
included the base diet alone or the base diet with a portion of the soybean meal substituted out for
abomasally infused raw isoleucine, leucine and methionine (raw EAA). The steer was restrictively
fed at 2 h intervals for the final 24 hours of each period to establish steady state AA absorption from
the gut. On day 10 of each period, the steer received a 2 h infusion of a complete mixture of AA
that was isotopically labeled, and blood was collected over a 4 h period. Plasma was assessed for
isotopic enrichment using a gas chromatograph linked with an isotope ratio mass spectrometer via
a combustion oven (Sessions, 2006).

This isotope based approach is based on the use of the variation of AA clearance rates from blood
as a representation of the variation of AA absorption rates from the intestinal tract in steady state
conditions. The model is a two pool model where one pool represents the free AA pool in blood

and the other represents body protein. Table 1 shows the AA entry rate into blood of the base diet
treatment and the abomasally infused isoleucine, leucine and methionine treatment.
Bioavailability values were computed for each AA by determining the entry rate of each AA from
soybean meal. The difference between the raw EAA entry rate and the calculated soybean meal entry
rate from the raw EAA treatment yields the contribution of AA solely from the raw EAA infusion.
This value was divided by the infused AA concentration to achieve bioavailability.
Based on this data, bioavailability values for leucine and methionine are just above 100% (122.9
and 121.5%, respectively) while isoleucine is roughly 89.5% bioavailable. Corn silage, chopped
hay and dry matter intake changed between the two periods which could have introduced variability
by affecting microbial protein yield, leading to the over/under predicted bioavailability values. This
data represents a single steer and two treatments so the variability in reported values needs to be
statistically quantified by using several animals and protein sources. However, even with this small
data set, estimates of error are lower than those reported by Borucki et al. (2008) for a bolus isotope
technique (SE=13.7). More accurate values should result from a two hour infusion of isotope as
the sampling period is 4 h whereas the bolus dose only allows a sampling period of 30 min after
the initial bolus before the isotope enrichment returns to baseline. An extension of this research
will be conducted that will result in 6 observations for 6 treatments which should provide more
representative bioavailability estimates. The estimates of bio-availability for each EAA reported
here are not dependent upon in situ based estimates of ruminally undegraded protein, but are direct
measurements in the animal. With further investigation, this method could provide a useful tool to
develop AA digestibility values for main ingredients used in ruminant diets.
Table 1. Entry rates of amino acids (AA) for the base diet and raw essential amino acid diet.
AA Raw EAA STD CV Base diet STD CV AA amount Bio-

entry rate entry rate infused availability
(mmol/min) (mmol/min) (g/d) (%)
Isoleucine 2.75 0.28 10.21 1.28 0.04 3.19 48.08 89.5

Leucine 2.73 0.24 8.91 1.65 0.09 5.31 28.33 122.9
Methionine 1.10 0.09 8.81 0.46 0.03 7.15 17.08 121.5
References
Borucki, C.S.I., H. Lapierre, L.E. Phillip, P.W. Jardon and R. Berthiaume, 2008. Towards non-invasive methods to
determine the effect of treatment of soya-bean meal on lysine availability in dairy cows. Animal 22: 224-234.
Khezri, A., S. Baker and A. Khatibi, 2011. Improving the efficiency of dietary nitrogen use in dairy farms to minimize
environmental pollution. SAADC 2011 Strategies and Challenges for Sustainable Animal Agriculture-Crop
Systems 3: 731-736.
Sessions, A.L., 2006. Isotope-ratio detection for gas chromatography. Journal of Separation Science 29: 1946-1961.

Production factors have a larger impact than genetic selection for lamb
growth
K.R. Kelman1,2*, C. Alston3, D.W. Pethick1,2 and G.E. Gardner1,2
1Australian Cooperative Research Centre for Sheep Industry Innovation, Australia;2Murdoch
University, School of Veterinary and Life Sciences, Western Australia, 6150, Australia;3Griffith
Social and Behavioural Research College, Griffith University, Mt. Gravatt, Queensland, 4122,
Australia; k.kelman@murdoch.edu.au
Abstract
Growth and factors which influence it, such as nutrition and genetic selection for growth, are key
profit drivers in animal production. Nutrition can vary substantially between sites of lamb rearing,
so it was hypothesised that the magnitude of the impact for production factors, would be greater
than for genetic selection. Weight data from 17,525 lambs produced over five years and eight sites
was analysed using a linear mixed effects model in SAS with fixed effects for production factors
(site, year of birth, sex, birth type-rear type, age of dam, sire type, dam breed within sire type) and
genetic selection for growth at key time points (weaning and post weaning breeding values). As
hypothesised, production factors had a greater impact on lamb growth than genetic selection, with
the impact of site of rearing as much as double the impact of genetic selection and as much as nine
times the impact of other production factors.
Keywords: lamb, breeding value, growth
Introduction
Growth is a key profit driver in animal production. Therefore, factors which influence growth have
the potential to impact profitability. In lamb, growth is influenced by both nutrition and genetic
selection. As environmental nutrition can vary substantially between sites of lamb rearing, it was
hypothesised that the magnitude of the impact for production factors, including site, would be greater
than for genetic selection for growth using sire breeding values.

The design of the Sheep Cooperative Research Centre Information Nucleus Flock experiment has
been presented by Fogarty et al. (2007) and Van der Werf et al. (2010). Each year between 2007 and
2011 approximately 3,500 lambs were produced from Merino and cross bred ewes from artificially
inseminated mating at eight research sites across Australia (Kirby NSW, Trangie NSW, Cowra NSW,
Rutherglen VIC, Hamilton VIC, Struan SA, Turretfield SA, and Katanning WA), representing a broad
range of environments and production systems. The lambs were progeny of 435 key industry sires
representing major production types in the Australian sheep industry. The sires used had divergent
Australian Sheep Breeding Values (ASBV) for birth weight (BWT), weaning weight (WWT) and
post weaning weight (PWWT). Lambs were reared under extensive pasture grazing conditions and
fed pellets, hay or grain when feed supply was limited (Ponnampalam et al., 2014). Lamb weights
were recorded at birth, weaning and approximately every two weeks thereafter resulting in weight
data from 17,525 lambs with 141,206 weight recordings. Lamb weight at birth, 100 days (weaning)
and 150 days (post weaning) were analysed using a linear mixed effects model in SAS with fixed
effects for production factors (site, year of birth, sex, birth type-rear type, age of dam, sire type and
dam breed within sire type). Lamb identification, sire identification and dam identification by year
were included as random terms. To assess the impact of genetic selection for growth, sire ASBVs
for growth (BWT, WWT, and PWWT) were included separately as covariates in the model.

Lamb weight was influenced by both production and genotypic factors and as hypothesised, the
magnitude of the effects varied (Table 1). Site of rearing had the largest impact on lamb weight at
100 and 150 days, being 1.7 and 2.0 times the magnitude of the effect of the WWT and PWWT
ASBVs, respectively. Furthermore, the effect of site was as much as 9.2 times the magnitude of effect
of other production factors. Sire type had also had a large impact on weight at 100 and 150 days,
with progeny of Terminal sires being heavier at these time points. At birth, lamb birth type had the
greatest impact on lamb weight, being 1.8 times the magnitude of the effect of the BWT ASBV and
as much as 14.5 times the magnitude of effect of other production factors. Site of rearing also had
a relatively large effect at birth, however the results indicate that sibling competition is more likely
to impact nutrition in utero than the site of rearing. Overall the impact of sire ASBVs for growth
had a relatively small effect on progeny weight compared to the effect of production factors such as
site, birth type-rear type combination or sire type. While producers cannot control site environment
elements they are able to select for growth using sire ASBVs and genotype. At 100 and 150 days the
magnitude of difference between sire types is close to double the magnitude of the growth ASBV
effect. Thus producers will have a large impact on lamb growth by using the high growth Terminal
sire type, and a greater impact by combining this with the use of growth ASBVs.
Table 1. Magnitude of effect for production and for genetic effects on lamb weight (kg).1
Birth weight Weight day 100 Weight day 150
Site 1.19 10.26 13.20

Year 0.12 2.72 2.79
Sex 0.32 1.56 1.76
Birth type-rear type 1.74 9.42 8.81
Dam age 0.50 3.50 2.20
Sire type 0.53 9.21 10.44
Dam breed-Sire type 0.46 4.52 4.74
BWT 0.95 – –
WWT – 5.98 –
PWWT – – 6.62
1 Australian sheep breeding values: BWT = birth weight; WWT = weaning weight; PWWT = post weaning weight; – =
not modelled.
References
Fogarty, N.M., R.G. Banks, J.H.J Van der Werf, A.J. Ball and J.P. Gibson, 2007. The information nucleus – a new
concept to enhance sheep industry genetic improvement. Proc. Ass. Adv. Anim. Breed. Genet. 17: 29-32.
Ponnampalam, E.N., K.L. Butler, R.H. Jacob, D.W. Pethick, A.J. Ball, J.E. Hocking Edwards, G. Geesink and D.L.
Hopkins, 2014. Health beneficial long chain omega-3 fatty acid levels in Australian lamb managed under extensive
finishing systems. Meat Science 96(2, Part B): 1104-1110.
Van der Werf, J.H.J, B.P. Kinghorn and R.G. Banks, 2010. Design and role of an information nucleus in sheep breeding
programs. Animal Production Science 50(12): 998-1003.

Increasing growth breeding values in Merino lambs decreases
the non-esterified fatty acid response during feed deprivation
S.M. Stewart*, D.W. Pethick, G.E. Gardner and P. McGilchrist
School of Veterinary and Life Sciences, Murdoch University, South Street, 6150 Murdoch, WA,
Australia; s.stewart@murdoch.edu.au
Abstract
Under current Australian industry pre-slaughter guidelines, lambs may be off feed for up to 48
hours prior to slaughter. This aim of this study was to examine the effect of growth using estimated
breeding values for post-weaning weight (PWT) on the non-esterified fatty acid (NEFA) response
to feed deprivation. Eighty nine lambs from Merino dams with Merino or Terminal sires with a
range in breeding values for PWT were used in this experiment. Blood samples were collected via
jugular catheters at time 0, 24, 36 and 48 hours of feed deprivation for the determination of NEFA
concentration. NEFA concentration was higher in Merino lambs at 36 and 48 hours of feed deprivation
compared to Terminal sired lambs. There was a negative association between NEFA concentration
during feed deprivation and increasing PWT but only in Merino sired lambs at 48 hours of feed
deprivation. This effect accounted for the siretype difference in NEFA concentration at 36 and 48
hours. Further work is required to understand the impact of feed deprivation on fat metabolism in
animals selected for high genetic growth.
Keywords: feed restriction, growth, lipolysis, non-esterified fatty acids, sheep
Introduction
Under current Australian industry pre-slaughter guidelines, lambs may be off feed for up to 48 hours
prior to slaughter. Recent work by Stewart et al. (unpublished data) indicates that significant adipose
tissue turnover occurs in prime lambs pre-slaughter but the role of fasting per se and genotype in
young animals has not been studied. Animals with high metabolic requirements for growth have
increased adipose tissue mobilisation and plasma non-esterified fatty acid concentrations (NEFA)
in response to periods of nutritional restriction (Foot and Russel, 1979). Therefore it is hypothesised
that selection for increasing growth using the Australian Sheep Breeding Value for post-weaning
weight (PWT) will increase NEFA response to feed deprivation.

Lambs (n=89) from Merino dams artificially inseminated using Terminal and Merino sires with a
broad range in PWT breeding values were used in this experiment. Lambs were randomly allocated
to three groups, balanced for sex, siretype and sire. On day 0, lambs were assigned to individual pens
and fed the same ration (ME 11 MJ/kg DM, CP 14.5% DM) prior to insertion of jugular cannulas
on day 5. On day 6 lambs were taken off feed and resting blood samples collected at time 0, 24, 36
and 48 hours of feed deprivation for the determination of plasma NEFA concentration. Lambs were
slaughtered and carcass data collected on day 30. NEFA concentration was analysed using linear
mixed effect models with fixed effects for siretype, sex and time off-feed. Covariates were then
incorporated including PWT and body composition indicators obtained from carcases post slaughter.

NEFA concentration increased with time off feed (P<0.05). NEFA concentrations in Merino sired
lambs was 13 and 22% higher (P<0.05; Figure 1) at 36 and 48 hours of feed deprivation compared
to Terminal sired lambs. Contrary to our hypothesis, there was a negative association between
NEFA concentration and increasing PWT during feed deprivation. NEFA concentration decreased

2.0
**
**
NEFA (mmol/l)
1.5
1.0
0.5
0.0
0 24 36 48
Hours off feed
Figure 1. Effect of time off-feed (hours) on non-esterified fatty acid (NEFA)concentration (mmol/l)
in Merino (grey bars) and Terminal (black bars) sired lambs. **P<0.01.
by 23% (P<0.05; Figure 2) as PWT increased from -1 to 12 kg, but only in Merino sired lambs at
48 hours of feed deprivation. Furthermore, when PWT was included in the model, it accounted for
the difference found between Terminal and Merino siretypes at 36 and 48 hours of feed deprivation.
This difference in growth impetus between siretypes may also explain the reduced NEFA response
in high growth Terminal sired lambs compared to Merino lambs at 48 hours of feed deprivation.
The mechanism driving this is unclear; however higher rates of protein turn-over are observed in
high growth animals (Oddy et al., 1995). During starvation this may result in an elevated supply of
gluconeogenic amino acids being available for glucose homeostasis (Heitmann and Bergman, 1980),
thus reducing the reliance on fat mobilisation. Further studies under commercial slaughter conditions
are needed to understand the effects of fasting versus other factors such as acute pre-slaughter stress.
1.8
1.5
NEFA (mmol/l)
1.2
0.9
0.6
-2 2 6 10 14 18
PWT
Figure 2. Association between non-esterified fatty acid (NEFA) (mmol/l) and post-weaning weight
(PWT) at 48 hours off-feed. Lines represent LS means ± S.E. Black dots denote Merino and black
triangles Terminal sire residuals from the response surface.
Acknowledgements
This research was funded by the Meat and Livestock Australia Science and Innovation award for
young people in science. The authors wish to thank the Australian Meat Processors Corporation and
the Australian Cooperative Research Centre for Sheep Industry Innovation for funding the Ph.D.
candidature scholarship and research. The technical staff at Murdoch University and the Katanning
research station are thanked for their invaluable assistance in data collection, sample processing
and on farm management.
References
Foot, J.Z. and A. Russel, 1979. The relationship in ewes between voluntary food intake during pregnancy and forage
intake during lactation and after weaning. Animal production 28: 25-39.
Heitmann, R. and E. Bergman, 1980. Integration of amino acid metabolism in sheep: effects of fasting and acidosis.
American Journal of Physiology 239: E248.
Oddy, V., P. Speck, H. Warren and P. Wynn, 1995. Protein metabolism in lambs from lines divergently selected for
weaning weight. Journal of Agricultural Science 124: 129-137.

Energy and protein restriction in the goat mammary gland: proteomics
and metabolomics profiling
M. Palma1, L.E. Hernandez-Castellano2,3, A.M. Ferreira1,4, P. Nanni5, J. Grossmann5, A. Arguello1,
N. Castro1, J. Capote6, M Matzapetakis1 and A.M. Almeida7,8*
1ITQB/UNL – Instituto de Tecnologia Química e Biológica, Oeiras, Portugal; 2Department of Animal
Science, Universidad de Las Palmas de Gran Canaria, Arucas, Gran Canaria, Spain; 3present
address: Veterinary Physiology, Vetsuisse Faculty, University of Bern, Bern, Switzerland; 4ICAAM
– Instituto de Ciencias Agrarias e Ambientais Mediterranicas, Evora, Portugal; 5ETH Zurich,
Functional Genomics Center, Zurich, Switzerland; 6ICIA – Instituto Canario de Investigaciones
Agrarias, Valle Guerra, Tenerife, Spain; 7IBET – Instituto de Biologia Experimental e Tecnologica,
Oeiras, Portugal; 8Ross University School of Veterinary Medicine, St. Kitts and Nevis (West Indies);
adealmeida@rossvet.edu.kn
Abstract
Seasonal weight loss (SWL) and particularly energy and protein restriction are pressing issues in
animal production. Animals selected in SWL prone areas are well adapted to SWL. Understanding
the molecular mechanisms of SWL adaptation is of high importance in animal selection. We aimed
study the effect of SWL, on the mammary gland secretory tissue proteome and metabolome in 2
goat breeds from the Canary Islands with different levels of tolerance to SWL: Majorera (tolerant)
and Palmera (susceptible). Within each breed, goats with the same age and stage of lactation were
divided into two groups (n=5): control (constant weight) and underfed (15% liveweight reduction).
At day 22, mammary gland biopsies were extracted and proteomics and metabolomics profiles
obtained. Proteomics: Protein extracts were obtained and trypsin digested using the FASP protocol.
Peptides were loaded onto reverse-phase C18 columns and analysed on an LTQ-Orbitrap Velos
mass spectrometer. Protein identification and label free quantification were performed using Mascot
and Progenesis software. Metabolomics:aqueous fractions were obtained by tissue aqueous/organic
extraction. 1H NMR spectra were collected from the aqueous extract of the mammary gland using
a 800 MHz Bruker AvanceII+ spectrometer. Regarding the proteomics component, 1,010 proteins
were identified, from which 96 were considered statistically different among groups. SWL lead to an
increase of proteins related to apoptosis and stress processes in both breeds. Moreover, both breeds
showed a decrease in the number of proteins related to protein, carbohydrates and fat biosynthesis.
When both breeds were compared after SWL, Majorera breed showed higher expression of immune
system related proteins compared to Palmera breed. In contrast, Palmera breed showed higher
expression of proteins related to apoptosis, ketone bodies formation (fat liver) and protein metabolic
processes compared to Majorera breed. Regarding the Metabolomics component, we were able
to identify 47 different compounds in the aqueous fraction of mammary gland extracts. Lactose,
glutamate, glycine, lactate and glucose were found to be the most abundant. Statistical evaluation
using principal component analysis and partial least squares revealed differences between control
and underfed animals, although no differences between breeds were observed. In conclusion, the
two goat breeds have a different metabolism reaction to SWL, highlighting differences particularly
related to the immune system and apoptosis.
Keywords: mammary gland, dairy goat, seasonal weight loss
Introduction
Animal production is particularly important in tropical countries where the goat plays a major role
in food security. SWL (seasonal weight loss) is one of the major problems in animal production,
as affected animals may lose up to 30% of their body weight with severe consequences. The use
of supplementation is one proposed solution to SWL, however, it is very expensive and difficult

to implement. The use of local breeds known to be tolerant to SWL is increasing, being much
less expensive and easy to implement. The Canary Islands are a subtropical archipelago, with a
huge biodiversity and different climates. In these Islands, several local goat breeds may be found
developed from common ancestors imported from Northern Africa and the Iberian Peninsula. Two
interesting breeds are the Majorera (from the arid Island of Fuerteventura) and the Palmera (from
the humid island of La Palma). The first are characterized by their adaptation to semiarid climates.
Contrarily, Palmera goats are adapted to rainy climates, and more susceptible to SWL. In recent
years, Proteomics and transcriptomics have become increasingly relevant tools used to characterize
important physiological processes such as the adaptation to SWL. In this work, the influence of
feed restriction on the mammary gland proteome and metabolome of Majorera and Palmera goat
breeds were analysed.

Experimental design has previously been described (Lerias et al., 2013, 2015). Briefly, within each
breed, goats with the same age and stage of lactation were divided into two groups (n=5): control
(constant weight) and underfed (15% liveweight reduction). At day 22, mammary gland biopsies
were extracted and kept at -80 °C until Omics analysis. Proteomics Analysis: Protein extracts were
obtained and trypsin digested using the FASP protocol. Peptides were loaded onto reverse-phase C18
columns and analysed on an LTQ-Orbitrap Velos mass spectrometer. Protein identification and label
free quantification were performed using Mascot and Progenesis software. Metabolomics: Aqueous
fractions were obtained by tissue aqueous/organic extraction. 1H NMR spectra were collected from
the aqueous extract of the mammary gland using a 800 MHz Bruker AvanceII+ spectrometer.

Proteomics analysis revealed a total of 1,010 identified proteins. Of these, 96 were found to have
differential expression between the existing groups. With the metabolomics analysis, we were able to
identify 47 different compounds in the mammary gland. Proteomics analysis show that SWL caused
an expression increase in cellular stress and apoptosis pathways in both breeds. Also an expression
decrease was noticed for proteins involved in to protein, carbohydrates and lipid metabolisms as a
consequence of undernutrition. Interestingly, the tolerant breed, showed higher expression of immune
system related proteins compared to the SWL sensitive breed. Conversely, the latter also showed
an increased expression of apoptotic pathway proteins. Metabolomics analysis identified lactose,
glutamate, glycine, lactate and glucose as the most abundant peptides. Significant differences were
observed, in mammary gland biopsies between control and restricted-fed animals of both breeds.
Interestingly little differences were found between breeds. In conclusion, the two goat breeds have
a different metabolism reaction to SWL, highlighting differences particularly related to the immune
system and apoptosis.
References
Lerias, J.R., L.E. Hernández-Castellano, A. Morales-Delanuez, S.S. Araújo, N. Castro, A. Argüello, J. Capote and A.M.
Almeida, 2013. Body live weight and milk production parameters in the Majorera and Palmera goat breeds from
the Canary Islands: influence of weight loss. Trop. Anim. Health Prod. 45(8): 1731-1736.
Lerias, J.R., R. Peña, L.E. Hernández-Castellano, J. Capote, N. Castro, A. Argüello, S.S. Araújo, Y. Saco, A. Bassols
and A.M. Almeida, 2015. Establishment of the biochemical and endocrine blood profiles in the Majorera and
Palmera dairy goat breeds: the effect of feed restriction. J. Dairy Res. 82(4): 416-425.

Estimation of endogenous urinary N excretion in lactating dairy cattle
H. Lapierre1*, J.W. Spek2, C.E. Galindo1 and D.R. Ouellet1
1Agriculture and Agri-Food Canada, Sherbrooke, QC, Canada J1M 0C8; 2Wageningen UR Livestock
Research, 6708 WD, Wageningen, the Netherlands; helene.lapierre@agr.gc.ca
Abstract
The estimation of endogenous urinary N excretion in dairy cows, included in the estimation of protein
maintenance requirement, is revised combining reported data (from Spek et al., 2013) and estimations
of urinary excretion of N metabolites. It averages 0.043 g N/kg BW/d or 6.46 g CP/kg BW0.50/d
and would represent the requirement, assuming an efficiency of 100% as these metabolites are end-
products of metabolism. Although this is a 60% increment over Swanson (1977) estimation, this
would have a limited impact on essential amino acid (AA) requirement, as most of the N-metabolites
excreted are derived from non-essential AA.
Keywords: cattle, endogenous, urinary, nitrogen
Introduction
To estimate endogenous urinary (Endo-Uri) excretion included in metabolisable protein (MP)
requirement (rqt), most of the models used to balance dairy rations apply 2.75 g net protein/kg
BW0.50 (Swanson, 1977) or a rqt of 4.1 g MP/kg BW0.50 (0.67 efficiency). This value was estimated
from studies in cattle fed for weeks low-N diets with adequate energy. Under these experimental
conditions, this estimation of Endo-Uri includes purine derivatives (PD) originating from absorbed
microbial protein synthesis (PDfromMCP), whereas such low N feeding conditions may result in
underestimation of excretion of other endogenous N-metabolites. The amino acid (AA) composition
used currently for Endo-Uri is that of the empty body; identification of the N fractions constituting
Endo-Uri will allow a better estimation of its AA composition. Therefore, our objective was to
quantify the sources of Endo-Uri and compare with data reported on urinary N excretion to determine
the validity of these assumptions.

From a database built to study urinary excretion of total N (UN) and urea N (UUN) in lactating
dairy cattle (Spek et al., 2013), data from 11 studies (50 diets) reporting UN, UUN and PD urinary
excretions were used. Cows averaged (mean ± SD) 627±33 kg BW, 609±118 g N intake/d, 196±59
g urinary N/d, and 32.0 ± 7.3 kg milk/d; estimations of MP supply was made using the Dutch feed
tables (CVB, 2007). Based on literature, estimations of the fractions of urinary N excretion were
calculated. First, the Endo-Uri ‘measured’ (Endo-Urimeas) was calculated as the reported non-UUN
excretion minus PDfromMCP plus endogenous urea-N excretion (0.1 g N/kgBW0.60). The PDfromMCP
were estimated from MCP, using 85% digestibility of MCP purines, 83% recovery of absorbed
PD and a ratio of 11.6 g N in PD/100 g of N in MCP. The Endo-Uri estimate (Endo-Uriest) was
calculated as the sum of the excretions of endogenous urea-N, endogenous PD (0.385 × BW0.75
mmol/d), creatinine (24.8 mg creatinine/kg BW/d), creatine (0.36 × creatinine excretion), hippuric
acid (0.26 × non-UUN) and 3methylHis (50.5 + 3.536 × BW µmol/d). Endo-Uriest was calculated
for each treatment mean. The coefficient of determination was determined by regression of Endo-
Uriest against Endo-Urimeas. The mean square prediction error (MSPE) was decomposed into error
in central tendency (ECT) or mean bias, error due to the regression (ER) or linear bias, and error
due to disturbance (ED) or random.

Results are reported in Table 1. The mean of Endo-Uriest averaged 102% of Endo-Urimeas, with a
good correlation (r=0.95). However, ER represented 77% of MSPE, whereas ED represented 19%
of MSPE. From these estimations, Endo-Uriest averages 0.043 g N/kg BW/d or 6.46 g CP/kg BW0.50
and would represent the rqt, assuming an efficiency of 100% as these metabolites are end-products
of metabolism (Sauvant et al., 2015). This value is about 60% higher than the value proposed by
Swanson (1977), but is in agreement with the 0.05 g N/kg BW/d proposed in the new French system
Systali (Sauvant et al., 2015). In addition, knowledge of the origin of the metabolites constituting
Endo-Uri indicates that it has a limited impact on essential AA rqt. Besides the contribution of Met
as a methyl donor, only Arg is involved in creatine and creatinine synthesis, His into very limited
3methylHis excretion whereas the best AA composition for endogenous urea excretion, which
represents 18% of Endo-Uri, would probably be the empty body composition. Although mean of
Endo-Uriest is in the good range, more investigation is needed to correct the mean bias which results
in an overestimation at low Endo-Urimeas and overestimation at high Endo-Urimeas.
Table 1. Urinary excretion of N metabolites in lactating dairy cows.1
Item Status2 Mean SD % Endo-Urimeas % non UUN
non UUN M 44.9 19.4

PDfrom MCP M 23.1 5.7 51.4
non urea endo M 21.8 18.2
Endo-Urimeas 26.6 18.2
endogenous urea E 4.8 0.1 18.1
PDendogenous E 2.7 0.1 10.3 6.1
3methylHis E 0.1 0.01 0.4 0.2
creatinine E 5.9 0.3 22.0 13.1
creatine E 2.1 0.1 8.0 4.7
hippuric acid E 11.5 5.0 43.4 25.7
Endo-Uriest 27.2 5.0 102.1 101.2
1 UUN = urea N; PD = purine derivatives; PD

from MCP = originating from absorbed microbial protein synthesis; Endo-Uri
= endogenous urinary; Endo-Urimeas = Endo-Uri ‘measured’; Endo-Uriest = Endo-Uri estimate.
2 M = measured, i.e. reported or directly calculated from diet parameters; E = estimated as indicated in the text.
Acknowledgements
Financial support from The Dairy Farmers of Canada, Agriculture and Agri-Food Canada and Adisseo
France S.A.S. is acknowledged.
References
Centraal Veevoederbureau (CVB), 2007. Table of feedstuffs. Information about composition, digestibility and feeding
value. Centraal Veevoederbureau, Lelystad, the Netherlands.
Sauvant, D., G. Catalapiedra-Hijar, L. Delaby, J.-B. Daniel, P. Faverdin and P. Nozière, 2015. Actualisation des besoins
protéiques des ruminants et détermination des réponses des femelles laitières aux apports de protéines digestibles
dans l’intestin. INRA Productions Animales 28: 347-368.
Spek, J.W., J. Dijkstra, G. van Duinkerken, W.H. Hendriks and A. Bannink, 2013. Prediction of urinary nitrogen and
urinary urea nitrogen excretion by lactating dairy cattle in northwestern Europe and North America: a meta-analysis.
Swanson, E.W. 1977. Factors for computing requirements of protein for maintenance of cattle. Journal of Dairy
Science 60: 1583-1593.

Representation of essential amino acid use by the portal drained viscera
and liver in cattle
A.J. Myers1*, M.D. Hanigan1, J. Castro Marquez1, R.R. White1, H. Lapierre2, R. Martineau2 and
J. France3
1Virginia Tech, Blacksburg, VA 24060, USA; 2Agricultural and Agri-Food Canada, Sherbrooke, QC,
J1M 0C8, Canada; 3Centre for Nutrition Modelling, Department of Animal and Poultry Science,
University of Guelph, Guelph, ON, NIG 2W1, Canada; adelynm2@vt.edu
Abstract
The objective of this work was to evaluate an integrated model of portal drained viscera (PDV) and
liver (LIV) utilisation of essential amino acids (EAA). Predictions of utilisation using a previously
derived model and constants for PDV resulted in concordance correlation coefficients (CCC)
ranging from 0.26 to 0.75. Rederivation of rate constants on an extended dataset using the original
model resulted in CCC from 0.74 to 0.87. Modification of the model using variable rate constants
improved CCC from 0.75 to 0.91. Using the previously derived fixed constants for the LIV model
resulted in CCC from -0.03 to 0.30. Rederivation of the constants yielded CCC of -0.03 to 0.29.
Modification of the model to utilise variable rate constants resulted in CCC of 0.14 to 0.56. The
newly derived models accurately predicted EAA use by PDV and LIV, and can be used for calculating
post-splanchnic EAA availability.
Keywords: milk yield, mechanistic model, splanchnic, liver
Introduction
Predicting milk protein yield from absorbed EAA supply and post absorptive efficiency is a commonly
proposed goal. Equations have been derived to determine milk yield as a function of animal and
diet factors, including EAA supply. However, these approaches have rarely gone beyond empirical
representations of tissue EAA demand. As such, the interactions among competitive post-absorptive
tissues have been incompletely characterised in extant models. The PDV and LIV tissue beds are
significant EAA sinks that impact net supply to mammary. Hanigan et al. (1998) modelled PDV and
LIV EAA utilisation (utilisation = inputs – outputs) as a function of absorbed EAA, blood flow, and
blood EAA concentrations with fixed clearance rate constants. The objective of the current work
was to evaluate the Hanigan model against a larger dataset and to formulate revised equations if
warranted. We hypothesized that mechanistic models of PDV and LIV EAA utilisation could predict
PDV and LIV EAA use with acceptable accuracy and precision.

Data were collected from 44 studies conducted in dairy cows, from 1974 to 2012, reporting blood
flows (BF, l/h) and arterial ([A]), portal ([VP]), and hepatic venous EAA concentrations ([VH], µM).
Absorbed (Abs) EAA were predicted from digestible flows (NRC, 2001). Predicted PDV utilisation
was calculated as: (Abs + [A] × BFP) – ([VP] × BFP), where [VP] was predicted from Hanigan et al.
(1998). LIV utilisation was predicted in a similar manner from portal and arterial fluxes. The models
were assessed using the reported rate constants (Han98), rate constants refitted to the current data
(Han16), or a modified representation with variable clearance rate constants fitted to the current data
(Myr16). Parameter estimates were derived by non-linear, least-squares regression (R Core Team,
2015). Model errors were summarized as CCC. Proportions of mean squared error (MSE) attributable
to mean bias, slope bias, and dispersion error were calculated to evaluate potential biases.

Refitting the static rate constants for PDV and LIV EAA utilisation (Han16) improved predictions
compared with Han98 rate constants (Table 1). The CCC increased for PDV, ranging from 0.26 to
0.75 for Han98 and from 0.74 to 0.87 for Han16. Although the CCC for LIV estimates from Han16
were improved in comparison to Han98 for many EAA, the overall fit was poor compared to the
PDV reflecting low utilisation compared to overall supply.
Slope bias was apparent for both models with respect to arterial EAA concentrations. The PDV
model was also biased against absorbed EAA. A 2nd rate constant was added to the PDV model
(Equation 1), and the rate constant in the LIV model was represented as a linear function of arterial
concentrations to reduce bias (Equation 2):
[ A]* BFp Abs
=[VP ] + (1)
K1 + BFp K 2 + BFp
([ A]* BFa ) + ([VP ]* BFp )

[VH ] = (2)
( K 3 + K 4*[ A]) + ( BFp + BFa )
Where, BFp = portal BF, Abs = absorption, and BFa = arterial BF.
The modifications improved predictions resulting in CCC ranging from 0.75 to 0.91 for PDV, and
from 0.14 to 0.56 for LIV. Although the Myr16 models were an improvement, residual analysis
demonstrated modest (<40% of MSE) but significant (P<0.1) slope biases for predicting LIV His,
Ile, Leu, Thr, and Val uptake. However, Ile, Leu, and Val uptakes are very small, thus the problem
is insignificant for these three. There was no significant slope bias for the PDV model (Equation 1).
Using the integrated models, fractional splanchnic removal of absorbed EAA ranged from 0.78 to
2.02. Fractional splanchnic removal of total EAA supply (absorbed plus arterial) ranged from 0.068
to 0.41. Future work will incorporate a mammary component into this framework to better estimate
mammary EAA uptake and net efficiency of EAA deliverance to that tissue. In general, the revised
models accurately predicted EAA utilisation by PDV and LIV, and could be used to predict supply
of EAA to mammary tissue.
Table 1. Concordance correlation coefficients for predictions of essential amino acids (EAA) use by
portal drained viscera (PDV) and liver (LIV).
AA Han98 Han16 Myr16 Han98 Han16 Myr16
PDV LIV
Arg 0.54 0.74 0.75 0.29 0.29 0.43

His 0.26 0.74 0.78 0.05 0.27 0.14
Ile 0.53 0.86 0.87 -0.04 0.05 0.16
Leu 0.47 0.84 0.91 -0.06 0.05 0.42
Lys 0.75 0.84 0.90 -0.13 -0.05 0.38
Met 0.36 0.79 0.80 -0.03 -0.03 0.18
Phe 0.41 0.84 0.85 -0.03 0.24 0.56
Thr 0.57 0.87 0.88 -0.03 -0.12 0.18
Val 0.44 0.83 0.86 -0.07 0.07 0.29

References
Hanigan M.D., J. France, D. Wray-Cahen, D.E. Beever, G.E. Lobley, L. Reutzel and N.E. Smith, 1998. Alternative
models for analysis of liver and mammary transorgan metabolite extraction data. British Journal of Nutrition 79:
63-78.
National Research Council (NRC), 2001. Nutrient requirements of dairy cattle (7th Ed.). National Academy Press,
R Core Team, 2015. R: a language and environment for statistical computing. R Foundation for Statistical Computing,
Vienna, Austria. Available at: https://www.R-project.org.

Modelling homeorhetic trajectories of milk component yields, body
composition and dry matter intake in dairy cows: influence of parity,
phenotypic potential and breed
J.B. Daniel1,2, N.C. Friggens1, H. Van Laar2, K.L. Ingvartsen3 and D. Sauvant1
1UMR Modélisation Systémique Appliquée aux Ruminants (MoSAR), INRA-AgroParisTech, 16 rue
Claude Bernard, 75231 Paris cedex 05, France; 2Trouw Nutrition R&D, P.O. Box 220, 5830 AE
Boxmeer, the Netherlands; 3University of Aarhus, Faculty of Agricultural Sciences, Research Center
Foulum, P.O. Box 50, 8830 Tjele, Denmark; jean-baptiste.daniel@trouwnutrition.com
Abstract
The prediction of nutrient partitioning is important in predicting dairy cow performance. Partitioning
is largely affected by physiological state and production potential. Therefore, a model was developed
that aims to provide a dynamic framework to predict a consistent set of reference performance patterns
(milk component yields, body composition change, dry matter intake) sensitive to physiological
status across a range of milk production potentials (within and between breed). Two large datasets
in which the plane of nutrition was similar for all animals were used for model calibration. The first
dataset, weekly data of Holstein cows differing in their production level, was used to calibrate the
effect of phenotypic potential in the model. A second dataset was used to calibrate the between-
breed effect (Holstein, Danish Red and Jersey) on the mobilization and reconstitution of body
composition and on the yield of individual milk components. Overall quality of the model calibration
in predicting DMI, milk and milk component yields and contents, empty body weight and body
condition score was assessed through the root mean square prediction error (RMSPE, expressed as
a ratio of the observed mean) and concordance correlation coefficient (CCC). The overall RMSPE
and CCC averages across variables predicted were 0.05 and 0.77, respectively. The model predicts
a mutually consistent set of performance through lactation and pregnancy across a large range of
animal production potential at different parity.
Keywords: milk composition, model, energy, protein
Introduction
To predict animal performance, two types of regulation need to be considered: homeostatic regulation
(e.g. driving responses to dietary changes), and homeorhetic regulation, the coordinated changes
in metabolism to support a physiological state (Bauman and Currie, 1980). This paper focusses on
the second type of regulation and describes a dynamic model of the lactation which predicts dairy
cow performance as well as the flow and partition of net energy for dairy cows of different parity,
phenotypic potential and breed.
Model description
The structure of the model is characterized by 2 sub-models, a regulating sub-model of homeorhetic
control which sets rules of partition and size of flows along the lactation, and an operating sub-model
that translates these rules into animal performance. The regulating sub-model describes lactation as the
result of 3 driving forces (=purposes): (1) use of previously acquired resources through mobilization;
(2) acquisition of new resources with a priority of partition towards milk; and (3) subsequent use
of resources towards body reserves gain. The dynamics of these 3 driving forces were adjusted
separately for fat (milk and body), protein (milk and body) and lactose (milk). Milk yield is then
predicted from lactose and protein yields with an empirical equation developed from literature data.
The model predicts desired dry matter intake (DMI) as an outcome of net energy requirements, for
a given dietary net energy content. The parameters controlling milk component yields and body

composition changes were calibrated using 2 datasets in which the plane of nutrition was the same for
all animals. The first dataset, weekly data of Holstein dairy cows, was used to calibrate the effect of
phenotypic production level in the model. A second dataset was used to calibrate the between-breed
effect (Holstein, Danish Red and Jersey) on the mobilization/reconstitution of body composition
and on the yield of individual milk components.

The quality of the model calibration in predicting DMI, milk yield, milk component yields and
contents, empty body weight and body condition score was assessed through the root mean square
prediction error (RMSPE, expressed as a ratio of the observed mean) and concordance correlation
coefficient (CCC). Overall, the average RMSPE across variables was 0.05 (range from 0.03 for BCS
to 0.10 for milk protein content) and the average CCC was 0.77 (range from 0.51 for milk lactose
content to 0.94 for milk yield). The lactation and intake curves simulated here were in agreement
with published average curves or equations (Faverdin et al., 2007; NRC, 2001). Overall the effect of
animal production potential on performance was consistent with existing genetic correlations (e.g.
between energy balance and milk yield or DMI and milk yield). These calibrations showed that the
model framework was able to adequately simulate milk yield, milk component yield, body protein
and lipid changes and DMI throughout lactation for primiparous and multiparous cows of different
breeds, and differing in their production level.
References
Bauman, D.E. and W.B. Currie, 1980. Partitioning of nutrients during pregnancy and lactation: a review of mechanisms
involving homeostasis and homeorhesis. J. Dairy Sci. 63: 1514-1529.
Faverdin, P., R. Delagarde, L. Delaby and F. Meschy, 2007. Alimentation des vaches laitières. In: Alimentation des
bovins, ovins et caprins. Besoins des animaux – Valeur des aliments – Tables INRA 2007, mise à jour 2010.
Editions Quae, Versailles, France, pp. 23-58.

Milk metabolites as biomarkers of energy balance in goats during
lactation
P. Criscioni1, T. Larsen2 and C. Fernández1*
1Animal Science Department, ACUMA Research Center, Polytechnic University of Valencia, 46022
Valencia, Spain; 2Dpt of Animal Science, Faculty of Sciences and Technology, Aarhus University,
8830 Tjele, Denmark; cjfernandez@dca.upv.es
Abstract
The aim of this work was to identify milk metabolites as potential biomarkers of physiological
imbalance throughout lactation in goats. Twenty Murciano-Granadina goats were fed with a mixed
diet (forage:concentrate = 40:60) with energy and protein value of 17 MJ/kg DM and 164 g/kg DM,
respectively. During peak, mid and late lactation, the goats were allocated to individual metabolism
cages (energy balance and milk metabolites) and heat production was determined during 24 hours
using a mobile open-circuit respirometry system connected to a head box. Stage of lactation altered
metabolisable energy intake, that was greater in early lactation than late and heat production higher
in early than later. Milk urea was higher at late lactation than earlier (7.2 vs 6.4 mM, respectively).
The greater values obtained in milk β-hydroxybutyrate during early and mid lactation (142 µM on
average) than late (66 µM) revealed a potential use for monitoring mobilization of body tissue (-62
early vs 11 mid vs 46 late kJ/kg0.75 BW, respectively).
Keywords: heat production, goat, milk metabolites
Introduction
Several indicators are being used on-farm to identify cows with metabolic diseases (Larsen et
al., 2016) but the information available in goats is scarce. Reducing the degree of physiological
imbalance in individual goats will reduce the risk of disease and, thereby, improve production. An
index for physiological imbalance, based on several metabolites in milk, will more directly relate
to mechanisms associated with the development of several diseases during early lactation. The aim
of this work was to identify milk metabolites as potential biomarkers of physiological imbalance
throughout lactation in goats under negative and positive energy balance.

Twenty Murciano-Granadina goats were homogenously selected (44.5±3.02 kg of body weight;
640±45.4 kg of milk in previous lactation), and fed a mixed diet in pens ad libitum; alfalfa hay
(40%) and concentrate (60%) with energy and protein value of 17 MJ/kg DM and 164 g/kg DM,
respectively. During peak, mid and late lactation, the goats were allocated to individual metabolism
cages as follow: 10 days of adaptation and 5 days of collections samples (feed intake, refusal, total
faecal and urine output, and milk yield). Heat production was determined during 24 hours using a
mobile open-circuit respirometry system connected to a head box (Fernández et al., 2015). Data
were analysed with a linear model and stage of lactation was the fixed effect.

Stage of lactation altered (P<0.05) energy balance, milk performance and composition with exception
of milk glucose and uric acid. Metabolisable energy intake was greater in early lactation than late
and heat production higher in early than later (Table 1). Milk urea was higher at late lactation than
mid (7.2 vs 6.3 mM, respectively) indicating protein was supplied in excess at late lactation and
it was catabolized into urea by the liver. The greater values obtained in milk β-hydroxybutyrate

during early and mid lactation (142 µM on average) than late (66 µM) revealed a potential use for
monitoring mobilization of body tissue (-62 early vs 11 mid vs 46 late kJ/kg0.75 BW, respectively).
Milk isocitrate apparently reflects the energy balance even better and might be followed further in
the future, possibly in combination with other milk variables (Larsen et al., 2016).
Table 1. Daily energy partitioning (kJ/kg0.75 of BW), milk yield and metabolites of Murciano-
Granadina goats (n=20) during lactation.
Stage of lactation SEM P-value
Early Middle Late Stage
Energy balance
Gross energy intake 1,925a 1,811a 1,598b 37.20 0.001
Energy in faeces 563a 559a 431b 18.22 0.003
Energy in urine 43b 56a 37b 1.92 0.001
Energy in methane 96ab 107a 84b 2.63 0.002
Metabolisable energy intake 1,223a 1,090ab 1,046b 25.75 0.011
Heat production 796a 676b 643b 17.25 0.001
Energy in milk 489a 403b 357b 11.39 0.001
Recovered energy in tissue -62c 11b 46a 6.04 0.001
Milk yield and metabolites
Production, kg/d 2.9a 1.8b 1.6b 0.11 0.001
Dry matter, % 13.6b 14.5a 13.3b 0.14 0.001
Fat, % 4.6a 4.8a 4.0b 0.10 0.002
Protein, % 3.6b 4.2a 3.7b 0.05 0.001
Lactose, % 4.7b 4.8ab 4.9a 0.03 0.058
β-hydroxybutyrate, µM 132a 153a 66b 8.9 0.001
Glucose, mM 0.13 0.14 0.13 0.005 0.582
Glucose 6P, mM 0.15b 0.21a 0.14b 0.01 0.007
isoCitrate, mM 0.14a 0.10b 0.08b 0.001 0.001
Uric acid, µM 134 153 133 10.1 0.643
Milk urea, mM 6.6ab 6.3b 7.2a 0.12 0.007
References
Fernández, C., M.C. López and M. Lachica, 2015. Low cost open-circuit hood system for measuring gas exchange in
small ruminants: from manual to automatic recording. Journal of Agricultural Science 153: 1302-1309.
Larsen, T., L. Alstrup and M.R. Weisbjerg, 2016. Minor milk constituents are affected by protein concentration and
forage digestibility in the feed ration. Journal of Dairy Research 83: 12-19.

Effect of grain increase in the diet on brush border enzymes activity
in cattle
P. Górka1*, A. Błońska1, B.L. Schurmann2, M.E. Walpole2, S. Li3, J.C. Plaizier3, Z.M. Kowalski1
and G.B. Penner2
1Department of Animal Nutrition and Dietetics, University of Agriculture in Krakow, 30-059 Krakow,
Poland; 2Department of Animal and Poultry Science, University of Saskatchewan, 51 Campus Dr,
Saskatoon, SK, S7N 5A8, Canada; 3University of Manitoba, Winnipeg, MB, R3R 3N2, Canada;
p.gorka@ur.krakow.pl
Abstract
The objective of this study was to determine changes in the activity of main brush border enzymes in
cattle in response to an abrupt but moderate increase in the proportion of grain in the diet. Twenty-five
Holstein steer calves were assigned to 1 of 5 treatments and fed control diet (CON; 92% chopped
grass hay and 8% mineral-vitamin supplement on DM basis) or moderate grain diet (MGD; 50%
chopped grass hay, 42% rolled barley grain and 8% mineral-vitamin supplement) that was fed for
3, 7, 14, or 21 d. Lactase activity in the proximal jejunum increased linearly and maltase activity
in duodenum tended to increase linearly with advancing days on MGD and tended to (P≤0.10) be
greater in the proximal jejunum for MGD compared to CON. Aminopeptidase N activity in the
proximal jejunum increased cubically with advancing days on MGD and tended to be greater for
MGD as compared to CON. Dipeptidylpeptidase IV activity in ileum decreased on d 14 after the
shift to the MGD and then increased. These data indicate that adaptation to a MGD involves increase
in the activity of intestinal brush border enzymes and these changes respond gradually over time.
Keywords: concentrate, adaptation, small intestine, digestive enzymes
Introduction
Diets high in rapidly fermentable carbohydrates are commonly fed to dairy and beef cattle in order
to increase dietary energy density. However, postruminal nutrient digestion (especially of starch)
may limit the efficiency of this feeding strategy (Harmon, 2009) likely due to limited activity of
intestinal brush border enzymes (Brake et al., 2014). The objective of this study was to determine
changes in the activity of main brush border enzymes in cattle in response to an abrupt but moderate
increase in the proportion of grain in the diet.

Twenty-five Holstein steer calves (213±23 kg; 5 to 7 months of age) were blocked by BW and within
block randomly assigned to 1 of 5 treatments: the control diet (CON; 92% chopped grass hay and
8% mineral-vitamin supplement on DM basis) or moderate grain diet (MGD; 50% chopped grass
hay, 42% rolled barley grain, and 8% mineral-vitamin supplement) that was feed for 3 (MGD3),
7 (MGD7), 14 (MGD14), or 21 d (MGD21). Intestinal epithelium from the duodenum, proximal
jejunum (2 m from the duodenum) and ileum was sampled at the end of the study and activities of
lactase, maltase, dipeptidylpeptidase IV, aminopeptidase A and aminopeptidase N were determined.
Data were analysed as randomized complete block design using the Mixed procedure of SAS.
Polynominal contrasts were used to determine linear, quadratic, or cubic changes that occurred with
advancing days on MGD whereas an orthogonal contrast was used to compare enzymes activities
between CON and MGD.

Lactase activity in the proximal jejunum increased (from 24.65 to 38.21 U/g of protein; P=0.02)
linearly and maltase activity in duodenum tended to increase (from 10.94 to 13.48 U/g of protein;
P=0.08) linearly with advancing days on the MGD. Furthermore, maltase activity in the proximal
jejunum tended to be greater for MGD compared to CON (18.67 and 27.44 U/g of protein for
CON and MGD, respectively; P=0.09). Notably, maltase activity was on average similar for the
investigated sections of the small intestine to lactase activity, indicating low activity of maltase in
ruminating cattle. Aminopeptidase N activity in the proximal jejunum increased cubically (from
13.78 to 25.82 U/g of protein; P=0.01) with advancing days on MGD and tended to be greater
for MGD as compared to CON (13.78 and 19.58 U/g of protein for CON and MGD, respectively;
P=0.09). Dipeptidylpeptidase IV activity in ileum tended to decrease from 20.38 for CON to 11.76
for MGD14 and then increased to 22.34 U/g of protein for MGD21 (quadratic, P=0.07).
Low efficiency of postruminal nutrient digestion in cattle (namely starch) is widely known. This
is attributed predominantly to insufficient pancreatic enzymes secretion and/or nutrient absorption
(Harmon, 2009). However, it has been shown that brush border enzyme activities may be also an
important factor limiting efficiency of intestinal nutrient digestion in cattle (Brake et al., 2014; Gilbert
et al., 2015). This study indicates that adaptation to MGD involves increase in the activity of main
intestinal brush border enzymes and these changes respond gradually over time, which may limit
efficiency of nutrient utilisation in cattle.
Acknowledgements
Funded by Ministry of Science and Higher Education of Poland (BM-4234/KŻZiP/2013)
References
Brake, D.W., E.C. Titgemeyer and D.E. Anderson, 2014. Duodenal supply of glutamate and casein both improve
intestinal starch digestion in cattle, but by apparently different mechanisms. Journal of Animal Science 9: 4057-4067.
Gilbert, M.S., A.J. Pantophlet, H. Berends, A.M. Pluschke, J.J. van den Borne, W.H. Hendriks, H.A. Schols and W.J.
Gerrits, 2015. Fermentation in the small intestine contributes substantially to intestinal starch disappearance in
calves. Journal of Nutrition 145: 1147-1155.
Harmon, D.L., 2009. Understanding starch utilization in the small intestine of cattle. Asian-Australian Journal of
Animal Sciences 22: 915-922.

Effects of rumen thermodynamics on volatile fatty acid production
and interconversion in dairy cattle
L.B. Harthan*, R.R. White and M.D. Hanigan
Department of Dairy Science, Virginia Tech, 2080 Litton Reaves, Blacksburg, VA 24061, USA;
lbh25@vt.edu
Abstract
Models used to predict ruminal volatile fatty acid (VFA) production currently have large errors and
limited representations of VFA interconversion. To help refine such models, this study investigated
the role of rumen thermodynamics in ruminal VFA production and interconversion. Using a replicated
3×3 Latin square design, 6 cannulated steers were continuously ruminally infused with 3 different
treatments: a dilute blend of HCl and H3PO4 to drop the rumen pH by 0.5 (LowpH); propionate
(HighVFA); and distilled water as a control (CON). Continuous 13C-labeled acetate, propionate,
and butyrate infusions were used to assess VFA dynamics. VFA production, absorption, and
interconversion fluxes were differentially regulated by the LowpH and HighVFA treatment groups
compared to CON. Results indicate that ruminal VFA production, absorption, and interconversion
fluxes are regulated in part by rumen thermodynamics.
Keywords: rumen, VFA, thermodynamics, dairy, modelling
Introduction
Recent studies have demonstrated that interconversion among volatile fatty acid (VFA) can account
for a significant portion of the 50 to 65% prediction error in current VFA models. VFA interconversion
may be controlled by rumen thermodynamic state,which is controlled in part by rumen pH and VFA
concentration. It was hypothesized that increased ruminal propionate would result in increased
conversion of propionate to other VFA, while a lowered ruminal pH would result in a greater flux of
acetate and butyrate to propionate. The results of this study will contribute to improving the accuracy
and precision of ruminal VFA models by improving understanding of thermodynamic control on
VFA production, absorption, and interconversion.

Six ruminally cannulated Holstein steers (294±33 kg initial BW) were used in a replicated 3 ×
3 Latin square design. Animals were cared for and housed at the Virginia Tech dairy complex in
Blacksburg, VA in accordance with the Virginia Tech Institutional Animal Care and Use Committee.
The trial consisted of 3, 10 day periods, with 3 treatments applied across each period. Treatments
were continuously infused into the rumen using clinical infusion pumps (Plum LifeCare 5000; Abbott,
Lake Bluff, IL, USA) and included: a dilute blend of HCl and H3PO4 to drop the rumen pH by 0.5
(LowpH), propionate (HighVFA), and distilled water as a control (CON). Each period included
a 7 day adjustment period followed by 3 days of sampling. These 3 sampling days were used for
continuous ruminal infusions of Na-2-13C Acetate, Na-2-13C propionate, and Na-2-13C butyrate
(Cambridge Isotope Laboratories, Tewksbury, MA, USA) for 6 hours. Each steer was infused with
each isotope once during each period and 12 h were allowed between isotope infusions. Continuous
LiCo-EDTA infusion was used to estimate rumen liquid outflow and liquid volume was determined
from a bolus dose of polyethylene glycol. Rumen fluid was sampled before the start of the infusions
and every hour for 12 hours to assess VFA concentrations and enrichment, Co concentration, and
pH. A non-linear, fully exchangable, 3 pool dynamic model was used to estimate VFA production,
absorption, and interconversion fluxes for each treatment according to Nolan et al. (2014). Model
fitting was performed using R Statistical Software (R Core Team, 2015).

The LowpH treatment resulted in increased production of all 3 VFA, increased acetate absorption,
and decreased absorption rates of propionate and butyrate compared to CON (Table 1). The LowpH
treatment increased conversion of propionate to acetate and propionate to butyrate relative to
CON. The HighVFA treatment increased production of propionate and butyrate and decreased
acetate production compared to CON. Absorption of acetate and propionate increased relative to
CON, while absorption of butyrate decreased. The HighVFA treatment also resulted in different
VFA interconversion fluxes, most notably decreased acetate to butyrate, and increased propionate
to butyrate and propionate to acetate. All fluxes were estimated precisely by the model (P<0.05)
with the exception of the conversion of butyrate to propionate which was not statistically different
from 0 in all models. Together these data suggest that rumen VFA regulation is more complex than
thermodynamics alone, and is likely mediated through factors such as rumen microbial function.
Table 1. Rumen volatile fatty acid (VFA) production, absorption, and interconversion fluxes (mol/h).1
Treatments P-values
LowpH HighVFA CON Treatment Period
Production
Acetate 0.518±0.017a 0.419±0.016b 0.450±0.016c <0.001 0.573
Propionate 0.0911±0.0152a 0.145±0.015b 0.0742±0.0149c <0.001 <0.001
Butyrate 0.181±0.050a 0.121±0.050b 0.100±0.050c <0.001 <0.001
Absorption
Acetate 0.345±0.042a 0.272±0.042b 0.130±0.042c <0.001 <0.001
Propionate 0.0849±0.0255a 0.153±0.025b 0.120±0.025c <0.001 <0.001
Butyrate 0.0780±0.0189a 0.0541±0.0187b 0.170±0.019c <0.001 <0.001
Interconversion
Acetate to butyrate 0.430±0.084a 0.256±0.084b 0.471±0.084c <0.001 <0.001
Acetate to propionate 0.0727±0.0338a 0.0955±0.0337b 0.0596±0.0337c <0.001 0.978
Butyrate to acetate 0.472±0.077a 0.284±0.077b 0.365±0.077c <0.001 <0.001
Butyrate to propionate 0.0993±0.216a 0.640±0.211b 0.0308±0.2107a <0.001 <0.001
Propionate to acetate 0.0570±0.0135a 0.0792±0.0134b 0.0299±0.0134c <0.001 <0.001
Propionate to butyrate 0.0493±0.0149a 0.0447±0.0148a 0.0308±0.0148b <0.001 <0.001
1LowpH = ruminal infusion of HCl/H3PO4 blend to drop pH by 0.5; highVFA = ruminal infusion of 20% expected
daily production of propionate; CON = ruminal infusion of water. Treatment LSmeans reported as values ± SEM.
Acknowledgements
Financial support for this work was provided by the John Lee Pratt Endowment Animal Nutrition
Program and the Virginia Agricultural Council.
References
Nolan, J., R. Leng, R. Dobos and R. Boston, 2014. The production of acetate, propionate and butyrate in the rumen of
sheep: fitting models to 14C-or 13C-labelled tracer data to determine synthesis rates and interconversions. Animal
Production Science 54(12): 2082-2088.
R Core Team. 2015. R: a language and environment for statistical computing. R Foundation for statistical computing,
Vienna, Austria.

Mammary and whole body energy metabolism in lactating cows fed
high-roughage diets
K. Higuchi1*, F. Ohtani1, Y. Kobayashi1, I. Nonaka1, O. Enishi1, M. Sutoh1 and K. Yayou2
1NARO Institute of Livestock and Grassland Sciences, 2 Ikenodai, Tsukuba, Ibaraki 305-0901,
Japan; 2National Institute of Agrobiological Sciences, 2 Ikenodai, Tsukuba, Ibaraki 305-8602,
Japan; higuchik@affrc.go.jp
Abstract
To estimate the energy efficiency for milk production of cows receiving high-roughage diet, energy
metabolism experiment at whole-body and mammary gland levels of cows fed 3 diets containing 60%
of roughage with different forages was conducted. The result was concluded that energy efficiency
at mammary gland was almost 0.9, while metabolisable energy (ME) efficiency for milk production
and ME supply toward mammary gland depended on feed source.
Keywords: lactating cow, energy metabolism, mammary metabolism
Introduction
It is well known that a higher proportion of dietary roughage influences chewing activity and ruminal
volatile fatty acid production in cow. The dietary effect of roughage can also alter energy utilisation
for milk production. The aim of the present study was to estimate the energy efficiency for milk
production of cows fed high-roughage diets by measuring energy metabolism at whole-body and
mammary gland levels.

Four Holstein lactating cows (191 days in milk) fitted with an ultrasound flow probe around the left
external pudic artery were housed in a temperature and humidity controlled-room and fed 3 diets: a
IH diet (45% Italian ryegrass silage, 15% alfalfa hay cube and 40% concentrate mix; crude protein
(CP) 18.1%; neutral detergent fibre (NDF) 46.8%), a CH or a CL diet (high or low CP design using
50% corn silage, 10% alfalfa hay cube and 40% concentrate mix; CP 17.6 vs 13.5%; NDF 39.9 vs
38.8%) over 21-day periods according to 2 incomplete 3×3 Latin square design. All the diets were
fed satisfying the total digestible nutrients requirement for a Holstein cow producing 22 kg milk/
day (NARO, 2007). The rates of CP sufficiency for the requirement in the IH, CH, and CL diets
were 132, 121, and 99%, respectively. The energy balance trials were made using the open circuit
respiratory chambers with digestion trial apparatus (Higuchi et al., 2010; Iwasaki et al., 1982). The
energy expenditure of the udder was calculated from their oxygen consumption using arterio-venous
difference technique measuring mammary blood flow and blood oxygen concentration of carotid
artery and mammary vein. The energy uptake of mammary gland was estimated as the sum of energy
expenditure of the udder and milk energy. Mammary energy efficiency was estimated as the ratio of
milk energy to mammary energy uptake. The data were analysed according to the following model
using GLM procedure of SAS 9.4: Yijk = µ + Pi + Fj + Ck + eijk; where Yijk is the variable analysed,
µ is the overall mean; Pi, Fj, Ck are the effects of the period, feed, and cow, respectively; and eijk
is the residual error. With a significant effect of feed, Tukey’s multiple comparison test was done.

Body weight (BW) tended to be higher in IH, and dry matter (DM) intake was statistically highest
in IH, but these differences were small (Table 1). Milk yield in CH tended to be higher while no
difference was detected in milk component between the diets. Total chewing time of IH was higher

than that of CH. Digestibilities of DM, CP, fibre and starch were highest (P<0.03) in IH and gross
energy intake was highest in CH. Energy partitioning for faeces was lowest and for urine and retention
were highest in IH. There were no differences in energy partitioning for methane, heat and milk
although a tendency in milk partition was detected in CH. ME intake was lowest in CL diet. When
assuming ME requirement for maintenance as 486.6 kJ/kgBW0.75 (NARO, 2007), ME efficiency
for milk production (kl) ranged 0.61 to 0.71, the value being highest in IH. Lower mammary blood
flow (tendency), and lower mammary energy expenditure and uptake were detected in IH. There
were no differences in mammary energy efficiency estimated at 0.87-0.88. In conclusion, energy
efficiency at mammary gland was almost 0.9, while ME efficiency for milk production and ME
supply toward mammary gland depended on feed source.
Table 1. Performance and energy metabolism of whole-body and mammary gland.1
CL CH IH SEM P-value
Body weight (BW), kg 516 512 524 2 0.062

Dry matter intake (DMI), kg/day 15.6b 15.7b 15.7a 0 0
Milk yield, kg/day 21.5 23.5 22 0.4 0.066
Total chewing time, min/kg DMI 36ab 35b 44a 2 0.038
Gross energy (GE) intake, kJ/kgBW0.75/day 2,738b 2,809a 2,733b 12 0.021
Energy partition for, %GE intake
Faeces 36.4a 34.7a 29.5b 0.7 0.004
Urine 2.0c 2.6b 3.4a 0.1 0.001
Methane 6.2 5.6 6.7 0.3 0.117
Heat 32.4 31.8 31.4 0.3 0.177
Milk 23.4 24.7 23.2 0.4 0.084
Retention -0.5b 0.5b 5.8a 0.6 0.003
Metabolisable energy intake, kJ/kgBW0.75/day 1,515b 1,602a 1,650a 16 0.011
kl 0.61b 0.64b 0.71a 0.01 0.015
Mammary blood flow, l/min 5.4 5.3 4.6 0.1 0.098
Mammary energy expenditure, kJ/kgBW0.75/day 97ab 102a 88b 1 0.041
Mammary energy uptake, kJ/kgBW0.75/day 763 824 739 19 0.165
Mammary energy efficiency 0.872 0.877 0.882 0.002 0.174
1 Means with different superscripts within the same row significantly differ (P<0.05).
References
Higuchi, K., Y. Kobayashi, I. Nonaka, and O. Enishi, 2010. A new data acquisition system for respiration trial system
on metabolism laboratory in National Institute of Livestock and Grassland Science. Bulletin of National Institute
of Livestock and Grassland Science 10: 15-27.
Iwasaki, K., T. Haryu, R. Tano, F. Terada, M. Itoh and K. Kameoka, 1982. New animal metabolism facility, especially
the description of respirational apparatus. Bulletin of National Institute of Animal Industry 39: 41-78.
National Agriculture and Food Research Organization (NARO), 2007. Japanese feeding standard for dairy cattle. Japan
Livestock Industry Association, Tokyo, Japan.

Estimation of duodenal endogenous protein flow in dairy cattle:
a regression approach
H. Lapierre1*, M.D. Hanigan2 and D.R. Ouellet1
1Agriculture and Agri-Food Canada, Sherbrooke, QC, J1M 0C8, Canada; 2Virginia Polytechnic
Institute and State University, Blacksburg, VA, USA; helene.lapierre@agr.gc.ca
Abstract
The endogenous N (EN) fraction of the duodenal flow does not represent a net supply to the animal.
However, it is difficult to measure EN so that total measured N flows can be adequately corrected.
The estimation of duodenal EN flow has been revised to integrate studies that had been published
since the release of NRC in 2001. Based on these studies, duodenal EN flow (g N/d) = 15.4 (± 2.6)
+ 1.21 (± 0.24) × dry matter intake (DMI; kg/d). Compared with the value proposed by NRC, this
equation increases EN estimation at low DMI and decreases it at high DMI, the breaking point
being at DMI = 22.3 kg/d.
Keywords: dairy cattle, duodenal, endogenous, protein
Introduction
Duodenal flow of N comprises 3 fractions: undegraded dietary, microbial and endogenous (EN).
The contribution of EN may represent between 15 and 20% of duodenal N flow (Ouellet et al.,
2002, 2010). This fraction is important to acknowledge and quantify as it does not constitute a net
protein supply to the ruminant, but a recycling of amino acid previously absorbed and used from
arterial blood to synthesize EN. Not all models used to balance dairy rations adequately represent this
recycled contribution to duodenal protein flow, but those who do generally use the value proposed
by NRC (2001) of 1.9 g N/kg dry matter intake (DMI). Our objective was to revise this estimation,
including new information published since NRC (2001) and using only cattle data.

Due to the technical challenge of assessing EN flow, observed data are relatively scarce in the
literature. Nevertheless, duodenal EN flow has been determined in growing and mature cattle through
different methods. A limited database was constructed containing physiological status, DMI, body
weight (BW), and reported EN. These data were used to develop relationships between EN and
DMI by regression.

A summary of the observed duodenal EN flows is presented in Table 1. The data from Ørskov et al.
(1986) indicated EN flow from the rumen and abomasum even when cattle received only ruminal
volatile fatty acid infusions. The data from steers fed at low intakes indicated a disproportionate
ratio of duodenal EN relative to DMI when compared to cows fed at higher intakes. Collectively
these data support the inclusion of an intercept when expressing the duodenal EN relative to DMI
which was not adopted by the NRC. The physiological status (growing vs mature) was not significant
(P=0.29) when included in the regression. The resulting equation is: duodenal EN (g N/d) = 15.4
(± 2.6) + 1.21 (± 0.24) × DMI (kg/d), with both the slope and the intercept being highly significant
(P≤0.01). Compared with this equation, the current estimation of duodenal EN in NRC (2001) of 1.9
g N/kg DMI would underestimate EN in cattle with a DMI lower than 22.3 kg/d and overestimate
when DMI is higher than that.

Table 1. Duodenal endogenous nitrogen (EN, g N/d) flows from cattle as reported.
Animal DMI BW EN Method2 Reference

kg/d1 kg
2 dairy cows 8.34 500 18.8 15N Brandt et al. (1980)

2 dairy cows 0/3.4 675 15.4 Rumen VFA inf. Ørskov et al. (1986)
4 dairy cows 14.4 625 34.0 15N-Leu dilution Ouellet et al. (2002)
4 dairy cows 17.6 607 40.0 15N-Leu dilution Ouellet et al. (2010)
Dairy cows 18.0 600 40.9 Meta-analysis Marini et al. (2008)
Dairy cows 17.9 597 29.5 Meta-analysis Sauvant et al. (2013)
3 steers 2.86 300 23.0 RDP-free diet Hart and Leibholz (1990)
2 steers 0/2.2 278 11.6 Rumen VFA inf. Ørskov et al. (1986)
4 steers 0/1.8 278 13.3 Rumen VFA inf. Ørskov et al. (1986)
4 steers 3.14 424 24.4 RDP-free diet Hannah et al. (1991)
4 steers 3.37 372 26.2 RDP-free diet Lintzenich et al. (1995)
Growing cattle 6.82 359 24.2 Meta-analysis Marini et al. (2008)
1 When VFA were infused in the rumen, DMI=0 /kg of DM infused in the rumen.
2 VFA inf. = volatile fatty acid infusion; RDP = rumen degradable protein.
Acknowledgements
Financial support from The Dairy Farmers of Canada and Agriculture and Agri-Food Canada is
acknowledged.
References
Brandt, M.W., K. Rohr and P. Lebzien, 1980. Determination of endogenous protein-N in duodenal chyme of dairy
cows using of N-15. Journal of Animal Physiology and Animal Nutrition 44: 26-27.
Hannah, S.M., R.C. Cochran, E.S. Vanzant and D.L. Harmon, 1991. Influence of protein supplementation on site and
extent of digestion, forage intake, and nutrient flow characteristics in steers consuming dormant bluestem-range
forage. Journal of Animal Science 69: 2624-2633.
Hart, F.J. and J. Leibholz, 1990. A note on the flow of endogenous protein to the omasum and abomasum of steers.
Animal Production 51: 217-219.
Lintzenich, B.A., E.S. Vanzant, R.C. Cochran, J.L. Beaty, R.T.J. Brandt and G. St-Jean, 1995. Influence of processing
supplemental alfalfa on intake and digestion of dormant bluestem-range forage by steers. Journal of Animal
Science 83: 1187-1195.
Marini, J.C., D.G. Fox and M.R. Murphy, 2008. Nitrogen transactions along the gastrointestinal tract of cattle: a meta-
analytical approach. Journal of Animal Science 86: 660-679.
Ørskov, E.R., N.A. McLeod and D.J. Kyle, 1986. Flow of nitrogen from the rumen and abomasum in cattle and sheep
given protein-free nutrients by intragastric infusion. British Journal of Nutrition 56: 241-248.
Ouellet, D.R., R. Berthiaume, G. Holtrop, G.E. Lobley, R. Martineau and H. Lapierre, 2010. Effect of method of
conservation of timothy on endogenous nitrogen flows in lactating dairy cows. Journal of Dairy Science 93:
4252-4261.
Ouellet, D. R., M. Demers, G. Zuur, G. E. Lobley, J. R. Seoane, J. V. Nolan and H. Lapierre, 2002. Effect of dietary
fiber on endogenous nitrogen flows in lactating dairy cows. Journal of Dairy Science 85: 3013-3025.

Evaluation of the INRA Systali digestive model through measured net
portal appearance of nutrients in ruminants
P. Nozière1*, J. Vernet1, F. Raulhac1, P. Chapoutot2, H. Lapierre3, D. Sauvant2 and I. Ortigues-Marty1
1INRA-Vetagrosup, UMR1213 Herbivores. 63122 Saint-Genès-Champanelle. France; 2INRA-
AgroParisTech, UMR MoSAR, 75231 Paris cedex 05, France; 3Agriculture and Agri-Food Canada,
Sherbrooke, QC, J1M 0C8, Canada; pierre.noziere@clermont.inra.fr
Abstract
The digestive model included in the renewed INRA feed unit system for ruminants allows to predict
absorbable flows of acetate, propionate, butyrate, glucose and amino acids. We aimed to assess this
model using a large database of measured net portal nutrient appearance. As expected, predicted
absorbable flows were systematically higher than measured net portal (absorbed) appearance, and
the relationships were consistent with current quantitative knowledge on metabolism of portal-
drained viscera.
Keywords: nutrient, ruminant, portal
Introduction
The INRA model of net energy and metabolisable protein supply has been renewed (Sauvant and
Nozière, 2016) to account for the effects of feeding level (FL), proportion of concentrate (PCO), and
rumen protein balance, on the main digestive processes: transit, ruminal and intestinal digestibility,
and partition of fermented organic matter (OM) towards microbial growth, volatile fatty acids (VFA)
and gas. It allows predicting the absorbable nutrient flows: total VFA, acetate (C2), propionate (C3)
and butyrate (C4), glucose (Glc), and amino acids (AA). We aimed to assess this renewed digestive
model by comparing predicted absorbable nutrient flows to their measured net portal appearance
(NPA) in ruminants.

The INRA Flora database (Vernet and Ortigues-Marty, 2006) gathering published results on
NPA of nutrients in ruminants has been used. All diets (n=761, FL = 2.1±0.84 DM intake %BW,
PCO=0.38±0.33, crude protein = 146±33 g/kg DM) have been characterised using the renewed INRA
digestive model (Systoolweb.fr application: Chapoutot et al., 2015). Predicted absorbable nutrients
were: total VFA (from rumen and large intestine), Glc, and AA. Total rumen VFA depend on OM
fermented in the rumen, PCO, and the fermentation products of silages, whereas molar proportions
of C2, C3, and C4 depend on digested NDF/digested OM, rumen starch digestibility, FL, and the
species (cattle vs sheep). Large intestine VFA depend on OM fermented in this compartment (14%
of apparently digested OM in total tract), with fixed molar proportions of 76% C2, 16% C3, and
5% C4. Glc corresponds to starch digested in the small intestine, and AA to the metabolisable
protein (PDI), accounting for dietary and microbial AA. Measured NPA were total VFA, C2, C3,
C4, β-OH-C4, Glc, and α-amino-N. Relationships between predicted absorbable nutrient flows
and their measured NPA were assessed, using variance-covariance models allowing dissociation
between within- and between-experiment variability (Sauvant et al., 2008). Normality of residuals
and interfering factors (species, intake level, diet composition) on slopes, LSMeans, and residuals
were checked systematically.

For each nutrient, only within-experiment relationships, which were very close to between-ones, are
presented (Table 1). The negative intercept for Glc (the only one significant) is consistent with the net

Table 1. Means ± standard errors (SE) and within-experiment relationships between measured net
portal appearance (NPA) of nutrients (Y) and predicted absorbable nutrient flows (X) in mmol C
or N/d/kg body weight.
Measured NPA Predicted absorbable nexp3 ntrt4 Intercept ± SE Slope ± SE RMSE5

flows
acetate (105±53) acetate (139±51) 37 95 10.6±9.4NS 0.69±0.07*** 10.7

propionate (54±35) propionate (66±28) 38 100 -3.5±4.4NS 0.89±0.07*** 8.5
butyrate (13±12) butyrate (41±20) 32 85 -1.5±1.6NS 0.37±0.04*** 2.9
β-OH-butyrate (22±13) butyrate (41±20) 33 87 2.2±3.4NS 0.48±0.08*** 4.8
glucose (-3±14) glucose1 (34±25) 46 117 -15.1±1.8*** 0.38±0.05*** 7.7
α-amino N (12±6) N_PDI2 (21±7) 43 122 0.95±1.59NS 0.52±0.07*** 2.9
1 Starch digestible in small intestine.

2 N (CP/6.25) from dietary and microbial protein digestible in the intestine.
3n
exp = number of experiments.
4 n = number of treatments.
trt
5 RMSE = root mean square error.
***P<0.001; NSP>0.05.
basal uptake of glucose with non-starch diets. As expected, for each nutrient, predicted absorbable
flows were higher than their measured NPA, and the slopes, reflecting an average marginal net portal
recovery lower than unity, are consistent with current quantitative knowledge on metabolism of portal-
drained viscera (PDV). Indeed, the slopes for glucose and AA, representing a portal recovery of 38
and 71% (assuming total AA = 1.37 α-amino N) of the absorbable flow, indicate important usage
of these nutrients by the PDV. These relationships, though very close to those previously derived
from INRA (2007) (Nozière et al., 2010), are based on a more analytical prediction of absorbable
flows. In particular, the previous calculation of fermented organic matter for VFA prediction did not
properly dissociate ruminal vs intestinal fermentations, whereas this partition is better represented
in the renewed model. Few interfering factors were detected on LSMeans for C3 and C4 (negative
effect of NDF, species [cattle>sheep], positive effect of FL for C4) and on slopes for α-amino N
(positive effect of NDF), which deserve further analyses.
Acknowledgements
Financial support from Inzo and Limagrain is acknowledged.
References
Chapoutot, P., O. Martin, P. Nozière and D. Sauvant, 2015. Systool web, a new one-line application for the French
INRA ‘Systali’ project. In: Book of Abstracts of the 66th Annual Meeting of the European Federation of Animal
Science. Wageningen Academic Publishers, Wageningen, the Netherlands, pp. 265.
INRA, 2007. Alimentation des bovins, ovins et caprins – Besoins des animaux – Valeurs des aliments – Tables INRA
2007. Editions Quae, Versailles, France, 307 pp.
Nozière P., I. Ortigues-Marty, C. Loncke and D. Sauvant, 2010. Carbohydrate quantitative digestion and absorption in
ruminants: from feed starch and fibre to nutrients available for tissues. Animal 4: 1057-1074.
Sauvant, D. and P. Nozière, 2016. Quantification of the main digestive processes in ruminants: the equations involved
in the renewed energy and protein feed evaluation systems. Animal 10: 755-770.
Sauvant, D, P. Schmidely, J.J. Daudin and N.R. St Pierre, 2008. Meta-analyses of experimental data in animal nutrition.
Animal 2: 1203-1214.
Vernet, J. and I. Ortigues-Marty, 2006. Conception and development of a bibliographic database of blood nutrient
fluxes across organs and tissues in ruminants: data gathering and management prior to meta-analysis. Reproduction
Nutrition Development 46: 527-546.

Are circulating blood metabolite concentrations related to changes in
absorption in ruminants?
I. Ortigues-Marty1*, J. Vernet1, S. Ashaba1, H. Lapierre2 and P. Nozière1
1INRA, UMR1213 Herbivores, 63122 Saint-Genès-Champanelle, France; 2Agriculture and Agri-
Food Canada, Sherbrooke, QC, J1M 0C8, Canada; isabelle.ortigues@clermont.inra.fr
Abstract
To define the limits of the interpretation of blood profile in ruminants, relationships between arterial
concentrations and net portal appearance or net splanchnic release of acetate, butyrate, propionate,
lactate, glucose, β-hydroxy-butyrate, α-amino-N, ammonia and urea-N were studied by meta-analysis
on published results. Concentrations of the major volatile fatty acids reflected their net portal supply.
By contrast, animal characteristics but not nutrient supply influenced concentrations of L-lactate,
α-amino-N and ammonia. Glucose concentrations varied with both net portal supply and animal
characteristics. Concentrations of the other metabolites linearly reflected their net splanchnic release.
Keywords: blood profile, arterial concentration, portal absorption, net splanchnic release
Introduction
The concentrations of blood metabolites in peripheral blood vessels are often used as indicators of
the nutritional status of the animals. Such information is easy to obtain from a simple blood sample,
even on farm, and interpreted in relation to feed supply. However, circulating concentrations are
the result of both nutrient supply and utilisation by the body tissues. Nutritional management of
animals could benefit from dissociating the impacts of feed intake and nutrient use on circulating
blood metabolites. Hence, the purpose of the work is to establish by meta-analysis the relationships
between metabolite arterial concentrations and their net portal absorption (NPA) or net splanchnic
release (NSR) in ruminants.

The meta-analysis was developed from the Flora database (Vernet and Ortigues-Marty, 2006) which
gathers data from publications on splanchnic fluxes in ruminants. These studies involve sheep and
cattle in different physiological states (growing, adult either lactating or non-productive), fed at
frequent intervals a range of intakes (from fasting to ad libitum) of diets with widely different forage
to concentrate ratios. Intra-study variance-covariance models were developed according to Sauvant
et al. (2008). The response variables (Y) were the arterial concentrations of metabolites (mM). The
independent variables (X) were the NPA or NSR of acetate, butyrate, propionate, lactate, glucose,
β-hydroxy-butyrate (BHB), α-amino-N, ammonia and urea-N (mmol/h/kg live weight). The GLM
(Minitab 16) response equation was: Y = αi + α + β X + e, with α = overall intercept, αi = effect
of the experimental study i on the intercept α, β = slope and e = error. Normality of residuals and
interfering factors were checked systematically. The effect of animal characteristics (species, animal
type, physiological state and energy balance) was analysed on LSMeans and residuals.

Results (Table 1) indicate that changes in arterial concentrations of acetate, propionate, and butyrate
reflect modifications in their NPA and hence are related to diet composition and intake. For two of
the metabolites mainly synthesized by the liver, urea-N and BHB, arterial concentrations varied
significantly with NSR, interpreted as changes in their recycling and/or synthesis by visceral tissues.
Concentrations of L-lactate, α-amino-N and ammonia were affected by animal characteristics

Table 1. Responses of arterial concentrations (mM) to net portal absorption (NPA) or net splanchnic
release (NSR) of metabolites (mmol/h/kg BW) in ruminants.
Nutrient Predictor Ntrt1 Intercept ± SE2 Slope ± SE2 RMSE
Acetate NPA 191 0.734±0.051*** 0.274±0.026*** 0.153

NSR 103 0.838 ±0.069*** 0.204±0.026NS 0.160
Propionate NPA 209 0.017±0.002*** 0.042±0.002*** 0.007
NSR 112 0.032±0.002*** 0.164±0.022*** 0.006
Butyrate NPA 175 -0.0003±0.001NS 0.141±0.005*** 0.004
NSR 91 0.006±0.001*** 0.379±0.020*** 0.005
L-lactate NPA 282 0.616±0.017*** -0.094±0.079NS 0.091
NSR 190 0.577±0.007*** 0.045±0.078NS 0.092
Glucose NPA 310 3.762±0.011*** 0.485±0.117*** 0.158
NSR 192 3.615±0.056*** 0.041±0.082NS 0.156
BHB NPA 117 0.340±0.028*** 0.507±0.128*** 0.083
NSR 72 0.243±0.03 *** 0.501±0.059*** 0.058
NH3-N NPA 317 0.200±0.005*** 0.002±0.008 NS 0.018
NSR 232 0.209±0.001*** -0.029±0.022NS 0.019
α-amino-N NPA 187 3.063±0.058*** 0.191±0.130NS 0.182
NSR 123 3.354±0.025*** -0.025±0.169NS 0.155
Urea-N NPA 305 6.085±0.296*** -3.52±0.638*** 1.543
NSR 223 4.738±0.237*** 5.696±0.379*** 1.063
1 Ntrt = number of treatments.

2 ***P<0.001; NS = not significant.
(results not shown) and not by food supply. Glucose concentrations were not only affected by animal
characteristics but were also linearly related to its NPA, suggesting an impact of starch bypass or
intestinal glucose metabolism. Hence, the impact of tissue metabolism is more important than
metabolite or precursor availability on the concentrations of these metabolites. Finally, α-amino-N
concentrations, which reflect a heterogeneous group of amino acids, was not related to NPA, in
contrast to concentrations of individual essential amino acids which were related to their duodenal
flow (Patton et al., 2015). These results indicate the limits of interpretation of concentrations of
circulating metabolites measured in experimental work or on-farm.
Acknowledgements
Financial support from Inzo and Limagrain is acknowledged.
References
Patton, R.A., A.N Hristov, C. Parys and H. Lapierre, 2015. Relationships between circulating plasma concentrations
and duodenal flows of essential amino acids in lactating dairy cows. Journal of Dairy Science 98: 4707-4734.
Sauvant, D, P. Schmidely, J.J. Daudin and N.R. St Pierre, 2008. Meta-analyses of experimental data in animal nutrition.
Animal 2: 1203-1214.
Vernet, J. and I. Ortigues-Marty, 2006. Conception and development of a bibliographic database of blood nutrient
fluxes across organs and tissues in ruminants: data gathering and management prior to meta-analysis. Reproduction
Nutrition Development 46: 527-546.

Proteomics of total and phosphorylated proteins in skeletal muscle of
Angus and Nellore cattle
R.T.S. Rodrigues1,2,3, M.L. Chizzotti2,3*, C.E. Vital1, M.T. Baracat-Pereira1, E. Barros1,3, R.A.
Gomes2,3, K.C. Busato3 and M.M. Ladeira3
1Nubiomol, Universidade Federal de Viçosa, 36570-900 Viçosa, Brazil; 2DZO, Universidade Federal
de Viçosa, 36570-900 Viçosa, Brazil; 3DZO, Universidade Federal de Lavras, 37200-000 Lavras,
Brazil; mariochizzotti@ufv.br
Abstract
The aim of this study was to evaluate differences in the proteome and phosphoproteome of the skeletal
muscle between Nellore and Angus cattle. Proteins were extracted from Longissimus dorsi muscle
and separated by 2-D electrophoresis, and identified by mass spectrometry. Nellore had greater
abundance of tropomyosin 1 and troponin T, and Angus had higher abundance of prohibitin and
HSPA9. Nellore had greater phosphorylation of myosin regulatory light chain 2 and Angus had higher
phosphorylation of troponin T and phosphoglucomutase-1. The proteome and phosphoproteome
of the skeletal muscle of Nellore and Angus differed for proteins related to apoptosis inhibition,
mitochondrial organization, glucose metabolism and contractile regulation.
Keywords: actomyosin complex, apoptosis, beef, posttranslational modifications
Introduction
Metabolism is deeply controlled by interactions between proteins and metabolic pathways in which
proteins are the key molecules. The phosphorylation is one of the main post-translational changes
affecting the structure and protein activity. Zebu (Nellore) meat is considered leaner and less tender
than taurine (Angus), which might be related to differences in the proteome and phosphoproteome
of the skeletal muscle.

Proteins were extracted from Longissimus dorsi muscle of seven Nellore and seven Angus bulls,
immediately after slaughter, and separated by two-dimensional electrophoresis using strips of 24 cm
at pH 4 to 7, and analysed using the Image Master 2D Platinum Software 7.0 (P<0.05). The Pro-Q
Diamond stain was used for phosphorylated proteins analysis. Differentially abundant proteins
were digested with trypsin and mass spectra of the peptides were obtained using matrix assisted
laser desorption/ionization time-of-flight mass spectrometry. Proteins were identified and validated,
respectively, using the Mascot and the Scaffold to 90% in the Bovidae database.

In relation to proteome, Angus had greater abundance of prohibitin (2.2 fold; important in
mitochondrial organization and inhibition of apoptosis), and HSPA9 (2.0 fold; responsible for
apoptosis inhibition and correlated with beef tenderness according to Guillemin et al., 2011). Nellore
had higher abundance of tropomyosin 1 (2.9 fold) and fast skeletal muscle troponin-T (TNNT3, 2.0
fold), which are related to the stability of actin filaments and regulation of muscle contraction and
more abundant in tough beef (Chaze et al., 2013). Regarding the phosphoproteome, Angus had greater
abundance of phosphoglucomutase-1 (1.9 fold), which is more active when phosphorylated, and is
related to glycolitic metabolism and faster rate of muscle pH decline in post mortem (Anderson et
al., 2014), and greater phosphorylation of troponin T (3.6 fold) which reduces ATPase activity and
Ca2+-sensitivity and may increases its cleavage by calpain (Streng et al., 2013). Nellore had higher

phosphorylation of myosin regulatory light chain 2 (1.5 fold), that is linked to pre slaughter stress,
increased sarcomere shortening, and lower meat tenderness (Franco et al., 2015). The proteome
and phosphoproteome of the skeletal muscle of Nellore and Angus differed for proteins related to
apoptosis inhibition, mitochondrial organization, glucose metabolism and contractile regulation.
Acknowledgements
This study was supported by CNPq, FAPEMIG, CAPES, and NUBIOMOL (Brazil).
References
Anderson, M.J., S.M. Lonergan, E. Huff-Lonergan, 2014. Differences in phosphorylation of phosphoglucomutase 1 in
beef steaks from the longissimus dorsi with high or low star probe values. Meat Science 96: 379-384.
Chaze, T., J.F. Hocquette, B. Meunier, G. Renand, C. Jurie, C. Chambon, L. Journaux, S. Rousset, C. Denoyelle, J. Lepetit
and B. Picard, 2013. Biological markers for meat tenderness of the three main French beef breeds using 2-DE and
MS approach. In: F. Toldra and L.M.L Nollet (eds.). Proteomics in Foods. Springer, New York, USA, pp. 127-146.
Franco, D., A. Mato, F.J. Salgado, M. López-Pedrouso, M. Carrera, S. Bravo, M. Parrado, J.M. Gallardo and C. Zapata,
2015. Tackling proteome changes in the longissimus thoracis bovine muscle in response to pre-slaughter stress.
Journal of Proteomics 122: 73-85.
Guillemin, N., M. Bonnet, C. Jurie, and B. Picard, 2011. Functional analysis of beef tenderness. Journal of Proteomics
75: 352-365.
Streng, A.S., D. Boer, J. Velden, M.P Dieijen-Visser and W.K.W.H. Wodzig, 2013. Posttranslational modifications of
cardiac troponin T: an overview. Journal of Molecular and Cellular Cardiology 63: 47-56.

Diet-dependent energy metabolism of neonatal calves determined by
indirect calorimetric measurements
C.T. Schäff, J. Gruse, M. Derno and H.M. Hammon*
Leibniz-Institute for Farm Animal Biology (FBN), Institute of Nutritional Physiology ‘Oskar Kellner’,
18196 Dummerstorf, Germany; hammon@fbn-dummerstorf.de
Abstract
After birth, energy supply changes from parenteral carbohydrate supply to oral fat and carbohydrate
supply by colostrum. Besides nutrients, colostrum provides a wide range of bioactive substances
like immunoglobulins and growth factors. These promote health and the development of the
gastrointestinal tract, the first site of nutrient absorbance, thus regulating energy supply. Energy
expenditure (EE) can be calculated from data provided by indirect calorimetric measurements, but
such data are rare for neonatal calves. Therefore, this study aimed to gain data on substrate utilisation
and EE in neonatal male calves (n=14) during the first week of life when fed either colostrum or a
formula similar in macronutrient composition but almost without bioactive substances for the first
two days of life. For indirect calorimetric as well as physical activity measurements calves were
placed in respiration chambers on day 2 and 7 of life. We found significant differences within the
respective day and between the two days for EE, fat and carbohydrate oxidation (COX) and respiratory
quotient (RQ) in all calves. Fat oxidation and EE decreased, whereas RQ and COX increased from
day 2 to 7. Only COX differed according to initial diet. Activity was highest at evening feeding and
when the light turned on in the morning. Our data indicate that ontogenetic maturation influences
EE and substrate oxidation whereas initial diets only influence COX. EE decreases with advanced
adaptation to extra-uterine life and thus more mature metabolic processes. Diurnal rhythms of EE
and COX become more apparently with increasing age.
Keywords: colostrum, energy expenditure, newborn calves
Introduction
Neonatal calves’ energy supply changes with birth from parenteral supply of carbohydrates, mainly
glucose, by the placenta to oral fat and carbohydrate supply, mainly triglycerides and lactose,
by colostrum. Besides nutrients, colostrum provides a wide range of bioactive substances like
immunoglobulins, cytokines, hormones and growth factors. The latter promote the development of
the gastrointestinal tract, which is the first site of nutrient absorbance. Therefore, maturation of the
gastrointestinal tract improves energy supply. Energy expenditure (EE) can be calculated from data
provided by indirect calorimetric measurements. Such data, which will clarify substrate utilisation
immediately after birth, are rare for neonatal calves. Thus, this study aimed to gain data on substrate
utilisation and EE in neonatal calves during the first week of life when fed either colostrum or a milk-
based formula without bioactive substances but a comparable nutrient content. We hypothesized that
substrate utilisation and EE change with age and may be affected by diet fed immediately after birth.

German Holstein male calves were fed two diets, either pooled colostrum (n=7) or a formula (n=7)
for the first two days of life. Individual formulas for day 1 and 2, corresponding to colostrum milking
1 and 3 after calving, consisted of lactalbumin powder, whey powder, refined palm and coconut
fat, casein powder, monocalcium phosphate, calcium carbonate and sodium chloride (Gruse et al.,
2015). The nutrient composition was comparable to colostrum but it almost totally lacked bioactive
substances. From day 3 until the end of the study at day 8 of life (end of calorimetric measurement)
calves were fed a commercial milk replacer (150 g powder/l water). Amounts fed were 10% of birth
weight on day 1 and 12% from day 2 on. Calves were fed twice daily by nipple bottle. Refused

amounts of feed were tube-fed for the first two days to ensure complete feed intake. For indirect
calorimetric measurements calves were placed in respiration chambers on day 2 and 7 of life under
constant climate conditions (21 °C). After gas exchange equilibration time, CO2 production and
O2 consumption were measured every six minutes for 21 hours to calculate EE, fat (FOX) and
carbohydrate oxidation (COX) and respiratory quotient (RQ) (calculated as CO2 production per O2
consumption). In addition, physical activity in the chamber was measured by infrared sensor (Derno
et al., 2013). Data were hourly averaged and analysed by mixed model of SAS 9.3 with group, day,
hour and the respective interactions as fixed effects.

Our data revealed differences (P<0.05) for day, hour, day × hour interaction and group × day × hour
interaction for EE, FOX, COX and RQ, but no differences for group or group × day interaction
except for COX (group P<0.05). EE and FOX decreased, whereas RQ and COX increased from
day 2 to 7 (Table 1).
COX was higher in formula-fed than in colostrum-fed calves (P<0.05), although lactose intake was
comparable between groups. In both groups COX was higher and FOX was lower on day 7 than on
day 2 (P<0.001). EE was higher at birth than at one week of age, when adaptation to extra-uterine
life is advanced and metabolic processes are more mature. Furthermore, diurnal rhythms of EE and
COX became more apparently with increasing age. This is in contrast to FOX showing far less daily
variation on day 7 than on day 2. Activity measured in the chamber changed within day and showed
a similar pattern on both days. We found differences for hour and group × day × hour interaction
(P<0.05). Activity was highest at evening feeding and when lights turned on in the morning. Our
data indicate that ontogenetic maturation influences EE and substrate oxidation, but there is less
influence of different initial diets, especially on FOX and EE.
Table 1. Indirect calorimetric measurements for calves fed colostrum (COL) or formula (FOR) for
the first two days of life.1
COL min COL max FOR min FOR max SEM
Day 2
EE (kJ/kg body weight 0.75/d) 662.00 859.40 773.79 901.71 28.62
FOX (g/h) 8.26 12.25 8.55 11.11 0.59
COX (g/h) 7.88 13.03 10.68 17.06 1.42
RQ 0.77 0.81 0.79 0.83 0.01
Day 7
EE (kJ/kg body weight 0.75/d) 550.69 703.99 554.54 717.81 20.33
FOX (g/h) 5.71 6.86 5.47 6.89 0.59
COX (g/h) 12.14 18.99 13.19 20.76 1.53
RQ 0.82 0.86 0.84 0.87 0.01
1 EE = energy expenditure; FOX = fat oxidation; COX = carbohydrate oxidation; RQ = respiratory quotient.
References
Derno, M., G. Nürnberg, P. Schön, A. Schwarm, M. Röntgen, H.M. Hammon, C.C. Metges, R.M. Bruckmaier and B.
Kuhla, 2013. Short-term feed intake is regulated by macronutrient oxidation in lactating Holstein cows. Journal
of Dairy Science 96: 971-980.
Gruse, J., S. Görs, A. Tuchscherer, W. Otten, J.M. Weitzel, C.C. Metges, S. Wolffram and H.M. Hammon, 2015. The
effects of oral quercetin supplementation on splanchnic glucose metabolism in 1-week-old calves depend on diet
after birth. Journal of Nutrition 145: 2486-2495.

Amino acid composition of rumen microorganisms in cattle
M. Sok1,2, D.R. Ouellet2, J. Firkins3, D. Pellerin1 and H. Lapierre2*
1Université Laval, Québec, QC, G1V 0A6, Canada; 2Agriculture and Agri-Food Canada,
Sherbrooke, J1M 0C8, QC, Canada; 3The Ohio State University, Columbus 43210, Ohio, USA;
helene.lapierre@agr.gc.ca
Abstract
Because microbial protein constitutes more than 50% of the protein digested by the dairy cow, its
amino acid (AA) is needed to adequately estimate AA supply. Our objective was to update the AA
composition of the rumen bacteria flowing to the duodenum using only studies from cattle and
compare it with protozoa, based on published literature (61 and 26 reported means, respectively). The
AA (or true protein) fraction represented 0.77 of bacteria crude protein. The updated AA composition
of bacteria is close to that proposed by Clark et al., 1992. For all AA, the composition of protozoa (g
AA/100 g AA) differed from bacteria. Therefore, we need to consider the contribution of protozoa
to the duodenal microbial protein flow to adequately estimate AA duodenal flow.
Keywords: cattle, amino acid, bacteria, protozoa
Introduction
One of the most used references to assess the amino acid (AA) composition of duodenal microbial
protein (MCP) flow is from Clark et al. (1992) reporting the AA composition of rumen bacteria (7 of
the 18 studies were in sheep). It is however well known that part of MCP flowing at the duodenum
also includes protozoa, which may contribute 10 to 30% of duodenal MCP (Sylvester et al., 2005);
but their AA composition differed from that of bacteria (Martin et al., 1996). Therefore, our objectives
were: (1) to update the AA composition of rumen bacteria using only data from cattle; (2) build a
database to estimate the AA composition of rumen protozoa; and (3) determine if AA composition
of protozoa and bacteria differs.

Based on an initial search on Scopus.com completed with a thorough revision of referenced
manuscripts, 26 publications (61 treatments; between 1967 and 2014) reporting data from cattle
studies were selected to build the bacteria database and 10 publications (26 treatments; between
1971 and 2006) for the protozoa database. Concentrations of Met and Cys were only kept for studies
specifying that the sulfur groups had been protected prior to hydrolysis. The AA concentrations
outside 2 SD from means were not included for all AA except Cys and Met for which the range
was 1 SD due to large variation. The reported AA concentrations were standardised to g AA/100 g
AA. As the addition of formaldehyde to the ruminal samples may alter true protein/crude protein
(TP/CP) and AA composition, 5 publications reporting use of formaldehyde were excluded from
the bacteria database. When reported, the ratio TP/CP was standardised to yield g true protein/g CP.
The AA composition of bacteria and protozoa was compared by an analysis of variance including
the effect of the type of microorganism and the treatment for which the rumen samples were taken.

For purpose comparison, concentrations of Cys and Trp observed in the current study were added to
Clark et al. (1992) data and values of other AA adjusted to yield a sum of 100 g AA/100 g AA. The
ratio TP/CP for bacteria was lower (P=0.02) when formaldehyde was used (0.69 vs 0.77±0.027). All
AA differed between the 2 populations of microorganism: for essential AA, Trp (1.76) showed the

highest ratio and Lys (0.78) the lowest ratio bacteria AA/protozoa AA (Table 1). These data clearly
indicate the importance of acknowledging the presence of protozoa in duodenal protein flow to
correctly predict the AA duodenal flow.
Table 1. Amino acid (AA) composition of rumen bacteria and protozoa (g AA/100 g AA) in cattle.
AA Bacteria Protozoa P-value2
Mean cv Reference1 Mean cv
Ala 7.2 8.0 7.5 8.0 8.0 <0.0001

Arg 4.6 7.8 5.1 7.8 7.8 0.003
Asx 11.8 5.1 12.2 5.1 5.1 <0.0001
Cys 1.4 17.5 1.6 17.5 17.5 <0.0001
Glx 13.0 5.8 13.1 5.8 5.8 <0.0001
Gly 5.4 11.7 5.8 11.7 11.7 <0.0001
His 1.9 16.4 2 16.4 16.4 <0.0001
Ile 5.5 7.0 5.7 7.0 7.0 <0.0001
Leu 7.5 6.6 8.1 6.6 6.6 0.18
Lys 7.8 12.3 7.9 12.3 12.3 <0.0001
Met 2.4 15.7 2.6 15.7 15.7 <0.0001
Phe 5.2 7.0 5.1 7.0 7.0 0.0004
Pro 3.6 8.9 3.7 8.9 8.9 0.04
Ser 4.5 8.0 4.6 8.0 8.0 <0.0001
Thr 5.6 6.4 5.8 6.4 6.4 <0.0001
Trp 1.4 4.5 1.5 4.5 4.5 <0.0001
Tyr 5.2 11.4 4.9 11.4 11.4 <0.0001
Val 6.0 9.6 6.2 9.6 9.6 <0.0001
1 From Clark et al. (1992).

2 P-value of the difference between the AA composition of bacteria vs protozoa when both analyses were conducted in
the same study (21 comparisons).
Acknowledgements
The financial support of the Dairy Farmers of Canada, Agriculture and Agri-Food Canada and the
Natural Sciences and Engineering Research Council of Canada is acknowledged.
References
Clark J.H, T.H. Klusmeyer and M.R. Cameron, 1992. Microbial protein synthesis and flows of nitrogen fractions to
the duodenum of dairy cows. Journal of Dairy Science 75: 2304-2323.
Martin, C., L. Bernard and B. Michalet-Doreau, 1996. Influence of sampling time and diet on amino acid composition
of protozoal and bacterial fractions from bovine ruminal contents. Journal of Animal Science 74: 1157-1163.
Sylvester J.T., S.K.R. Karnati, Z. Yu, C.J. Newbold and J.L. Firkins, 2005. Evaluation of a real-time PCR assay
quantifying the pool size and duodenal flow of protozoal nitrogen. Journal Dairy Science 88: 2083-2095.

Ruminal absorption kinetics of D and L-lactate in lactating dairy cows
under washed rumen conditions
A.C. Storm*, T. Larsen and M. Larsen
Department of Animal Science, Aarhus University, Foulum, 8830 Tjele, Denmark;
adam.storm@anis.au.dk
Abstract
The study aimed to investigate the effect of increasing ruminal D- and L-lactate concentration
on fractional absorption rate of D- and L-lactate, and volatile fatty acid (VFA). Five multiparous
Danish Holstein dairy cows surgically prepared with ruminal cannulas were used for a washed
rumen study. Treatments were three ruminal buffers containing the same initial concentrations of
VFA (50, 33, 2, 12, and 2 mM acetate, propionate, isobutyrate, butyrate, and valerate, respectively),
and increasing concentrations of both D- and L-lactate (1, 6, and 11 mM, respectively). The three
buffers were introduced into a temporarily emptied and washed rumen for 30 min in a Latin-square
design. The results shows that the fractional absorption rate of VFA was unaffected by the increasing
ruminal concentrations of lactate, and that the fractional absorption rate of D/L-lactate increases
with decreasing ruminal lactate concentration. This indicates that both D and L-lactate is efficiently
absorbed at low ruminal concentrations (~1 mM), but that part of the absorption mechanism gets
saturated at increasing levels.
Keywords: D-lactate, dairy cow, absorption kinetic, ruminal absorption
Introduction
Ruminal microorganisms produce two isomeric forms of lactate, D and L, when fermenting the
feed. Lactate fermentation is enhanced by increasing dietary concentrations of easily fermentable
carbohydrates, which is also correlated to the risk of sub-acute ruminal acidosis (SARA). L-lactate
is also a product of other mammal metabolic pathways, whereas D-lactate only is of microbial
origin. Thus, D-lactate found in the blood or urine is of microbial origin and D-lactate is therefore
a potential biomarker of SARA. L-lactate is one of the quantitatively most important hepatic
glucogenic precursors (~25% of net output) and hepatic extraction is approximately 12% of total
influx. However, there is no specific enzyme responsible for hepatic use of D-lactate, and D-lactate
is thus mainly excreted into urine.
Ruminal absorption of L-lactate is presumably facilitated through monocarboxylate transport proteins

located in the apical membrane of the ruminal epithelium. However, it is not clear if D-lactate is
absorbed as efficiently as L-lactate or if there is an upper limit to the absorption capacity of the
rumen. The objective of the present study was to determine the fractional absorption rate of D- and
L-lactate at increasing ruminal concentrations of lactate, and potential impact on volatile fatty acid
(VFA) absorption.

Five multiparous Danish Holstein dairy cows surgically prepared with ruminal cannula were used
for the present washed rumen study. Treatments were three ruminal buffers containing the same
initial concentrations of VFA (50, 33, 2, 12, and 2 mM acetate, propionate, isobutyrate, butyrate,
and valerate, respectively), and increasing concentrations of D and L-lactate (1, 6, and 11 mM).
The three buffers (Low, Medium and High) were introduced into a temporarily emptied and washed
rumen for 30 min in a replicated 3 × 3 Latin-square design. In between each treatment buffer, a
washing-buffer without VFA and lactate was applied for 20 min to wash out residues of the previous

buffer in the rumen and epithelium. Experimental setup, chemical analysis of VFA and L-lactate
followed Storm et al. (2011). D-lactate was analysed by a novel enzymatic-fluorometric procedure
where intrinsic sample L-lactate dehydrogenase activity was eliminated prior to analysis (T. Larsen,
unpublished). Samples of ruminal buffers were obtained at 9, 20, 30 min after administration of the
treatment buffers and upon evacuation of the buffers. Ruminal lactate and VFA concentrations were
logarithmic transformed prior to fitting to a first order kinetic model. The fractional absorption rates
were subjected to statistical analysis using the MIXED procedure of SAS with lactate level as fixed
effect and cow as random effect. Data are presented as least squares means ± the standard error of
the mean and significance is declared at P≤0.05.

The fractional absorption rate of D-lactate was 64.6%/h at the low level of ruminal D-lactate (1
mM), but decreased to ~29%/h, when the initial ruminal concentration was 6 or 11 mM (Table 1).
The absorption of L-lactate followed the same pattern as for D-lactate. This indicates that D- and
L-lactate are absorbed by similar mechanisms, if not the same. Data also indicates that at least two
rate limiting absorption mechanisms are involved in the absorption of D- and L-lactate. The first
mechanism has a greater rate of absorption compared to the second one, but is saturated when the
lactate concentration increases, whereas the second mechanism is not saturated and driven by mass
action. Thus, data could be described by a model including a Michaelis-Menten kinetics combined
with a first order kinetics.
The fractional absorption rates of VFA confirm similar absorption rates for the quantitatively most
important VFA. Compared to other VFA’s, markedly lower absorption rates were observed for the
branched-chain isobutyrate and markedly greater absorption rates for the more lipophilic valerate.
This indicates that the lipophilicity of VFA affects the absorption rate; thus, confirming passive
diffusion as a major route of ruminal VFA absorption. Absorption rates of VFA were not affected
by increasing ruminal lactate concentrations; thus, indicating that ruminal lactate and VFA are not
competing for the same absorption mechanism.
Table 1. Fractional absorption rate (k, %/h) of D- and L-lactate, and volatile fatty acid from the
washed rumen of dairy cows challenged with increasing DL-lactate concentration.
Item Rumen buffer concentration of D- and L-lactate SEM P-values
Low, 1 mM Medium, 6 mM High, 11 mM
kD-lactate 64.6a 29.4b 28.4b 6.86 <0.01

kL-lactate 70.2a 15.6b 12.0b 11.2 <0.01
kacetate 77.9 77.8 81.4 7.08 0.55
kpropionate 81.9 82.3 87.8 7.91 0.29
kisobutyrate 37.1 39.2 42.4 4.64 0.17
kbutyrate 77.8 80.2 85.3 7.81 0.08
kvalerate 108 112 117 9.47 0.25
References
Storm, A.C., M.D. Hanigan and N.B. Kristensen, 2011. Effects of ruminal ammonia and butyrate concentrations
on reticuloruminal epithelial blood flow and volatile fatty acid absorption kinetics under washed reticulorumen
conditions in lactating dairy cows. Journal of Dairy Science 94: 3980-3994.

Exchanging fat for lactose in milk replacer stimulates de novo lipogenesis
in calves
J.J.G.C. van den Borne1*, E. Labussière2,3, M. Mielenz4,5, H. Sauerwein4 and W.J.J. Gerrits1
1Wageningen University, Animal Nutrition Group, P.O. Box 338, 6700 AH Wageningen, the
Netherlands; 2INRA-UMR, 1348 Pegase, 35590 Saint-Gilles, France; 3Agrocampus Ouest-UMR,
1348 Pegase, 35590 Saint-Gilles, France; 4University of Bonn, Institute of Animal Science,
Physiology & Hygiene Unit, Katzenburgweg 7-9, 53115 Bonn, Germany; 5Leibniz Institute for
Farm Animal Biology (FBN), Institute of Nutritional Physiology Oskar Kellner, Wilhelm-Stahl-Allee
2, Dummerstorf 18196, Germany; j.vandenborne@schippers.eu
Abstract
Previous studies showed that heavy milk-fed calves often develop problems with glucose homeostasis
and do not utilise dietary carbohydrates for body fat deposition. This suggests that calves may
lack the metabolic capacity for synthesizing fatty acids from glucose. Poor glucose utilisation for
growth may also be explained by the large supply of dietary fatty acids, allowing synthesis of body
fat exclusively from dietary fatty acids. The main objective of the current study was therefore to
assess energy balance, body fat deposition and de novo fatty acid synthesis when exchanging fat for
lactose in milk replacer for veal calves. Sixteen Holstein-Friesian calves (121±3 kg) were assigned
to either a high-fat (HF; 30% fat) or a low-fat (LF; 10% fat) milk replacer for 12 weeks. This large,
iso-energetic exchange between fat and lactose did not affect growth performance, protein and energy
retention. The respiratory quotient exceeded unity during several hours per day in LF calves, but not
in HF calves. The difference in isotope recovery in expired CO2 between two glucose isotopomers,
as a proxy for de novo lipogenesis, was greater in LF calves (12.9%) than in HF calves (1.4%),
indicating increased fatty acid synthesis from glucose in LF calves. Expression of genes involved
in fatty acid synthesis, desaturation and elongation, and triacylglycerol synthesis was greater in
LF calves than in HF calves, and this was most pronounced in retroperitoneal adipose tissue. In
conclusion, these findings indicate that calves are able to synthesize fatty acids from glucose when
a low-fat milk replacer is fed.
Keywords: fatty acid synthesis, gene expression, glucose metabolism, milk replacer, veal
Introduction
Previous studies have shown that heavy milk-fed calves often develop problems with glucose
homeostasis (e.g. Hugi et al., 1997) and do not utilise dietary carbohydrates for body fat deposition
(Van den Borne et al., 2007). This suggests that calves may lack the metabolic capacity for synthesizing
fatty acids de novo from glucose, which could explain the high responses of plasma glucose and
insulin after feeding as well as the large proportion of dietary carbohydrates oxidized. Alternatively,
the large supply of fatty acids from milk replacer can be directly used for body fat deposition, hence
obviating fatty acid synthesis from glucose. The main objective of the current study was therefore
to assess energy balance, body fat deposition and de novo fatty acid synthesis when exchanging fat
for lactose in milk replacer for veal calves.

Sixteen Holstein-Friesian male calves (121±2 kg) were assigned to a low-fat (LF) diet (n=8) or a
high-fat (HF) diet (n=8). Treatments were iso-nitrogenous, and lactose and fat were iso-energetically
exchanged based on digestible energy. The feeding level was 2.25× the energy requirements for
maintenance, and treatments were maintained for 13 weeks including one week of energy balance
measurements in respiration chambers (n=6 per treatment). Also, recovery of 13C in expired CO2

was measured after oral supplementation with [2-13C] and [U-13C]glucose on separate days, as the
difference in recovery between both isotopomers serves as a proxy for fatty acid synthesis de novo
(Van den Borne et al., 2007). At the end of the study, calves were killed and mRNA expression of genes
involved in fat metabolism was measured in liver, Rectus Femoris and retroperitoneal adipose tissue.

Average daily gain (1.4 kg), nitrogen retention (0.84 g/kg0.75/d) and energy retention (592 kJ/
kg0.75/d; Table 1) did not differ between LF and HF calves. Apparent total tract nitrogen digestibility
was greater in HF calves (86.9%) than in LF calves (81.2%). The respiratory quotient exceeded
unity during 22% of the day in LF calves, but not in HF calves. The difference in isotope recovery
in expired CO2 between the two isotopomers was greater in LF calves (12.9%) than in HF calves
(1.4%), indicating increased de novo fatty acid synthesis in HL calves. mRNA expression of genes
in involved in fatty acid synthesis, desaturation and elongation, and triacylglycerol synthesis was
substantially greater in HL calves than in HF calves in all tissues, and this was most pronounced in
retroperitoneal adipose tissue (data not shown).
In conclusion, a large, iso-energetic exchange between fat and lactose did not affect whole-body
nitrogen and energy retention in milk-fed calves. Calves are able to synthesize fatty acids from
glucose when a high-lactose, low-fat milk replacer is fed, as indicated by: (1) respiratory quotient
exceeded unity; (2) reduced recovery of labelled glucose ([2-13C] vs [U-13C]) in expired CO2; and
(3) increased expression of genes involved in de novo lipogenesis.
Table 1. Effects of an iso-energetic exchange between dietary fat and lactose on energy partitioning
(kJ/kg BW0.75/day) and isotope recovery in expired air (% of dose).
Item Treatment1 SEM P-value
HF LF
Digestible energy intake 1,379 1,375 30 0.949

Metabolisable energy intake 1,309 1,297 31 0.861
Heat production 695 728 21 0.468
Total energy retained 614 570 23 0.379
Energy retained as protein 128 120 8 0.641
Energy retained as fat 486 450 16 0.313
Respiratory quotient 0.88 0.99 0.01 <0.001
13C recovery from [U-13C]glucose 80.4 73.7 1.7 0.039
13C recovery from [2-13C]glucose 79.0 60.8 2.4 0.022
1 HF = high-fat diet; LF = low-fat diet.
References
Hugi, D., R.M. Bruckmaier, and J.W. Blum, 1997. Insulin resistance, hyperglycemia, glucosuria, and galactosuria
in intensively milk-fed calves: dependency on age and effects of high lactose intake. Journal of Animal Science
75: 469-482.
Van den Borne, J.J.G.C., G.E. Lobley, M.W.A. Verstegen, J.-M. Muijlaert, S.J.J. Alferink, and W.J.J. Gerrits, 2007.
Body fat deposition does not originate from carbohydrates in milk-fed calves. Journal of Nutrition 137: 2234-2241.

Effect of plasma factors on in vitro activation and proliferation of
leukocytes from peripartal cows
S. Wang1, S. Meese1, S.E. Ulbrich2, M. Röntgen3, M. Kreuzer1 and A. Schwarm1*
1ETH Zurich, Institute of Agricultural Sciences, Animal Nutrition, Zurich, Switzerland; 2ETH Zurich,
Institute of Agricultural Sciences, Animal Physiology, Zurich, Switzerland; 3FBN, Institute of Muscle
Biology & Growth, Dummerstorf, Germany; angela.schwarm@usys.ethz.ch
Abstract
During early lactation, the immune function of ruminants is often compromised. Lactation promotes
a change in plasma concentrations of immunoregulatory metabolites and hormones (hereafter called
immune modulators). In the present study the causality between important immune modulators
[β-hydroxybutyrate (BHB), cortisol, prolactin, adrenaline (isoproterenol) and insulin] and in vitro
responses of immune cells from peripartal cows were investigated. Peripheral blood mononuclear
cells (PBMC) were derived from four multiparous dairy cows 25±9 days before and 14±4 days after
calving. The activation and proliferation of PBMC did not differ between treatments with either high
or low concentrations of cortisol (45 vs 20 nmol/l), prolactin (300 vs 20 ng/ml) and isoproterenol
(130 vs 70 ng/l). Amounts of 2 instead of 0.5 mmol/l BHB reduced PBMC proliferation by tendency
in early lactation, and reduced PBMC activation in dry and early lactating cows. Amounts of 0.2
instead of 0.7 ng/ml of insulin impaired PBMC proliferation by tendency in early lactation. These
results suggest that cortisol, prolactin and isoproterenol alone are unrelated to immune system
depression in early lactation. The impaired proliferation of PBMC from early-lactating cows
under high concentrations of BHB (immune suppressor) or low concentrations of insulin (immune
promoter) supports a causality between these plasma factors and the response of immune cells in
the in vivo situation.
Keywords: immune cell, cell cycle, early lactation, metabolic load, immune modulator
Introduction
The immune system makes up less than 5% of all body tissues, but is ranked second in priority in
getting access to nutrients once the supply of the central nervous system is secured (Elsasser et al.,
2008). The metabolic priority decreases with physiological status such as pregnancy and lactation,
and is regulated by cytokines and the endocrine-immune gradient (Nelson et al., 2002). Lactation
promotes a change in plasma concentrations of metabolites and hormones. In peripartal dairy cows,
plasma factors with suppressing effects on immune function (e.g. ketones like BHB) increase
whereas, except of prolactin, those like insulin with promoting effects decrease. The present study
aimed to elucidate the influence of some important metabolites and hormones on immune cells from
peripartal dairy cows.

Blood from four dairy cows in their 2nd to 4th lactation was collected 25±9 days before and 14±4
days after parturition from the Vena jugularis into vacutainer tubes with sodium heparin. The
PBMC were prepared by density-gradient centrifugation. The activation and proliferation ability of
PBMC treated with the modulators at two concentrations in the presence or absence of the mitogen
phytohaemagglutinin (PHA, 4 µg/ml) were assessed by measuring oxygen consumption rate (OCR)
after 24 h (as a measure of ATP production; Schwarm et al., 2013) and the MTT (3-(4,5-dimethyl-
2-thiazolyl)-2,5-diphenyl-2H-tetrazolium bromide)-reducing activity after 72 h of incubation,
respectively. The concentrations of each modulator were set to: BHB (0.5 vs 2 mmol/l), cortisol
(45 vs 20 nmol/l), prolactin (300 vs 20 ng/ml), isoproterenol (130 vs 70 ng/l) and insulin (0.2 and

0.7 ng/ml). The activation was calculated as the difference between OCR (nmol/min/107 cells) of
PBMC in the presence and absence of PHA divided by the OCR of PBMC in the absence of PHA.
Student’s paired t-test was carried out to evaluate the dose-dependent effect of plasma factors within
time points. The level of significance was set to P<0.05 and 0.05≤P<0.10 was considered as a trend.

The activation and proliferation of PBMC did not differ between treatments with either high or low
concentrations of cortisol (45 vs 20 nmol/l), prolactin (300 vs 20 ng/ml) and isoproterenol (130 vs 70
ng/l). Amounts of 2 instead of 0.5 mmol/l BHB reduced PBMC proliferation by tendency (P<0.10)
in early lactation, and reduced PBMC activation during dry-off and early lactation (0.79 vs 0.94
and 0.82 vs 0.87 for days -25 and +14, respectively). Amounts of 0.2 instead of 0.7 ng/ml of insulin
impaired PBMC proliferation by tendency (P<0.10) in early lactation. Unexpectedly, the high insulin
level led to even lower (P<0.05) PBMC activation during dry-off compared to the low insulin level
(0.88 vs 1.02), whereas no such difference was observed in early lactation. These results suggest
that cortisol, prolactin and isoproterenol alone are unrelated to immune system depression in early
lactation. The impaired proliferation of PBMC from early-lactating cows under high concentrations
of BHB (immune suppressor) or low concentrations of insulin (immune promoter) supports that
there is a causality between these plasma factors and the response of immune cells in the in vivo
situation. The impaired PBMC proliferation at high BHB concentrations can be attributed to the
reduced PBMC activation. In contrast, the impaired PBMC proliferation at low insulin concentrations
cannot be related to an impaired PBMC activation. Thus, activated cells must have aborted the cell
cycle. The observation that proliferation of PBMC from dry cows was not impaired when treated
with high concentrations of BHB or low concentrations of insulin, suggests that in isolated PBMC
the imprinting from in vivo conditions for utilising nutrients had a greater impact than the in vitro
treatment.
Acknowledgements
This study was supported by the German Research Foundation and the China Scholarship Council.
The help of co-workers at Strickhof and ETH Zurich is greatly acknowledged.
References
Elsasser T.H, T.J. Caperna, C.J. Li, S. Kahl and J.L. Sartin, 2008. Critical control points in the impact of proinflammatory
immune response on growth and metabolism. Journal of Animal Science 86: E105-E125.
Nelson R.J., G.E. Demas, S.L. Klein and L.J. Kriegsfeld, 2002. Energetics and immune function. Seasonal patterns of
stress, immune function, and disease. Cambridge University Press, Cambridge, UK, pp. 151-189.
Schwarm, A., T. Viergutz, B. Kuhla, H.M. Hammon and M. Schweigel-Röntgen, 2013. Fuel feeds function: energy
balance and bovine peripheral blood mononuclear cell activation. Comparative Biochemistry and Physiology,
Part A 164: 101-110.

Energy requirements for maintenance and weight gain during
pregnancy in dairy goats
I.A.M.A. Teixeira*, C.J. Härter, L.D. Lima, H.G.O. Silva, D.S. Castagnino, A.R. Rivera and K.T.
Resende
Department of Animal Science, School of Agrarian Sciences and Veterinary Medicine,
Universidade Estadual Paulista – UNESP, Campus Jaboticabal, São Paulo, 14884-900, Brazil;
izabelle@fcav.unesp.br
Abstract
The aim of this study was to determine maintenance energy requirements during pregnancy in dairy
goats through the use of the comparative slaughter technique. Gestational length had no significant
effect on maintenance energy requirements (NEm). The NEm obtained was 72.9 kcal/kg0.75 of maternal
body weight (MBW), the metabolisable energy for maintenance was 118 kcal/kg0.75 of MBW, and
the km was 0.619. Gestational length had no significant (P>0.05) effect on maternal body weight
gain. The kg obtained was 0.632.
Keywords: energy demand, gestation, doe, maternal body weight gain
Introduction
Important physiological changes occur in the maternal body during pregnancy; such as increase in
blood volume, enhanced intestinal absorption, and adaptations by the respiratory and cardiovascular
systems (Mattison et al., 1990). Thus, it is suggested that the maintenance requirements of pregnant
females are different than those of non-pregnant ones. The current feeding systems used for goats
estimate maintenance energy requirements during pregnancy based on data from adult, non-pregnant
animals (NRC, 2007). Therefore, the aim of this study was to determine maintenance energy
requirements during pregnancy in dairy goats.

The study was arranged in a randomized block design, using a 3×3 factorial scheme: 3 gestational
lengths (80, 110 and 140 days of pregnancy) and 3 feeding regimes (no restriction – 0%, 20% and
40% restriction). As single pregnancies are less common in goats, only does carrying twins were
included in this study. Fifty-four dairy goats pregnant with twins and of similar body weight (BW)
were randomly distributed into 3 groups according to gestational length (80, 110 and 140 days of
pregnancy). Each group was divided into 6 blocks containing 3 animals, and each doe within a block
was subjected to a different feeding regime. Comparative slaughter technique was used to estimate
maintenance energy requirements. The maternal bodies (MB = whole goat body – mammary gland
and pregnant uterus) were ground and homogenized, and the samples freeze-dried. The energy content
of the samples was determined by a bomb calorimeter (Parr Instrument Co., Moline, IL, USA). Heat
production (HP, kcal/kg0.75 of MB weight) was considered as the difference between metabolisable
energy intake (MEI, kcal/kg0.75 of maternal body weight (MBW)) and the energy retained in the
maternal body and pregnancy products (RE, kcal/kg0.75 of MBW). The antilog of the intercept of the
linear regression between the log of HP and MEI was used to estimate the net energy requirement for
maintenance (NEm, kcal/kg0.75 of MBW). The metabolisable energy for maintenance (MEm, kcal/
kg0.75 of MBW) was considered as the value at which HP was equal to MEI. The regression slope
of RE against MEI above maintenance was assumed to be the partial efficiency of ME utilisation
for maternal body energy gain (kg). ME required for maintenance (km) was estimated as NEm/MEm.
The data was analysed as mixed models using SAS (version 9.4; SAS Inst. Inc., Cary, NC, USA),
in which the blocks were considered as a random effect. Significance was considered at P<0.05.

Although physiological changes take place in the maternal body, gestational length had no significant
effect on NEm. The NEm obtained was 72.9 kcal/kg0.75 of MBW, which is greater than the values
reported for younger female Saanen goats (61 kcal/kg BW0.75) weighing 30 to 45 kg (Almeida et
al., 2015), indicating that pregnant adult does require greater maintenance energy than growing
female goats. The MEm obtained was 118 kcal/kg 0.75 of MBW, which is similar to the 120 kcal/kg
BW0.75 reported by NRC (2007). However, it is important to point out that MBW is not the same
as total goat BW during pregnancy, thus it is necessary to be taken into account in diet formulation.
The km observed herein (0.619) was similar to previously reported values for growing Saanen goats
(Almeida et al., 2015); however, it is lower than the km of 0.678 reported by Sahlu et al. (2004) for
non-lactating goats. Gestational length had no significant effect on kg. The kg obtained was 0.632,
which is greater than the 0.39 reported by Sahlu et al. (2004). This suggests that pregnant goats
are more efficient at accumulating body reserves; which will be mainly used at the beginning of
lactation, when the animal is in negative energy balance.
Acknowledgements
The authors would like to thank the State of São Paulo Research Foundation (FAPESP) for the
financial support (Project No. 2013/04758-5, 2006/60480-2).
References
Almeida, A.K., K.T. Resende, N. St-Pierre, S.P. Silva, D.C. Soares, M.H. Fernandes, A.P. Souza, N.C. Silva, A.R. Lima
and I.A. Teixeira, 2015. Energy requirements for growth in male and female Saanen goats. Journal of Animal
Science 93(8): 3932-3940.
Mattison, D.R., E. Blann and A. Malek, 1990. Physiological alterations during pregnancy: impact on toxicokinetics.
Symposium: pharmacokinetics in developmental toxicity. Fundamental Applied Toxicology 16: 215-218.
National Research Council (NRC), 2007. Nutrient requirements of small ruminants sheep, goats, cervids and new
world camelids. National Academy Press, Washington, DC, USA.
Sahlu, T., A.L. Goetsch, J. Luo, I.V. Nsahlai, J.E. Moore, M.L. Galyean, F.N. Owens, C.L. Ferrell and Z.B. Johnson,
2004. Nutrient requirements of goats: developed equations, other considerations and future research to improve
them. Small Ruminant Research 53: 191-219.

Part 2.
Physiological aspects of protein and energy metabolism and
nutrition: monogastrics
Changes in muscle metabolism in Iberian and Landrace × Large-White
pigs fed lysine deficient diets
P. Palma-Granados, A. Haro, I. Seiquer, L. Lara, J.F. Aguilera and R. Nieto*
Department of Physiology and Biochemistry of Animal Nutrition, Estación Experimental del Zaidín
(CSIC), Profesor Albareda, 18008, Granada, Spain; rosa.nieto@eez.csic.es
Abstract
Previous studies showed differential effects of Lys deficiency on performance of Iberian and Landrace
gilts reared in similar conditions. The objective of the present work was to investigate Lys deficiency
effects on muscle characteristics of an obese (Iberian) and a conventional pig genotype (Landrace ×
Large-White, LLW). We used 32 barrows, 16 from each breed, randomly assigned to 2 experimental
diets in a factorial arrangement (2 breeds × 2 diets; 8 pigs per treatment combination). Diets were
isoenergetic and with identical composition (180 g CP /kg DM, 14 MJ metabolisable energy) except
for Lys content: 0.98% Lys, Lys adequate diet (LA) and 0.47% Lys, Lys deficient diet (LD). Initial
and final body weight was 10 and 25 kg, respectively. Performance was reduced in both pig types
when fed LD diets, particularly in LLW pigs (P<0.05). When fed LD diets, intramuscular fat (IMF)
content increased in L. dorsi of Iberian and in B. femoris of both pig genotypes P<0.05). Oleic acid
increased (P<0.05) and polyunsaturated fatty acid decreased (P<0.01) in L. dorsi and B. femoris
of pigs of both genotypes fed LD diets. Proportion of type I muscle fibres increased in L. dorsi of
both pigs types fed LD diets (P<0.001). Feeding Lys deficient diets could be a suitable strategy for
increasing IMF content in obese and lean pigs. Nevertheless, further research is needed to deep our
knowledge on the mechanisms involved in these changes.
Keywords: lysine deficiency, pig genotype, intramuscular fat, fatty acids, muscle fibres
Introduction
Previous studies showed that the effects on growth and body protein retention of Lys deficient diets
were less pronounced in Iberian than in Landrace gilts reared in similar conditions (Rivera-Ferre et
al., 2006). Additionally, a reduced Lys intake has been shown to influence intramuscular fat (IMF)
in pigs and to affect other parameters of muscle metabolism (Katsumata et al., 2005) although
mechanisms involved are not fully understood. The objective of the present work was to investigate
the effect of dietary Lys deficiency on muscle characteristics in an obese (Iberian) and a conventional
pig genotype (Landrace × Large-White, LLW) under identical experimental conditions.

Thirty two barrows, 16 from each breed, were randomly assigned to each of two experimental diets
in a 2 × 2 factorial arrangement (2 breeds × 2 diets; 8 pigs per treatment combination). Diets-based
on barley, maize, gluten meal and soybean meal were isonitrogenous and isoenergetic (180 g CP /kg
DM, 14 MJ metabolisable energy) and of identical AA composition except for the Lys content: 0.98%
Lys, Lys adequate (LA); and 0.47% Lys, Lys deficient (LD). Pigs were housed in individual 2 m2 pens.
Initial body weight (BW) was 10.3±0.2 kg. Pigs were fed at 85% of ad libitum intake of the Iberian
genotype, of greater intake capacity. Daily feed allowance was adjusted weekly on BW basis. At 25 kg
BW pigs were slaughtered and L. dorsi and B. femoris muscles rapidly dissected and stored at -20 °C.
IMF was determined by Folch extraction and fatty acid (FA) profile analysed by gas chromatography.
Assessment of oxidative fibre proportions in L. dorsi muscle was determined by NADH dehydrogenase
activity. Results were analysed by two-way ANOVA following GLM procedure of SAS, according
to the factorial design. When interaction between main factors was significant, statistical differences
among treatment combinations were assessed by Least Square Mean t-test (P<0.05).

Growth rate was reduced in both pig types when fed LD diets, particularly in LLW pigs (21.1 vs
15.4, and 23.4 vs 12.6 g/kg BW per day, for Iberian and LLW pigs fed LA and LD diets, respectively;
interaction genotype × diet, P<0.05). When fed LD diets, IMF content increased in L. dorsi of
Iberian and in B. femoris of both pig genotypes (P<0.01; Table 1). Oleic acid increased (P<0.05)
and polyunsaturated FA decreased (P<0.01) in L. dorsi and B. femoris of pigs of both genotypes
fed LD diets. Proportion of oxidative fibres in L. dorsi muscle increased both in Iberian and LLW
pigs fed LD diets (by 23%, on average; P<0.001).
Feeding Lys deficient diets could be a suitable strategy for increasing IMF content in obese and
lean pigs, although further research is needed to deep our knowledge on the mechanisms involved
in these changes and on their effects upon particular muscles.
Table 1. Intramuscular fat concentration (g/100 g muscle) and FA profile (g/100 g FA) of L. dorsi
and B. femoris muscles of Iberian and Landrace × Large-White pigs (LLW) fed adequate (LA) or
lysine deficient (LD) diets.1
Iberian LLW SEM P-value2
LA LD LA LD Genotype Diet G×D
L. dorsi
IMF 3.58a 6.46b 2.47a 3.26a 0.40 <0.001 <0.001 <0.05
C18:1 n-9 39.4 42.2 37.3 39.0 0.80 <0.01 <0.01 NS
C18:2 n-6 8.03 5.29 10.2 8.35 0.40 <0.001 <0.001 NS
SFA 39.2 39.8 37.6 40.4 0.92 NS NS NS
MUFA 49.5 52.8 47.6 47.7 1.17 <0.01 NS NS
PUFA 11.3 7.30 14.8 11.8 0.65 <0.001 <0.001 NS
B. femoris
IMF 3.36 3.91 2.35 2.97 0.20 <0.001 <0.01 NS
C18:1 n-9 39.0 41.2 35.5 37.7 0.94 <0.01 0.05 NS
C18:2 n-6 9.85 7.57 13.4 11.1 0.57 <0.001 <0.001 NS
SFA 37.1 37.4 36.4 37.2 0.76 NS NS NS
MUFA 48.9 51.4 45.1 46.5 1.30 <0.01 NS NS
PUFA 14.0 11.2 18.5 16.3 0.91 <0.001 <0.01 NS
1 IMF = intramuscular fat; SFA = saturated fatty acids; MUFA = monounsaturated fatty acids; PUFA = polyunsaturated
fatty acids.
2 NS = P>0.05; means bearing different superscript letter differ.
Acknowledgements
This work was funded by Spanish MINECO Grant AGL2011-25360.
References
Katsumata, M., S. Kobayashi, M. Matsumoto, E. Tsuneishi and Y. Kaji, 2005. Reduced intake of dietary lysine
promotes accumulation of intramuscular fat in the Longissumus dorsi muscle of finishing gilts. Animal Science
Journal 76: 237-244.
Rivera-Ferre, M.G., J.F. Aguilera and R. Nieto, 2006. Differences in whole-body protein turnover between Iberian and
Landrace pigs fed adequate or lysine deficient diets. Journal of Animal Science 84: 3346-3355.

Effect of dietary live yeast supplementation on thermal heat
acclimatization in finishing male pigs
E. Labussière1,2*, S. Dubois1,2, M. Castex3 and D. Renaudeau1,2
1INRA, UMR 1348 PEGASE, Domaine de la Prise, 35590 Saint-Gilles, France; 2Agrocampus Ouest,
Domaine de la Prise, UMR 1348 PEGASE, 35000 Rennes, France; 3Lallemand SAS, 19 rue des
Briquetiers, 31702 Blagnac, France; etienne.labussiere@rennes.inra.fr
Abstract
Pigs subjected to heat stress decrease their feed intake that causes important economic losses. The
objectives of the experiment were to determine the effects of dietary live yeast supplementation
(without or with Saccharomyces cerevisiae boulardii CNCM I-1079) on energy metabolism of
finishing boars (n=10) at thermoneutrality or during heat stress challenge in respiration chamber.
Nitrogen and energy balances were measured individually during three periods of 7, 7 and 6 days
with ambient temperature of 22, 28 and 28 °C, respectively. Dietary live yeast supplementation
increased dry matter intake from 2.29 to 2.65 kg/d, whereas heat stress decreased feed intake. These
variations were associated with an increased number of meals when diet was supplemented (6.8 vs
5.5 meals/day) and a significantly increased eating speed of the control diet between periods 1, 2 and
3. Metabolisable energy intake was higher when diet was supplemented whereas total heat production
was not affected. Finally, energy retention was higher with dietary live yeast supplementation but
tended to decrease when ambient temperature increased.
Keywords: heat production, feeding behaviour, energy balance
Introduction
Pigs subjected to heat stress decrease their feed intake that causes important economic losses in the
swine industry because of the related effects on growth performance (Renaudeau et al., 2011). In
dairy cows, there are indications that live yeast may help animals to cope with heat stress because
of improved homeothermia regulation (Salvati et al., 2015). The objectives of the experiment were
to determine the effects of dietary live yeast supplementation on energy metabolism of finishing
boars, submitted to heat stress challenge.

Five replicates with two Pietrain × (Landrace × Large White) littermates each were used. Half of
the animals was fed a standard fattening diet and the others received the same diet supplemented
with 0.005% Saccharomyces cerevisiae boulardii (CNCM I-1079, 1×106 cfu/g of feed) during two
weeks. Pigs were then housed in a metabolism cage placed in an open-circuit respiration chamber
during three consecutive periods of 7, 7 and 6 days, respectively when they received the same diets.
During the first period, the ambient temperature (AT) was kept constant at 22 °C (thermoneutrality).
Then the AT increased during two days up to 28 °C and was then maintained constant. Animals were
weighed at the beginning and the end of each period. Feed was proposed ad libitum and feeding
behaviour was continuously recorded. Nitrogen and energy balances were measured individually
per period. Gas exchanges were continuously measured in order to calculate heat production (HP)
and analyse its kinetic. The variance of the data was partitioned according diet, period, interaction
and replicate, considering repeated measurements.

At thermoneutrality, initial body weight of boars averaged 101 kg but boars that received supplemented
diet exhibited higher growth rate during subsequent periods (Table 1). Total dry matter intake (DMI)
increased from 2.29 to 2.65 kg/d when diet was supplemented, but decreased between period 1 and
periods 2 and 3 because of heat stress. Nevertheless, the decrease in DMI between periods 1 and 2
was lower when diet was supplemented compared to the control diet and to values from the literature
(-6 vs -15%; Le Bellego et al., 2002). The number of meals increased when diet was supplemented
(6.8 vs 5.5 meals/day; P=0.02) whereas pigs fed the control diet exhibited a significantly increased
feeding rate between periods 1, 2 and 3. Metabolisable energy intake was higher when diet was
supplemented but decreased when AT increased. Total HP was not affected by dietary treatments
whereas it decreased when AT increased. Finally, energy retention was higher with dietary live yeast
supplementation but tended to decrease when AT increased.
To conclude, dietary supplementation of Saccharomyces cerevisiae boulardii CNCM I-1079 in

finishing male pigs increased feed intake at thermoneutrality and tended to reduce the effect of high
ambient temperature through better acclimatization.
Table 1. Effect of dietary live yeast addition on energy metabolism in entire male pigs submitted to
heat stress.1
Period (ambient temperature) rsd P-value
1 (22 °C) 2 (28 °C) 3 (28 °C) Diet Period D×P
BW (kg) 1.0 0.41 <0.01 0.01

Non supplemented diet 100.6a 108.0bc 115.1de
Supplemented diet 102.1ab 110.9cd 119.5e
DMI (kg/d) 0.21 0.01 <0.01 0.27
Non supplemented diet 2.55 2.16 2.15
Supplemented diet 2.76 2.51 2.68
ME intake (MJ/kg BW0.60/d) 0.17 <0.01 <0.01 0.35
HP (MJ/kg BW0.60/d) 0.06 0.11 <0.01 0.11
RE (MJ/kg BW0.60/d) 0.14 <0.01 0.06 0.59
1rsd = residual standard deviation; D×P = interaction diet × period; BW = body weight; DMI = dry matter intake; ME
= metabolisable energy; HP = heat production; RE = retained energy.
References
Le Bellego, L., J. van Milgen and J. Noblet, 2002. Effect of high temperature and low-protein diets on the performance
of growing-finishing pigs. Journal of Animal Science 80: 691-701.
Renaudeau D., J.L. Gourdine and N.R. St-Pierre, 2011. A meta-analysis of the effects of high ambient temperature on
growth performance of growing-finishing pigs. Journal of Animal Science 89: 2220-2230.
Salvati G.G.S., N.N. Morais Junior, A.C.S. Melo, R.R. Vilela, F.F. Cardoso, M. Aronovich, R.A.N. Pereira and M.N.
Pereira, 2015. Response of lactating cows to live yeast supplementation during summer. Journal of Dairy Science
98: 4062-4073.

Citrulline and arginine synthesis in perinatal and young pigs
J.C. Marini1,2*, U. Agarwal2, J. Robinson2, Y. Yuan2, I.C. Didelija2, B. Stoll2 and D.G. Burrin2
1Section of Critical Care Medicine, Baylor College of Medicine, Houston, TX 77030, USA; 2USDA/
ARS Children’s Nutrition Research Center, Baylor College of Medicine, Houston, TX 77030, USA;
marini@bcm.edu
Abstract
The endogenous synthesis of arginine (ARG) is a multiorgan process in which citrulline (CIT)
synthesized in the gut is used by the kidney to produce ARG. Because the enzymes needed to convert
CIT into ARG (ASS and ASL) are present in the neonatal gut, this intestinal-renal axis is thought
not to be functional early in life. High plasma CIT concentrations, however, have been reported
in many species during the neonatal period. Using stable isotopes we determined that there is a
substantial production of CIT in premature and neonatal pigs. In addition, we also determined that
CIT is released by the gut and utilised by the kidneys to produce ARG without apparent difference
between neonatal and young pigs. We confirmed the presence of ASS and ASL in the small intestine
of neonates, but these two enzymes were absent in the gut of young pigs. The lack of co-localization
of ASS and ASL with the enzymes used for CIT synthesis prevents the neonatal gut from producing
ARG. Taken together these data demonstrates a functional intestinal-renal axis for the ‘de novo’
synthesis of ARG in the neonate.
Keywords: interorgan, metabolism, neonate, premature, tracers
Introduction
The endogenous synthesis of arginine (ARG) relies on a multiorgan process in which citrulline
(CIT) is synthesized by the gut and utilised by two renal enzymes (argininosuccinate synthase and
lyase, ASS and ASL) to produce ARG. Because ASS and ASL are present in the gut of neonates,
it is believed that during this period piglets are able to synthesize ARG in this organ, and thus that
this intestinal-renal axis is not functional. However, this is not consistent with the high plasma CIT
concentrations seen in neonates. To address this apparent paradox we measured CIT production in
premature, neonatal and young pigs. In addition, we determined the site of synthesis and utilisation
of CIT in neonatal and young pigs.

In experiment 1, premature (10 d preterm and 1.0±0.1 kg BW; n=14), neonatal (8 d and 2.5±0.2
kg BW; n=6) and young pigs (34 d and 8.5±0.3 kg BW; n=5) were studied. Pigs were fitted with
chronical catheters for infusion and sampling. An (ureido)[15N]CIT tracer was infused to determine
the production of this amino acid. The animals were fasted, with the exception of the premature pigs
which received total parenteral nutrition. The infusion lasted 4 h and was performed in conscious
animals.
In experiment 2, neonatal (8 d and 2.5±0.2 kg BW; n=4) and young pigs (30 d and 7.5±0.3 kg BW;
n=5) were anesthetized and catheters placed in the carotid artery and the jugular, portal, and renal
veins. (ureido)[15N]CIT was infused in the jugular vein to determine the site of production and
utilisation of CIT. The infusion lasted 4 h and was performed in unconscious fasted animals. Gut
samples were collected to determine by immunohistochemistry (IHC) the localization of the enzymes
for CIT synthesis (CPS 1 and OTC) and its conversion to ARG (ASS and ASL).

Neonatal pigs produced more CIT than preterm and young animals (74±4, 44±2 and 45±3 µmol/kg/h,
respectively; P<0.001). This was also true for the neonatal and young pigs in experiment 2 (66±7
and 41±3 µmol/kg/h, respectively; P<0.001). When compared to arterial enrichments, we observed
a dilution of the CIT tracer in the portal vein of neonates (P<0.003) and young pigs (P<0.001;
Figure 1A). However, no dilution of the tracers was evident in renal plasma (P>0.20). This indicates
an endogenous production and release of CIT by the portal drained viscera, but not by the kidney.
An increase (P<0.01) in renal plasma [15N]ARG (compared to arterial) was measured in both age
groups, indicating the utilisation of plasma CIT for the synthesis of ARG by the kidney. However,
no difference (P>0.20) between arterial and portal plasma ARG was observed suggesting that CIT
is not utilised by the gut (Figure 1B).
Gut IHC analysis indicated the presence of ASS and ASL in neonates, but not in young pigs. These
enzymes were localized toward the tip of the villus whereas the enzymes involved in the synthesis
of CIT (CPS1 and OTC) were localized towards the crypts. This lack of co-localization has also
been observed in rats (De Jonge et al., 1998) and humans (Kohler et al., 2008).
Taken together these data demonstrate that neonates (and also premature) pigs produce and export
CIT to the peripheral circulation suggesting that the intestinal-renal axis for the ‘de novo’ synthesis
of ARG is functional early in life. Despite the presence of ASS and ASL in the gut of neonates, the
lack of co-localization with CPS1 and OTC prevents the synthesis of ARG and explains the high
levels of circulating CIT observed in neonates of different species.
A B
Figure 1. [15N]citrulline and [15N]arginine enrichment in arterial (A), portal (P) and renal (R) blood
of 8 and 30 d old pigs. Bars are means ± SEM. Asterisks denote significant differences (P<0.01).
Acknowledgements
This work was supported by federal funds from the United States Department of Agriculture,
Agricultural Research Service under Cooperative Agreement No. 58-6250-6-001.
References
De Jonge, W.J., M.A. Dingemanse, P.A.J. de Boer, W.H. Lamers and A.F.M. Moorman, 1998. Arginine-metabolizing
enzymes in the developing rat small intestine. Pediatr Res 43: 442-451.
Kohler, E.S., S. Sankaranarayanan, C.J. van Ginneken, P. van Dijk, J.L.M. Vermeulen, J.M. Ruijter, W.H. Lamers
and E. Bruder, 2008. The human neonatal small intestine has the potential for arginine synthesis; developmental
changes in the expression of arginine-synthesizing and -catabolizing enzymes. BMC Developmental Biology 8: 107.

Dietary citrulline supplementation is an efficient strategy to increase
arginine availability
J.C. Marini1,2*, U. Agarwal2 and I.C. Didelija2
1Section of Critical Care Medicine, Baylor College of Medicine, Houston, TX 77030, USA; 2USDA/
ARS Children’s Nutrition Research Center, Baylor College of Medicine, Houston, TX 77030, USA;
marini@bcm.edu
Abstract
Arginine (ARG) is a conditionally essential amino acid that during high metabolic demand can
become essential in many different species, including humans. Thus supplementation with ARG
and its endogenous precursor (citrulline; CIT) has been proposed to increase ARG availability. To
determine which amino acid is more efficient at increasing ARG availability, mice were fed diets
supplemented with ARG or CIT. After a 2-week adaptation period, the kinetics of ARG and CIT
were determined while undergoing intragastric infusion of nutrients to mimic continuous feeding.
ARG supplementation failed to increase its flux due to an increase in its first pass metabolism. The
marginal contribution of dietary ARG to its flux was 31%. In contrast all (105±4%) the supplemented
CIT was accounted for as an increase in the CIT flux. Furthermore, approximately 59% of this CIT
was utilised for the ‘de novo’ synthesis of ARG resulting in a linear increase in the flux of ARG
(P<0.001). In conclusion, CIT supplementation is a better alternative to increase ARG availability,
than ARG supplementation itself.
Keywords: metabolism, first pass, requirements, tracer
Introduction
Arginine (ARG) is a conditionally essential amino acid that during high metabolic demand (rapid
growth, pregnancy) can become essential in many species, including humans. Because the synthesis
of its immediate precursor (citrulline; CIT) is more or less constant, the endogenous synthesis of
ARG cannot be upregulated to meet its demand. Therefore ARG supplementation seems to be the
obvious choice to improve ARG availability, restore function and maximize growth. CIT, however,
may be a better option since its first pass metabolism is negligible. To test the hypothesis that dietary
CIT supplementation is a better alternative than ARG to increase ARG availability, mice were fed
diets containing different amounts of these two amino acids.

Six-week old male mice (C57BL6/J; n=8) were fitted with gastric catheters and, after recovery
from surgery, fed one of the seven experimental diets for two weeks. The basal diet contained 2.5 g
ARG/kg diet, which we previously showed was sufficient to meet the ARG requirement for growth
(Marini et al., 2015). In addition, three supplementation levels of ARG or CIT (2.5, 7.5 and 12.5
g each/kg diet) were fed. After the 2-week feeding period, mice were infused with stable isotopes
to determine ARG and CIT fluxes and their interconversions. Mice were infused i.g. with liquid
diets with identical amino acid composition to the experimental diets (basal diet 75 µmol ARG/
kg/h; ARG and CIT supplemented diets: 75, 225 and 375 µmol additional ARG or CIT/kg/h). An
additional ARG tracer infused i.g. allowed for the determination of first pass metabolism (FPM)
and escape (FPE) of dietary ARG.

Dietary ARG supplementation failed to increase the flux of ARG (P=0.4) and CIT (P=0.93; Figure
1A and 1B, respectively). Dietary CIT supplementation, on the other hand, increased the flux of

both ARG and CIT (P<0.001; Figure 1A and 1B). The FPM of dietary ARG by the gut and liver
increased from 32±3% for the basal diet to more than 60% for the higher ARG supplementation
levels. The marginal ARG FPE was 31±3% (R2=0.84; Figure 2A). The recovery of the dietary
CIT supplement in the circulation (CIT flux) was 105±4% (R2=0.96; Figure 2B). Approximately
59±4% of the supplemented CIT was utilised for the synthesis of plasma ARG, thus the de novo
ARG synthesis pathway contributed from 9±1% to almost 30±1% of the ARG flux. These kinetic
changes translated in a greater (P<0.01) plasma arginine concentration response to citrulline than
to arginine supplementation.
In conclusion, CIT supplementation is a more efficient way to increase ARG availability in mice.
Whereas the marginal efficiency of ARG supplementation was ~30%, the efficiency of dietary CIT
to produce circulating ARG was ~60%. Furthermore, the fraction of CIT that could not be accounted
for as plasma ARG was likely utilised for the local synthesis of ARG at tissue level. The widespread
localization of the enzymes that catalyze this reaction and the fact that the only known fate for CIT
is its conversion into ARG, ensures that all supplemental CIT is converted into ARG.
A B
1000 600
800 500
Arginine Flux
Citruline Flux
(µmol/kg/h)
(µmol/kg/h) 400
600
300
400
200
200 100
0 0
Arginine 75 150 300 450 75 75 75 75 Arginine 75 150 300 450 75 75 75 75
Citruline 0 0 0 0 0 75 225 350 Citruline 0 0 0 0 0 75 225 350
Supplementation (µmol/kg/h) Supplementation (µmol/kg/h)
Figure 1. (A) Arginine and (B) citrulline fluxes in mice supplemented intragastrically with arginine
and citrulline. Values are means ± SEM.
A B
200 600
160
Flux (µmol/kg/h)
FPE (µmol/kg/h)
400
120
80
200
40
0 0
0 100 200 300 400 500 0 100 200 300 400 500
Supplementation (µmol/kg/h) Supplementation (µmol/kg/h)
Figure 2. Dietary arginine first pass escape (FPE) as function of arginine supplementation (panel A)
and citrulline flux as function of citrulline supplementation. Values are means ± SEM. The regression
equations are shown in the graphs.
Acknowledgements
This work was supported by NIH grant R01GM108940.
References
Marini, J.C., U. Agarwal and I.C. Didelija, 2015. Dietary arginine requirements for growth are dependent on the rate
of citrulline production in mice. J. Nutr. 145: 1227-1231.

Lowering the dietary protein content in piglets: how far can we go?
A.J.M. Jansman1*, H. van Diepen1, M. Rovers2 and E. Corrent3
1Wageningen UR Livestock Research, P.O. Box 338, 6700 AH Wageningen, the Netherlands; 2Orffa
Additives B.V., Vierlinghstraat 51, 4251 LC Werkendam, the Netherlands; 3Ajinomoto Eurolysine
s.a.s. 153 Rue de Courcelles, 75817 Paris Cedex 17, France; alfons.jansman@wur.nl
Abstract
We investigated the effects of stepwise lowering the dietary crude protein (CP) content from 172
to 130 g/kg, while balancing the levels of all essential amino acids via their supplementation in
free form, on the growth performance of piglets in the post-weaning period (8-25 kg BW). It was
concluded that the dietary CP level for piglets in the post weaning phase can be reduced till about
160 g/kg without negative effects on the growth performance when diets are adequately balanced
for the first five limiting amino acids (Lys, Met + Cys, Thr, Trp and Val). A further reduction of
the dietary CP content, while maintaining the dietary amino acid profile for essential amino acids,
reduced growth performance of piglets.
Keywords: essential amino acids, requirements, piglets
Introduction
Nitrogen efficiency of pigs can be improved by reducing the dietary crude protein (CP) content, while
maintaining the concentrations of essential amino acids by the use of free amino acids using the ideal
amino acid profile (Chung and Baker, 1992). It is not known, however, whether the afore mentioned
concept also applies for post weaning piglets provided with very low CP diets supplemented with
up to ten amino acids in free form. The aim of the present study was therefore to determine the
effects of stepwise lowering the dietary CP content to 130 g/kg, while balancing the levels of all
essential amino acids via their supplementation in free form, on the growth performance of piglets
in the post-weaning period.

Five experimental diets were formulated with a decreasing CP level of 172, 159, 148, 138 to 129
g/kg for treatments V, IV, III, II and I, respectively (Table 1). In addition, a diet was evaluated with
130 g CP per kg, devoid of soybean meal, and also supplemented with free non-essential amino
acids up to the levels of the diet containing 159 g CP per kg (treatment IV). The level of SID lysine
was 1.00 g per kg in all experimental diets.
Table 1. Experimental treatments.
Group Crude protein (g/kg) Free amino acid supplementation
I 129 Lys, Met + Cys, Thr, Trp, Val, Ile, His, Phe, Leu, Tyr
II 138 Lys, Met + Cys, Thr, Trp, Val, Ile, His, Phe, Leu, Tyr
III 148 Lys, Met + Cys, Thr, Trp, Val, Ile, His, Phe
IV 159 Lys, Met + Cys, Thr, Trp, Val
V 172 Lys, Met + Cys, Thr, Trp
VI 130 Lys, Met + Cys, Thr, Trp, Val, Ile, His, Phe, Leu, Tyr and Asp,
Glu, Gly, Pro, Ser, Pro till level in IV

The levels of essential amino acids in all diets were in proportion or excess relative to the concentration
of SID lysine according to the current recommendations as far as available. Ratio to Lys on a SID
basis was 60% for Met + Cys, 65% for Thr, 22% for Trp, 70% for Val, 53% for Ile, 100% for Leu,
32% for His, 95% for Phe + Tyr and 42% for Arg. Each treatment group consisted of 64 piglets
divided over eight pens with eight piglets each. Feed intake (FI), body weight gain (BWG) and feed
conversion ratio (FCR) were measured as the response criteria over an experimental period of four
weeks, starting one week post-weaning (8-25 kg BW). The results were statistically analysed using
ANOVA and treatment means were compared using the least significant difference test.

Over the complete experimental period, FI, BWG and FCR were significantly influenced by the
dietary treatments (P<0.05) (Table 2). The FI was numerically lowest in treatment I and VI and
highest in treatment II (P<0.05). The FI in treatment V did not differ significantly from the intake of
the other treatments (P>0.05). The BWG was highest in treatments IV and V and lowest in treatments
I and VI (P<0.05). Compared to treatment V, BWG was significantly lower in treatments I and VI
(P<0.05). The reduced performance of the piglets in treatments I, II, III and VI compared to V might
be related to a suboptimal provision of Phe + Tyr and/or Arg relative to the actual requirements of
the piglets for these amino acids and/or due to a marginal supply of non-essential amino acids in the
low protein diets, which was as low as 49 g/kg in the diet for treatment I. In contrast, Gloaguen et al.
(2014) decreased dietary CP content from 176 to 135 g/kg with no effect on the growth performance
of post weaning piglets.
Table 2. Feed intake, body weight gain and feed conversion ratio over weeks 0-4 of the experimental
period.1
FI (kg/d) %2 BWG (g/d) %2 FCR %2
I 0.793a 97 480a 88 1.652d 111

II 0.850b 104 526b 96 1.617c 108
III 0.813ab 100 526b 96 1.546b 104
IV 0.835b 102 564c 103 1.481a 99
V 0.816ab 100 548bc 100 1.491a 100
VI 0.786a 96 488a 89 1.612c 108
P-value 0.01 <0.001 <0.001
LSD 0.038 27 0.028
1 FI = feed intake; BWG = body weight gain; FCR = feed conversion ratio.
2 Values relative to the values of treatment V (=100%).
References
Chung, T.K. and D.H. Baker, 1992. Ideal amino acid pattern for 10-kilogram pigs. Journal of Animal Science 70:
3102-3111.
Gloaguen, M., N. Le Floc’h, E. Corrent, Y. Primot and J. van Milgen, 2014. The use of free amino acids allows
formulating very low crude protein diets for piglets. Journal of Animal Science 92: 637-644.

Does a reduction in dietary protein affect immune status of pigs kept
under different sanitary conditions?
Y. van der Meer1,2,4*, W.J.J. Gerrits1, A.J.M. Jansman3, B. Kemp4 and A. Lammers4
1Animal Nutrition Group, Wageningen University, Wageningen, the Netherlands; 2De Heus
Animal Nutrition, Ede, the Netherlands; 3Wageningen UR Livestock Research, Wageningen, the
Netherlands; 4Adaptation Physiology Group, Wageningen University, Wageningen, the Netherlands;
yvonne.vandermeer@wur.nl
Abstract
Low sanitary conditions can lead to immune stimulation resulting in associated nutrient costs. This
study was conducted to gain knowledge about the interactions between dietary protein level, amino
acid profile, and immune system activation in pigs kept under different sanitary conditions. Ten week
old boars were kept under low or high sanitary conditions and received one of four diets in a 2×2
setting: variable in protein (low or normal level), and amino acid profile (basal or supplemented).
At 13, 18 and 24 weeks of age blood samples were collected for analyses of immune parameters.
Haptoglobin and IgG against keyhole limpet hemocyanin were higher for low sanitary conditions.
Low protein diets reduced haptoglobin concentration and increased white blood cell counts especially
for low sanitary pigs. Increase of dietary concentration of methionine, threonine and tryptophan did
not compensate for this effect.
Keywords: pig, sanitary conditions, protein, immune system
Introduction
There is a strong incentive to increase efficiency of the conversion of proteins into edible products.
In pig nutrition, reduction of dietary crude protein (CP) content can contribute to increase this
efficiency. It is generally perceived that dietary deficiencies due to this reduction can be compensated
for by supplementing essential amino acids (AA) to the diet (Gloaguen et al., 2014; Kerr and Easter,
1995), thus maintaining animal performance at lower CP intake. However, the optimal dietary AA-
profile is influenced by animal and environmental conditions. Low sanitary conditions often lead
to stimulation of the immune system, resulting in associated nutrient costs, including AA costs. As
increased AA costs and the thrive to reduce dietary CP level are contradictory, we need to increase
our knowledge on the interactions between AA nutrition and immune system activation in pigs kept
in different sanitary conditions.

In total 612 pigs divided over 68 pens (nine boars/pen) were subjected to one of 8 treatments in a
2×2×2 factorial arrangement. Pigs were kept in two sanitary conditions: low (LSC) vs high (HSC)
and offered one of four diets ad libitum: with a low CP (LP) or normal CP (NP) level (80 vs 100%
NRC recommendations), and with a basal (AA-B) or supplemented (AA-S) AA profile. The AA-B
profile was designed to meet AA requirements for body protein deposition and basal maintenance
purposes. In AA-S diets concentrations of Met, Thr and Trp were increased by 20% compared with
the concentrations in AA-B diets. At 13, 18 and 24 weeks of age, blood samples of two pigs per pen
were taken for analysis of haptoglobin concentration, natural antibodies IgG and IgM against keyhole
limpet hemocyanin (KLH), and white blood cell (WBC) numbers. Pigs were kept for a complete
growing-fattening period and were slaughtered at a weight of on average ± 110 kg.

Haptoglobin levels (LSC: 0.92 g/L, HSC: 0.78 g/L) and IgG levels against KLH (LSC: 3.53 HSC:
3.08) were higher for LSC compared with HSC pigs (P<0.0001, P=0.04, respectively; Table 1),
indicating that degree of immune activation differed among sanitary conditions (SC). LSC pigs fed
NP, but not LP diets, had higher haptoglobin values compared with LSC pigs fed LP diets (interaction
CP×SC; P=0.010). The WBC number was higher in AA-S fed pigs compared with AA-B fed pigs
when kept at LSC, but not at HSC (interaction SS×AA, P=0.04). Pigs fed NP diets had a lower WBC
number compared with pigs fed LP diets (P=0.02). Number of platelets in pigs fed AA-S diets was
higher compared with pigs fed AA-B diets (P=0.002).
In conclusion, LP diets reduced levels of immune molecules like haptoglobin and increased production
of WBC, especially in pigs kept in LSC. Additional dietary supplementation of Met, Thr and Trp was
not clearly compensating for these effects. Therefore, these results could indicate that reduced CP
levels in pig diets may influence responses of the immune system and resistance against infections.
Table 1. Blood parameters of pigs kept under low and high sanitary conditions, fed low or normal
protein diets in combination with basal or supplemented amino acid (AA) profile.
Item4 LSC1 HSC1 SEM P-values
LP2 NP2 LP NP
SC×AA
SC×CP
AA5
SC5
CP5
B-AA3 S-AA3 B-AA S-AA B-AA S-AA B-AA S-AA
Haptoglobin 0.98 0.82 1.14 1.14 0.8 0.74 0.63 0.76 0.16 0.004 0.25 0.91 0.01 0.42
IgG-KLH 3.46 3.71 3.52 3.41 3.11 3.01 2.99 3.2 0.28 0.04 0.75 0.62 0.45 0.87
IgM-KLH 7.31 7.01 7.32 7.13 7.08 7.15 7.27 7.08 0.2 0.68 0.47 0.07 0.91 0.32
WBC 22.1 24.2 20.9 22.3 22.5 23.1 22.6 21.4 1.31 0.89 0.02 0.15 0.6 0.04
Platelets 542 624 521 623 471 588 544 726 94.8 0.92 0.23 0.002 0.14 0.46
1 LSC = low sanitary conditions; HSC = high sanitary conditions.

2 LP = low protein diet, 80% of NRC (2012) advise; NP = normal protein diet (NRC, 2012).
3 B-AA = basal AA-profile; S-AA = supplemented AA-profile with 20% extra Met, Thr and Trpt.
4 Hapto = haptoglobin concentration in g/l serum; IgG-KLH and IgM-KLH = IgG and IgM titer against KLH; WBC =
white blood cell count in 109/l blood.

5 SC = sanitary conditions; CP = crude protein level; AA = amino acid profile.
Acknowledgements
Financial support of the Dutch Feed4Foodure consortium is gratefully acknowledged.
References
Gloaguen, M., N. Le Floc’h, E. Corrent, Y. Primot and J. van Milgen, 2014. The use of free amino acids allows
formulating very low crude protein diets for piglets. Journal of Animal Science 92: 637-644.
Kerr, B.J. and R.A. Easter, 1995. Effect of feeding reduced protein, amino acid-supplemented diets on nitrogen and
energy balance in grower pigs. Journal of Animal Science 73: 3000-3008.
National Research Council (NRC), 2012. Nutrient requirements of swine. National Academy Press, Washington, DC,
USA.

Dietary ammonia appearance in portal blood of pigs fed diets deficient
in non-essential amino acid nitrogen is incomplete
W.D. Mansilla1, A.K. Agyekum2, C.L. Zhu1, J.K. Htoo3, C.M. Nyachoti2 and C.F.M. de Lange1*
1Department of Animal Biosciences, University Guelph, Guelph, ON, N1G 2W1, Canada; 2Department
of Animal Science, University of Manitoba, Winnipeg, MB, R3T 2N2, Canada; 3Evonik Nutrition &
Care GmbH, 63457, Hanau-Wolfgang, Germany; cdelange@uoguelph.ca
Abstract
Dietary ammonia has been shown to be utilised efficiently as a source of nitrogen (N) for endogenous
synthesis of non-essential amino acids (NEAA) when pigs are fed diets deficient in NEAA-N. An
experiment was conducted to understand ammonia-N absorption and liver ammonia-N metabolism
in pigs fed diets deficient in NEAA-N. Four crossbred gilts were surgically fitted with 4 catheters (i.e.
portal, hepatic and mesenteric veins, and carotid artery). A basal diet was formulated to be deficient
in NEAA-N and supplemented with cornstarch (Control) or ammonium citrate (Ammonia) to supply
2.72% extra crude protein. After 7 days of 8 h feeding, a saline solution of ρ-amino hippuric acid
(PAH) was infused into the mesentertic vein to determine portal and hepatic vein plasma flows.
Blood samples were collected at 0, 30, 60, 90, 120, 180, 240, 300, 390 and 480 min after feeding,
and analysed for plasma ammonia, urea and PAH concentrations. Total ammonia-N appearance in
portal vein and hepatic ammonia-N uptake were higher for Ammonia than Control. Total urea-N
appearance in portal blood was similar for both treatments, but hepatic urea-N release tended to be
higher for Ammonia. The increase in ammonia-N appearance in portal blood (Ammonia vs Control)
accounted for only 23.8% of dietary ammonia-N intake. These results indicate that the portal vein
drained organs use dietary ammonia for synthesis of N-metabolites other than urea.
Keywords: ammonia nitrogen, non-essential amino acids, pigs, portal vein
Introduction
Inclusion of crystalline amino acids (AA) allows formulation of diets that better match the animal’s
AA requirements while decreasing dietary protein content. Diets that are highly supplemented with
crystalline AA, however, may be deficient in total nitrogen (N) for endogenous synthesis of non-
essential amino acids (NEAA). Previous studies have shown that dietary ammonia, in the form of
ammonium citrate, appears to be an effective source of N for endogenous synthesis of NEAA. The
objective of the present study was to further understand dietary ammonia absorption and ammonia-N
metabolism in the portal vein drained organs (PVDO) and liver of growing pigs.

Four gilts of about 24 kg body weight were fitted with 4 catheters (i.e. carotid artery, and portal,
hepatic and mesenteric veins; Rerat et al., 1991) for infusion and serial blood sampling. A purified
basal diet was formulated to be deficient in NEAA-N, but to meet requirements for all essential
AA. Cornstarch (Control) or ammonium citrate (Ammonia) was added to the basal diet to maintain
or supply 2.72% extra crude protein, respectively. Pigs were restricted fed at 2.8 × maintenance
metabolisable energy requirements in 3 equal meals every 8 h for 7 d. On the last day, a saline
solution of ρ-amino hippuric acid (PAH) was infused continuously to determine blood flow in the
portal and hepatic veins. Blood samples were taken at 0, 30, 60, 90, 120, 180, 240, 300, 390 and 480
min after feeding and blood plasma was analysed for ammonia and urea-N, and PAH concentration.
Pigs were assigned to the dietary treatments following a cross-over design.

Plasma blood flow in portal and hepatic veins were calculated considering the dilution of PAH
in blood compared to the infusate. Hepatic artery plasma flow was calculated as the difference
between hepatic and portal vein blood flows. Metabolites (e.g. urea or ammonia-N) input or output
was calculated from blood flow to or from the PVDO or liver, and corresponding metabolite
concentrations. The balance of specific metabolites in either the PVDO or the liver was calculated
by subtracting output from input.

One observation per treatment was lost due to incomplete feed intake before blood sampling was
started. For Ammonia treatment, ammonia-N balance in the PVDO increased during the first 3 h
after feeding. At 480 min after feeding, values became similar to time 0 min for both treatments.
Ammonia-N balance in the liver decreased at 30 min for Ammonia compared to Control and remained
lower until 3 h after feeding. At the end of the sampling period, ammonia-N balance in the liver
returned to similar values compared to time 0 min for both treatments.
Urea-N balance in PVDO was not different between Control and Ammonia, indicating that the
intestinal wall is not using dietary ammonia-N for urea synthesis. Total urea-N balance in the liver
increased for Ammonia compared to the Control. Urea-N balance, returned to similar values compared
to time 0 for both treatments.
Ammonia-fed pigs consumed ~1,720 mg of ammonia-N per meal but only 410 mg of ammonia-N
was recovered in the portal vein per feeding period. This amount represents only 24% of total
dietary ammonia-N intake, implying that the intestinal wall is using ammonia-N for the synthesis
of nitrogenous metabolites other than urea (e.g. NEAA including citrulline). In pigs on Ammonia,
liver urea-N output approached PVDO ammonia-N output. In pigs on Control, liver urea-N output
did not increase in agreement with the high km for urea synthesis (Haussinger, 1983).
1,500 Control
Ammonia N/urea-N (mg)
1000 Ammonia
500
-500
-1000
Ammonia-N Ammmonia-N Urea-N Urea-N
appearance in appearance in appearance in appearance in
portal vein hepatic vein portal vein hepatic vein
Figure 1. Overall (8 h) balance (output minus input) of ammonia and urea-N in portal vein drained
organs and liver.
References
Haussinger, D., 1983. Hepatocyte heterogeneity in glutamine and ammonia metabolism and the role of an intercellular
glutamine cycle during ureogenesis in perfused rat liver. European Journal of Biochemistry 133: 269-275.
Rerat, A., C. Simoes-Nunes, F. Mendy, P. Vaissade, and P. Vaugelade, 1991. Splanchnic fluxes of amino acids after
duodenal infusion of carbohydrate solutions containing free amino acids or oligopeptides in the non-anaesthetized
pig. British Journal of Nutrition 68: 111-138.

The effect of the rate of starch digestion on energy efficiency in low and
high performing piglets
R.J.J. van Erp1*, H.M.J. van Hees1, R.T. Zijlstra3 and W.J.J. Gerrits2
1Trouw Nutrition R&D, Veerstraat 38, 5831 JN Boxmeer, the Netherlands; 2Animal Nutrition Group,
Wageningen University, P.O. Box 338, 6700 AH Wageningen, the Netherlands; 3Department of
Agricultural Food, and Nutritional Science, University of Alberta, Edmonton, AB, T6G 2P5, Canada;
rik.van.erp@trouwnutrition.com
Abstract
Differences in growth performance of piglets during lactation might be related to differences in
insulin sensitivity. Therefore glucose metabolism may be affected differently in low performing (LP)
and high performing pigs (HP) at weaning when feeding either a slowly digestible starch (SDS) or
rapidly digestible starch (RDS). In a 2×2 factorial block design 30 LP pigs and 30 HP pigs equal
in birth weight were housed in one of four respiration chambers and fed diets containing either
RDS or SDS. In the first week feed was available ad libitum. In the second week feed supply was
restricted to 65% of the observed feed intake in the first week. Energy and nitrogen balances were
calculated for both weeks. Results were used to calculate incremental efficiencies of gross energy
(GE) and metabolised energy (ME) for growth. Incremental efficiency of GE for growth was higher
when feeding RDS compared with SDS, particularly so in LP pigs (diet×pig type; P=0.05). The
incremental efficiency of ME for growth tended to be lower with SDS compared with RDS (74.1
vs 75.6; P=0.05) but was not affected by growth retardation during lactation.
Keywords: growth retardation, energy balance, restricted feeding
Introduction
Pigs equal in birth weight sometimes perform differently pre weaning for no obvious reasons (Paredes
et al., 2012). Little is known about physiological differences between these types of pigs but it might
be related to differences in energy metabolism and insulin sensitivity (Paredes et al., unpublished
data). To investigate this, differences in energy expenditure and efficiency between low performing
pigs and high performing pigs at weaning were studied. It was hypothesized that energy efficiency
of low performing pigs improves when feeding a slowly digestible starch as a result of a lower and
prolonged insulin response.

From a subpopulation of pigs equal in birth weight (1.36±0.13 kg), thirty low performing pigs (LP;
6.41±0.54 kg) and thirty high performing pigs (HP; 9.56±0.69 kg) were selected at weaning. In a 2×2
factorial block design, sixteen groups of five piglets (8 × LP and 8 × HP) were housed in one of four
identical climate respiration chambers for two weeks, and fed diets containing 40% rapid digestible
starch (Remyline AAX-DR rice starch, RDS) or slowly digestible starch (Nastar Pea starch, SDS).
In the first week, feed was available ad libitum. In the second week, feed supply was restricted to
65% of the observed feed intake in the first week. Measurements were conducted in two age groups
(9 and 11 week of age). Energy and nitrogen balances were calculated for both weeks. Incremental
efficiencies of gross energy (GE) and metabolised energy (ME) for growth were calculated as the
difference in energy retention between the first and second week divided by the respective difference
in GE or ME intake. Energy balance data were analysed using a general linear model with diet, pig
type, block, and age group as fixed effects.

Preliminary results and discussion
When feed was available ad libitum GE intake tended to be higher for SDS compared with RDS
(1,595 vs 1,654 kJ/kg0.75; P=0.08; Table 1), and energy losses with faeces and urine were higher
for SDS compared with RDS (169 vs 146 kJ/kg0.75; P=0.02). Also protein accretion tended to be
higher for SDS compared with RDS (315 vs 297 kJ/kg0.75; P=0.07). Protein to fat ratio tended to
be higher for LP pigs fed SDS compared with LP pigs fed RDS while protein to fat ratio tended
to be lower for HP pigs fed SDS compared with HP pigs fed RDS (diet×pig type; P=0.09). The
incremental efficiency of GE for growth was higher for RDS than for SDS, particularly so in LP pigs
(diet×pig type; P=0.05). The incremental efficiency of ME for growth tended to be lower for SDS
compared with RDS (74.1 vs 75.6; P=0.05). The absence of the interaction effect diet × type for the
incremental efficiency of ME for growth indicates that more SDS is available for fermentation in the
large intestine of LP pigs resulting in a higher loss of microbial biomass with faeces. In conclusion,
the incremental efficiency of ME for energy retention is not affected by growth retardation during
lactation, but is higher for RDS compared with SDS.
Table 1. The effect of slowly digestible (SDS) and rapidly digestible (RDS) starch in low performance
(LP) and high performance (HP) pigs on energy balance and incremental efficiency for growth
corrected for gross energy intake when feed was available ad libitum or feed supply was restricted.
Figures are expressed as kJ/kg0.75 body weight per day.1
HP LP SEM P-value
RDS SDS RDS SDS Diet Type D×T
Ad libitum feeding
GE intake 1,582 1,682 1,607 1,626 30.3 0.08 0.62 0.21
Faeces + urine 144 163 148 175 8.19 0.02 0.36 0.63
ME intake 1,432 1,513 1,452 1,445 31.5 0.49 0.49 0.69
HPtot 860 867 853 865 8.34 0.30 0.58 0.81
ER 571 645 599 580 26.4 0.32 0.49 0.11
Erp 297 320 297 309 8.66 0.07 0.52 0.50
Erf 274 325 302 272 18.9 0.60 0.50 0.06
Restricted feeding
GE intake 1,042 1,106 1,060 1,076 18.6 0.06 0.76 0.22
Faeces + urine 99.3 113 96.4 107 4.81 0.03 0.39 0.71
ME intake 937 986 958 963 19.6 0.49 0.93 0.83
HPtot 736 733 735 737 9.27 0.95 0.87 0.80
ER 201 254 223 226 16.1 0.12 0.87 0.16
Erp 186 194 184 194 6.61 0.19 0.88 0.84
Erf 15.2 59.9 39.7 32.0 10.4 0.11 0.88 0.03
Incremental efficiency for energy retention
GE 68.5 68.1 68.8 64.5 0.86 0.02 0.09 0.05
ME 74.9 74.5 76.3 73.7 0.67 0.05 0.72 0.14
1 GE = gross energy; ME = metabolised energy; HP = total heat production; ER = total energy retention; ER = energy
tot p
retained as protein; ERf = energy retained as fat.
References
Paredes, S.P., A.J.M. Jansman, M.W.A. Verstegen, A. Awati, W. Buist, L.A. den Hartog, H.M.J. van Hees, N. Quiniou,
W.H. Hendriks and W.J.J. Gerrits, 2012. Analysis of factors to predict piglet body weight at the end of the nursery
phase. Journal of Animal Science 90(9): 3243-3251.

Influence of deoxynivalenol (DON) on the piglet’s immune system with
due regard to sodium sulfite decontaminated feed: in vivo results
A.T. Tran1, M. Paulick1, A. Berk1, J. Kluess1*, J. Frahm1, D. Schatzmayr2 and S. Dänicke1
1Institute of Animal Nutrition (FLI), Bundesallee 50, 38116 Braunschweig/Germany; 2BIOMIN
Research Center, Technopark 1, 3430 Tulln, Österreich; jeannette.kluess@fli.bunde.de
Abstract
Pigs respond very sensitive to the presence of the mycotoxin deoxynivalenol (DON) in the diet,
especially with a reduction in feed intake and with an immuno-modulation. A recent report suggested
that sodium sulfite (Na2SO3, SoS) treatment of DON contaminated maize as feed ingredient reduced
the toxicity of DON as proven by an improved feed intake. The aim of the present study was to
investigate the effects of feeding a DON contaminated diet, either untreated or SoS treated on the
hematology, biochemistry and also on oxidative stress in piglets. Twenty castrated male piglets
(7.57±0.92 kg BW) were equally divided into four experimental groups and fed with different diets
as follow: CON- (control diet), CON+ (diet containing SoS treated maize), DON- (diet containing
untreated contaminated maize; 6 mg DON/kg feed), and DON+ (diet containing SoS treated
contaminated maize; 0.8 mg DON/kg feed). After 37 days, blood samples were taken to examine
the haematological, biochemical parameters, nitric oxide (NO) production and ferric reducing
ability of plasma (FRAP). There was no effect of DON and SoS on the red blood count and the NO
production. In the white blood cells, DON and SoS alone reduced the concentration of lymphocytes
and monocytes. Interestingly, a combined effect of DON and SoS compensated this haematological
parameter. DON alone induced an increase of aspartate aminotransferase whereas SoS reduced the
Bilirubin concentration. Besides, the SoS treated piglets showed no effect on the expression of FRAP.
A significant increase of FRAP was found in DON-fed piglets.
Keywords: deoxynivalenol, sodium sulfite, nitric oxide, FRAP, aspartate aminotransferase
Introduction
Pigs respond very sensitive to the presence of the mycotoxin deoxynivalenol (DON) in the diet,
especially with a reduction in feed intake and often an immuno-modulation (Dänicke and Brezina,
2013). A recent report suggested that sodium sulfite (Na2SO3, SoS) treatment of DON-contaminated
feed reduced the toxicity of DON as proven by an improved feed intake (Paulick et al., 2015). Thus,
we investigated the effects of feeding a DON-contaminated diet, either untreated or SoS treated, on
haematology, clinical biochemistry and redox status of weaning piglets.

Twenty piglets (7.57±0.92 kg BW) were equally divided into four experimental groups based on
following diets: CON- (control diet with 10% maize), CON+ (diet with 10% maize wet-conserved
with 5 g SoS/kg), DON- (diet with 10% contaminated maize untreated; 6 mg DON/kg feed), and
DON+ (diet with 10% contaminated maize wet-conserved with 5 g SoS/kg; 0.8 mg DON/kg
feed). After 37 days, blood samples were taken for analysing haematological profile, biochemical
parameters, nitric oxide (NO) production and ferric reducing ability of plasma (FRAP). Data were
statistically analysed with a two-factorial ANOVA (feed, SoS, feed × SoS) and differences (Student’s
t-test) were considered significant at P≤0.05.

Results
There was no effect of DON or SoS treatment on red blood cell counts. Leukocyte differential counts
showed a significant interaction for DON and SoS (P<0.01): in CON-fed groups SoS treatment
decreased total leukocytes, lymphocytes, neutrophils and monocytes, whereas in DON-fed groups
the opposite effect was detected. Clinical blood chemistry was not altered except for a significant
DON-induced increase of aspartate aminotransferase and a SoS-related bilirubin reduction (Table 1).
Investigation of the animal’s redox state showed no alteration of NO production, but FRAP was
strongly increased in DON-fed pigs, irrespective of SoS treatment (Figure 1).
Table 1. Effect of deoxynivalenol (DON) and sodium sulfite (SoS) in piglet diets on leukocytes counts
and selected parameters of clinical chemistry.1
DON SoS Leukocytes Lymphocytes Segmented neutrophils AST Bilirubin

(mg/kg) (g/kg) (103/µl) (103/µl) (103/µl) (U/l) (µmol/l)
– – 18.66a 11.91a 4.70ab 36.8b 3.09

– 5 13.42b 8.74b 3.68b 39.7ab 2.43
6 – 14.42b 8.37b 4.85ab 49.6a 3.01
6 5 20.8a 12.17a 6.79a 48.9a 2.57
PSEM 1.42 0.94 0.75 3.509 0.244
ANOVA (P-value)
DON 0.28 0.96 0.04 <0.01 0.91
SoS 0.69 0.75 0.53 0.75 0.04
DON × SoS <0.01 <0.01 0.06 0.62 0.66
1 AST = aspartate aminotransferase; PSEM = pooled standard error of means.
ANOVA P-value ANOVA P-value

DON 0.45 DON <0.01
SoS 0.8 –SoS
SoS 0.46
DONxSoS 0.68 +SoS DONxSoS 0.91
25
500
20
Nitric oxide (mM)
400
FRAP (µM)
15
300
10
200
5 100
0 0
CON DON CON DON
Figure 1. Effect of DON and SoS treatment on redox status of weaning piglets: NO production
(left panel) was not affected, whereas FRAP (right panel) increased significantly in DON-fed pigs,
irrespective of SoS treatment.

Conclusions
Our results indicate that both DON and SoS treatment had a significant impact on physiological
parameters of piglets in vivo: leukocytes were reduced with CON+ diet and DON- diet, but DON-
deactivation with SoS (DON+) was able to recover leukocyte counts. DON elevated the antioxidant
ability (FRAP) of piglets after five weeks, which might have been due to a feedback regulation of
a DON-induced rise in reactive oxygen species. Further analyses elucidating the observed effects
are in progress.
Acknowledgements
This project was financially supported by BIOMIN Holding GmbH, Tulln/Austria.
References
Dänicke, S. and U. Brezina, 2013. Kinetics and metabolism of the Fusarium toxin deoxynivalenol in farm animals:
consequences for diagnosis of exposure and intoxication and carry over. Food and Chemical Toxicology 60: 58-75.
Paulick, M., I. Rempe, S. Kersten, D. Schatzmayr, H.E. Schwartz-Zimmermann and S. Dänicke, 2015. Effects of
increasing concentrations of sodium sulfite on deoxynivalenol and deoxynivalenol sulfonate concentrations of
maize kernels and maize meal preserved at various moisture content. Toxins 7: 791-811.

Impact of dietary crude protein and amino acid restriction on the amino
acid deposition rate and profile in the empty body of modern Swiss
Large White pigs
I. Ruiz-Ascacibar1,2, P. Stoll1 and G. Bee1*
1Agroscope, Institute for Livestock Sciences, La Tioleyre 4, 1725 Posieux, Switzerland; 2ETH
Zurich, Institute of Agricultural Sciences, Universitätsstrasse 2, 8092 Zurich, Switzerland;
giuseppe.bee@agroscope.admin.ch
Abstract
The increasing concern about the environmental impact and the high cost of protein sources, make
the reduction of the dietary protein in pig diets a desirable target. In order to revisit the amino
acid (AA) composition in the empty body (EB) of pigs and to deliver updated values for feeding
recommendations, an experiment was performed with 32 entire male, castrated and female pigs,
each. From 20-140 kg body weight (BW), they were either allocated to a control (C) grower-finisher
diet formulated according to Swiss feeding recommendations, or a low protein grower-finisher diet
(R; 80% of crude protein, lysine (LYS), methionine (MET) + cystine (CYS), threonine (THR) and
tryptophan (TRP) of diet C). The LYS, MET, CYS and THR content of the EB was determined at
20, 60, 100 and 140 kg BW on 4 pigs per diet and sex. The AA-to-LYS ratios were also examined.
The AA deposition rate was not (P>0.05) affected by gender. R-pigs had lower LYS, MET, CYS and
THR deposition rate at 20 to 60 and 20 to 100, but not 20 to 140 kg BW. Except for the THR:LYS
ratio, which was 6.6% greater (P=0.05) in the EB of R-pigs at 60 kg BW, the AA profile was neither
affected (P>0.05) by the diet nor gender. The results obtained with the R-diet indicate the potential
to reduce the AA supply in the latter stage of growth without a major impact on the deposition of
the AA in the EB.
Keywords: amino acid deposition, amino acid restriction, pigs
Introduction
Pig production is an important contributor of nitrogen excreted by farm animals. Thus, there is
an increasing interest in reducing and optimizing the dietary crude protein supply in grower and
finisher diets of pigs. This can be achieved by reducing the dietary crude protein supply provided
that essential amino acid (AA) requirements of the pig are covered (Van Milgen et al., 2012).
Results from a current project (I. Ruiz-Ascacibar, P. Stoll, M. Kreuzer, V. Boillat, P. Spring and G.
Bee, unpublished data) revealed that over the last decades protein accretion rates as well as nitrogen
efficiency markedly improved in modern pig genotypes. It is unclear whether these changes altered
the AA composition and/or AA profile of the EB, both traits being a prerequisite for deriving accurate
AA feeding recommendations. The current results are part of a larger study, aiming to assess the
nutrient composition of the EB of pigs and to deliver updated values for feeding recommendations.

An experiment was conducted with 32 entire male (EM), castrated (CA) and female pigs (FE), each.
From 20 kg BW onwards, they either were allocated to a C-grower-finisher diet formulated according
to Swiss feeding recommendations, or to a R-grower-finisher diet (80% of crude protein, lysine (LYS),
methionine (MET) + cystine (CYS), threonine (THR) and tryptophan (TRP) of diet C). Pigs had ad
libitum access to the diets and fresh water. The AA composition of the EB was determined at 20,
60, 100 and 140 kg BW on 4 pigs per diet and sex. Pigs were stunned, exsanguinated and de-haired.
Organs and empty intestinal tract from each animal were homogenized together. Left carcasses were
frozen until grinding. The LYS, MET, CYS and THR content was analysed in each fraction and

their concentrations used to calculate their level in the EB. The AA concentrations of the 20 kg BW
pigs were used as basis to calculate the daily AA deposition rate from 20 to 60 (period 1), 20 to 100
(period 2) and 20 to 140 kg BW (period 3). The AA-to-LYS ratios were also examined. Statistical
comparisons were made by ANOVA followed by a Tukey test to identify differences between the
experimental groups using SYSTAT 13 (SYSTAT Software, Inc.). In the statistical model diet, sex
and diet × sex interaction were considered as fixed effects.

Only from 20 to 100 kg BW there was a sex effect on the AA deposition rate as the daily amount of
deposited LYS was lower (P=0.05) in FE (8.50 g/d) than EM (10.27 g/d), with intermediate values
for CA (10.03 g/d). In agreement, I. Ruiz-Ascacibar et al. (unpublished data) reported greater protein
deposition in CA and EM than in FE at 100 kg BW. In the BW range from 20 to 60, 20 to 100 and
20 to 140 kg, pigs fed the C-diet deposited daily the following amounts of LYS: 9.87, 10.67 and 9.89
g, of MET: 2.31, 2.20 and 2.09 g, of CYS: 0.89, 0.98 and 0.85 g, and of THR: 5.28, 5.94 and 5.36
g. Except for MET in the second period (20 to 100 kg BW), LYS, MET, CYS and THR deposition
rate was on average 28 and 24% lower (P<0.05) in the first and second period, respectively. No
differences in the deposition rate of these AA were observed in the third period. Except for the THR-
to-LYS ratio, which was greater (P=0.05) in the EB of R-pigs at 60 kg BW, the AA-to-LYS ratios
were affected by neither the diet nor the sex at any BW. The ratios of MET, CYS and THR to LYS
were 20.7, 9.9 and 55.8 at 20 kg, 22.1, 9.0 and 56.0 at 60 kg, 20.6, 8.8 and 55.9 at 100 kg and 21.1,
9.6 and 53.6 at 140 kg, respectively. The MET-to-LYS and the (MET+CYS)-to-LYS ratio was 16
and 2% lower, respectively, than that reported by NRC (2012) whereas the THR-to-LYS ratio was
with 11% slightly greater.
In conclusion, these new data using the serial slaughter technique suggest slight shifts in the AA
pattern of the EB of modern pigs, this shift being unaffected by the plane of nutrition. Furthermore,
the results obtained with the R-diet indicate that a marked reduction in the AA supply, especially
in heavier pigs (>100 kg BW), can be envisaged without a major impact on the deposition of LYS,
MET, CYS and THR in the EB. These findings are in agreement with previous results of I. Ruiz-
Ascacibar et al. (unpublished data), where a 20% reduction of dietary protein, LYS, MET, CYS TRP
and THR supply had no effect on EB protein deposition after 100 kg BW.
References
National Research Council (NRC), 2012. Nutrient requirements of swine (11th rev. Ed.). National Academy Press,
Van Milgen J., M. Gloaguen, N. Le Floc’h, L. Brossard, Y. Primot and E. Corrent, 2012. Meta-analysis of the response
of growing pigs to the isoleucine concentration in the diet. Animal 6: 1601-1608.

Effect of dietary proteins on immunity and metabolism in mice
S.K. Kar1*, A.J.M. Jansman2, L. Kruijt2, N. Benis1 and M.A. Smits1,2,3
1Host Microbe Interactomics, Wageningen University, De Elst 1, 6708 WD, Wageningen, the
Netherlands; 2Wageningen UR Livestock Research, Wageningen University, De Elst 1, 6708 WD,
Wageningen, the Netherlands; 3Central Veterinary Institute, Wageningen University, Wageningen,
the Netherlands; soumya.kar@wur.nl
Abstract
We investigated the effects of six dietary protein sources on a range of physiological and immunological
parameters in mice as measured locally (ileal tissue and content) and systemically (blood and urine).
Genome-wide transcriptome analysis showed down regulation of a number of immune (T-cell
related) and metabolic genes in ileal mucosa of mice fed with the soybean meal based (SBM) diet
as compared to other diets. SBM also distinctively influenced intestinal microbiota composition as
well as the metabolic profiles of amines in blood and urine. The host response of SBM deviated
strongly from the other protein sources in its capacity to interact with host metabolism and immunity.
Keywords: ileum, metabolic, microbiota, transcriptome
Introduction
Total or partial replacement of traditional protein resources for non-traditional sources in animal
feeds may contribute to solving the problem of future dietary protein shortages. However, before
new protein sources can be safely exploited in animal feeding practice, more knowledge is needed
on their interaction with the host, especially in the gastrointestinal (GI) tract. Previous studies have
suggested that dietary proteins can affect physiological, metabolic and immune functioning of the GI
tract (Jahan-Mihan et al., 2011). The objective of the current study was to investigate and compare
the effect of the use of various dietary protein sources in diets for mice on several metabolic and
immune parameters.

Thirty-six male C57BL/6J mice were stratified according to bodyweight and litter of origin into six
dietary treatment groups. The mice were fed for three weeks with semi-synthetic diets (formulated
to be nutrient sufficient) containing one of the following protein sources at an inclusion level of 30%
(as fed basis): soybean meal (SBM), casein, delactosed whey powder, spray dried plasma protein,
wheat gluten meal, or yellow meal worm. Ileal tissue and digesta, urine and blood samples were
taken. We have performed genome-wide transcriptomic profiling, 16S rRNA gene sequencing and
untargeted gas chromatography-mass spectrometry to evaluate effects of dietary protein source on
ileal mucosal response, residing microbiota and systemic amine metabolites, respectively. The SBM
diet served as reference to make comparisons with the other experimental diets.

The animals appeared to be healthy during the trial. Genome-wide transcriptome analysis showed
down regulation of a number of immune (T-cell related) and metabolic genes at ileal mucosae of
mice fed with SBM compared to the other experimental diets (Table 1).

Table 1. Summary of results of local and systemic responses to diets prepared with different protein
sources, i.e. soybean meal (SBM), casein (CAS), delactosed whey powder (DWP), spray dried plasma
protein (SDPP), wheat gluten meal (WGM), or yellow meal worm (YMW).1
Biological ~omics profile Significant SBM CAS DWP SDPP WGM YMW
level observation
Local Ileal T cell and metabolic ↓# ↑* ↑* ↑* ↑* ↑*

transcriptomic
gene
Actinobacteria (%)ϕ
Ileal microbiota 3.7 10.6 28.4 7.1 2.1 2.9
Bacteroidetes (%)ϕ
(top 3 phyla) 55.9 16.8 16.2 15.7 34.8 7.7
Firmicutes (%)ϕ 37.0 64 51.2 72.9 58.3 73.3
Systemic Metabolomics Blood Il↑# AbA↑* AbA↑* Ps↑* Ms↑* Mh↑*
Urine Pa↑# AbA↑* AbA↑* Mh↑* Mt↑* Mh↑*
1 ↓ = down; ↑ = up; # compared to other experimental diets;* compared to SBM; ϕ relative abundance; Il = isoleucine; Pa
= pipecolic acid; AbA = α-aminobutyric acid; Ps = putrescine; Ms = 1-methionine sulfoxide; Mt = 3-methoxytyramine;

Mh = 1-methylhistidine.
It is not known which component(s) in the SBM source is responsible for this ‘(T-cell) immune
suppressing’ activity. In addition, it is not known whether there is a direct effect of (digested) SBM
components on the intestinal immune cells or whether it is an indirect effect. The observation
that the SBM source also distinctively influenced the composition (and diversity) of the intestinal
microbiota, argues for the latter option. Among others, SBM-based diet also induced the blooming
of microbes belonging to Bacteroidetes phyla. The high non-starch polysaccharides content in the
SBM-based diet could have created favourable conditions for increasing the abundance of members
in the phyla Bacteroidetes. It is reported that Bacteroides species play critical roles in initiating
the degradation of complex substrates such as plant cell walls, starch particles and mucin (Flint
et al., 2012). Interestingly, we detected increased concentration of blood isoleucine in SBM-fed
mice which could be associated with the metabolic capacity of Bacteroidetes phyla to synthesise
isoleucine from 2-methyl-butyrate (Robinson and Allison, 1969). Taken together, the response of
SBM deviated strongly from diets with other protein sources in its capacity to interact with host
metabolism and immunity. The knowledge generated in this study may help to formulate healthy
diets for monogastrics with alternative protein sources.
Acknowledgements
The authors acknowledge the financial support from the Wageningen UR ‘IPOP Customized Nutrition’
programme, the graduate school (WIAS) and industrial partners Nutreco and Darling Ingredients Inc.
References
Flint, H.J., K.P. Scott, S.H. Duncan, P. Louis and E. Forano, 2012. Microbial degradation of complex carbohydrates
in the gut. Gut Microbes 3: 289-306.
Jahan-Mihan, A., B.L. Luhovyy, D.E. Khoury and G.H. Anderson, 2011. Dietary proteins as determinants of metabolic
and physiologic functions of the gastrointestinal tract. Nutrients 3: 574-603.
Robinson, I.M. and M.J. Allison, 1969. Isoleucine biosynthesis from 2-methylbutyric acid by anaerobic bacteria from
the rumen. Journal of Bacteriology 97: 1220-1226.

Methionine, leucine, isoleucine or threonine effects on mammary cell
signalling and pup growth in lactating mice
G.M. Liu1, M.D. Hanigan2, X.Y. Lin1, K. Zhao1 and Z.H. Wang1*
1Animal Science and Technology College, Shandong Agriculture University, 271018, China P.R.;
2Department of Dairy Science, Virginia Tech, Blacksburg, VA 24061, USA; zhwang@sdau.edu.cn
Abstract
Two studies were undertaken to assess the effects of individual essential amino acid (EAA)
supplementation of a protein deficient diet on lactational performance in mice using litter growth
rates as a response variable. The first was designed to establish a dietary protein response curve,
and the second to determine the independent effects of Leu, Ile, Met, or Thr supplementation of a
protein deficient diet. Supplementation with Ile, Leu, and Met increased litter weight gain by 11, 9,
and 10%, respectively, as compared to the protein deficient diet. These responses are supported by
independent phosphorylation responses for mTOR and 4eBP1. Incorporation of a multiple limiting
EAA concept into milk protein response models will help improve milk protein yield predictions,
thus allowing derivation of diets that will result in increased postabsorptive N efficiency and reduced
N excretion by dairy animals.
Keywords: single amino acid, litter growth rate, cell signalling, mice
Introduction
Individual essential amino acid (EAA) have different effects on signaling proteins and casein
fractional synthesis rates of mammary tissue in vitro (Appuhamy et al., 2012). We hypothesized that
supplementation of a protein deficient diet with those specific EAA would independently improve
lactational performance through cell signalling responses. There is no whole animal research to
support this strategy. So, the objective of the present study was to investigate the effect of Leu, Ile,
Met, or Thr supplementation of a protein deficient diet on lactational performance in vivo.

In the first experiment, modified AIN-76a purified diets containing 6, 9, 12, 15, 18, 21, 24, or 27%
protein were fed for 17 d to lactating mice (n=10). Based on data from the first experiment, the
2nd experiment examined the effects of independent additions of Leu, Ile, Met, or Thr to the 15%
protein diet to achieve AA intakes for each equal to the intake of that AA from the 21% protein
diet as compared to the 15% and 21% protein diets. Twenty mice were assigned to each treatment.
Mammary tissue was collected and analysed for mTOR pathway cell signalling.

In the first experiment, litter weight gain was affected by dietary protein (P<0.01) at concentrations
less than 21%. The rate of infanticide was 100% for diets less than 12% protein (Table 1). In the 2nd
experiment, supplementation with Ile, Leu, or Met increased (P<0.01) litter weight gain by 11, 9,
and 10% respectively as compared to the protein deficient diet (Table 2). The results demonstrated
that milk production responses to supplementation of single AA are independent and do not adhere
to the concept of a single limiting AA as laid out by Mitchell and Block (1946). Supplementation
of Ile, Leu, Met, or Thr increased (P<0.05) phosphorylation of mTOR by 55, 34, 47, and 45%,
respectively, as compared to the low protein group. Phosphorylation of 4eBP1 increased in response
to Ile, Met, or Thr supplementation by 60, 40 and 51%, respectively (P<0.01). These results were
consistent with the findings described previously by Appuhamy et al. (2012) in vitro. There were
no significant effects of treatment on phosphorylation of S6K1, eIF2α, or eEF2.

Table 1. Effect of different protein treatment on litter weight gain of lactation mice.
Dietary protein concentration SEM P-value
6% 9% 12% 15% 18% 21% 24% 27%
Littter weight gain (g) 0 0 45.97c 54.87c 71.91b 87.85a 83.44a 85.67a 9.53 0.0001
Infanticide rate (%) 100% 100% 60% 60% 20% 20% 10% 10%
Table 2. Effect of Leu, Ile, Met, or Thr supplementation to diets of lactating mice on litter weight
gain and mammary cell signalling protein phosphorylation.
Dietary protein 21% 15% 15% 15% 15% 15% SEM P-value
AA supplement – +Leu +Ile +Met +Thr –
Littter weight gain (g) 84.84a 78.81ab 77.79b 78.73ab 70.78c 70.98c 8.16 0.001
P-mTOR/T-mTOR 1.13a 1.15a 0.98a 1.07a 1.06a 0.73b 0.09 0.03
P-4eBP1/T-4eBP1 0.91b 0.82b 1.2a 1.05ab 1.13a 0.75b 0.09 0.007
P-S6K1/T-S6K1 1.22 0.97 1.1 1.3 0.99 0.91 0.10 0.31
P-eEF2/T-eEF2 1.04 0.94 0.85 0.95 0.83 0.93 0.10 0.65
P-eIF2α/T-eIF2α 1.03 1.07 1.03 0.96 0.93 1.08 0.09 0.83
Acknowledgements
This work was supported by the Natural Science Fund of China (31372340).
References
Appuhamy, J.A.D.R.N., N.A. Knoebel, W.A.D. Nayananjalie, J. Escobar and M.D. Hanigan, 2012. Isoleucine and
leucine independently regulate mTOR signaling and protein synthesis in MAC-T cells and bovine mammary tissue
slices. Journal of Nutrition 142: 484-491.
Mitchell, H.H. and R.J. Block, 1946. Some relationships between the amino acid contents of proteins and their nutritive
values for the rat. Journal of Biological Chemistry 163: 599-620.

Effects of supplemental dietary leucine and immune system stimulation
on whole body protein turnover in starter pigs
M. Rudar, C.L. Zhu and C.F.M. de Lange*
Department of Animal Biosciences, University of Guelph, Guelph, ON, N1G 2W1, Canada;
cdelange@uoguelph.ca
Abstract
Immune system stimulation (ISS) adversely affects nitrogen and amino acid metabolism. The
objective of the study was to evaluate the effect of dietary leucine (Leu) supplementation on whole
body protein turnover in starter pigs before and after ISS. A total of 28 Yorkshire barrows (initial BW
= 14.46±0.73 kg) were assigned to one of three dietary treatments: (1) CON, 1.36% standardised
ileal digestible (SID) Leu (n=13); (2) LEU-M, 2.04% SID Leu (n=7); or (3) LEU-H, 2.72% SID
Leu (n=7), and infused continuously with 15N (0.13±0.01 mmol 15N/kg BW/d from 15N-glycine) to
determine whole body protein kinetics. The study consisted of a 72-h pre-challenge period followed
by a 36-h challenge period. At the end of the pre-challenge period, ISS was induced in all LEU-M
and LEU-H pigs and seven CON pigs with an intramuscular injection of bacterial lipopolysaccharide;
the remaining CON pigs were injected with saline. Feed refusals and faeces were collected to
determine digestible N intake and urine was collected to determine urinary urea- and ammonia-N
and 15N excretions. During the pre-challenge period, there was a linear reduction in whole body
protein synthesis and breakdown with increasing Leu intake. During the challenge period, whole
body protein synthesis and protein deposition were lower in ISS+ than in ISS- pigs whereas whole
body protein breakdown was not affected by ISS. Leucine intake did not affect any aspect of whole
body protein turnover in ISS+ pigs.
Keywords: leucine, protein turnover, disease, pigs
Introduction
Immune system stimulation (ISS) adversely affects nitrogen (N) and amino acid (AA) metabolism
and reduces productivity in starter pigs. Among the essential AA, Leu is a potent anabolic stimulus
and has a regulatory role in skeletal muscle and whole body protein turnover. The objective of
the study was to evaluate the effect of dietary Leu supplementation in pigs on whole body protein
turnover and protein deposition (PD) before and after ISS.

A total of 28 Yorkshire barrows (initial BW = 14.46±0.73 kg) were surgically fitted with jugular
vein catheters. Following surgery, pigs were housed individually in metabolism crates and adapted
to one of three dietary treatments: (1) CON, 1.36% SID Leu (n=13); (2) LEU-M (n=8), 2.04% SID
Leu; and (3) LEU-H, 2.72% SID Leu (n=7). Pigs were fed every 4 h according to their BW and
water was provided to pigs at a 3 to 1 ratio.
The study consisted of a 72-h pre-challenge period (six consecutive 12-h collections) followed by a
36-h challenge period (three consecutive 12-h collections). Throughout both the pre-challenge and
challenge periods, pigs were infused continuously with 15N (0.13±0.01 mmol 15N/kg BW/d from
15N-glycine). At the end of the pre-challenge period, ISS was induced in all LEU-M and LEU-H
pigs, and half of CON pigs, with a single intramuscular injection of bacterial lipopolysaccharide (30
µg/kg BW; ISS+; n=7, 8, and 7 for CON, LEU-M, and LEU-H pigs, respectively); the remaining
CON pigs were injected with saline (ISS-; n=6). For each 12-h collection, feed refusals and faeces
were collected to determine digestible N intake, and urine was collected to determine urinary urea-

and ammonia-N and 15N excretions. Whole body protein turnover was calculated according to the
end-product method (Waterlow et al., 1978). PD was calculated as the difference between whole
body protein synthesis and degradation.

During the pre-challenge period, there was a linear reduction in whole body protein synthesis and
breakdown with increasing Leu intake (P<0.01; Table 1). PD was higher in both CON and LEU-H
pigs than LEU-M pigs (P<0.05) and there was a linear reduction in the ratio between protein synthesis
and PD with increasing Leu intake (P<0.05). Given the high energetic cost of protein synthesis,
and the relationship between protein turnover and minimum AA catabolism, it can be implied that
feeding supplemental Leu improves the efficiency of using energy and AA for PD.
During the challenge period, whole body protein synthesis (203 vs 169 mmol N/kg BW/d) and PD
(64.9 vs 45.0 mmol N/kg BW/d) were lower in ISS+ than in ISS- pigs (P<0.05) whereas whole
body protein breakdown was not affected by ISS. The reduction in whole body protein synthesis
during ISS can largely be attributed to a reduction in muscle protein synthesis (Breuillé et al., 1998).
Leucine intake did not affect any aspect of whole body protein flux in ISS+ pigs. The ability of Leu
to modulate muscle protein synthesis is blunted during ISS (Lang and Frost, 2005), which appears
to extend to whole-body protein synthesis as well.
Table 1. Aspects of whole-body protein flux (mmol N/kg BW/d) in pigs fed increasing levels of SID
Leu above estimated requirements for maximum PD during the 72-h pre-challenge period.1
Item Treatment SE P-value
CON LEU-M LEU-H Trt Lin Quad
Protein flux 325 292 277 14 0.025 0.011 0.585

Digestible N intake 92.1 89.3 91.8 0.9 0.023 0.765 0.010
Urinary N excretion 15.7 19.5 17.2 1.2 0.038 0.310 0.028
Protein synthesis 309 273 260 14 0.024 0.012 0.483
Protein degradation 233 203 185 14 0.027 0.011 0.701
PD 76.4 69.8 74.6 1.2 <0.001 0.240 <0.001
Protein synthesis: PD 4.04 3.89 3.48 0.16 0.032 0.010 0.471
1 PD = protein deposition; Trt = treatment; Lin = linear; Quad = quadratic.
References
Breuillé, D., M. Arnal, F. Rambourdin, G. Bayle, D. Levieux and C. Obled, 1998. Sustained modifications of protein
metabolism in various tissues in a rat model of long-lasting sepsis. Clinical Science 94: 413-424.
Lang, C.H. and R.A. Frost, 2005. Endotoxin disrupts the leucine-signaling pathway involving phosphorylation of
mTOR, 4E-BP1, and S6K1 in skeletal muscle. Journal of Cell Physiology 203: 144-155.
Waterlow, J.C., M.H. Golden and P.J. Garlick, 1978. Protein turnover in man measured with 15N: comparison of end
products and dose regimes. American Journal of Physiology Gastrointestinal and Liver Physiology 235: G165-G174.

Roles of corticosterone and superoxide in the ubiquitin proteasome
system in heat-stressed chickens
K. Furukawa, M. Kikusato* and M. Toyomizu
Animal Nutrition, Life Sciences, Graduate School of Agricultural Science, Tohoku University, 1-1
Tsutsumidori-Amamiyamachi, Sendai 981-8555, Japan; kmotoi@bios.tohoku.ac.jp
Abstract
The present study investigated the roles of corticosterone and mitochondrial superoxide production in
heat stress (HS)-induced muscle protein degradation by animal- and cell culture-based experiments.
Chickens at 25 d of age were exposed to HS conditions (33 °C) for 0, 0.5, 1 or 3 d. The plasma
corticosterone concentration was increased after 0.5 d of HS treatment (P<0.05), and the plasma
Nτ-methylhistidine concentration was increased by 3 d of HS treatment (P<0.05). In birds exposed
to 0.5 d of HS treatment, the mRNA levels of atrogin-1 (P<0.05), and mitochondrial superoxide
production (P<0.05) were increased relative to their respective values in thermoneutral birds. In
avian muscle cells incubated under high temperature conditions (41 °C), mitochondrial superoxide
generation and atrogin-1 mRNA levels were increased, and cellular protein content was subsequently
reduced. The addition of physiological concentrations of corticosterone had little effect on these
alterations. These results suggest that HS-induced muscle protein degradation may be due to the
activation of ubiquitination by atrogin-1, a process in which mitochondrial superoxide production
may act as a major inducible factor.
Keywords: atrogin-1, Nτ-methylhistidine, FoxO3, cell cultivation, mitochondria
Introduction
Heat stress (HS) induces mitochondrial superoxide generation and muscle protein degradation in
broiler chickens. While plasma corticosterone secretion may be implicated in HS-induced protein
degradation, our recent investigations have demonstrated that, in cultured avian muscle cells, HS-
induced mitochondrial superoxide generation induces protein degradation, probably via protein
ubiquitination by up-regulating atrogin-1 gene transcription (Furukawa et al., 2015). In order to
clarify the differential roles of mitochondrial superoxide production and corticosterone in the onset
of HS-induced protein degradation, the present study initially generated a time-course of changes
in the circulating levels of corticosterone and plasma Nτ-methylhistidine concentrations and the
mRNA level of atrogin-1 and FoxO3, which is one of transcriptional factor of atrogin-1, during
hyperthermic treatment. Thereafter, cell culture experiments were conducted to determine whether
mitochondrial superoxide or corticosterone instigates protein degradation in HS-treated muscle cells.

Animal study: 25-d-old male chickens (Ross) were randomly divided into two groups, each of which
were kept at either thermoneutral (24 °C) or HS conditions (33 °C). Birds were euthanized after 0,
0.5, 1 and 3 d of HS treatment, and the pectoralis superficialis muscles and plasma were collected.
Plasma Nτ-methylhistidine levels, which are used to evaluate protein degradation, and corticosterone
concentrations were determined by a HPLC method and a ELISA kit, respectively. Real-time RT-
PCR analysis was conducted to measure the mRNA levels of atrogin-1 and FoxO3, with the values
normalized to the levels of 18S-rRNA. The rates of superoxide generated from isolated muscle
mitochondria were fluorometrically determined.
Cell culture study: Isolated muscle cells were incubated at 37 °C in medium supplemented with
physiologically relevant levels of corticosterone (0 to 30 ng/ml) or were exposed to HS (41 °C)

conditions for 1 or 6 h, and a superoxide scavenger, Tempol, was added if necessary. After the
treatments, cellular protein content and mRNA levels were determined.

Plasma corticosterone and Nτ-methylhistidine concentrations were increased (P<0.05) after 0.5 d
and 3 d of HS treatment, respectively. In birds exposed to 0.5-d HS treatment, the mRNA levels of
atrogin-1 (P<0.05) and FoxO3 (P=0.09), as well as mitochondrial superoxide production (P<0.05),
were increased compared to their levels in thermoneutral birds. In avian muscle cells, the addition
of corticosterone at physiological levels increased the FoxO3 mRNA level (P<0.05) but did not
change the atrogin-1 mRNA level or cellular protein content. HS-treated cells showed increases in
mitochondrial superoxide generation and atrogin-1 mRNA levels compared to normal cells (P<0.05),
and a subsequently decreased cellular protein content. The addition of 20 ng/ml corticosterone
to HS-treated cells increased the level of FoxO3 mRNA (P<0.05), but it did not result in further
alterations to atrogin-1 mRNA levels, mitochondrial superoxide generation or cellular protein content.
The addition of Tempol to the HS/corticosterone-treated cells, restored the rate of mitochondrial
superoxide generation, the atrogin-1 mRNA level and cellular protein content to near-normal values.
These results suggest that mitochondrial superoxide rather than corticosterone may play an important
role in the induction of muscle protein degradation, probably via up-regulation of ubiquitination
by atrogin-1.
References
Furukawa, K., M. Kikusato, T. Kamizono, H. Yoshida and M. Toyomizu, 2015. Possible involvement of mitochondrial
reactive oxygen species production in protein degradation induced by heat stress in avian muscle cells. Journal
of Poultry Science 52: 260-267.

A comparison of reactive oxygen species regulation in skeletal muscle
with different muscle fibre compositions from heat-stressed birds
M. Kikusato* and M. Toyomizu
Tsutsumidori-Amamiyamachi, Sendai 981-8555, Japan; kmotoi@bios.tohoku.ac.jp
Abstract
The present study investigated differences in reactive oxygen species generation and elimination
between several skeletal muscle types with different muscle fibre compositions from broiler chickens
exposed to heat stress (HS) conditions. HS treatment (34 °C) of birds for 12 hours resulted in increases
in lipid peroxidation levels, compared to thermoneutral birds, in pectoralis superficialis muscle (type
II: 100%) (P<0.05) and extensor digitorum longus (EDL) muscle (type II: 87%) (P=0.08) but did
not change the levels in gastrocnemius muscle (type II: 70%). Mitochondrial superoxide production
was increased (P<0.05) in response to HS treatment in pectoral superficial muscle, whereas this
increase was not observed in gastrocnemius muscle. HS treatment did not alter mRNA levels of
Cu/Zn-superoxide dismutase, catalase, or glutathione peroxidase in either pectoralis superficialis or
gastrocnemius muscles. The Mn-SOD mRNA level did not respond to HS treatment in pectoralis
superficialis muscle, whereas it was augmented (P<0.05) by HS treatment in gastrocnemius muscles.
The mRNA level of avian uncoupling protein in pectoralis superficialis muscle was down-regulated
(P<0.05) in response to HS treatment, whereas a decrease did not occur in gastrocnemius muscles.
We suggest here that avian skeletal muscles with a large amount of glycolytic muscle fibres could be
less tolerant to HS-induced oxidative damage and mitochondrial superoxide production than those
with fewer glycolytic muscle fibres.
Keywords: avUCP, Mn-SOD, antioxidant defense system
Introduction
In broiler chickens, HS causes oxidative damage to several tissues. Our previous studies have shown
that HS-induced oxidative damage in the pectoralis superficialis muscle is due to an overproduction
of reactive oxygen species (ROS). Skeletal muscles are composed of two major muscle fibre types
that are referred as glycolytic (type II) and oxidative (type I) muscle fibres. While a major difference
between oxidative and glycolytic muscles is considered to be their mitochondrial content, recent
investigations have revealed that muscles with different muscle fibre compositions have different
bioenergetic characteristics including mitochondrial ROS generation and antioxidant capacity. Based
on these findings, it can be suggested that HS-induced oxidative damage in other muscles could be
attenuated by regulating ROS generation and/or up-regulating antioxidant capacity. Therefore, the
present study investigated differences in ROS homeostasis between various types of skeletal muscle
isolated from HS-treated chickens.

Ten 3-week old chickens (Ross strain, Gallus gallus) were randomly divided into two groups. One
of the two groups was maintained at thermoneutral conditions (24 °C) while the other group was
exposed to HS conditions (34 °C) for 12 hours. After the HS treatment, the birds were euthanized by
decapitation, and the pectoralis superficialis, extensor digitorum longus (EDL) and gactrocnemius
muscles were excised. Muscle lipid peroxidation was measured using thiobarbituric acid. The
superoxide generated from isolated muscle mitochondria was fluorometrically determined using
Amplex®Red. Real-time RT-PCR analysis was conducted to determine the mRNA levels of
genes related to ROS regulation: Cu/Zn-superoxide dismutase (Cu/Zn-SOD), Mn-SOD, catalase

(CAT), glutathione peroxidase (GPx) as well as avian uncoupling protein (avUCP) and PGC-1α, a
transcriptional cofactor of avUCP and Mn-SOD. The expression values were normalized to that of
18s-ribosomal protein. Student’s t-test was conducted to analyse the statistical differences between
thermoneutral and HS-treated groups in each muscle.

HS treatment increased the lipid peroxidation levels of pectoralis superficialis (type II: 100%, Ueda
et al., 2005; P<0.05) and EDL (type II: 87%, Williams and Dhoot, 1992; P=0.08) muscles relative
to the respective levels in thermoneutral birds, but it did not change the levels in gastrocnemius
muscle (type II: 70%). The level of the HS-induced increase in lipid peroxidation increased with
the percentage of glycolytic muscle fibre among muscles tested. While HS treatment increased
(P<0.05) mitochondrial superoxide production in pectoralis superficialis muscle, this did not occur
in gastrocnemius muscle. The mRNA level of avUCP, which regulates mitochondrial superoxide
production, in pectoralis superficialis muscle was down-regulated in response to HS treatment
(P<0.05) but the decrease was not observed in gastrocnemius muscle. The level of Mn-SOD mRNA
in pectoralis superficialis muscle did not change in response to HS treatment, while the level in
gastrocnemius muscles was augmented by HS treatment (P<0.05). HS treatment did not alter the
mRNA levels of Cu/Zn-SOD, CAT, GPx and PGC-1α in either of these muscles.
The present study demonstrates that avian skeletal muscles that contain a high percentage of glycolytic
muscle fibres could be less tolerant to HS-induced mitochondrial ROS generation than muscles with
lower percentages of glycolytic muscle fibres, and thereby exhibit a greater level of oxidative damage.
Transcription regulation of avUCP and Mn-SOD genes in the muscles may play an important role in
the induction of mitochondrial ROS production and oxidative damage due to HS treatment. Further
comparisons of the molecular machinery governing mRNA expression in the different muscles are
required to elucidate the mechanisms underlying HS-induced oxidative disturbance.
Acknowledgements
Grant-in-Aid: no. 25850182 (Japan Society for the Promotion of Science, Japan).
References
Ueda, M., K. Watanabe, K. Sato, Y. Akiba and M. Toyomizu, 2005. Possible role for avPGC-1alpha in the control of
expression of fiber type, along with avUCP and avANT mRNAs in the skeletal muscles of cold-exposed chickens.
FEBS Letters 579: 11-17.
Williams, K. and G.K. Dhoot, 1992. Heterogeneity and distribution of fast myosin heavy chains in some adult vertebrate
skeletal muscles. Histochemistry 97: 361-370.

Breast muscle protein turnover in broiler breeder parent stock
K. Vignale1,2, J.V. Caldas1,3, J.A. England1, N. Boonsinchai1, P. Sodsee1, M. Putsakum1, E.D.
Pollock1, S. Dridi1, C.M. Owens1 and C.N. Coon1*
1University of Arkansas, Center of Excellence for Poultry Science, Fayetteville, AR 72701, USA;
2current address: Kemin Industries, Des Moines, IA 50317, USA; 3Cobb – Vantress, P.O. Box 1030,
Siloam Springs, AR 72761-1030, USA; ccoon@uark.edu
Abstract
A study was conducted to evaluate the effect of four different feeding regimens on breast muscle
protein turnover in broiler breeder Cobb-500 parent stock (PS) pullets and breeder hens. The four
feeding regimens based on body weight (BW) curves utilised for the study were as follows: everyday
feeding, skip-a-day feeding (Cobb Standard BW curve), lighter BW (BW curve 20% under) and
heavier BW (BW curve 20% over). Each pullet feeding regimen (Treatment) consisted of 150
d-old pullet chicks that were provided a different feeding regimens from 4 to 20 wk of age. Protein
turnover was determined in PS pullets/breeders at 6, 10, 12, 16, 21, 25, 31, 37, 46, and 66 wk of
age. A completely randomized design was used with a 4×10 factorial arrangement (four feeding
regimens, 10 ages), each pullet represented a replicate. Five pullets/breeders at each age were given
an intravenous flooding-dose of 15N phenylalanine (150 mM, 40 atom percent excess) at a dose of 10
ml/kg for the determination of fractional synthesis rate (FSR). After 10 min, birds were euthanized
and the breast muscle (pectoralis major) excised for protein turnover. Excreta was also collected from
each pullet or breeder for 3-methylhistidine analysis. All birds were scanned with a dual energy x-ray
absorptiometry. The FSR in breast muscle of pullets significantly increased from 6 to 12 wk and then
decreased significantly for 31 wk-old breeders. FSR in breeder breast muscle increased significantly
from 31 to 66 wk. Breast muscle fractional degradation rate (FDR) significantly increased from 21
to 31 wk (peak egg production) (P<0.001), then significantly decreased at 66 wk (P<0.0001). There
was a large increase in breast muscle FDR during the transition for the pullet to sexual maturity with
continuing increases in breast muscle FDR through peak egg production
Keywords: fractional degradation rate, fractional synthesis rate, protein turnover, 15N phenylalanine,
GC-MS
Introduction
Skeletal muscle protein turnover in broiler breeders has been studied by Ekmay et al. (2012, 2013).
The researchers showed that the fractional degradation rate (FDR) of breast meat increased at
sexual maturity and then the fractional synthesis rate (FSR) and FDR declined with additional egg
production. The objectives of the present study were: to determine the effect of four different feeding
regimens: skip-a-day (SKIP), everyday (ED), heavier BW (HBW) and lighter body weight (LBW)
on breast muscle protein turnover in pullets and hens from broiler breeder parent stock during and
after sexual maturity.

The effect of four different feeding regimens on breast muscle protein turnover in broiler breeder
Cobb-500 parent stock (PS) pullets and breeder hens was evaluated. The four feeding regimens based
on BW curves utilised for the study were as follows: ED, SKIP (Cobb Standard BW curve), LBW
(BW curve 20% under) and HBW (BW curve 20% over). Each pullet feeding regimen (Treatment)
consisted of 150 d-old pullet chicks that were provided a different feeding regimens from 4 to 20
wk of age. Protein turnover was determined in PS pullets/breeders at 6, 10, 12, 16, 21, 25, 31, 37,
46, and 66 wk of age. A completely randomized design was used with a 4×10 factorial arrangement

(four feeding regimens, 10 ages), each pullet represented a replicate. Analysis of variance was
performed using JMP pro 11 software (second edition, 2014). Five pullets/breeders at each age were
given an intravenous flooding-dose of 15N phenylalanine (150 mM, 40 atom percent excess) at a
dose of 10 ml/kg for the determination of FSR. After 10 min, birds were euthanized and the breast
muscle (pectoralis major) excised and frozen in liquid nitrogen for protein turnover. Excreta was also
collected from each pullet or breeder for 3-methylhistidine analysis. All birds were scanned with a
dual energy x-ray absorptiometry. Protein synthesis and degradation was determined via GC-MS.

FSR in breast muscle of pullets significantly increased from 6 to 12 wk (3.62 to 10.93%/d; P=0.01)
and then decreased significantly for 31 wk-old breeders (5.77%/d; P=0.01). FSR in breeder breast
muscle increased significantly from 31 to 66 wk (5.77 to 11.76%/d; P=0.002). Breast muscle FDR
significantly increased from 21 wk (5.70%/d) to 25 wk (13.81%/d, first egg) and 31 wk (22.46%/d,
peak egg production) (P<0.001), then FDR significantly decreased at 66 wk (6.53%/d; P<0.0001)
(Figure 1). The present results are in agreement with Ekmay et al. (2012, 2013). The researchers
showed that the FDR of breast meat increased at sexual maturity. There was a large increase in
breast muscle FDR during the transition for the pullet to sexual maturity with continuing increases
in breast muscle FDR through peak egg production and a simultaneous loss in breeder lean mass
during this same time period. Since protein turnover is energetically expensive, it is believed that
broiler breeders rely on skeletal muscle tissue as a source of nutrients for egg production.
30
25
20
%/d
15
10
0
6 10 12 16 21 25 31 37 46 66
Age (week)
ED SKIP LBW HBW
Figure 1. Skeletal muscle fractional degradation rate for broiler breeder pullets and hens by age
and feeding regimen (SKIP = skip-a-day; ED = everyday; HBW = heavier BW; LBW = lighter BW).
References
Ekmay, R.D., C. Salas, J. England, S. Cerrate and C.N. Coon, 2012. The impact of diet and age on protein turnover
and its underlying mechanisms in broiler breeders. Poult. Sci. 91, Suppl. 1: 142.
Ekmay, R.D., C. Salas, J. England, S. Cerrate and C.N. Coon, 2013. The effects of age, energy and protein intake
on protein turnover and the expression of proteolysis-related genes in the broiler breeder hen. Comparative
Biochemistry and Physiology Part B, Biochemistry & Molecular Biology 164: 38-43.

De novo lipogenesis in broiler breeder hens
N. Boonsinchai, K. Hilton*, G. Mullenix, J.V. Caldas, A. Magnuson, J.A. England and C.N. Coon
University of Arkansas, Center of Excellence for Poultry Science, 1260 W Maple, Fayetteville, AR
72701, USA; kmhilton@email.uark.edu
Abstract
The de novo lipogenesis (DNL) is important in fat deposition and efficiency of animal production. Two
experiments were conducted using Cobb 500 hens for the purpose of gaining a better understanding
of DNL with non-lipid precursors and to evaluate the differences between young and older breeder
hens. Hens (25 wk old) in experiment 1 were dosed (50 mg/d) with U-13C Glucose, L-13C Alanine,
or L-13C Leucine for 14 d. Hens (28 and 40 wk of age) in experiment 2 were dosed with 40 mg/d
of U-13C Glucose for 14 d. Eggs and abdominal fat samples from the breeder hens were saved at
different times. The enrichment and concentrations of labelled palmitic acid (LPA) were analysed.
In experiment 1, the enrichment of LPA in the yolk, derived from glucose and alanine significantly
decreased (P<0.0001) from d1 (0.578 vs 0.381%) to d10 (0.032 and 0.047%), respectively, whereas
the enrichment of LPA derived from leucine significantly increased (P<0.0001) from d1 (0.112%)
to d10 (0.355%). In experiment 2, young hens showed significantly lower concentration of LPA
in the yolk on d1 (3.774 vs 2.706 mg/egg; P<0.05), but significantly higher on d7 (0.042 vs 0.178
mg/g; P<0.05) as compared to older hens, respectively, while there was no difference on d14. LPA
was only detected on d1 in abdominal fat of young hens in a noticeable amount (1.139 mg/g) and
the concentration decreased to lower levels on d14 (0.188 mg/g). These results indicate that breeder
hens use amino acids as precursors for DNL and the rate in young hens is higher than the older hens.
Keywords: de novo lipogenesis, non-lipid precursors, fatty acid synthesis, enrichment
Introduction
The de novo lipogenesis (DNL) and the synthesis of triglycerides are important factors in fat
deposition and efficiency of animal production. The precursors for DNL can be either glucose,
acetyl-CoA, or amino acids which can come from both food and breakdown of muscle tissue by
protein turnover (Murphy, 2006). Fractional protein degradation rate in breeder hens has been
shown to increase significantly at sexual maturity, and gradually decrease after peak production
(Vignale, 2014), suggesting that young breeder hens may use muscle protein for both yolk and
albumen formation more than older hens. The objectives of the present study were to determine if
non-glucose precursors can be used for fatty acid synthesis and to investigate difference in DNL
between young and old breeder hens.

The first experiment, 18 Cobb 500 hens (25 wk of age) were assorted into three groups of six hens
each. Four hens in each group were dosed (50 mg/hen/d) with U-13C glucose, L-13C alanine, or
L-13C leucine for 14 d, and two hens were used as control. After 14 d of dosing, enrichment in
eggs collected on d1, d5 and d10 were analysed. All hens were euthanized on d10 and abdominal
fat samples were taken. The second experiment compared the rate of DNL between young (28 wk)
and older (40 wk) breeder hens. Fifteen hens from each age group were used; twelve hens were
dosed with 40 mg/hen/d of U-13C glucose for 14 d, and the remainder of hens were used as control.
After 14 d of dosing, the enrichment in eggs saved on d1, d7, and d14 was determined. For each
sampling time, three hens from each treatment and one hen from control group were euthanized
and fat samples were taken. Fatty acid methyl esters were prepared and the percentage enrichment

and concentration (mg/egg) of labelled palmitic acid analysed using gas chromatography equipped
with flame-ionization detector and mass spectrometer.

In experiment 1, the concentration of labelled palmitic acid (LPA) in the yolk, which was derived
from U-13C glucose or L-13C alanine significantly decreased (P<0.0001) from d1 (5.578 and 4.041
mg/egg) to d5 (0.234 and 1.768 mg/egg) and were both almost negligible on d10 (0.338 and 0.085
mg/egg), respectively, whereas the concentration of labelled palmitic acid derived from L-13C
leucine significantly increased (P<0.0001) from d1 (0.713 mg/egg) to d10 (2.466 mg/egg) (Figure
1). These results show that young hens use amino acids not only for protein synthesis but also lipid
synthesis. The conversion of glucogenic amino acids (alanine and glutamic acid) into lipids in fat and
lean chickens was reported by Geraert et al. (1990). The different conversion patterns of leucine and
alanine into LPA in present research could be because leucine is a ketogenic amino acid which can
be converted into acetyl-CoA or acetoacetate which can either be used for energy purpose or fatty
acid synthesis (Margaret and John, 2013). In experiment 2, on d1 the total amount of LPA per egg
was significantly higher in 40 wk hens compared to 28 wk hens (3.774 vs 2.706 mg/egg; P<0.05).
However, on d7, the concentrations of labelled palmitic acid in the yolk were significantly lower
in 40 wk old hens compared to 28 wk hens (1.842 vs 3.018 mg/egg; P<0.05), while there was no
difference on d14. This data suggest that DNL for egg lipid in young hens is higher than in older
hens, which is consistent with results from Salas (2011) who proposed that young breeder hens use
DNL as a main source of egg yolk lipids while older breeder hens utilised more dietary and body
fat to make the egg yolk. Unlike the yolk, abdominal fat of young hens contained 10 fold labelled
palmitic acid (1.139 vs 0.138 mg/g on d1; P<0.01) compared to 40 wk hens, which decreased
continuously by d of sampling, and was almost negligible on d14 (0.188 vs 0.032 mg/g; P<0.001).
The total amounts of LPA in the abdominal fat of the hens is shown in Figure 2 based on concentration
per gram. These results were in agreement with Buyse et al. (2004) who proposed that the rate of
glucose oxidation increased with age of broilers, indicating less DNL in older chicks. These results
indicate that breeder hens use amino acids and glucose as precursors for DNL, and rate of DNL in
young hens is higher than in the older hens.
5
Labeled palmitic acid (mg/egg)
0
Day 1 Day 5 Day 10
Figure 1. Concentration of palmitic acid derived from labelled glucose, alanine and leucine.

6 90
80
Labeled palmitic acid (mg/egg) 5
70
4 60
50
3
mg/fat
40
2 30
20
1
10
0 0
Day 1 Day 7 Day 14
Age of bird (day)
mg/egg (28 wk) mg/egg (40 wk) mg/fat (28 wk) mg/fat (40 wk)
Figure 2. Concentration of labelled palmitic acid in total egg yolk and abdominal fat pad of young
and old breeder hens.
References
Buyse, J., B. Geypens, R.D. Malheiros, V.M. Moraes, Q. Swennen and E. Decuypere, 2004. Assessment of age-related
glucose oxidation rates of broiler chickens by using stable isotopes. Life Sciences 75: 2245-2255.
Geraert, P.A., M.L. Grisoni, S. Guillaumin, C. Law and M. Larbier, 1990. Amino acids as energy sources in genetically
fat and lean chickens. In: G. Lubec and Rosenthal, G.A. (eds). Amino acids, chemistry, biology and medicine.
ESCOM Science Publishers B.V., Leiden, the Netherlands, pp. 1125-1131.
Margaret, E.B., and T.B. John, 2013. Chapter 14: Amino acid metabolism. In: M.H. Stipanuk and Caudill, M.A.
(eds.). Biochemical, physiological, and molecular aspects of human nutrition. Elsevier Saunders, Saint Louis,
MO, USA, pp. 287-330.
Murphy, E.J., 2006. Stable isotope methods for the in vivo measurement of lipogenesis and triglyceride metabolism.
J. Anim. Sci. 84(E. Suppl.): E94-E104.
Salas, J.C., 2011. Determination of metabolizable energy requirements and mechanisms of energy mobilization towards
lipid egg formation in broiler breeder hens. PhD thesis, University of Arkansas, Fayetteville, AR, USA.
Vignale, K.L., 2014. Protein turnover in broilers, layers, and broiler breeders. PhD thesis, University of Arkansas,
Fayetteville, AR, USA.

Application of compartmental analysis to study nitrogen kinetics in
broilers
A.R. Troni1*, M.H. Green2, J.L. Ford2, N.K. Sakomura1, R.M. Suzuki1, D.M.B. Campos3, N.J.
Peruzzi1 and L.G. Pacheco1
1Universidade Estadual Paulista, 14884-900 Jaboticabal, SP, Brazil; 2Pennsylvania State University,
University Park, 16801 PA, USA; 3Universidade Federal de São Carlos, Lagoa do Sino, 18290-000
Buri, SP, Brazil; allan.reis.troni@gmail.com
Abstract
The aim of this study was applying model based compartmental analysis to the data to describe
nitrogen (N) kinetics in broilers at three ages. Male broilers (n=136) were fed diets naturally enriched
with 15N and were slaughtered during either an initial phase (0 to 14 d), growth phase (14 to 42 d) and
final phase (35 to 70 d). Samples of feed, jejunum, liver, plasma, breast, feathers and excreta were
collected at slaughter, and isotope concentration as a function of time was determined by isotope ratio
mass spectrometry. A model with five compartments, five components and 19 adjustable parameters
was developed to fit the data. Results confirmed the key role of liver and plasma in N metabolism,
with N transit times of 42 and 22 min, respectively, in the first phase, 10 h 48 min and 5 h 1 min
in the second phase, and 11 h 37 min and 10 h 42 min in the third phase. The high efficiency of
broilers in converting feed to animal growth was reflected on transit times to peripheral tissues of
10 d 4 h and 5 d 17 h in pectoralis major muscle and feathers, respectively in the first phase, 19 d 2
h and 10 d 21 h in the second phase, and 29 d 14 h and 28 d 19 h in the third phase. This application
of modelling enhances understanding of the dynamics of N transfer between tissues in broilers and
the changes in kinetics over time.
Keywords: metabolism, modelling, nutrition, stable isotopes
Introduction
It is important to understand nutrient metabolism and utilisation in order to optimize the production of
foods of animal origin. In the case of broilers, some information has been published on the impact of
nitrogen (N) metabolism on nutrient requirements and regarding the use of different food sources, but
nutrient kinetics and interrelationships among the various tissues have not been extensively studied.
Mathematical modelling of tracers kinetic data can be used to obtain information on the dynamic
behaviour of biological systems (Atkins, 1969). The aim of this study was to describe and quantify
N dynamics in broilers at three ages using 15N as a metabolic tracer and then applying model-based
compartmental analysis to the data.

Male broilers (Cobb 500®, n=136) were fed diets naturally enriched with 15N according to
requirements (Rostagno et al., 2011). To collect data, broilers were slaughtered during an initial
phase (0 to 14 d), a growth phase (14 to 42 d) and a final phase (35 to 70 d) in compliance the Unesp
Ethics Committee No. 9999/14. Samples of feed, jejunum, liver, plasma, breast, feathers and excreta
were collected at slaughter, and isotope concentration in function of time was determined by IRMS.
A compartmental model was developed using the Windows version of the Simulation, Analysis and
Modeling software (WinSAAM, v.3.0.8) and the kinetic parameters describing N metabolism were
determined according to Cifelli et al. (2007).

N transit and residence times are shown in Table 1. Mean transit time corresponds to a single pass
of N into a compartment before it exits reversibly or irreversibly; residence time is the total time
that N remains in a compartment during successive passages until it is irreversibly lost from the
system. Results show that, in the final stage of life, N remains in the pectoralis muscle for a long
time (>64 d). This is not surprising since this muscle is responsible for most of the system’s protein
turnover (Murray et al., 2012), because the flow of free amino acids is associated with the return of
nitrogenous material for the plasma. Transit and residence times were lower in organs that digest,
metabolise and transport N (jejunum, liver and plasma), as they are structures that prepare the N to
its final disposal destination (muscles and feathers). Once N enters feathers, it does not contribute
effectively to the turnover, as there is loss and subsequent replacement of this tissue (Leeson and
Walsh, 2004). The development of this compartmental model allowed us to quantify the kinetic
parameters and contribute to the understanding of protein metabolism during growth in broilers.
Table 1. Kinetic parameters for 15N in broiler chickens.1
Compartment Transit time (days:hours:minutes) Residence time (days:hours:minutes)
Initial Growth Final Initial Growth Final
Jejunum 0:01:39 0:02:14 0:02:18 0:01:18 0:02:18 0:23:59

Liver 0:00:42 0:10:48 0:11:37 0:22:58 0:11:50 1:03:27
Plasma 0:00:22 0:05:01 0:10:42 0:13:42 1:08:46 2:02:30
Breast 10:04:48 19:02:46 29:14:01 16:05:01 46:19:12 64:19:41
Feathers 5:17:08 10:21:43 28:19:15 11:03:15 13:11:25 31:14:01
1 Initial = 0-14 days; growth = 14-42 days; final = 35-70 days.
Acknowledgements
This project was funded by Fapesp N. 2013/25761-4 and Capes N. 99999.002424/2015-00.
References
Atkins, G.L., 1969. Multicompartment models for biological systems. Methuen & Co. ltd, London, UK, 153 pp.
Cifelli, C.J., J.B. Green and M.H. Green, 2007. Use of model-based compartmental analysis to study vitamin A kinetics
and metabolism. Vitamins and Hormones 75: 161-195.
Leeson, S. and T. Walsh, 2004. Feathering in commercial poultry I. Feather growth and composition. World’s Poultry
Science Journal 60: 42-51.
Murray, R.K., D.K. Granner and P.A. Mayes, 2012. Harpers illustrated biochemistry (29th Ed.). McGraw Hill
Professional, New York, NY, USA, 704 pp.
Rostagno, H.S., L.F.T. Albino, J.L. Donzele, P.C. Gomes, R.F. Oliveira, D.C. Lopes, A.S. Ferreira, S.L.T. Barreto and
R.F. Euclides, 2011. Tabelas brasileiras para aves e suínos: composição de alimentos e exigências nutricionais.
Universidade Federal de Viçosa, Viçosa, Brazil, 252 pp.

Incorporation of 15N from l-threonine in plasma of broilers
R.M. Suzuki1, N.K. Sakomura1*, J.A. Bendassolli2, J.C. Denadai3, A.R. Troni1, D.M.B. Campos4,
L.G. Pacheco1 and P.M. Júnior1
1São Paulo State University – Jaboticabal, SP, Brazil; 2University of São Paulo – Piracicaba, SP,
Brazil; 3São Paulo State University – Botucatu, SP, Brazil; 4Federal University of São Carlos –
Lagoa do Sino, SP, Brazil; sakomura@fcav.unesp.br
Abstract
The aim of the study was to compare the incorporation of L-[15N] threonine in plasma of broilers in
two different phases: starter (4-16 d) and finisher (32-44 d). A total of 102 male broilers were fed a
diet enriched with L-[15N] threonine for five days and then the animals were slaughtered daily over
the experimental period. Blood samples were collected and the isotope concentration was determined
by an isotope ratio mass spectrometer. Initial, maximum and final enrichment values measured in
δ15N for starter and finisher phases, respectively, were 3.74 and 0.99; 33.88 and 80.48; 3.74 and
28.40‰. The turnover constants determined were 0.589 and 0.322 in the starter and finisher phase,
respectively. These results showed the incorporation of L-[15N] threonine is higher in the starter phase.
Keywords: metabolism, stable isotopes, amino acid-labelled
Introduction
Stable isotopes have contributed to knowledge of the metabolism of nutrients, as has turnover,
which includes the synthesis and breakdown of a body component. Plasma has been widely used in
live animals for analysing tissue samples, and reflects isotopic enrichment of the diet (Hobson and
Clark, 1992). Plasma amino acid concentration is related to performance (Ishibashi et al., 1998).
Therefore, the incorporation of L-[15N] threonine in plasma of broiler chickens was studied and
compared for two different phases.

The incorporation of L-[15N] threonine by plasma was analysed in two different phases: starter
(4 to 16 days) and finisher (32 to 44 days) in 102 broiler chickens male Cobb 500®. The diet was
formulated according to the requirements (Rostagno et al., 2011). In the first five days of each
experimental period (4 to 9 days and 32 to 37 days), the diet was enriched with L-[15N] threonine
(69.6 mmol of L-[15N] threonine/kg). Plasma samples (three replications each) were collected at 0
(standard enrichment), 6, 12, 24, 48, 72, 96, 120, 126, 132, 144, 168, 182, 206, 240, 264, 288 hours.
Enrichment values expressed in δ15N were determined in by isotope ratio mass spectrometry and fitted
to an exponential model using the software OriginPro 8 (Origin Lab Inc.).The biological parameters
of the model proposed by Ducatti et al. (2002) were adopted including the turnover constant.

Figure 1 shows the mean enrichment of plasma. The values of initial enrichment obtained in the
starter and finisher phases were 3.74 and 0.99‰, respectively. The maximum enrichment in the
starter phase was 33.88‰ reached at 96 h, while in the finisher phase it was 80.48‰ at 120 hours.
The utilisation of L-[15N] threonine was different in these phases, considering the rate of protein
synthesis decreases with age while the rate of degradation increases, reducing the growth rate (Griffin
and Goddard, 1994). Hence, the decay of enrichment is less accentuated in the finisher phase due
to its slower incorporation shown by the turnover constants (kinitial=0.589 and kfinal=0.322). The
lowest enrichment in the starter phase was 3.74‰ at 264 h and 28.40‰ at 240 hours in the finisher

80 Phases
Starter
70 Finisher
60
δ 15N (‰)
50
40
30
20
10
0
-1 0 1 2 3 4 5 6 7 8 9 10 11 12 13
Time (days)
Figure 1. Mean enrichments of plasma in broilers chickens in two different phases
phase. In the finisher phase, the enrichment did not return to its initial value. It can be explained by
the 15N degraded in the others tissues by passing the plasma or due to a 15N excess, since the amount
of L-[15N] threonine provided was based on daily body weight of the bird. In the starter phase, the
final value returned to the beginning value representing that all 15N in this phase was excreted or
deposited. Therefore, considering both phases, the incorporation of L-[15N] threonine was greater
in the starter phase.
Acknowledgements
This project was funded by Fapesp No. 2013/25761-4.
References
Ducatti, C., A.S. Carrijo, A.C. Pezzato and P.F.A. Mancera, 2002. Modelo teórico e experimental da reciclagem do
carbono-13 em tecidos de mamíferos e aves. Scientia Agricola 59: 29-33.
Griffin, H.D. and C. Goddard, 1994. Rapidly growing broiler (meat-type) chickens: their origin and use for comparative
studies of regulation of growth. International Journal of Biochemistry 26: 19-28.
Hobson, K.A. and R.G. Clark, 1992. Assessing avian diets using stable isotopes I: turnover of 13C in tissues. Condor
94: 181-188.
Ishibashi, T., Y. Ogawa, T. Itoh, S. Fujimura, K. Koide and R. Watanabe, 1998. Threonine requirements of laying hens.
Poultry Science 77: 998-1002.
Rostagno, H.S., L.F.T. Albino, J.L. Donzele, P.C. Gomes, R.F. Oliveira, D.C. Lopes, A.S. Ferreira, S.L.T. Barreto and
R.F. Euclides, 2011. Tabelas brasileiras para aves e suínos: composição de alimentos e exigências nutricionais.
Universidade Federal de Viçosa, Viçosa, Brazil, 252 pp.

Biomarkers of optimum dietary branched chain amino acids for the best
growth performance in pigs
E.A. Soumeh1, M.S. Hedemann1, H.D. Poulsen1, E. Corrent2, J. van Milgen3 and J.V. Nørgaard1*
1Dept. of Animal Science, Aarhus University, Foulum, 8830 Tjele, Denmark; 2Ajinomoto Eurolysine
S.A.S., 75817 Paris Cedex 17, France; 3INRA, UMR1348 PEGASE, 35590 Rennes, France;
janvnoergaard@anis.au.dk
Abstract
The objective of the current study was to identify biomarkers of branched chain amino acids (BCAA)
intake status that could be linked to the best animal growth performance. Three dose-response
studies were carried out to collect blood and urine samples from pigs fed diets with increasing levels
of isoleucine (Ile), valine (Val), or leucine (Leu) followed by a non-targeted LC-MS approach to
characterize the metabolic profile of plasma and urine, when the level of BCAA in the diet is optimum
for the best animal growth performance. The LC-MS method could separate and identify the plasma
and urine metabolites which were discriminating the pigs fed optimum dietary level of Ile, Val, and
Leu. Among the discriminating plasma and urine metabolites, that correlated closely to the highest
animal growth performance, were plasma glycocholic acid and taurocholic acid as biomarkers of the
optimum Ile level in the diet, and plasma creatine and urinary 2-aminoadipic acid, ascorbic acid, and
choline as biomarkers of optimum Leu level. No good biomarkers were identified in the Val study.
Keywords: branched chain amino acid, biomarker, growth performance, metabolomics, pigs
Introduction
The existing estimates of branched chain amino acids (BCAA) requirements have been obtained
by empirical approach in dose-response experiments using animal growth performance (e.g. feed
intake, weight gain, and feed efficiency) as response criteria. Previous dose-response studies of our
lab demonstrated that 0.52 standardised ileal digestible (SID) isoleucine:lysine (Ile:Lys), 0.70 SID
valine (Val):Lys, and 0.93 SID leucine (Leu):Lys are the minimum Ile, Val, and Leu requirements,
respectively, to support the best growth performance of weaned piglets (Soumeh et al., 2014, 2015a,b).
Currently, the metabolic profile of pigs when feeding optimum levels of BCAA for the best growth
performance is unknown. Nutritional metabolomics has recently become a powerful approach to
provide a comprehensive overview of metabolic alterations in response to specific feed intake which
cannot be measured by traditional assays. In the current study, it was hypothesized that feeding
increasing levels of Ile, Leu, or Val would result in appearance of unique metabolites in blood and
urine which correlate with animal growth performance. The identification and characterization of
these biomarkers could be used to further improve the methods for estimating BCAA requirements
or protein quality.

Three dose-response experiments studied growth performance of pigs (n=96 in each study), which
were fed with increasing levels of SID Ile:Lys (0.42, 0.46, 0.50, 0.54, 0.58, and 0.62); SID Val:Lys
(0.58, 0.62, 0.66, 0.70, 0.74, and 0.78); and SID Leu:Lys (0.70, 0.80, 0.90, 1.00, 1.10, and 1.20).
The experiments started 1 week after weaning. At d 8 and 15 of each experiment, after an overnight
fast, pigs were supplied with 25 g/kg BW0.75 of feed and blood and urine samples were collected
3 h later from 8 pigs per treatment. Blood and urine samples were analysed by a HPLC-MS in a
non-targeted metabolomics approach to study the metabolic profile. The data acquired from HPLC-
MS were subjected to multivariate data analyses. Principle component analyses (PCA) and partial
least squares regression (PLS) were used for visualizing data, clustering metabolites and pattern

recognition. The discriminating metabolites were chosen using scaled regression coefficients plots.
The metabolites were subjected to statistical analyses using the MIXED procedure of SAS, where
the diet was included in the model as fixed effect, and room and period as random effects. Day of
sampling was included in the model as fixed effect, and the average of sampling days 8 and 15 was
therefore considered for each metabolite. Initial body weight of the pigs was included in the model
as a covariate.

Distinct grouping according to experimental diets were observed by PCA score plots and the PLS
models showed that the main differences in plasma and urine metabolites originated from AA and
their derivatives and from bile acids when pigs were fed increasing dietary levels of BCAA. Results
showed that concentrations of plasma hypoxanthine and tyrosine were higher, while concentrations
of glycocholic acid, tauroursodeoxycholic acid, and taurocholic acid were lower, when the dietary
level of Ile was optimum for animal growth. Urinary hippuric acid was up-regulated by the optimum
Ile level; while plasma hippuric acid was down-regulated by the optimum dietary Val. Plasma
3-methyl-2-oxovaleric acid and creatine were lowest when dietary Leu was optimum for animal
growth. The optimum level of Leu in the diet up-regulated urinary ascorbic acid and choline, while
2-aminoadipic acid, acetyl-DL-valine, Ile, 2-methylbutyrylglycine, and Tyr were down-regulated.
Among the discriminating metabolites to increasing dietary level of Ile, Val, and Leu obtained from
PLS regression coefficients, those which showed similar pattern to animal growth performance traits
were identified as biomarkers of dietary optimum BCAA for the best animal growth performance.
Plasma glycocholic acid and taurocholic acid were regarded as biomarkers of the optimum Ile level
in the diet and plasma creatine and urinary 2-aminoadipic acid, ascorbic acid, and choline were
regarded as biomarkers of optimum dietary Leu level. The optimum Val level in the diet had a less
pronounced metabolic response reflected in plasma or urine than other BCAA. In conclusion, several
metabolites in both blood and urine were identified as being biomarkers for optimal growth in pigs
fed increasing levels of BCAA. These biomarkers were used as response criteria in a short-term
dose-response studies to evaluate the BCAA requirements (Nørgaard et al., 2016).
References
Nørgaard, J.V., E.A. Soumeh, M. Curtasu, H.D. Poulsen, E. Corrent, J. van Milgen and M.S. Hedemann, 2016. Blood
biomarkers in short-term studies on amino acid requirement in pigs. In: J. Skomial and H. Lapiere (eds.). Energy
and protein metabolism and nutrition. EAAP Scientific Series No. 137. Wageningen Academic Publishers,
Wageningen, the Netherlands, pp. 349-350.
Soumeh, E.A., J. van Milgen, N.M. Sloth, E. Corrent and H.D. Poulsen, 2015a. The optimum ratio of standardized ileal
digestible leucine to lysine for 8 to 12 kg female pigs. Journal of Animal Science 93: 2218-2224.
Soumeh, E.A., J. van Milgen, N.M. Sloth, E. Corrent, H.D. Poulsen and J.V. Nørgaard, 2014. The optimum ratio of
standardized ileal digestible isoleucine to lysine for 8-15 kg pigs. Animal Feed Science and Technology 198:
158-165.
Soumeh, E.A., J. van Milgen, N.M. Sloth, E. Corrent, H.D. Poulsen and J.V. Nørgaard, 2015b. Requirement of
standardized ileal digestible valine to lysine ratio for 8- to 14-kg pigs. Animal 9: 1312-1318.

Potential contribution of net portal absorption of volatile fatty acids to
energy expenditure in Iberian gilts fed acorn
L. González-Valero, M. Lachica, J.M. Rodríguez-López, L. Lara and I. Fernández-Fígares*
Department of Animal Nutrition, Estación Experimental del Zaidín, CSIC, Camino del Jueves s/n,
18100 Armilla, Granada, Spain; ifigares@eez.csic.es
Abstract
The aim of this work was to determine the potential contribution of net portal-drained viscera
(PDV) flux of volatile fatty acids (VFA) to whole body heat production (HP) in Iberian gilts fed
with acorn, and to find out the effect of acorn feeding over time. Two sampling periods (P-I and P-II)
were done with six gilts (34 kg average body weight) set up with three catheters: in carotid artery
and portal vein for blood sampling, and ileal vein for para-aminohippuric acid (PAH) infusion to
measure portal plasma flow. Pigs were fed at 2.5× metabolisable energy for maintenance (MEm) a
commercial diet in two portions, at 09.00 (0.25) and 15.00 h (the remaining 0.75). The day previous
to P-I, pig diet changed to 2.4 kg of acorn. After feeding 0.25 of ration a serial blood collection (n=10)
followed for 6 h. Following identical protocol, one week later P-II was done. PDV HP calculated
from O2 consumption was 22.8% greater (P<0.05) in P-II than in P-I but net PDV flux of VFA was
not affected. Potential contribution of VFA to whole body HP was almost identical in both periods
(5.7%), representing 10.9% of MEm. Pigs adapted for one week to an acorn diet had increased PDV
HP without an increase in the net PDV flux of VFA, indicating that nutrient(s) other than VFA may
be responsible of the increased PDV HP.
Keywords: energy, pig, portal-drained viscera, volatile fatty acid
Introduction
Most consumers appreciated Iberian pig (Sus mediterraneus) cured products come from animals that
nowadays are reared intensively using commercial diets up to about 100 kg body weight (BW), and
subsequently transferred to the Mediterranean forest for the final fattening period where they face a
radical dietary shift eating acorns from Quercus sp. and grazing available pasture. The acorn is very
palatable but with low protein content, unbalanced amino acid profile, and high amount of condensed
tannins (particularly in the hulls) bound to dietary and endogenous proteins; conversely, it is rich in
starch and lipids. Studies on portal-drained viscera (PDV) metabolism in native breeds are scarce.
Our aim was to determine the potential contribution of net PDV flux of volatile fatty acids (VFA)
to whole body heat production (HP) in Iberian gilts fed acorn, and to find out its effect over time.

Two sampling periods were done with six gilts set up with three catheters: in carotid artery, and portal
and ileal veins. Pigs were fed at 2.5× metabolisable energy for maintenance (MEm) a commercial
diet (145 g crude protein (CP)/kg DM and 14.3 MJ ME/kg DM) in two portions, at 09.00 (0.25)
and 15.00 h (the remaining 0.75) during the whole study. The day previous to the first sampling
period (P-I), diet changed to 2.4 kg of acorn (54.7 g CP and 18.86 MJ ME intakes). Forty five min
prior to blood sampling a pulse dose of PAH (15 ml, 2% w/v) was infused into ileal vein followed
by continuous infusion (0.8 ml/min). Blood was taken simultaneously from carotid and portal vein
0.5, 1, 1.5, 2, 2.5, 3, 3.5, 4, 5 and 6 h after feeding 0.25 of daily ration. O2 content, haemoglobin
saturation and haematocrit were measured. Pigs continued the acorn diet for one week, then the
second sampling period (P-II) was done following identical protocol. Plasma was analysed for PAH
and VFA (acetic, propionic and butyric acids). Portal plasma flow (PPF), net PDV flux of VFA and
PDV HP were obtained. Assumed values of MEm and kg for Iberian gilts were 422 kJ/kg0.75 BW/d

and 0.582, respectively (Nieto et al., 2012). Time of sampling was not statistically significant and
data were pooled and subjected to ANOVA-I.

PPF and PDV HP values (Table 1) were similar for P-I to Iberian and for P-II to Landrace gilts fed
a commercial diet under identical feeding schedule (González-Valero et al., 2016). PDV HP was
22.8% greater (P<0.05) in P-II than in P-I although net PDV flux of VFA was only slightly greater
(4.6%). Potential contribution of VFA to whole body HP was almost identical in both periods (5.7%),
representing 10.9% of MEm which is less than half of the value reported for Meishan pigs fed high
fibre diet (Yen et al., 2004). Pigs adapted for one week to an acorn diet increased PDV HP with no
increase in the net PDV flux of VFA, indicating that nutrient(s) other than VFA may be responsible
of the increased PDV HP.
Table 1. Portal plasma flow (PPF), portal-drained viscera (PDV) heat production (HP), net PDV
flux of volatile fatty acids (VFA), potential VFA HP, and their contribution to MEm and whole body
HP in Iberian pigs (34 kg average BW; n=6) fed acorns at identical intake level in the 1st (P-I) and
2nd (P-II) sampling period (mean ± SEM for 10 measurements).
P-I P-II SEM P-value
PPF (ml/min) 622 855 18.38 0.001

PDV HP (kJ/h)1 46.8 60.6 1.331 0.002
Net PDV flux of VFA (mmol/h) 22.7 23.8 1.680 0.767
Acetate 13.2 16.0 1.149 0.265
Propionate 8.62 6.35 0.608 0.111
Butyrate 0.91 1.40 0.082 0.025
Total VFA HP (kJ/h)2 26.7 26.7 1.905 0.987
Acetate 11.5 14.0 1.004 0.265
Propionate 13.2 9.69 0.928 0.111
Butyrate 1.98 3.04 0.179 0.025
Total VFA HP/MEm3 0.113 0.104
Total VFA HP/whole body HP4 0.057 0.056
1 Energy equivalent: O2 = 20.4 kJ/l.

2 Energy equivalents: acetic = 874 kJ/mol; propionic = 1,527 kJ/mol; butyric = 2,180 kJ/mol.
3 ME = 422 kJ/kg0.75 BW/d (Nieto et al., 2012).
m
4 Whole body HP = [(ME intake – ME ) × (1 – k )] + ME ; k = 0.582 (Nieto et al., 2012).
m g m g
Acknowledgements
Grant AGL2006-05937/GAN from the Ministry of Science and Education of Spain (FEDER).
References
González-Valero, L., J.M. Rodríguez-López, M. Lachica and I. Fernández-Fígares, 2016. Contribution of portal-drained
viscera to heat production in Iberian gilts fed a low-protein diet: comparison to Landrace. Journal of Science of
Food and Agriculture 96: 1202-1208.
Nieto, R., L. Lara, R. Barea, R. García-Valverde, M.A. Aguinaga, J.A. Conde-Aguilera and J.F. Aguilera, 2012.
Response analysis of the Iberian pig growing from birth to 150 kg body weight to changes in protein and energy
supply. Journal of Animal Science 90: 3809-3820.
Yen, J.T., V.H. Varel and J.A. Nienaber, 2004. Metabolic and microbial responses in western crossbred and Meishan
growing pigs fed a high-fiber diet. Journal of Animal Science 82: 1740-1755.

Effect of dietary net energy and digestible lysine levels on growth
performance of growing pigs
J.K. Htoo1* and J. Morales2
1Evonik Nutrition & Care GmbH, 63457, Hanau-Wolfgang, Germany; 2PigChampPro Europa, SL,
Segovia, Spain; john.htoo@evonik.com
Abstract
A 28-d experiment was conducted to evaluate the effect of dietary net energy (NE) and standardised
ileal digestible (SID) Lys levels on performance of 23 to 45 kg pigs. A total of 384 mixed-sex pigs
(initial BW of 23.3 kg) were assigned to 6 dietary treatments with 8 pen replicates (4 barrows and
4 gilts/pen) per treatment using a 2×3 factorial design with 2 levels of SID Lys (0.97 and 1.06%)
and 3 levels of NE (9.65, 10.00 and 10.35 MJ/kg). There was no SID Lys×NE interaction for any
performance parameter (P>0.05). Overall, feed intake was not affected by the dietary treatments
(P>0.05). Increasing SID Lys from 0.97 to 1.06% increased final BW, average daily gain (ADG) and
feed:gain (G:F) (P<0.05). Increasing dietary NE levels did not affect ADG (P>0.05) but increased
G:F (P=0.011). Based on these results the ADG and G:F of 23 to 43 kg pigs maximized when the
diet contains 1.06% SID Lys and SID Lys:NE of 1.02 to 1.06 g/MJ.
Keywords: lysine, net energy, pig, growth
Introduction
Supplying adequate dietary amino acids (AA) particularly lysine (Lys), the first limiting AA, is
important to achieve maximum pig performance. Because dietary energy content can influence
feed intake, it has been suggested to maintain an optimum Lys to energy ratio in pig diets. Among
the energy systems, diet formulation based on net energy (NE) considers heat lost associated with
metabolism and ensures consistent performance (Le Bellego et al., 2001). Studies evaluating the
optimal dietary NE levels and standardised ileal digestible (SID) Lys:NE ratios in diets for pigs are
scarce. Thus, the objective of this study was to evaluate the effect of dietary NE and SID Lys levels
on growth performance of approximately 23 to 43 kg pigs.

A 28-d experiment was conducted with 384 mixed-sex pigs (PIC GP1050; initial BW of 23.3±0.4 kg)
by assigning to 6 dietary treatments with 8 pen replicates (4 barrows and 4 gilts/pen) per treatment
using a 2×3 factorial design with 2 levels of SID Lys (0.97 and 1.06%) and 3 levels of NE (9.65, 10.00
and 10.35 MJ/kg). Diets were formulated based on corn, soybean meal, wheat bran and soybean oil
using the analysed contents of AA and NE, and published SID coefficients of AA (AMINODat® 4.0.
Platinum version, 2010; Evonik Degussa GmbH, Hanau-Wolfgang, Germany.) for ingredients to meet
the ideal AA ratios. The contents of AA and NE in the ingredients were analysed by near infrared
technology. Pigs had free access to feed (mash) and water. Individual BW and feed disappearance
were recorded weekly to calculate average daily feed intake (ADFI), average daily gain (ADG) and
feed:gain (G:F). Data were analysed by ANOVA using the GLM procedure of SAS and the model
included initial BW as a covariant. Orthogonal-polynomial contrasts were used to determine effects
of dietary SID Lys, NE and their interaction.

The analysed contents of CP and total AA in the experimental diets were consistent and close to
calculated values. Performance results are given in Table 1. There were no SID Lys×NE interaction
effects for any performance parameter (P>0.05). For the overall 28 d, the ADFI was not affected

Table 1. Effects dietary lysine and net energy (NE) concentrations on performance and energy
efficiency of growing pigs (d 0-28).1,2
Initial BW Final BW ADFI ADG G:F SID Lys NE

(kg) (kg) (kg) (kg) (g/kg gain) (MJ/kg gain)
SID Lys (%)

0.97 23.3 42.0 1.305 0.668 0.513 18.99 19.56
1.06 23.3 43.0 1.321 0.704 0.533 19.93 18.79
NE (MJ/kg)
9.65 23.2 42.3 1.330 0.679 0.512 19.89 18.92
10.00 23.3 42.5 1.319 0.687 0.521 19.53 19.26
10.35 23.3 42.6 1.291 0.691 0.536 18.95 19.35
SID Lys×NE
0.97 9.65 23.3 42.0 1.319 0.670 0.509 19.16 19.05
10.00 23.3 41.5 1.292 0.650 0.505 19.29 19.89
10.35 23.3 42.4 1.302 0.684 0.525 18.51 19.74
1.06 9.65 23.2 42.6 1.340 0.689 0.515 20.62 18.78
10.00 23.3 43.6 1.345 0.723 0.538 19.76 18.64
10.35 23.3 42.9 1.279 0.699 0.547 19.39 18.95
SEM 0.401 0.449 0.024 0.016 0.009 0.356 0.346
P-values
SID Lys 0.889 0.008 0.386 0.009 0.011 0.003 0.010
NE 0.979 0.750 0.240 0.748 0.046 0.039 0.427
SID×NE 0.971 0.138 0.276 0.141 0.363 0.390 0.373
1Data are least square means.

2 ADFI = average daily feed intake; ADG = average daily gain; G:F = feed:gain; SID = standardised ileal digestible;
Lys = lysine.
by the dietary treatments (P>0.05). Increasing SID Lys from 0.97 to 1.06% increased final BW
(P=0.008), ADG (P=0.009) and G:F (P=0.011) which agrees with Warnants et al. (2003) who
reported that at least 1.07% SID Lys is required to optimize ADG and G:F of 31 to 50 kg pigs. The
ADG was not affected by the dietary NE levels (P>0.05). The ADG of pigs maximized when the
diet contains 1.06% SID Lys and 10.00 MJ/kg NE, which corresponds to SID Lys:NE of 1.06 g/MJ.
Similarly, Zhang et al. (2011) reported that a dietary NE of 9.87 MJ/kg, i.e. SID Lys:NE of 1.03 g/
MJ, was optimum for 20 to 50 kg pigs. The G:F optimized at the highest NE level, i.e. 10.35 MJ/kg
NE and 1.06% SID Lys, which corresponds to SID Lys:NE of 1.02 g/MJ. Increasing dietary NE level
decreased the amount of SID Lys need/kg BW gain from 19.89 to 19.53 and 18.95 MJ/kg (P=0.039)
indicating improved Lys utilisation. Increasing SID Lys from 0.97 to 1.06% decreased (P=0.010)
the amount of NE need from 19.56 to 18.79 g/kg BW gain. Based on these results the ADG and G:F
of 23 to 43 kg pigs maximized at a dietary SID Lys of 1.06% and SID Lys:NE of 1.02 to 1.06 g/MJ.
References
Le Bellego, L., J. van Milgen, S. Dubois and J. Noblet, 2001. Energy utilization of low-protein diets in growing pigs.
Warnants, N., M.J. Van Oeckel and M. De Paepe, 2003. Response of growing pigs to different levels of ileal standardised
digestible lysine using diets balanced in threonine, methionine and tryptophan. Livestock Production Science 82:
201-209.
Zhang G., X. Yi, L. Chu, N. Lu, J. K. Htoo and S. Qiao, 2011. Effects of dietary net energy density and standardized
ileal digestible Lysine: net energy ratio on the performance and carcass characteristic of growing-finishing pigs
fed low crude protein diets. Agricultural Sciences in China 10(4): 602-610.

Prediction of true and apparent ileal digestibility of amino acids of wheat
for broiler chickens
O. Lasek*, R. Augustyn and J. Barteczko
Department of Animal Nutrition and Dietetics, University of Agriculture in Krakow, 32-120 Krakow,
Poland; o.lasek@ur.krakow.pl
Abstract
The aim of the study was to evaluate true (TD) and apparent (AD) ileal digestibility of amino acids
(AA) of nine wheat cultivars for broilers. The experiment was carried out with 110 broiler chickens
divided randomly in 10 groups of 11 birds each. Broilers were fed ad libitum with nine wheat cultivars
and also a protein-free diet. In vivo digestibility was measured by the indicator method with Cr2O3
as marker. The content of basic nutrients, AA as well as detergent and dietary fibre, starch and sugar
differed among cultivars. The TD and AD of AA depended on chemical composition, especially on
detergent and dietary fibre. Regression equations with only three independent variables enabled a
good prediction of AA digestibility.
Keywords: regression equation, amino acids, wheat cultivars, chicken broilers
Introduction
Wheat is one of the major cereals used in poultry nutrition in Europe. It is generally admitted that
there is no problem when wheat is given to adult birds. However, performances of broilers are
often unsatisfactory due to the variability in nutrient content (especially anti-nutritional factors) and
digestibility among wheat cultivars (Steenfeld, 2001). Due to this variation an accurate prediction of
nutrient digestibility of wheat cultivars for broiler chickens is required, including the digestibility of
amino acids (AA). Ileal digestibility is currently the preferred approach to estimate AA availability
(Lemme et al., 2004). Therefore, the aim of the study was to determine true (TD) and apparent (AD)
ileal digestibility of AA of nine wheat cultivars for broiler chickens in order to propose equations
to predict TD and AD from chemical composition.

The experiment was carried out with 110 broiler chickens of line Ross 308 at the age of 42-49 days.
Broilers were randomly divided into 10 groups (11 birds each) and fed with nine wheat cultivars:
Vinjett, Napola, Bryza, Zebra, Bombona, Muza, Mikula, Satyna, Torka or a protein free diet. The
feeds were offered ad libitum. The experiment lasted 7 d and included 3 d of adaptation, 3 d of proper
period and 10 h of fast, 1.5 h of free access to food for filling the digestive tract and 2.5 h of fast
prior to slaughter (on d 49). For determining TD, endogenous AA were measured in broilers fed the
protein-free diet. Basic nutrients as well as soluble (SDF) and insoluble dietary fibre (IDF), neutral
detergent fibre (NDF), acid detergent fibre (ADF), acid detergent lignin (ADL), starch, sugars, Cr2O3,
and AA in wheat cultivars were analysed according to standard methods (AOAC, 2005). Additionally,
thousand-grain mass (TGM) was determined. Relationships between the digestibility of AA and the
chemical composition as well as TGM were calculated using the REG procedure of SAS (2002).

Chemical composition varied widely among wheat cultivars. The mean ± SD of the determined
parameters amounted to: starch 673.8±49.4 g/kg DM; crude protein 136.1±10.4 g/kg DM; ether
extract 17.9±3.1; sugars 52.1±13.2; crude ash: 18.9±1.0; crude fibre 25.0±2.9 g/kg DM; ADF
39.5±3.1 g/kg DM; NDF 118.2±9 g/kg DM; IDF 112.6±8.3 g/kg DM; SDF 20.3±7.6 g/kg DM

and TGM 45±3.57 g. The digestibility of AA was positively correlated with AA content and higher
correlation was found for TD than AD. Short et al. (1999) showed that AD of AA in wheat grain
increased with CP concentration. According to Lemme et al. (2004) AA digestibility, both TD and
AD, increases also as the concentration of particular AA increases. Regression equations with the
AA content as independent variable are of little pratical use, because AA analysis is too expensive.
Furthermore, AA digestibility was reduced when more detergent or dietary fibre was present in the
grain. Also in swine, AA digestibility decreased as NDF content in wheat bran increased (Huang et
al., 2001). According to Steenfeldt (2001), the dietary fibre content was responsible for lower nutrient
utilisation of wheat diets by broiler chickens. The best predictors of AA digestibility (apparent as
well as true) were: content of TDF, IDF, ADL, Ash and also TGM (Table 1). The content of IDF in
wheat grain was the best fibre parameter to predict AA digestibility.
In conclusion, regression equations based on basic nutrients, fibre content and TGM, are suggested
to predict AA digestibility of wheat grains in broiler chickens.
Table 1. Regression equations to predict ileal apparent (AD) and true digestibility (TD) of amino
acids of wheat grains (n=9).1,2
AD, % Equation to predict ileal AD R2 RSD P-value
LYS 62.25±11.46 166.97 – 2.65×TGM – 38.31×ADL + 11.26×EE 0.84 4.84 <0.001

MET 78.85±9.55 67.76 – 8.06×IDF + 55.89×Ash – 23.30×ADL 0.82 4.27 <0.001
CYS 77.08±6.56 94.32 – 0.74×TGM – 3.01×TDF + 31.12×Ash 0.68 3.93 <0.001
THR 66.77±9.00 -178.75 – 2.30×TGM + 14.18×EE – 2.57×CP 0.71 5.14 <0.001
TD, % Equation to predict ileal TD R2 RSD P-value
LYS 81.90±11.45 113.71 – 2.09×TGM – 31.87×ADL + 39.47×Ash 0.82 5.14 <0.001

MET 85.97±9.09 71.52 – 7.34×IDF + 53.71×Ash – 24.43×ADL 0.82 4.08 <0.001
CYS 83.96±6.12 104.46 – 0.72×TGM – 2.95×TDF + 28.01×Ash 0.70 3.53 <0.001
THR 82.03±8.42 11.16 – 1.49×TGM + 58.53×Ash + 0.72×S 0.69 4.99 <0.001
1 The contents of nutrients are expressed in %.

2 TGM = thousand-grain mass; EE = ether extract; TDF = total dietary fibre; IDF = insoluble dietary fibre; ADL = acid
detergent lignin; CP = crude protein; S = starch; RSD = residual standard deviation.
References
Association of Official Analytical Chemists (AOAC), 2005. Official methods of analysis (18th Ed.). AOAC, Washington,
DC, USA.
Huang S.X., W.C. Sauer and B. Marty, 2001. Ileal digestibilities of neutral detergent fiber, crude protein, and amino acids
associated with neutral detergent fiber in wheat shorts for growing pigs. Journal of Animal Science 79: 2388-2396.
Lemme A., V. Ravindran and W.L. Bryden, 2004. Ileal digestibility of amino acids in feed ingredients for broilers.
World’s Poultry Science Journal 60: 423-437.
SAS, 2002. The SAS System, Version 9.1. SAS Institute Inc., Cary, NC, USA.
Short, F.J., J. Wiseman and K.N. Boorman, 1999. Application of a method to determine ileal digestibility in broilers
of amino acids in wheat. Animal Feed Science and Technology 79: 195-209.
Steenfeldt, S., 2001. The dietary effect of different wheat cultivars for broiler chickens. British Poultry Science 42:
595-609.

Replacing dietary non-essential amino acids with ammonia nitrogen
does not alter amino acid profile of retained body protein in growing pigs
fed a diet deficient in non-essential amino acid nitrogen
W.D. Mansilla1, J.K. Htoo2 and C.F.M. de Lange1*
1Department of Animal Biosciences, University Guelph, Guelph, ON, N1G 2W1, Canada; 2Evonik
Nutrition & Care GmbH, 63457, Hanau-Wolfgang, Germany; cdelange@uoguelph.ca
Abstract
Supplementing ammonia-nitrogen (N) in diets deficient in non-essential amino acid N (NEAA-N)
improves body weight (BW) gain in growing pigs. A serial slaughter study was conducted to
determine the effect of feeding ammonia-N on body protein gain and carcass amino acid (AA)
profile of pigs fed diets deficient in NEAA-N. In total, 32 Yorkshire barrows with initial BW of
16.3 kg were used. Eight pigs were euthanized at the beginning of the experiment to estimate initial
whole body AA composition; the remaining pigs were assigned to 3 different dietary treatments. A
basal cornstarch and casein-based diet, not deficient in essential AA (EAA) but low in crude protein
(CP), was formulated and supplemented with cornstarch (Control) or with 2 different N-sources (i.e.
ammonia and NEAA) supplying 2.7% additional CP. Pigs were restricted fed at 3.0× maintenance ME
requirements during 3 consecutive weeks. At the end of the experiment, whole viscera and carcass
were ground and analysed for N and AA content. Whole body protein deposition was increased
with ammonia and NEAA treatments (tAmmonia and tNEAA, respectively) compared to Control.
Body protein AA profile in Control had lower levels of Leu, Lys, Met + Cys and Phe compared to
both N supplementations but it was similar for tAmmonia and tNEAA. De novo NEAA synthesis
was increased for tAmmonia compared to tNEAA. These results show that ammonia is an efficient
N source and can be used for de novo synthesis of NEAA in pigs.
Keywords: ammonia-nitrogen, non-essential amino acids, pigs, protein retention
Introduction
In a previous study, it was shown that supplementing ammonia-nitrogen (N) in diets deficient in
non-essential amino acid N (NEAA-N) improves body weight (BW) gain and N retention in growing
pigs. In that study and when N was supplemented as either ammonium citrate or a mix of crystalline
NEAA, increments in BW gain were similar. The objective of the present study was to further
understand the effect of supplementing dietary ammonia-N to pigs fed diets deficient in NEAA-N
on the AA profile of retained body protein.

A total of 32 Yorkshire barrows, divided in two equal blocks (BW = 16.3±1.0 kg, mean ± SD) were
used. Four pigs per block were used to estimate initial whole body protein mass and AA profile; the
remaining pigs were assigned to 3 different dietary treatments. Pigs were housed individually and
fed restricted at 3.0 x maintenance ME requirements (191 kcal/kg BW0.60; NRC, 2012), and BW
was monitored weekly during 3 consecutive weeks.
A basal diet containing casein and crystalline EAA as the only N sources was formulated. The
basal diet exceeded requirements for all EAA (NRC, 2012), but was low in CP content (N × 6.25 =
8.01%). Cornstarch (Control) and 2 different sources of N (di-ammonium citrate and a NEAA mix;
tAmmonia and tNEAA, respectively) were added to the basal diet supplying 2.7% extra CP. The
total amount of standardised ileal digestible (SID) NEAA in tNEAA was based on the NEAA profile
of body protein (Mahan and Shields, 1998) to minimize the need for endogenous NEAA synthesis.

On the last day of the performance trial, pigs were killed to determine final whole body protein
mass and AA profile. For each pig, carcasses and pooled visceral organs were weighed and then
frozen. Frozen carcasses and visceral organs were ground, thoroughly mixed, and subsampled for
subsequent analyses of dry matter, CP and AA contents.
Protein AA profile was calculated as the proportion (%) of individual AA in carcass and viscera
protein, respectively. The AA profile of retained protein in carcass + viscera was calculated as the
ratio between the increment of total AA mass (i.e. AA retention) and the increment of total protein
mass (i.e. protein retention) × 100%. For each EAA, utilisation efficiency was calculated as whole
body AA retention divided by SID AA intake × 100%. The minimum de novo synthesis of individual
NEAA (g/d) was calculated as daily whole body AA retention minus daily SID AA intake.

Crude protein content of whole carcass was lower for Control (37.2±2.3%) compared to tAmmonia
and tNEAA, while it was similar for tAmmonia and tNEAA (44.2±2.1%). Whole body protein
retention increased with adding either ammonia or non-EAA compared to the control diet, but it
did not differ between tAmmonia and tNEAA. The EAA contents in retained carcass protein were
decreased for Iso, Leu, Lys, Met+Cys and Phe as compared to Control, but there were no differences
between tAmmonia and tNEAA (Table 1). Total N and EAA utilisation efficiency was increased with
adding N to Control, but it did not differ between tAmmonia and tNEAA. The de novo synthesis of
NEAA was increased for tAmmonia compared to tNEAA.
Table 1. Retained body protein (g/d) and AA profile of retained protein (% of CP) in carcass +
viscera of pigs fed diets deficient in NEAA-N (Control) or Control supplemented with ammonia-N
(tAmmonia) or a mix of NEAA (tNEAA).
Carcass + viscera Control tAmmonia tNEAA SEM P-values
Control vs tAmmonia
N-supplemented vs tNEAA
diets
Final Body weight (kg) 24.8 27.5 27.2 0.5 <0.01 0.64
Retained body protein 44.2 84.4 76.2 6.9 <0.01 0.38
Arginine 6.76 6.57 6.85 0.24 0.86 0.38
Histidine 2.75 3.19 3.08 0.18 0.09 0.64
Isoleucine 3.32 3.69 3.80 0.17 0.05 0.61
Leucine 6.17 6.88 7.08 0.25 0.01 0.54
Lysine 5.81 6.82 7.11 0.30 <0.01 0.47
Methionine + cysteine 2.74 3.05 3.24 0.12 0.01 0.25
Methionine 1.78 2.04 2.17 0.10 0.01 0.34
Phenylalanine 3.39 3.63 3.74 0.12 0.05 0.50
Threonine 3.63 3.80 3.88 0.13 0.22 0.62
Valine 4.21 4.48 4.60 0.15 0.08 0.54
References
Mahan, D.C. and R.G. Shields, 1998. Essential and nonessential amino acid composition of pigs from birth to 145
kilograms of body weight, and comparison to other studies. Journal of Animal Science 76: 513-521.
National Research Council (NRC), 2012. Nutrient requirements of swine. National Academy Press, Washington, DC,
USA.

The protein requirement before and after implantation in mink
C.F. Matthiesen1*, C. Larsson1, P. Junghans2 and A.-H. Tauson1
1Department of Veterinary Clinical and Animal Sciences, Faculty of Health and Medical Sciences,
University of Copenhagen, Grønnegårdsvej 3, 1870 Frederiksberg C, Denmark; 2Leibniz Institute for
Farm Animal Biology, Institute of Nutritional Physiology ‘Oskar Kellner’, Dummerstorf, Germany;
cmt@sund.ku.dk
Abstract
The protein requirement in mink is relatively high, similar to other strict carnivores. However, the
requirement for gestation is incompletely known although the importance of adequate nutrient
provision is well recognized. The objective was to determine the protein requirement before (BEF)
and after (AFT) implantation in order to support high implantation and foetal survival rates. A total
of 106 females was used, out of which sixty-six were used BEF and the remaining 40 females were
used AFT. Experimental diets providing 10, 15, 20, 25, 30, 35, 40 and 45% of metabolisable energy
(ME) from protein were used BEF whereas the AFT study only included diets from 20 to 45% of ME
from protein. A non-invasive stable isotope technique, indicator amino acid oxidation (IAAO), was
evaluated and used to measure the protein requirement BEF and AFT implantation. The IAAO data
were combined with euthanisation data which included number of implantation sites and embryo/
foetal survival rates. In conclusion, implantation occurred at normal rates even at the lowest levels
of protein whereas BEF embryo survival tended (P=0.1) to be compromised when protein provision
was <20% of ME from protein. Protein provision from 20 to 45% of ME all supported implantation
and resulted in similar foetal survival rates in all feeding groups AFT. The IAAO concurred with
euthanisation data indicating that IAAO can be a valuable non-invasive tool to determine the protein
requirement in mink.
Keywords: gestation, strict carnivore, indicator amino acid oxidation, protein
Introduction
The mink is a strict carnivore and has as such a high protein requirement, which is incompletely
known for e.g. gestation. It has induced ovulation and delayed implantation with an embryonic
diapause lasting from a few days up to 12 weeks (Enders, 1952). The length of the embryonic
diapause is inversely related to embryo survival. Implantation occurs after the vernal equinox and
is followed by 31 days of ‘true gestation’. Gestation can be as short as 39 days with no embryonic
diapause, but is commonly around 51 days (Murphy, 1984). The objective of the present study was
to determine the protein requirement before (BEF) and after (AFT) implantation in female mink, and
to evaluate an indicator amino acid oxidation (IAAO) technique as a non-invasive tool to determine
these requirements.

A total of 106 females was used. The experiment was conducted using different dietary protein
provision BEF (10, 15, 20, 25, 30, 35, 40 and 45% of metabolisable energy (ME) from protein) and
AFT (20, 25, 30, 35, 40 and 45% of ME from protein) implantation. The BEF diets were fed for
32 days from mating until implantation was anticipated to be completed and the AFT diets were
fed for 20 days from anticipated time of implantation until euthanisation shortly before expected
parturition. The non-invasive oral stable isotope technique IAAO based on 13C kinetics in breath
CO2 was used to measure the protein requirement. The CO2 production was measured by means of
indirect calorimetry (IC) and samples of air from the respiration chamber were automatically drawn
every five minutes and analysed using an infrared 13C isotope analyser (IRIS). After a baseline

sample was taken, the mink was given an oral dose of 1-13C-L-leucine (5 mg/kg BW) administrated
in a small amount of Hills prescription diet feline a/d. The gas exchange measurement by IC and
IRIS started directly after the oral dose was given and lasted for 10 h. The cumulative recovery of
13C was calculated and used to estimate the protein requirements by a broken line approach. Each
female was measured once during a 4 weeks period in the BEF (early, mid or late measurements) and
once in a 3 weeks period in the AFT study. All females were euthanized to investigate the number
of implantation sites and embryo/foetal survival rates. The data were analysed using the MIXED
procedure in in SAS (version 9.3).

The results were evaluated from number of implantation sites, embryo/ foetal survival rates and
combined with IAAO data. All females except one had implantation sites at the time of euthanisation.
Number of implantation sites in BEF females were not affected by dietary protein provision whereas
embryo survival tended (P=0.1) to be affected BEF (Table 1).
The IAAO values (data not shown) were stable from mating until late March (early and mid-
measurements), indicating that the protein requirement was met which concurred with no effect of
protein provision on number of implantation sites even when protein provision was very low. The
IAAO data were higher at 10 and 15% of ME from protein in late measurements indicating a break
point between 15 and 20% of ME close to the time of implantation which is in agreement with
embryo survival rates BEF. Number of viable foetuses, foetal survival rates and IAAO data were not
affected by dietary protein provision in the AFT study, indicating that the protein requirement was
met. However, foetal survival rates were numerically lower at 20% of ME from protein which is in
line with previous findings by Vesterdorf et al. (2012). This indicates that the protein requirement is
fulfilled using protein levels ≥20% of ME from protein both before and after implantation.
Table 1. Number of implantation sites, viable embryos/foetuses and embryo/foetal survival rates as
an effect of protein provision before (BEF) and after (AFT) implantation.1,2
Metabolisable energy from protein (%) RR P-value
10 15 20 25 30 35 40 45
BEF (n=66)
Implantation sites 10 8.9 10.1 10.2 11.4 11.3 11.4 11.8 3.6 NS
Viable embryos 8.2 7.7 8.2 9.7 10.7 10.8 11.0 11.7 3.4 NS
Survival rate, % 84.6ab 75.5a 85.2ab 96.8b 94.2b 96.5b 97.1b 99.1b 18.2 0.1
AFT (n=40)
Implantation sites 8.7 8.3 10.6 10.0 9.5 9.0 2.8 NS
Viable foetuses 7.1 7.6 10.1 8.9 9.0 8.5 3.1 NS
Survival rate, % 81.3 91.4 95.7 89.1 95.4 93.1 19.6 NS
1 Values with different superscripts (a,b) in a row differ significantly (P<0.05).

2 RR = square root of residuals.
Acknowledgements
Financial support from Fur Animal Levy Fund, Denmark, Protein requirements and metabolism
in mink.

References
Enders, R.K., 1952. Reproduction in the mink (Mustela vison). Proceedings of the American Philosophical Society
96: 691-755.
Murphy, B.D., 1984. Embryo survival in mink. Blue book of Fur Farming 1984: 19-23.
Vesterdorf, K., A. Harrison, C.F. Matthiesen and A.-H. Tauson, 2012. Effects of protein restriction in utero on the
metabolism of mink dams (Neovison vison) and on mink kit survival as well as on postnatal growth. Open Journal
of Animal Sciences 2: 19-31.

Transgenic flax overexpressing polyphenols and inflammation: anti-
inflammatory mechanisms
M. Matusiewicz1*, I. Kosieradzka1, M. Zuk2, T. Niemiec1, A. Łozicki1, G. Halik1, M. Makarski1 and
J. Szopa2
1Department of Animal Nutrition and Biotechnology, Faculty of Animal Sciences, Warsaw University
of Life Sciences, Ciszewskiego 8, 02-786 Warsaw, Poland; 2Department of Genetic Biochemistry,
Faculty of Biotechnology, University of Wrocław, Przybyszewskiego 63/77, 51-148 Wrocław, Poland;
magdalena_matusiewicz@sggw.pl
Abstract
Anti-inflammatory action of transgenic W92/72 flaxseed cake overexpressing polyphenolic
antioxidants has been confirmed. The objective of this work is to explain possible anti-inflammatory
mechanisms. Contents of polyphenols in non-transgenic Linola and transgenic W92/72 flaxseed cake
were determined. For 14-week nutritional study four groups of rat obesity models were utilised.
Rats were given the diets: (1) standard (SD), (2) high-fat with pork lard (HFD) as well as high-fat
supplemented with 30% of seed cake of either (3) Linola flax (HF Linola) or (4) W92/72 flax (HF
W92). Accumulation of secoisolariciresinol diglucoside, caffeic, ferulic and coumaric acids, and their
glucosides in W92/72 seed cake increased significantly compared to Linola. Concentration of inactive
cytoplasmic nuclear factor-kappa B (NF-κB), transcription factor regulating immune response,
was increased in liver of HF W92 group compared to HF Linola and HFD. Significant relationship
between intake of polyphenols and content of cytoplasmic NF-κB was found. Active nuclear NF-κB
level in liver was not affected statistically significantly. Liver concentration of p53, which is tumor
suppressor, was higher in HF W92 group in comparison with SD and HFD, of which the differences
were not statistically significant. Liver contents of cyclooxygenase-2 and 5-lipoxygenase, the
enzymes of arachidonic acid cascade, responsible for synthesis of pro-inflammatory products, did
not differ statistically significantly. This work indicates that NF-κB and p53-dependent mechanisms
are involved in anti-inflammatory activity of W92/72 flax.
Keywords: GMO, flaxseed cake, phenolic acids, SDG, inflammatory state
Introduction
Flaxseed cake containing antioxidants is important dietary component. Anti-inflammatory action
of transgenic W92/72 flaxseed cake overexpressing polyphenolic compounds has been confirmed
(Matusiewicz et al., 2015). The aim of this work is the explanation of underlying anti-inflammatory
mechanisms.

Contents of polyphenols and their glucosides in non-transgenic Linola and transgenic W92/72 flaxseed
cake were determined using ultra-performance liquid chromatography (n=3). For present 14-week
nutritional study four groups (n=6) with standardised body weight of male Wistar-Crl:WI(Han)
rats, obesity models induced by high-fat diet feeding, were utilised. Rats were given ad libitum the
following diets: (1) standard (SD), (2) high-fat with pork lard (HFD) as well as high-fat supplemented
with 30% of seed cake of either (3) Linola flax (HF Linola) or (4) W92/72 flax (HF W92).
After completion of research, contents of nuclear factor-kappa B (NF-κB) in cytoplasmic and

nuclear liver protein fractions and level of p53 in liver extracts were measured using enzyme-linked
immunosorbent assay. Concentrations of cyclooxygenase-2 (COX-2) and 5-lipoxygenase (5-LOX)

in liver were evaluated by Western Blot. One-way analysis of variance was performed, correlation
coefficients were calculated.

Accumulation of all of the analysed antioxidants in W92/72 seed cake increased significantly (P<0.01)
compared to Linola. The content of secoisolariciresinol diglucoside (SDG), important lignan whose
rich source are flaxseeds, was increased by 172%. Also the phenolic acids level was increased –
caffeic acid by 33%, ferulic acid by 113% and coumaric acid by 219%. The concentration of caffeic
acid glucoside was 154% higher, ferulic acid glucoside level was elevated by 327% and coumaric
acid glucoside content was 28% higher.
Concentration of inactive cytoplasmic NF-κB, transcription factor regulating immune response,

was increased in HF W92 group compared to HF Linola and HFD (P<0.01) (Table 1). Significant
relationship (P<0.05 or P<0.01) between intake of polyphenols and content of cytoplasmic NF-κB
was found. Calculated correlation coefficient was 0.78 for intake of SDG, 0.70 for caffeic acid, 0.77
for ferulic acid, 0.78 for coumaric acid, 0.78 for caffeic acid glucoside, 0.79 for ferulic acid glucoside
and 0.69 for coumaric acid glucoside. Active nuclear NF-κB level was not affected statistically
significantly (Table 1). Concentration of p53, which is tumor suppressor, was higher in HF W92
group in comparison with SD and HFD, of which the differences were not statistically significant.
Level of p53 was not significantly correlated with polyphenol intake. Contents of COX-2 and 5-LOX,
the enzymes of arachidonic acid cascade, responsible for synthesis of pro-inflammatory products,
did not differ statistically significantly (data not presented).
This work indicates that NF-κB and p53-dependent mechanisms are involved in anti-inflammatory
activity of W92/72 flax. For cytoplasmic NF-κB concentration are responsible polyphenols being
direct objectives of transformation.
Table 1. The concentration of nuclear factor-kappa B (NF-κB) in the liver nuclear and cytoplasmic
fraction protein extract and p53 in the liver protein extract of rats fed different diets.1,2
SD HFD HF Linola HF W92 SEM P-value
NF-κB cytoplasmic 7.45A 13.53B 12.05B 18.15C 1.44 <0.001

NF-κB nuclear 14.25 11.78 12.85 10.67 1.39 0.333
p53 0.82a 0.99a 1.76ab 2.82b 0.53 0.056
1 Significant effect: values within a factor that do not share a common superscript differ significantly: a, b = P<0.05;
A, B = P<0.01.
2 SD = standard diet; HFD = high-fat with pork lard; HF Linola = high-fat diet supplemented with 30% of seed cake of
Linola flax; HF W92 = high-fat diet supplemented with 30% of seed cake of W92/72 flax.
Acknowledgements
Financial support: the National Science Centre, Poland, Project No. N N311 526540.
References
Matusiewicz, M., I. Kosieradzka, M. Sobczak-Filipak, M. Zuk and J. Szopa, 2015. Transgenic flax overexpressing
polyphenols as a potential anti-inflammatory dietary agent. Journal of Functional Foods 14: 299-307.

Rice diet containing high fat and rice hull affects the growth
performance of heat-exposed broiler chickens
F. Nanto, M. Kikusato, S. Ohwada and M. Toyomizu*
Tsutsumidori-Amamiyamachi, Sendai 981-8555, Japan; toyomizu@bios.tohoku.ac.jp
Abstract
The present study was conducted to investigate the effect of feeding two types of rice-based diet
containing whole-grain paddy rice (fat 11%) or dehulled rice (fat 6%) on the growth performance
and intestinal immunological functions of broiler chickens subjected to heat stress. 0-d-old male
chicks were randomly divided into three groups, and fed either a corn diet (CO, fat 6%), whole-
grain paddy rice diet (WR, fat 11%) or dehulled rice diet, (DR, fat 6%). Each diet contained 20%
crude protein and 3.1 kcal/g metabolisable energy. At 21 d of age, birds in the CO-fed group were
randomly divided into two groups, one of which was maintained at 24 °C, while the other group
and the WR- and DR-fed groups were kept at 33 °C for 7 days. In heat-stressed conditions, WR-fed
birds showed a significant reduction in body weight gain compared with that of the CO-fed group,
whereas a decrease was not observed in the DR-fed group. WR-fed heat-stressed birds showed
bacteria-derived endotoxin in the plasma. Gut mucosal morphological damage; plasma, liver and
mucosal lipid peroxidation; and the plasma ceruloplasmin concentration of these birds were increased
relative to the CO-fed heat-stressed birds. The results suggest that feeding broiler chickens a WR diet
that is high in fat enhances growth retardation and oxidative damage under heat stress conditions.
These effects might be due to impairment of the intestinal barrier and immune function.
Keywords: whole-grain paddy rice, heat stress, intestinal morphology
Introduction
Different production regions use various grains as feedstuff: in Japan the use of paddy rice as a
feedstuff is growing. To assess the utility of rice-feeding, not only its nutritional value but also its
effects on physiological metabolism need to be understood. Heat is a major stressor causing growth
retardation and high mortality. We have previously investigated the effects of feeding whole-grain
paddy rice (WR) or corn (CO) diets, which contained 10 or 6% fat, respectively, with a metabolisable
energy (ME) content of 3.1 kcal/g, on the growth performance of heat-stressed broiler chickens:
WR-fed birds exhibited poorer growth performance than CO-fed birds (Nanto et al., 2013). From
these results, we hypothesized that a high level of fat and/or rice hull in the diet might lead to the
poor growth observed in the WR-fed group. Therefore, the present study was conducted to clarify
the cause of the growth retardation observed in heat-stressed broiler chickens fed a high fat WR diet.

0-d-old male chicks (Ross strain) were randomly divided into three groups, and fed CO (fat 6%),
WR (fat 11%) or dehulled rice (DR; fat 6%) diet. Each diet contained 20% crude protein and 3.1
kcal/g ME. At 21 d of age, birds in the CO-fed group were randomly divided into two groups (n=8),
one of which was maintained at 24 °C, while the other group and WR- and DR-fed groups were
kept at 33 °C for 7 days. After the treatment, birds were euthanized by decapitation, and blood,
ileum tissue and liver were excised. Cross-sections of the ileum tissue were obtained, and the villus
height and crypt depth were measured. Mucosal and liver lipid peroxidation levels were determined
colorimetrically, with the values expressed as nmol of malondialdehyde (MDA) per equivalent
of g wet tissue. Plasma endotoxin and ceruloplasmin (Cer) concentrations were determined by a
chromogenic limulus amebocyte lysate assay and p-phenylenediamine colorimetric method, as

indexes of the inflammatory response and intestinal barrier dysfunction, respectively. Data were
analysed using one-way ANOVA with Tukey’s multiple comparison post tests.

Hyperthermic treatment resulted in a significant decrease in body weight gain (BWG) in the CO-fed
birds (325±26 g) compared with birds kept under thermoneutral conditions (519±9 g). Under heat-
stressed conditions, the BWG of the WR group (222±26 g) was significantly reduced relative to the
CO-fed birds, whereas no difference in BWG during the heat exposure period was observed between
the CO- and DR-fed groups (303±26 g). The ratio of villus height to crypt depth in the ileum of WR
birds (6.3±0.3) was significantly lower than that in the CO- (7.6±0.5) and DR-fed (8.9±0.4) birds
under heat stress conditions. The MDA content of the ileum mucosa of WR-fed birds (42.2±3.9 nmol/g
tissue) was significantly higher than in CO- (24.2±2.9 nmol/g tissue) and DR- (29.6±4.4 nmol/g
tissue) fed birds. The plasma endotoxin concentrations showed a similar pattern to the ileum MDA
content over the groups tested. Liver and plasma MDA contents and plasma Cer concentrations of
heat-stressed WR-fed birds were higher than those of heat-stressed CO- and DR-fed birds.
Conclusions
The present study demonstrates that feeding broiler chickens with a high fat WR diet enhances
growth retardation and oxidative damage under heat stress conditions, and that this might be due to
an impairment of the intestinal barrier and immune function.
References
Nanto, F., C. Ito, M. Kikusato and M. Toyomizu, 2013. Effect of paddy rice diets on performance in chickens under
thermoneutral and heat stress conditions. In: J.W. Oltjen, E. Kebreab and H. Lapierre (eds.). Energy and protein
metabolism and nutrition in sustainable animal production. EAAP Scientific Series No. 134. Wageningen Academic
Publishers, Wageningen, the Netherlands, pp. 505-507.

Lysine deficiency and genotype affect amino acid composition of carcass
protein of growing pigs
P. Palma-Granados, N. Hidalgo-Checa, L. Lara, J.F. Aguilera and R. Nieto*
Department of Physiology and Biochemistry of Animal Nutrition, Estación Experimental del Zaidín
(CSIC), Profesor Albareda, 18008, Granada, Spain; rosa.nieto@eez.csic.es
Abstract
Previous studies showed differential effects of Lys deficiency on growth and body protein retention
of Iberian and Landrace gilts of similar body weight (BW) reared in similar conditions. The objective
of the present work was to investigate the effects of genotype and Lys deficiency on carcass amino
acid (AA) composition of Iberian and Landrace × Large-White (LLW) pigs in similar experimental
conditions. Thirty two barrows, 16 from each breed, were randomly assigned to 2 experimental diets
in a factorial arrangement (2 breeds × 2 diets). Diets were isoenergetic and with identical composition
(180 g CP/kg DM, 14 MJ metabolisable energy) except for the Lys content (0.98% Lys, Lys adequate
diet (LA); and 0.47% Lys, Lys deficient diet (LD)). The experiment started at 10 and ended at 25
kg BW. Performance was reduced in both pig types when fed LD diets (P<0.05). Proportions of Ile,
Val and Phe were higher (7 to 13%, P<0.01) in carcass protein of Iberian compared to LLW pigs.
When fed LD diets, Leu decreased (9%, P<0.001) in carcass protein of pigs from both genotypes,
His proportion was reduced in Iberian but not in LLW pigs, whereas Lys proportion was reduced
only in LLW pigs (interaction genotype × diet, P<0.05). The results show that AA proportions in
carcass protein of growing pigs can be altered by factors like genotype and a single AA deficiency.
Keywords: amino acid deficiency, amino acid composition, body protein, Iberian pig
Introduction
Amino acid (AA) requirements in pigs could be influenced by many factors (sex, genotype, health
status) although it has been widely assumed that these factors do not necessarily affect AA composition
of body proteins. In addition, previous studies showed differential effects on growth and body protein
retention of Lys deficient diets in Iberian and Landrace gilts under similar experimental conditions
(Rivera-Ferre et al., 2006). The objective of the present work was to investigate the effects of genotype
and dietary Lys deficiency on AA composition of carcass protein of Iberian (an obese genotype) and
Landrace × Large-White (LLW) pigs maintained under identical experimental conditions.

Thirty two barrows, 16 from each breed, were randomly assigned to each of two experimental diets
in a 2 × 2 factorial arrangement (2 breeds × 2 diets; 8 pigs per treatment combination). Diets – based
on barley, maize, gluten meal and soybean meal – were isonitrogenous and isoenergetic (180 g CP
/kg DM, 14 MJ metabolisable energy) and of identical AA composition except for the Lys content
(0.98% Lys, Lys adequate (LA); and 0.47% Lys, Lys deficient (LD)). The rest of nutrients met or
exceeded recommendations (NRC, 2012). Pigs were housed in individual 2 m2 pens. Initial body
weight (BW) was 10.3±0.2 kg. Pigs were fed at 85% of ad libitum intake of the Iberian genotype
of greater intake capacity. Daily feed allowance was calculated weekly on BW basis. At 25 kg BW
pigs were slaughtered by exsanguination after electrical stunning. Carcass was stored at -20 °C until
analysis. After homogenization and freeze-drying, AA were determined after protein hydrolysis in
6 M HCl by high-performance liquid chromatography. Cys and Met were determined as cysteic
acid and methionine sulphone, respectively, after oxidation with performic acid before hydrolysis.
AA were expressed as g/kg carcass protein. Results were analysed by ANOVA using the GLM
procedure of SAS, according to the factorial design. When interaction between main factors was

significant, statistical differences among treatment combinations were assessed by Least Square
Mean t-test (P<0.05).

Growth rate was reduced in both pig types when fed LD diets, particularly in LLW pigs (interaction
genotype × diet, P<0.05; Table 1). Proportions of Ile, Val and Phe were higher (7 to 13%, P<0.01)
and those of Met and Thr tended to decrease (by 7 and 3%, respectively, P=0.06) in carcass protein
of Iberian compared to LLW pigs. When fed LD diets, Leu proportion decreased (9%, P<0.001)
and Arg tended to increase (3%, P=0.09) in carcass protein of pigs of both genotypes. However,
His proportion of carcass protein was reduced in Iberian but not in LLW pigs fed LD diets, whereas
Lys proportion was reduced only in LLW when fed LD diets (by 13%, interaction genotype × diet,
P<0.05). Present results show that proportions of AA in carcass protein of growing pigs can be altered
by factors like genotype and a single AA deficiency, which may have consequences on definition
of AA profile of Iberian pig diets.
Table 1. Amino acid concentration (g AA/kg CP) in the carcass of Iberian and Landrace × Large-
White pigs (LLW) fed adequate (LA) of lysine deficient (LD) diets.1
Iberian LLW SEM P-value
LA LD LA LD G D G×D
Arg 82.19 85.39 84.32 85.95 1.36 NS 0.091 NS

His 38.04a 32.85b 38.89a 37.42a 0.80 <0.01 <0.001 <0.05
Ile 33.61 33.84 31.85 30.44 0.81 <0.01 NS NS
Leu 67.58 63.46 66.76 60.24 1.29 NS <0.001 NS
Lys 66.34a 65.37a 64.85a 56.74b 1.45 <0.01 <0.01 <0.05
Met 19.48 17.74 20.23 19.75 0.70 0.062 NS NS
Phe 37.88 37.72 34.93 32.20 0.89 <0.001 NS NS
Thr 38.95 39.82 41.06 40.06 0.59 0.060 NS NS
Val 47.22 47.96 44.55 44.25 0.70 <0.001 NS NS
Cys 10.169 9.417 10.52 9.528 0.28 NS <0.01 NS
Gly 102.08 111.91 96.14 110.36 2.72 NS <0.001 NS
Pro 67.73 68.63 67.07 69.72 1.57 NS NS NS
1 Means bearing different superscript letter differ significantly; G = genotype; D = diet; NS: P>0.05.
Acknowledgements
This work was funded by Spanish MINECO Grant AGL2011-25360.
References
National Research Council (NRC), 2012. Nutrient requirement of swine (11th Ed.). The National Academy Press,
Rivera-Ferre, M.G., J.F. Aguilera and R. Nieto, 2006. Differences in whole-body protein turnover between Iberian and
Landrace pigs fed adequate or lysine deficient diets. Journal of Animal Science 84: 3346-3355.

Effect of copper nanoparticles and copper sulphate on metabolic rate
and growth of broiler embryos
A. Scott*, K.P. Vadalasetty and A. Chwalibog
Department of Veterinary Clinical and Animal Sciences, University of Copenhagen, Groennegaardsvej
3, 1870 Frederiksberg C, Denmark; abdullah.scott@sund.ku.dk
Abstract
Our previous experiment showed that in ovo administration of copper nanoparticles (Cu-NP) and
copper sulphate (CuSO4) remarkably improved body weight in growing chickens. The objective of
the present experiment was to elucidate potential effects of Cu-NP and CuSO4 on the metabolic rate
and development during embryogenesis. Fertilized broiler eggs were divided into six groups: control
not injected, placebo injected with demineralised water, CuSO4 (injected with 50 or 100 mg/kg)
and Cu-NP (injected with 50 or 100 mg/kg). Gaseous exchange was measured in an open-air-circuit
respiration unit and energy expenditure (EE) was estimated from day 10 to 19 of embryogenesis.
In-ovo injection of 50 mg/kg Cu-NP and CuSO4 during incubation significantly increased O2
consumption and EE compared with the control group, but Cu-NP had the highest effect on the
metabolic rate. However, organ weights relative to the yolk-free body weight were not affected by
the treatments. In addition, blood parameters did not show any changes. These results demonstrate
that Cu-NP can be a prospective replacer of CuSO4, which might be a successful alternative growth
promoter when it is provided in ovo.
Keywords: growth promoter, metabolic rate, gene expression
Introduction
In poultry, there is substantial interest in using Cu as an alternative to antibiotics that can produce
equivalent effects on chicken performance. In reality, feed mixtures are enhanced with high levels
of Cu as growth promoters (Karimi et al., 2011). The objective of the present study was to evaluate
the effects on embryo development after in ovo injection of copper nanoparticles (Cu-NP) and
copper sulphate (CuSO4). It has been demonstrated by Mroczek-Sosnowska et al. (2016) that a
better performance of chickens could be related to changes in the metabolic rate and development
of embryos. We also assumed that in ovo injection of Cu can be used in much smaller doses than
feeding Cu during a post-hatched period.

Broiler eggs (n=300) from Ross 308 (37-weeks old) breeder strain were obtained from a commercial
hatchery and randomly distributed in six groups: control (not injected), injected with demineralised
water, injected with 50 or 100 mg/kg Cu-NP and injected with 50 or 100 mg/kg CuSO4. Oxygen
consumption and carbon dioxide production were measured according to the procedure described by
Chwalibog et al. (2007) in an open-air circuit respiration unit. The hatched chickens were slaughtered
and dissected for blood and organ collection.

Oxygen-consumption showed increments during the incubation period from days 10 to 19 in all
groups. There was a significant increase (P<0.05) of O2 consumption in Cu-NP and CuSO4 groups
compared to the demineralised water and control groups. In-ovo injection of 50 mg/kg Cu-NP resulted
in remarkably (P<0.05) higher O2 consumption, particularly between days 16 to 19 compared to all
other groups. This might be due to a better bioactivity of Cu-NP, affecting the blood vessel formation

and growth to a higher level than CuSO4 (Mroczek-Sosnowska et al., 2015). Furthermore, the
biological effect of Cu-NP is linked directly to physicochemical characteristics and a better ability
to penetrate into the organism than Cu salts (Mamonova et al., 2013). The energy expenditure was
significantly higher (P<0.05) in the Cu-NP group compared to the other groups. However, increasing
metabolic rate did not affect the growth of broiler embryos probably because they already reached
maximum growth due to genetic modification. Blood analyses showed no significant differences
(P≥0.05) among treatments, although the cholesterol and triglycerol levels decreased in groups treated
with Cu-NP compared to the other groups. The yolk-free body weight and the relative organ weight
(heart, liver, breast, intestine) were not affected by in ovo injection of Cu-NP or CuSO4 (P>0.05),
but we observed an increase in the residual yolk sac (P<0.05) for treated groups, being highest in
the group injected with 50 mg/kg of Cu-NP.
The results from the current study indicated the impact of Cu-NP administrated in ovo on metabolic
rate of broiler embryos, which might be a partial explanation of the long-lasting effects on chicken
performance after hatching. Moreover, the in ovo application of Cu-NP could be considered as a
potential replacer of CuSO4 in the future.
References
Chwalibog, A., A.-H. Tauson, A. Ali, C. Matthiesen, K. Thorhauge and G. Thorbek, 2007. Gas exchange, heat
production and oxidation of fat in chicken embryos from a fast or slow growing line. Comparative Biochemistry
and Physiology Part A: Molecular and Integrative Physiology 146: 305-309.
Karimi, A., G. Sadeghi and A. Vaziri, 2011. The effect of copper in excess of the requirement during the starter period
on subsequent performance of broiler chicks. Journal of Applied Poultry Research 20: 203-209.
Mamonova, I.A., M.D. Matasov, I.V. Babushkina, O.E. Losev, Ye.G. Chebotareva, E.V. Gladkova and Ye.V. Borodulina,
2013. Study of physical properties and biological activity of copper nanoparticles. Nanotechnologies in Russia
8: 303-308.
Mroczek-Sosnowska, N., M. Łukasiewicz, A. Wnuk, E. Sawosz, J. Niemiec, A. Skot, S. Jaworski and A. Chwalibog,
2016. In ovo administration of copper nanoparticles and copper sulfate positively influences chicken performance.
Journal of the Science of Food and Agriculture 96: 3058-3062.
Mroczek-Sosnowska, N., E. Sawosz, K.P. Vadalasetty, M. Łukasiewicz, J. Niemiec, M. Wierzbicki, M. Kutwin, S.
Jaworski and A. Chwalibog, 2015. Nanoparticles of copper stimulate angiogenesis at systemic and molecular
level. International Journal of Molecular Sciences 16: 4838-4849.

The effect of the rate of starch digestion on diurnal heat production and
RQ in low and high performing piglets
R.J.J. van Erp1*, H.M.J. van Hees1, R.T. Zijlstra3 and W.J.J. Gerrits2
1Trouw Nutrition R&D, Veerstraat 38, 5831 JN Boxmeer, the Netherlands; 2Animal Nutrition Group,
Wageningen University, P.O. Box 338, 6700 AH Wageningen, the Netherlands; 3Department of
Agricultural Food, and Nutritional Science, University of Alberta, Edmonton, AB, T6G 2P5, Canada;
rik.van.erp@trouwnutrition.com
Abstract
Low performing pigs at weaning are associated with reduced insulin sensitivity at 6 weeks of age.
Therefore glucose metabolism at weaning may be affected differently in low performing (LP) pigs
compared with high performing (HP) pigs when feeding either a slowly digestible starch (SDS) or
rapidly digestible starch (RDS). In a 2×2 factorial block design 30 LP pigs and 30 HP pigs equal in
birth weight were housed in one of four respiration chambers and fed diets containing either RDS or
SDS. In the first week feed was available ad libitum. In the second week feed supply was restricted
to 65% of the observed feed intake in the first week. Heat production and RQ were analysed. Resting
metabolic rate and activity energy expenditure were calculated with penalized b spline regression
procedures. Greater levels of postprandial de novo acid synthesis and lower levels of fat oxidation
during fasting were observed with SDS compared with RDS. However, no differences in diurnal
heat production and respiratory quotient were found between LP and HP pigs.
Keywords: growth retardation, heat partitioning, pigs
Introduction
Growth retardation in early life can occur during lactation (Paredes et al., 2012). S.P. Paredes et al.
(unpublished data) demonstrated that growth retardation during lactation is associated with reduced
insulin sensitivity at 6 weeks of age. The aim of this study was to assess the effect of starch degradation
rate on heat partitioning and respiratory quotients of growth retarded piglets. We hypothesized that
the postprandial increase de novo fatty acid synthesis is lower when feeding slowly digestible starch
(SDS), particularly so in growth retarded piglets.

From a subpopulation of pigs equal in birth weight (1.36±0.13 kg), thirty low performing pigs (LP;
6.41±0.54 kg) and thirty high performing pigs (HP; 9.56±0.69 kg) were selected at weaning. In a 2×2
factorial block design, sixteen groups of five piglets (8 × LP and 8 × HP) were housed in one of four
identical climate respiration chambers for two weeks, and fed diets containing 40% rapid digestible
starch (Remyline AAX-DR rice starch, RDS) or slowly digestible starch (Nastar Pea starch, SDS). In
the first week feed was available ad libitum. In the second week feed supply was restricted to 65%
of the observed feed intake in the first week. Pigs did not receive feed during these measurements.
All measurements were conducted in two age groups (9 and 11 week of age). Heat production and
respiratory quotient (RQ) were analysed by indirect calorimetry. Resting metabolic rate (RMR) and
activity energy expenditure (AEE) were calculated by using penalized b-spline regression procedures
(Van Klinken et al., 2012). All data were analysed using a general linear model with diet, pig type,
block, and age group as fixed effects. Gross energy intake was included as covariable to adjust other
parameters to the same GE intake level.

When feed was available ad libitum, RMR was lower for HP pigs compared with LP pigs (740 vs
757 kJ/kg0.75; P=0.04). When feed supply was restricted RMR tended to be lower for HP compared
with LP pigs (608 vs 624 kJ/kg0.75; P=0.09). When feed was available ad libitum, HP pigs were more
active compared with LP pigs when fed the RDS diet, whereas no difference in AEE was observed
when the SDS diet was fed (diet × type; P=0.05). Figure 1 shows that when feed supply was restricted
AEE was higher for RDS compared with SDS at 4 and 5 hours after the afternoon feeding. RQ was
higher for RDS compared with SDS at two and three hours after each meal, reflecting a higher level
of de novo fatty acid synthesis on SDS diets. Also during the 8 hours prior to the morning feeding,
RQ was higher for SDS compared with RDS. Presumably, this is a combination of the oxidation
of glucose due to a slower digestion rate of starch compared with RDS, and oxidation of VFA as a
result of a higher level of fermentation with SDS. Circadian patterns of HPtot, AEE, RMR and RQ
did not differ between LP and HP pigs. In conclusion, SDS results in less de novo fatty acid synthesis
in the fed state and lower levels of fat oxidation during fasting compared with RDS. No differences
between HP pigs and LP pigs were observed.
HP*RDS LP*RDS
A 1000 HP*SDS LP*SDS B 350
* * **
HPtot (kJ/kg0.75)
300
AEE (kJ/kg0.75)
900
250
800 200
700 150
100
600 50
500 0
0 2 4 6 8 10 12 14 16 18 20 22 0 2 4 6 8 10 12 14 16 18 20 22
Hour Hour
C D
1.05 * * * * * * * * * * * *
RMR (kJ/kg0.75)
670
1.00
620
0.95
RQ
570 0.90
520 0.85
0 2 4 6 8 10 12 14 16 18 20 22 0 2 4 6 8 10 12 14 16 18 20 22
Hour Hour
Figure 1. (A) Circadian patterns of total heat production (HPtot), (B) activity energy expenditure
(AEE), (C) resting metabolic rate (RMR), and (D) respiratory quotient (RQ) of low performance (LP,
square) and high performance (HP, triangle) pigs fed either a slowly digestible starch diet (SDS,
solid) or rapidly digestible starch diet (RDS, open) when feed supply was restricted. Arrows indicate
feeding time. Asterisks indicate significant differences between diets within each time point (P<0.05).
References
Paredes, S.P., A.J.M. Jansman, M.W.A. Verstegen, A. Awati, W. Buist, L.A. den Hartog, H.M.J. van Hees, N. Quiniou,
W.H. Hendriks and W.J.J. Gerrits, 2012. Analysis of factors to predict piglet body weight at the end of the nursery
phase. Journal of Animal Science 90(9): 3243-3251.
Van Klinken, J.B., S.A. van den Berg, L.M. Havekes, K.W. van Dijk, 2012. Estimation of activity related energy
expenditure and resting metabolic rate in freely moving mice from indirect calorimetry data. PloS One 7(5):
36162-36162.

Interactions among leucine and threonine on growth and amino acid
metabolism in weaned piglets
A.G. Wessels1*, H. Kluge2, F. Hirche2, J. Bartelt3, E. Corrent4 and G.I. Stangl2
1Department of Animal and Food Science, Faculty for Veterinary Science, Autonomous University
of Barcelona, 08193 Bellaterra, Spain; 2Institute of Agricultural and Nutritional Sciences, Martin-
Luther University Halle-Wittenberg, 06120 Halle, Germany; 3Lohmann Animal Nutrition GmbH,
27472 Cuxhaven, Germany; 4AJINOMOTO EUROLYSINE S.A.S., 75817 Paris, Cedex 17, France;
anna.wessels@uab.cat
Abstract
High doses of dietary leucine (Leu) may modify the availability of other dietary amino acids (AA)
such as valine, isoleucine and tryptophan for protein synthesis. Since threonine (Thr) has an alternative
degradation pathway which is catalysed by the branched-chain keto acid dehydrogenase complex
(BCKDH), a study was performed to elucidate effects of dietary Leu in the availability of Thr. The
two-factorial study design included 2 levels of Leu (standardised ileal digestible (SID) Leu:Lys 100
versus 200%) and Thr (SID Thr:Lys 57 versus 65%) each, applied with low protein diets (15.6%
crude protein; SID Lys 0.93%) to weaned piglets. The experiment was divided into 2 parts: (1)
documentation of growth response over 42 days with 60 female piglets fed ad libitum; and (2) nitrogen
balance study with restricted feed application over 21 days to 20 male piglets and collection of urine
and faeces. Subsequently, the piglets of the balance study were sacrificed for sampling of blood and
tissues, which were analysed for free AA concentrations and activity of BCKDH, respectively. The
growth performance remained unaffected by dietary treatment. High intakes of dietary Leu reduced
the concentrations of plasma valine and isoleucine. Simultaneously, adequate intakes of Thr lowered
this effect in tendency. Neither the basal BCKDH activity in liver nor in cardiac- or skeletal muscle
was affected by dietary treatment. In presence of adequate intakes of Leu (SID Leu:Lys 100%) the
nitrogen retention was significantly improved by higher supplementation of Thr.
Keywords: low protein diet, amino acids, pig, nitrogen retention, BCKDH
Introduction
Generally, leucine (Leu) is found in abundant amounts in common feed ingredients used in practical
pig diets while the amounts of other essential amino acids (AA) do not fulfil the requirements.
High doses of dietary Leu may modify the availability of other AA such as valine, isoleucine and
tryptophan for protein synthesis by stimulating the common metabolism or due to the competition
for physiological transport systems. House et al. (2001) described alternative degradation pathway
for threonine (Thr) which is catalysed by the branched-chain keto acid dehydrogenase complex
(BCKDH) – an enzyme complex which degrades the branched-chain AA Leu, valine and isoleucine
irreversibly. The objective of the present study was to test whether high intakes of dietary Leu may
increase the degradation of Thr by stimulation of hepatic BCKDH, in which extent other AA are
reduced in blood plasma and if dietary AA imbalances affect the nitrogen retention on the weaning
period of pigs.

Possible interactions among Leu and Thr were analysed in a two-factorial study design. To this
end, 4 low-protein (15.6% crude protein; standardised ileal digestible (SID) Lys 0.93%) diets were
prepared with 2 levels each of Leu (SID Leu:Lys 100 versus 200%) and Thr (SID Thr:Lys 57 versus
65%). The experiment was divided into 2 parts. Within the first part 60 (n=15) female piglets with
an average body weight of 10.2 kg randomly allotted to one of the 4 treatment groups. These piglets

were single-housed and fed ad libitum over 42 days. Body weight and feed intake were recorded
weekly to calculate average weight gain and gain-to-feed ratio. Simultaneously, within the second
part, 20 (n=5) male piglets with a body weight of 10.1 kg were randomly allotted to one of the 4
treatment groups and placed in balance cages. These piglets were fed specified amounts of feed and
samples of urine and faeces were collected daily. After 21 days the piglets from the balance part
were sacrificed to collect samples. Blood plasma was collected to determine free AA concentrations.
Liver and kidney tissue, cardiac and skeletal muscle (longissimus dorsi, 7th rib) were analysed for
the basal activity of the enzyme BCKDH. Two-way ANOVA was performed using IBM SPSS.

There was no effect of the experimental diets on growth performance of the piglets (P>0.1).
Increasing dietary Leu levels resulted in higher plasma concentrations of Leu (P<0.001), and lower
concentrations of valine and isoleucine (P<0.001). Threonine is known to have beneficial effects on
the total amount of intestinal mucin, which improves the uptake of nutrients by the small intestine
(Montagne et al., 2004). Accordingly, the uptake of dietary AA tended to increase with higher intakes
of dietary Thr, since groups fed SID Thr:Lys 65% showed higher concentrations of branched-chain
AA in plasma compared to the low-Thr groups (P<0.1; Table 1). The BCKDH activity and the Thr
concentration in plasma were not altered by the dietary Leu level. It could be assumed that SID
Leu:Lys 200% is not sufficient to increase alternative degradation of Thr by BCKDH. Threonine
level increased nitrogen retention (P<0.05) only when Leu levels were adequate (SID Leu:Lys
100%). Except for the nitrogen retention, no interaction effects among dietary Leu and Thr were
found. Based on the results of the blood plasma analyses we conclude that higher supplementation
of Thr could weaken the physiological disadvantages caused by imbalanced diets and improves the
nutrient uptake.
Table 1. Effects of dietary threonine (Thr) and leucine (Leu) on plasma amino acid concentrations
(µmol/l) of piglets.1,2
SID Leu:Lys (%) 100 200 100 200 P-value
SID Thr:Lys (%) 57 57 65 65 Leu Thr Leu×Thr
Threonine 300±61a 260±91a 478±54b 445±96b 0.309 0.000 0.926

Valine 483±34b 246±41a 498±35b 288±25a 0.000 0.082 0.387
Isoleucine 207±14b 100±14a 222±22 b 116±16a 0.000 0.054 0.942
Leucine 227±37a 391±70b 230±28a 478±51b 0.000 0.058 0.076
BCAA 916±81b 737±119a 949±80a 882±90ab 0.010 0.049 0.200
1 Data are presented as means ± SD, values within a row with different superscripts differ significantly (Tukey-test,
P<0.05); n=5 per treatment.
2 BCAA = branched-chain amino acids; SID = standardised ileal digestible.
References
House, J.D., B.N. Hall and J.T. Brosnan, 2001. Threonine metabolism in isolated rat hepatocytes. American Journal
of Physiology, Endocrinology and Metabolism 281: E1300-E1307.
Montagne, L., C. Piel and J.P. Lallès, 2004. Effect of diet on mucin kinetics and composition: nutrition and health
implications. Nutrition Reviews 62: 105-114.

The optimal lysine requirement of modern genotype piglets
S. Millet1*, M. Aluwé1, E. Le Gall3, E. Corrent3, J. De Sutter2 and S. De Campeneere1
1Institute for Agricultural and Fisheries Research (ILVO), Animal Sciences Unit, Scheldeweg 68, 9090
Melle, Belgium; 2Orffa Belgium NV, Rijksweg 10G, 2880 Bornem, Belgium; 3Ajinomoto Eurolysine
S.A.S., 153 Rue de Courcelles, 75817 Paris Cedex 17, France; sam.millet@ilvo.vlaanderen.be
Abstract
In total 30 pens of 6 piglets each were divided over 5 dietary treatment groups (T1-T5). Diets were
formulated to design a dose-response to lysine (SID lysine was 8.50, 9.75, 11.00, 12.25, and 13.50 g/
kg, for T1-T5, respectively). Treatments were iso-energetic (net energy was 9.8 MJ/kg), and respected
the ideal amino acid profile for piglets. Performances improved linearly with increasing SID lysine
concentration. Within the measured range, a linear increase in feed intake (R2=0.66, P<0.001) and
daily gain (R2=0.86, P<0.001) and a linear improvement of feed conversion ratio (R2=0.85, P<0.001)
was observed with increasing lysine levels. Modelling with plateau models did not permit to find a
breakpoint within the measured range.
Keywords: amino acid, pig
Introduction
Over the last decades, studies have been performed on the lysine requirement of piglets. Since then,
genetics have evolved. Above, in Flanders, lysine levels in commercial diets are mostly below the
requirements as determined previously at our institute (Warnants et al., 2005). Therefore we designed
a trial to study the effect of dietary lysine concentration on performance of modern genotype (hybrid
sow × Piétrain boar) piglets between 4 and 9 weeks of age.

Animals and housing
The pigs were a crossing of a Piétrain boar and a hybrid sow (RA-SE genetics). In two blocks, each
time 90 piglets were divided over 15 pens of 6 pigs at weaning. Each pen consisted of 3 female
and 3 castrated male piglets. Each pen was randomly assigned to a treatment (T1-T5), with in total
3 pens per treatment and per block. One pen measured 1.0 m × 1.8 m (1.8 m2). The slatted floor
was covered with a synthetic coating. The temperature in the compartments ranged from 26 °C at
the start to 22 °C at the end of the experiments. The pigs had free access to water and were fed ad
libitum. Daily feed intake, daily gain and feed conversion ratio were determined for each treatment
group during the experimental period, from 4 to 9 weeks of age.
Diets
In total 30 pens of 6 piglets each (7.7±0.6 kg) were divided over 5 dietary treatment groups (T1-T5).
Diets were formulated to design a dose-response to standardised ileal digestible (SID) lysine (Table
1). Treatments were iso-energetic and respected the ideal amino acids profile for piglets (Gloaguen
et al., 2013).

Over all groups, bodyweight at 9 weeks was 21.7±2.6 kg. SID lysine level had a clear effect on
performances (Table 2). Within the measured range, an increase of dietary SID lysine concentration
with 1 g/kg led to an increased feed intake of 24 g (feed intake = 23.8x + 278.0, R2=0.66) and an

Table 1. Formulated nutrient concentration of the experimental diets (treatment 1-5).1
T1 T2 T3 T4 T5
Net energy, MJ/kg 9.8 9.8 9.8 9.8 9.8

Crude protein, g/kg 201 203 205 208 210
SID lysine, g/kg 8.50 9.75 11.00 12.25 13.50
1 SID = standardised ileal digestible.
Table 2. Effect of standardised ileal digestible lysine content on performances of piglets between 4
and 9 weeks of age.1,2
T1 T2 T3 T4 T5 SEM P-value
Daily gain, g 284a 333b 390c 437d 450d 12 <0.001

Daily feed intake, g 474a 509ab 546bc 587c 583c 10 <0.001
FCR, g/g 1.67a 1.53b 1.40c 1.35cd 1.30d 0.03 <0.001
1 Values with different superscript differ according to Tukey’s post-hoc test (P<0.05).
2 FCR = feed conversion ratio.
increased growth rate of 35 g per day (daily gain = 34.8x – 4.3, R2=0.86). The linear response was
even more clear with daily gain in function of daily lysine intake (y = 44.8x + 109.5, R2=0.95).
However, data modelling did not permit to define the requirement of the animals, as there was no
clear breakpoint.
Especially in piglets, energy intake is limiting growth. A considerable part of the ingested feed is
used for maintenance requirements. Surplus energy consumed can be used exclusively for growth.
The gain in growth speed was higher than the increase in feed intake, which suggests that this
growth was mainly an increase in muscle mass. The increase in the efficiency of the daily muscle
deposition resulted in a linear improvement of feed conversion ratio (FCR = -0.075x + 2.275,
R2=0.85, P<0.001) with increasing lysine levels. The results coincide with the findings of Warnants
et al. (2005). They suggest optimal SID lysine content of 12.6 (for FCR) or 12.3 (for DG) g/kg.
Similarly, Kendall et al. (2008) deducted a true ileal digestible lysine concentration of 13 g/kg. The
NRC (2012) recommendation for piglets between 11 and 25 kg is also 12.3 g/kg of SID lysine. These
levels are close to T4 and T5 of the present trial, which may explain why no breakpoint was found.
Conclusion
The results show that within the range of 8.50-13.50 g/kg, performances increase linearly with
increased dietary lysine level.
References
Gloaguen, M., N. Le Ffloc’h and J. van Milgen, 2013. Couverture des besoins en acides amines chez le porcelet alimenté
avec des régimes à basse teneur en protéines INRA Prod. Anim. 26(3): 277-288.
Kendall, D.C., A.M. Gaines, G.L. Allee and J.L. Usry, 2008. Commercial validation of the true ileal digestible lysine
requirement for eleven-to-twenty-seven-kilogram pigs. Journal of Animal Science 86: 324-332.
Warnants, N., M.J. van Oeckel, M. De Paepe and D. De Brabander, 2005. Aminozurenbehoeften van big tot vleesvarken.
Departement Dierenvoeding en Veehouderij, Melle, Belgium, pp. 19-32.

Part 3.
Animal product quality and health in the light of protein and
energy metabolism and nutrition
Lamb intramuscular fat percentage is correlated between muscles, with
whole body fatness, and with muscle oxidative capacity in the loin
G.E. Gardner*, D.W. Pethick and F. Anderson
School of Veterinary and Life Sciences, Murdoch University, Murdoch, WA 6150, Australia; Australian
Cooperative Research Centre for Sheep Industry Innovation, University of New England, Armidale,
NSW 2351, Australia; g.gardner@murdoch.edu.au
Abstract
This study assessed correlations for intramuscular fat percentage (IMF%), and oxidative capacity
between the M. longissimus lumborum (loin) and muscles in the fore and hind section, as well as
their association with whole carcase fatness. The IMF% correlations between the loin and the other
muscles of the carcase were strong, as was the correlation with carcase fatness, however muscle
oxidative capacity correlated poorly with IMF% in all muscles except the loin. This suggests that
IMF% in other muscles of the carcase can be predicted by IMF% in the loin, that this prediction will
be enhanced by accurate measurement of carcase fatness, but not by indicators of muscle oxidative
capacity.
Keywords: breeding values, computed tomography, leanness
Introduction
Eating quality of lamb meat is essential for maintaining consumer demand and is strongly associated
with intramuscular fat percentage (IMF%) (Pannier et al. 2014). On this basis work is underway to
develop systems for determining IMF% in Australian abattoirs using measurements taken from the
loin. This assumes that loin IMF% would correlate strongly to that evident within other muscles of
the carcase, an assertion supported by work in beef (Brackebusch et al., 1991), but not previously
explored in lamb.
To further enhance the prediction of IMF% in other muscles, alternative biochemical indicators
could be explored. Kelman et al. (2014) noted an association between IMF% in the loin and muscle
oxidative capacity. If this association extended to other muscles, this would imply that knowledge
of muscle type (indicated through colour) may further improve the prediction of IMF% in muscles
beyond the loin. Muscle oxidative capacity can be indicated by isocitrate dehydrogenase activity and
myoglobin concentration, with the red myoglobin pigment associated with colour in meat (Kelman
et al., 2014). Therefore we hypothesise that IMF% in lamb will correlate between different muscles
of the carcase, enabling eating quality prediction. Furthermore we expect that increasing muscle
oxidative capacity will be associated with increased IMF% in muscles other than just the loin.

Pasture fed lambs (n=360) were slaughtered at a target carcase weight of 23 kg in 2011. These lambs
were from the Katanning site of the Sheep Information Nucleus Flock and were the progeny of sires
divergent for numerous production traits, including carcase fatness. Within 4 hours postmortem,
muscle samples were collected from 3 sections of the carcase for analysis of myoglobin concentration,
and isocitrate dehydrogenase and lactate dehydrogenase activity: from the fore section, the M.
supraspinatus and M. infraspinatus; from the saddle section, the M. longissimus lumborum (loin); and
from the hind section, M. semimembranosus and M. semitendinosus. Within 72 hours post-mortem
carcases were CT scanned to determine their fat percentage (CTfat%), and then the muscles mentioned
above were sampled for IMF%. Multivariate analyses were used to determine partial correlations
for IMF% between muscles and with CTfat%, with these comparisons repeated to determine simple

correlations. Bivariate analyses we used to determine partial correlations between IMF%, myoglobin
concentration, and isocitrate dehydrogenase activity within individual muscles. The multivariate and
bivariate models were all corrected for fixed effects including sex, siretype, and dambreed.

As reported in Anderson et al. (2015), the correlation in IMF% between the loin to the other muscles
was fairly consistent (Table 1) with values ranging between 0.34 and 0.40 (partial correlation
coefficient). There was a strong correlation between the m. supraspinatus and the m. infraspinatus
in the fore section of the carcass, and the weakest correlations were those between the forequarter
and hindquarter muscles. Simple correlations demonstrated similar trends to the partial correlation
coefficients. The correlation of IMF% and CTfat% varied between muscles from 0.24 to 0.41
(partial correlation coefficient, Table 1), and was strongest in the loin. This supports the correlated
maturation of all fat depots throughout the body (Butterfield, 1988), and implies that a single point
measurement of IMF% within the loin is adequate for predicting IMF%, and therefore eating quality
within other muscles of the carcase. This prediction could be further enhanced with the inclusion
of an accurate measurement of carcase fatness, although it is likely that this would introduce bias
in instances where selection indexes simultaneously target a reduction in back-fat and an increased
IMF%, effectively uncoupling the biological correlation between these traits.
Within the loin there was a weak correlation (P<0.01) between IMF% and isocitrate dehydrogenase
activity and myoglobin concentration, with partial correlation coefficients of 0.14. Yet contrary to our
hypothesis, this association was not evident in any of the other muscles tested. Therefore knowledge
of muscle type, which could be indicated by its pigment colour association, is unlikely to further
enhance prediction of IMF% in the carcase.
Conclusion
Measurement of IMF% in the loin enables prediction of IMF% in other muscles, suggesting scope
for predicting their eating quality. Accurate measurement of whole carcase fatness will enhance this
prediction, however indicators of muscle oxidative capacity will not.
Table 1. Partial (above diagonal) and simple correlation coefficients (below diagonal) between
the IMF% in the m. semimembranosus (SM), m. semitendinosus (ST), m. supraspinatus (SS), m.
infraspinatus (IS) and m. longissimus lumborum (LL), and for % carcase fat (CT fat%) measured
using computed tomography in lamb.1
SM ST SS IS LL CT fat%
SM 1 0.43 0.3 0.38 0.4 0.3

ST 0.42 1 0.25 0.29 0.4 0.24
SS 0.41 0.3 1 0.68 0.34 0.29
IS 0.48 0.34 0.75 1 0.34 0.25
LL 0.45 0.47 0.45 0.45 1 0.41
CT fat% 0.24 0.32 0.36 0.31 0.48 1
1 All correlations are significantly different from zero (P<0.05).

References
Anderson, F., Pethick, D.W. and Gardner, G.E., 2015. The correlation of intramuscular fat content between muscles of
the lamb carcass and the use of computed tomography to predict intramuscular fat percentage in lambs. Animal
9: 1239-1249.
Brackebusch, S.A., Mckeith, F.K., Carr, T.R. and Mclaren, D.G., 1991. Relationship between Longissimus Composition
and the Composition of Other Major Muscles of the Beef Carcass. Journal of Animal Science 69: 631-640.
Butterfield, R., 1988. New concepts of sheep growth. University of Sydney, Sydney, Australia.
Kelman, K.R., Pannier, L., Pethick, D.W. and Gardner, G.E., 2014. Selection for lean meat yield in lambs reduces
indicators of oxidative metabolism in the longissimus muscle. Meat Science 96: 1058-1067.
Pannier, L., Gardner, G.E., Pearce, K.L., McDonagh, M., Ball, A.J., Jacob, R.H. and Pethick, D.W., 2014. Associations
of sire estimated breeding values and objective meat quality measurements with sensory scores in Australian lamb.
Meat Science 96: 1076-1087.

Alicar: a database for carcass characteristics, diet composition and
intake in ruminants
J. Vernet1,2*, M. Reichstadt1,2, M. Al-Jammas1,2 and I. Ortigues-Marty1,2
1 INRA, UMR1213 Herbivores, 63122 Saint-Genès-Champanelle, France; 2 Clermont
Université, VetAgro Sup, UMR1213 Herbivores, BP 10448, 63000 Clermont-Ferrand, France;
jean.vernet@clermont.inra.fr
Abstract
The Alicar database gathers published data on performance and carcass composition of ruminants
as influenced by dietary conditions. It was developed with the Merise Method. The environment is
APACHE, MySQL and PHP. The relational model of Alicar counts 11 tables: publication, experiment,
animal group, treatment, measurement-results, methods, feeds, ingredient composition and ration
intake, feed chemical composition, INRA feed specific code and meta-analysis code. Animals,
feeding conditions and methods are finely described. Currently, Alicar includes 111 publications in
cattle (794 treatments). Cattle data cover a range of animal maturing rates (mainly intermediate and
early), and production types (mainly meat and dual purpose). Carcass composition is from chemical
or physical measurements, and/or proxy traits.
Keywords: database, carcass characteristics, diet composition, intake, ruminants
Introduction
The amount of available published information on the impact of nutrition on carcass characteristics
in ruminant animals is large. The information can be aggregated in databases and used to develop
response equations by meta-analyses. The objective of the present work is to present the Alicar
database (alimentation, animal performances and carcass characteristics): (1) its structure as an
example of structuration of data from the literature for modelling purposes; and (2) its current
contents from cattle based publications.

The Alicar Database was developed with the Merise method, similarly to the Flora database (Vernet
and Ortigues-Marty, 2006) but with differences in the relational model. It was developed as part
of a full web data warehouse Nutriflux (Vernet et al., 2007). Hence, it has a SQL architecture and
the environment is APACHE, MySQL and PHP. The current cattle dataset in Alicar was built from
WOS based on the following criteria: carcass composition and dietary data (111 publications, 794
treatments). The distribution of data against animal factors modalities was studied. Descriptive
statistics were calculated for the different variables and the effects of the main animal factors were
studied by one way Anova (Minitab 17).

The Relational model of Alicar presents the different data tables and the relationships between them
(Figure 1).
First, 4 tables describe the publication (publication table), the experiment (experiment table), the
animals (animal-group table) and the experimental treatments (treatment table) (Figure 1). Groups
of animals are described as precisely as possible (species, breed, type of breed, sex, type of animal,
physiological stage). The measurement-result table describes all types of measurements. Methods
(method table) are indexed to evaluate the impact of methods on measurement-results. For the

Publication Experiment Animal-group Treatment Measurement-result Method
Feed Ingredient Meta-analysis-code

composition
of ration and
intake
INRA-feed- Feed-chemical-
specific-code composition
Figure 1 The Alicar relational model.
description of feeding characteristics, 3 tables are used to describe: the publication feeds and rations
(feed table), the ingredient composition of rations and intake (ingredient-composition-of-rations-
and-intake table), and the chemical composition of rations (feed-chemical-composition table). To
have a fine description of publication diets, diets are characterized according to INRA (2007) feed
tables, using the specific code of each relevant feeds (INRA-feed-specific-code table), with a link
to INRA (in press).
Current cattle data (Table 1) show a wide range of animal characteristics and performances. Maturing
rates are essentially intermediate (40.3%) and early (37.4%) followed by late (12.1%) and dairy
breeds (10.2%). Production type are mainly meat (60.2%), dual purpose (29.6%), and dairy (10.2%).
Physiological stages are first fattening (58.1%), then growth + fattening (27.1%), growth (11.7%)
and not reported (3.1%). Sexes are castrated males (implanted or not, 42.1%), then castrated males
(36.4%), males (7.2%), implanted females (3.9%) and the rest (10.4%). From those animals, average
daily gains, slaughter weights and carcass yield, varied with sex, maturing rate, production type
and stage. Carcass lipid and protein contents are reported in 19.8% of the publications, while proxy
traits in 87% (sub cutaneous fat thickness) or 58.5% (yield grade). They all show a wide range of
variation, and can be used for meta-analysis (Al-Jammas et al., 2016).
Table 1. Meta-design of animal performances and carcass traits in cattle (Alicar database).1,2
Ntreatments Mean Sd Min Max Sex Mat-rate Prod-ty Stage
ADG (g/d) 734 1,362 330 212 2,240 ***(28%) ***(13%) ***(4%) ***(3%)
FCR (kg/kg) 302 7.68 2.51 4.17 28.5 ***(9%) ***(5%) **(3%) **(3%)
SW (kg) 351 524 82 169 753 ***(28%) ***(15%) ***(6%) ***(19%)
HCW (kg) 715 326 54 94 516 ***(12%) ***(7%) ***(5%) ***(8%)
CY (%) 307 60.68 2.95 50.9 65.5 ***(34%) ***(42%) ***(26%) *(1%)
SCFT 682 11.84 4.02 0.8 35 ***(6%) ***(12%) NS ***(3%)
YG 486 2.85 0.54 1.05 4.7 *(1%) NS NS NS
Mar (10 points) 222 4.44 0.81 1.4 8.33 NS ***(6%) ***(6%) NS
LIP%CC 238 31.27 4.07 8.4 40.4 ***(16%) ***(28%) ***(5%) ***(9%)
Prot%CC 191 15.24 1.56 13.2 21.9 NS ***(9%) *(2%) ***(10%)
1 Mat-rate = maturing rate (early, intermediate, late; dairy breeds); Prod-ty = production type (meat, dairy, dual purpose);
Stage = physiological stage (growth, fattening, growth + fattening); ADG = average daily gain; FCR = feed conversion
ratio; SW = slaughter weight; HCW = hot carcass weight; CY = carcass yield; SCFT = sub cutaneous fat thickness;
YG = yield grade; Mar = marbling score; LIP%CC = lipid weight % cold carcass weight; Prot%CC = protein weight
% cold carcass weight.
2 ***P<0.001; **P<0.01; *P<0.1; values in parenthesis: adjusted R2.

References
Al-Jammas, M., J. Agabriel, J. Vernet and I. Ortigues-Marty, 2016. Effects of diet composition on carcass fat in beef
cattle: a meta-analysis. In: J. Skomial and H. Lapiere (eds.). Energy and protein metabolism and nutrition. EAAP
Scientific Series No. 137. Wageningen Academic Publishers, Wageningen, the Netherlands, pp. 233-234.
INRA, 2007. Alimentation des bovins, ovins et caprins. Tables INRA 2007, Editions Quae, Versailles, France.
INRA, in press. INRA 2016 Feed Unit System for Ruminants. Wageningen Academic Publishers, Wageningen, the
Netherlands.
Vernet, J. and I. Ortigues-Marty, 2006. Conception and development of a bibliographic database of blood nutrient fluxes
across organs and tissues in ruminants. Reproduction Nutrition Development 5: 527-546.
Vernet, J., J.P. Brun, I. Ortigues-Marty, 2007. Bibliographical database applied to ruminant nutrition. In: I. Ortigues-
Marty, N. Miraux and W. Brand-Williams (eds.). Energy and protein metabolism and nutrition. EAAP Scientific
Series No. 124. Wageningen Academic Publishers, Wageningen, the Netherlands, pp. 591-592.

Effects of diet composition on carcass fat in beef cattle: a meta-analysis
M. Al-Jammas, J. Agabriel, J. Vernet and I. Ortigues-Marty
INRA, UMR1213 Herbivores, 63122 Saint-Genès-Champanelle, France;
isabelle.ortigues@clermont.inra.fr
Abstract
To improve the ability of the MecSic model to predict carcass composition of beef cattle, the objective
was to identify the dietary characteristics that significantly influence the relationship between carcass
composition and metabolisable energy intake and that could ultimately be introduced into MecSic.
A meta-analysis was applied to 61 publications in finishing cattle. Results showed that the dietary
concentrations of fibre, starch and protein had an effect on the relationship. Therefore, at iso-energy
intake, the type of diets (concentrate vs forage) and the ratio protein /energy can modify carcass
composition of beef cattle.
Keywords: carcass fat, finishing cattle, energy, diet
Introduction
Carcass quality of beef cattle is an important criteria in the remuneration of producers. With the aim
of developing nutritional strategies towards improved carcass quality, models were developed to
predict carcass composition. The model MecSic (Hoch and Agabriel, 2004) simulates growth and
carcass composition driven by metabolisable energy (ME) intake (MEI) during the finishing period.
However, the simulations do not account for the source of dietary energy (fibre vs starch) nor the level
of protein intake at iso-energetic intakes, and their potential effects on tissue deposition for a given
type of animal. Preliminary work identified a possible indicator of the effects of diet composition
(a ratio of absorbed nutrients) on tissue deposition (Agabriel et al., 2013). As a first step towards
expanding these results, our objective was to identify the dietary characteristics that influence the
carcass fat content besides MEI by meta-analysis from published results.

61 publications (76 studies, 343 treatments) on finishing cattle were extracted from the Alicar database
(Vernet et al., 2016). All reported results on (1) diet composition and intake; and (2) chemical carcass
composition (lipid and/or protein), USDA yield grade or subcutaneous fat thickness (predictors
of carcass composition). Diets were characterized using INRA Feed Tables (2007, in press), and
animal breeds according to their maturing rates (early, intermediate or late). A within-study variance–
covariance model was developed (Minitab 16) according to Sauvant et al. (2008) as: carcass fat (%
of hot carcass weight) = α + αi (Maturing rates) + Maturing rates+ β MEI (kcal.d-1.kg BW-0.75) + e,
with α = overall intercept, αi = effect of the experimental study i on the intercept α, β = slope and
e = error. Maturing rate was introduced in the model following a preliminary principal component
analysis which showed its strong correlation with carcass fat. Presence of factors having a significant
impact on residuals and individual slopes was tested either by analysis of variance (for qualitative
factors such as animal breeds or sex) or regression (for quantitative factors such as diet composition).

The dataset covered a wide range of animals in terms of breeds (68% of beef breeds) and maturing
rates (46% of early maturing and 46% of intermediate maturing), with a majority of males (95% of
castrated males, implanted or not). The ranges of live animal and carcass characteristics were also
wide (average daily gain from 0.6 to 2.1 kg/d, hot carcass weight from 226 to 413 kg, and fat content

of hot carcass from 21 to 41%). Dry matter (DM) intake ranged from 5.2 to 13 kg/d, and dietary
concentrations of starch, neutral detergent fibre (NDF), protein digestible in the intestine (PDI) and
ME were 0-730, 105-613, 46-147 g/kg DM and 9-14 MJ/kg DM, respectively.
At similar MEI, the proportion of fat in the carcass was 0.6 and 2.1% higher (P<0.00) in early
maturing breeds than in intermediate and late maturing breeds respectively. Whatever the breed, MEI
was the primary driver of carcass composition (Figure 1A) but did not totally account for changes of
carcass fat concentration (Figure 1B). Residuals of the model were significantly related to the dietary
concentration of PDI (P=0.04) and digestible starch /NDF ratio (P=0.01). Individual slopes were
significantly affected by the dietary NDF and starch concentrations, as well as the starch/NDF ratio
(P=0.02) and digestible starch /ME ratio (P=0.01). In conclusion, carcass composition in finished
cattle varies with diet composition in addition to MEI. Potential indicators of these effects are the
composition of ME (fibre vs starch) and the protein/energy ratio. Response equations are needed in
order to improve the simulation de carcass composition from MEI (Agabriel et al., 2013).
38 38
35 35
Observed carcass fat (%)
32
Carcass fat (%)
32
29 29
26 26
23 23
20 20
20 23 26 29 32 35 38 150 200 250 300 350 400
Predicted carcass fat (%) MEI
Figure 1. (A) Observed vs predicted carcass fat, % and (B) intra-study relationships between carcass
fat % and metabolisable energy intake (MEI; kcal/d kg BW0.75).
References
Agabriel, J., M. Al-Jammas, I. Ortigues-Marty, C. Villetelle, P. Noziere and F. Garcia-Launay, 2013. Effects of diet
composition during the finishing period on protein and lipid deposit in young bulls. In: J.W. Oltjen, E. Kebreab and
H. Lapierre (eds.). Energy and protein metabolism and nutrition in sustainable animal production. EAAP Scientific
Series No. 134. Wageningen Academic Publishers, Wageningen, the Netherlands, pp. 319-320.
Hoch, T., and J. Agabriel, 2004. A mechanistic dynamic model to estimate beef cattle growth and body composition:
1. model description. Journal of Agricultural Systems 81: 1-15.
INRA, 2007. Alimentation des bovins, ovins et caprins; besoins des animaux-valeurs des aliments. Édition Quae,
Versailles, France, 311 pp.
INRA, in press. INRA 2016 Feed Unit System for Ruminants. Wageningen Academic Publishers, Wageningen, the
Netherlands.
Sauvant, D., P. Schmidely, J.J. Daudin and N.R. St-Pierre, 2008. Meta-analyses of experimental data in animal nutrition.
Animal 2: 1203-1214.
Vernet, J., M. Reichstadt, M. Al-Jammas and I. Ortigues-Marty, 2016. Alicar: a database for carcass characteristics,
diet composition and intake in ruminants. In: J. Skomial and H. Lapiere (eds.). Energy and protein metabolism
and nutrition. EAAP Scientific Series No. 137. Wageningen Academic Publishers, Wageningen, the Netherlands,
pp. 229-231.

Effect of dietary starch content on prediction of feed intake by six
different models for dairy cows
P. Nørgaard* and L.M. Jensen
Department of Veterinary Clinical and Animal Science, Faculty of Health and Medical Sciences,
University of Copenhagen, Grønnegaardsvej 3, 1, 1870 Frederiksberg, Denmark; pen@sund.ku.dk
Abstract
The objective of this investigation was to evaluate the effect of dietary starch content on the difference
between observed and predicted intake (residual) by dairy cows. The study was conducted on two
independent data sets, where dry matter intake (DMI) was predicted by the Dutch model from Zom,
the German, Austria and Swiss model from Gruber, the Finnish TDMI model from Huthanen, the
NRC model and the Scandinavian model from NorFor, and analysed on a data set consisting of 12
experiments including 917 lactating dairy cows fed 94 different rations ad libitum. Furthermore,
the prediction of daily net energy intake (NEI) by the NorFor intake model and NEI model by
Jensen was analysed on a data set consisting of 19 experiments including 812 lactating dairy cows
of different breeds fed 80 different rations ad libitum. Net energy intake was estimated by the use
of the NorFor digestive kinetic model. The relationship between residual daily DMI and NEI and
dietary starch content was analysed by simple linear regression with random effect of experiments.
In conclusion, the Zom model might be improved by including dietary starch and the NorFor model
appears to over-predict DMI with increasing content of starch in the ration.
Keywords: model evaluation, DMI, NorFor, TDMI, NRC
Introduction
The performance of high-yielding dairy cows is closely related with the intake of energy, which
depends on both animal and dietary characteristics. Recently several intake models have been
developed; the North American NRC (2001) model, the Finnish TDMI model by Huhtanen et al.
(2011), the Dutch model by Zom et al. (2012), the German, Austria and Swiss model by Gruber et
al. (2004), the Scandinavian model NorFor by Volden et al. (2011), and a new net energy intake
model net energy intake (NEI) described by Jensen (2015). The TDMI, Zom, Gruber, NorFor and
NEI models predict intake based on the digestibility of forage or forage NE value and the proportion
of concentrates. The NRC model is based on animal characteristics and milk yield, which is not
used in the Zom model. The Zom model uses content of crude fibre in feeds, and the TDMI model
includes the content of total acids in silage and metabolisable protein. The NorFor model includes
content of forage NDF and total dietary starch and sugar content. The NEI model predicts NEI as a
linear function of the dietary chewing index (Jensen, 2015). The objective of this investigation was
to evaluate the effect of dietary starch content on the difference between observed and predicted
(residual) intake by dairy cows fed different rations by use of different intake models.

The study was conducted on two independent data sets, where the five models predicting dry
matter intake (DMI) were analysed using intake data from 12 experiments including 917 primi- and
multiparous lactating dairy cows of different breeds fed 94 different rations ad libitum (data I), see
details by Jensen et al. (2015). The two models predicting NEI were analysed on 19 experiments
including 812 primi- and multi-parous lactating dairy cows of different breeds fed 80 different rations
ad libitum (data II) (Jensen, 2015). The daily intake of DM ranged between 13 to 25 kg and 15 to 29
kg in data I and II, respectively. The starch content (g/kg DM) ranged between 57 to 267 and 32 to
329 in data I and II, respectively. The intake of NE was estimated by the NorFor digestive kinetic

model. The relationship between residual daily DM and NE values and dietary starch content was
analysed by simple linear regression with random effect of experiments.

Increasing starch content caused a significantly over-prediction of DMI by the Zom model (P<0.001),
while the DMI was under predicted by the NorFor model (P<0.001), and there was a tendency of
over-estimation of DMI by the NRC model (P<0.1) (Table 1). The NEI model tended to over-predict
NEI at increasing starch content (g/kg DM) (P<0.1), where there was no significant effect of starch
content in DM on the residual NEI predicted by the NorFor model. However, NEI was not related
to the content of starch per MJ NE by the NorFor or NEI models. The R2 values of the six models
analysed with random effect of experiment ranged between 0.34 by residual DMI by the Gruber model
to 0.86 by the residual NEI for the NorFor model. This is associated with the difference between
rations, where the R2 values for the fixed part of the regression ranged between 0.001 for residual
DMI of the Gruber model to 0.27 by the Zom model, which can be associated to the variation of
starch content. In conclusion, the Zom model can be improved by including dietary starch into the
model, whereas the NorFor model appears to over-predict the effect of starch in the ration.
Table 1. The relationship between residual intake (observed – predicted) and the dietary content of
starch for six intake models with experiments as random variable.1
Model Data Starch Residual Intercept Slope RMSPE R2
I g/kg DM kg DM/d kg DM/d Fixed2 Random3
Gruber -1.4 -0.07 <0.01 1.80 0.001 0.34

NRC -0.4 -2.20** 0.01T 1.52 0.10 0.53
TDMI 0.1 0.89 -0.01 1.71 0.05 0.40
Zom -1.6 3.95*** -0.03*** 3.16 0.27 0.83
NorFor -0.2 -2.63*** 0.01*** 1.17 0.23 0.75
II g/kg DM MJ NE/d MJ NE/d
NorFor 2.0 3.78 0.012 10.9 0.03 0.86

NEI 2.0 4.78 -0.12T 11.1 0.14 0.94
II g/MJ NE MJ NE/d
NorFor 2.0 3.39 -0.04 10.9 0.03 0.86

NEI 2.0 4.60 -0.11 11.1 0.10 0.94
1 ***P<0.001; **P<0.01; TP<0.1.

2 Experiment as fixed effect.
3 Experiment as random effect.
References
Gruber, L., F.J. Schwarz, D. Erdin, B. Fischer, H. Spiekers, H. Steingass, U. Meyer, A. Chassot, T. Jilg, A. Omermaier
and T. Gruggenberg, 2004. Vorhersage der Futteraufnahme von Milchkühen – Datenbasis von 10 Forschungs- und
Universitätsinstituten Deutschlands, Österreichs und der Schweiz. 116. VDLUFA-Kongress, Rostock, VDLUFA-
Schriftenr 60: 484-504.

Huhtanen, P., M. Rinne, P. Mantysaari and J. Nousiainen, 2011. Integration of the effects of animal and dietary factors
on total dry matter intake of dairy cows fed silage-based diets. Animal 5: 691-702.
Jensen, L.M., 2015. Prediction of feed intake by dairy cows. Development of a new net energy intake model. Phd thesis,
Faculty of Health and Medical Sciences, University of Copenhagen, Copenhagen, Denmark, 132 pp.
Jensen, L.M., N.I. Nielsen, E. Nadeau, B. Markussen and P. Nørgaard, 2015. Evaluation of five models predicting feed
intake by dairy cows fed total mixed ration. Livestock Science 176: 91-103.
National Research Council (NRC), 2001. Nutrient requirements of dairy cattle (7th rev. Ed.), National Academy Press,
Volden, H., N.I. Nielsen, M. Åkerlind, M. Larsen, Ø. Havrevoll and A.J. Rygh, 2011. Prediction of voluntary feed intake.
In: H. Volden (ed.). The Nordic feed evaluation system 2011. Wageningen Academic Publishers, Wageningen,
the Netherlands, pp. 113-126.
Zom, R.L.G., G. André and A.M. van Vuuren, 2012. Development of a model for the prediction of feed intake by dairy
cows: 1. Prediction of feed intake. Livestock Science 143: 43-57.

The effect of silica-calcite sedimentary rock (opoka) in the diet on
texture parameter of selected muscule in broiler
M. Makarski1*, T. Niemiec1, A. Łozicki1, I. Kosieradzka1, D. Pietrzak2, L. Adamczak2, M. Matusiewicz1
and P. Koczoń3
1Warsaw University of Life Sciences, Animal Nutrition and Biotechnology, Nowoursynowska 166,
02-787 Warszawa, Poland, 2Warsaw University of Life Sciences, Food Technology, Nowoursynowska
166, 02-787 Warszawa, Poland, 3Warsaw University of Life Sciences, Chemistry, Nowoursynowska
166, 02-787 Warszawa, Poland; mateusz_makarski@sggw.pl
Abstract
Opoka is a silica-calcite sedimentary rock, that occurs mainly in South-Eastern Europe and Russia.
Primary components of opoka are silicon oxide (SiO2) and calcium carbonate (CaCO3). The effect
of diatomite (fossil shell) on mineral content in tibia bone in both broiler and cockerel has been
demonstrated in literature. Therefore, research on the effect of opoka enriched diet on chemical
composition and quality features of breast and leg muscles of broiler has been started. The study
was carried out on 42 Ross 308 chickens randomly assigned to 2 dietary treatments: I – control, II
– diet with 1% of diatomaceous earth. Birds were raised 42 days. Then the birds were slaughtered
and the carcasses were chilled for 12 h at 4 °C. Carcass analyses were conducted and samples of
breast and leg muscles were collected for further chemical analyses. 24 h post slaughter, samples of
breast and leg muscles were analysed due to pH24, technological parameters and proximate chemical
composition, using standard methods. The obtained data were statistically analysed by one-factorial
ANOVA using Statgraphics + Software (2009). Dietary silica-calcite rock added as 1% of the diet
caused statistically significant increase in ash and decrease in lipid content of leg muscles (P<0.01).
Furthermore, the addition of opoka rock to the diet increased water-holding capacity (WHC),
hardness, springiness, chewiness and cohesion of leg muscles (P<0.05).
Keywords: broiler, silica-calcite sedimentary rock, muscle, chemical composition, texture parameters
Introduction
Opoka is a silica-calcite sedimentary rock, that occurs mainly in South-Eastern Europe and Russia.
Primary components of Opoka are silicon oxide (SiO2) and calcium carbonate (CaCO3). Other
components are calcite, quartz, clay minerals and amorphous SiO. Opoka is quite similar to
diatomaceous earth (diatomite, DE) i.e. siliceous sedimentary rock, due to structure and chemical
composition. The effect of diatomite (fossil shell) on mineral content in tibia bone in both broiler
and cockerel has been demonstrated in literature (Adeyemo, 2013) and (Adebiyi, 2009), respectively.
Diatomaceous earth increases feed efficiency and eggs laying (Eshleman, 1966). Mathis and
McDougald (1995) found that feeding DE significantly improved metabolism in broilers. Therefore,
research on the effect of opoka enriched diet on chemical composition and quality features of breast
and leg muscles of broiler, has been started.

48 one-day-old ROSS 308 broiler chickens (40.9±1.5 g) were randomly assigned into 2 dietary
treatments: I – control, II – diet with 1% of opoka. Each block consisted of 24 birds. The chickens
were reared in individual pens for 42 days under standard conditions: temperature from 32 to 21 °C;
humidity 64-70%, light/dark cycle 20/4 h during the first 10 d, and 20/10 h to the end of experiment.
The animals had free access to water and feed. Diets, starter (to 14 days) and grower (from 15 to
42 days) were formulated in compliance with NRC requirements (NRC, 1994). The chickens in the
control group were fed ad libitum with the standard diet. Animals in the experimental group were

fed with the standard diet supplemented with 1% of silica-calcite sedimetary rock (opoka). At the
end of the experiment the broilers were fasted for 12 h and slaughtered. Carcasses were chilled at
4 °C for 12 h. 24 h post slaughter, samples of breast and leg muscles were analysed for: pH24 with
an Elmetron CP-411 pH-meter with a combined electrode; water-holding capacity (WHC) with
a modified blotting-paper method; proximate chemical composition (water, protein, fat and ash)
using standard methods; colour of breast skin and breast and thigh meat of chilled carcasses were
measured using a reflectance colorimeter (Chroma Meter CR,**b, Minolta, Japan). Values were
reported according to the CIE (2004) L* a* b* system, where L*, a* and b* represent lightness (or
brightness), redness-greenness and yellowness-blueness, respectively; texture parameters – using
a Zwick testing machine type 1120; collagen content – according to ISO 3496:2000. The obtained
data were statistically analysed by one-factorial ANOVA using Statgraphics + Software (2009).

Dietary silica-calcite rock added as 1% of the diet caused statistically significant increase in ash and
decrease in lipid content of leg muscles (P<0.01). Probably the concentration of ash in the muscles
of the legs of the animals receiving the opoka, increased by the compounds of magnesium, which
contributed to the retention of water in the intracellular fluid. Therefore the total water content in
the leg muscles was significantly higher in the animals receiving the opoka than the animals in the
control group. Less amount of fat in the muscles of the legs in animals with opoka, can also be seen
in the functioning of the liver, the main organ lying on the route of lipid distribution and homeostasis
of magnesium. It is an evidence suggesting that there is an association between dietary magnesium
deficiency and plasma lipids disorder. It has been shown that fat diets decrease magnesium absorption.
In most broiler diets, the main energy source is carbohydrate part of which participates in lipogenesis.
Rayssiguier (Rayssiguier et al., 1981) reported that in magnesium-deficient rats, hypertriglyceridemia
could have arisen as a consequence of increased synthesis of triglycerides in the liver, decreased
removal of lipids from the blood or a combination of both. It is likely that broilers receiving opoka
rich in magnesium compounds effectively metabolise fats in the liver thereby reducing adiposity
of leg muscles. Furthermore, the addition of opoka rock to the diet, increased WHC, hardness,
springiness, chewiness and cohesion of leg muscles (P<0.05). Technological quality refers to a several
meat attributes, mainly the sensoric (colour, tenderness) and the Physico-chemical (pH, cooking
loss, water-holding capacity, clinging, springiness, hardness, chewiness). Increased participation
of water and ash and reduced levels of fat in the muscle tissue of the legs of the animals receiving
opoka significantly improved technological features of the meat. The hardness of the meat with
increased chewiness depends inter alia on the fat and water contents in the meat and the structure of
the muscle fibres. The supplementation of broiler diet with opoka can be effectively used to modify
texture features (hardness, springiness, chewiness, cohesion) of tissue which serves to regulate the
nutritional and technological value of the chicken meat.
References
Adebiyi, O.A, O.A. Sokunbi and E.O. Ewuola, 2009. Performance evaluation and bone characteristics of growing
cockerel fed diets containing different levels of diatomaceous earth. Middle-East Journal of Scientific Research
4(1): 36-39.
Adeyemo, G.P., 2013. Growth performance of broiler chickens fed fossil shell growth promoter. Food and Nutrition
Sci. 4: 16-19.
Eshleman, J.C., 1966. Poultry feed containing about 1% diatomaceous earth. US Pat. No. 3,271,161.
Mathis, G.F. and L.R. McDougald, 1995. Improvement in feed conversion of broilers by feeding amorphous silica
from freshwater diatoms. Poultry Science 74, Suppl. 1: 147.
National Research Council (NRC), 1994. Nutrient requirements of poultry (9th rev. Ed.). National Academy Press,
Rayssiguier, Y., E. Gueux and D. Weiser, 1981. Effect of magnesium deficiency on lipid metabolism in rats fed a high
carbohydrate diet. J. Nutr. Ill: 1876-1883.

Muscle anserine content is associated with pork meat quality and
carnosine synthase gene expression
M.F. Palin1*, J. D’Astous-Pagé2, R. Blouin2, S. Cliche3, F. Fortin4, B. Sullivan5 and C. Gariépy3
1Sherbrooke R&D Centre, Agriculture and Agri-food Canada (AAFC), 2000 College Street,
Sherbrooke, QC, J1M 1Z7, Canada; 2Département de Biologie, Université de Sherbrooke, 2500
Blvd Université, Sherbrooke, QC, J1K 2R1, Canada; 3Saint-Hyacinthe R&D Centre, AAFC, 3600
Casavant Blvd W, St-Hyacinthe, QC, J2S 8E3, Canada; 4Centre de Développement du Porc du
Québec (CDPQ), 2590 Blvd Laurier, Québec, QC, G1V 4M6, Canada; 5Canadian Centre for Swine
Improvement (CCSI), 960 Carling Av. Ottawa, ON, K1A 0C6, Canada; mariefrance.palin@agr.gc.ca
Abstract
Pork meat quality may be improved by increasing muscle carnosine content (β-alanyl-L-histidine), yet
no information is available on the potential of anserine (β-alanyl-1-methyl-L-histidine) in improving
meat quality. Study objectives were: (1) to assess anserine content in the longissimus muscle of 85
Duroc (DD), 92 Landrace (LL) and 105 Yorkshire (YY) pigs; (2) to look for associations between
anserine content and meat quality traits; and (3) to study the expression of genes related with
carnosine and anserine metabolism. Pigs were slaughtered at 120 kg and muscle samples collected
immediately. Meat quality measurements were collected 24 h post mortem. Muscle anserine content
was quantified by HPLC. Meat quality parameters included water holding capacity, colour, shear
force and pH 24 h. The mRNA abundance of selected genes was measured by qPCR assays. There
was a breed effect for muscle anserine content with the highest values being observed in YY pigs.
For each breed, pigs were grouped in 3 categories based on their anserine content (Low, Medium
and High). In DD pigs, carnosine synthase (CARNS1) mRNA abundance was lower in the High
anserine group than in the Low and Medium groups. DD and LL pigs with High anserine content
had higher pH 24 h and lower colour L* values than pigs in the Low group. DD pigs from the High
group had lower drip loss and freezing loss compared with the Low and Medium groups. This study
shows that high muscle anserine content is associated with improved pork meat quality and lower
muscle CARNS1 mRNA abundance.
Keywords: anserine, carnosine, gene expression, meat quality, pig, skeletal muscle
Introduction
Carnosine (β-alanyl-L-histidine) and anserine (β-alanyl-1-methyl-L-histidine) are naturally-occurring
dipeptides found in meat, poultry and some fish. The pH-buffering, carbonyl scavenging and
antioxidant properties (Boldyrev et al., 2013) of carnosine may bring advantages in terms of pork
quality. In many aspects, anserine shows similar properties to carnosine but no information is available
on its potential to improve pork meat quality. Study objectives were: (1) to assess anserine content
in the longissimus muscle of different pig breeds; (2) to look for associations between anserine
content and meat quality traits; and (3) to study the expression of genes related with carnosine and
anserine metabolism.

A total of 85 Duroc (DD), 92 Landrace (LL) and 105 Yorkshire (YY) pigs entered the Deschambault
test station (Quebec) at 10-16 days old. At slaughter (120 kg liveweight), longissimus muscle samples
were collected immediately and meat quality was measured 24 h post mortem. Anserine content was
quantified by HPLC. Meat quality parameters included meat colour, water holding capacity, shear
force and pH. The mRNA abundance of carnosine/anserine-related genes was measured with qPCR
assays. An ANOVA was conducted to determine differences in muscle anserine content among breeds.
Using the FASTCLUS and DISCRIM procedures of SAS, pigs were grouped in 3 categories based

on their muscle anserine content (Low, Medium and High). The effect of anserine content category
on mRNA abundance and meat quality traits was then assessed using a one-way ANOVA (3 levels)
and means compared using multiple comparisons with a Tukey adjustment.

There was a breed effect for muscle anserine content (P<0.01) with the highest values being observed
in YY (21.41 mg/100 g) when compared with LL (20.32 mg/100 g; P≤0.05) and DD (19.92 mg/100
g; P<0.01) pigs. In YY pigs, there was no anserine content group effect for most of meat quality
parameters shown in Table 1, except for shear force. In DD pigs, the mRNA abundance of CARNS1
was lower in the High anserine content group than in the Low and Medium groups (P≤0.05). In
DD and LL pigs, lower colour L* values were found in the High anserine content group than in the
Low group (Table 1). DD and LL pigs with High anserine content had higher pH 24 h values when
compared with the Low group. DD pigs from the High group had lower drip loss and freezing loss
when compared with the Low and Medium groups. LL pigs with High muscle anserine content had
lower cooking loss values when compared with pigs in the Low group. A significant anserine content
group effect (P≤0.05) was observed in LL pigs for freezing loss values (Low vs Medium, P≤0.05).
For DD, LL and YY (data not shown) pigs, lower shear force values were found in the Low anserine
content group than in the Medium or High (P≤0.05, in LL only) groups.
This study shows that high muscle anserine content is associated with improved pork meat quality
parameters, as shown with better water holding capacity, lower colour L* and higher pH 24 h values.
Pigs with low anserine content had slightly more tender meat (e.g. lower shear force values), but
reported differences are unlikely to be detectable by pork consumers.
Table 1. Meat quality parameters in the longissimus muscle of Low, Medium and High groups of
muscle anserine content for Duroc and Landrace pigs.1
Parameters2 Duroc Landrace
Low Medium High SEM Low Medium High SEM

n=20 n=42 n=23 n=18 n=42 n=32
Anserine 16.17b 19.57a 23.82a 0.47 15.61b 19.76b 23.72a 0.38

Colour L* 53.18a 52.03ab 50.13b 0.81 54.87a 53.38ab 52.83b 0.60
Cooking loss (%) 18.13 18.47 16.77 0.74 19.63a 18.86ab 18.54b 0.34
Drip loss (%) 3.30a 3.63a 1.99b 0.41 4.33 3.98 3.43 0.53
Freezing loss (%) 9.56a 9.78a 7.83b 0.55 8.81b 10.10a 9.29ab 0.43
pH 24h 5.65b 5.63b 5.80a 0.06 5.53b 5.58ab 5.61a 0.02
Shear force (kg) 2.19b 2.61a 2.51ab 0.15 2.51b 2.68b 3.10a 0.11
1 Data represent mean values with their SEM; different letters within row differ at P≤0.05.
2 Anserine (mg/100 g muscle); Colour L*, measured with a Chroma Meter CR-300 colorimeter; Warner-Bratzler shear
force; GP = glycolytic potential (µmol of lactate eq/g tissue).
Acknowledgements
This work was financially supported by AAFC (J-000383), Swine Innovation Porc, Canada Pork
International, Canadian Pork Council, CDPQ and CCSI.
References
Boldyrev, A.A., G. Aldini and W. Derave, 2013. Physiology and pathophysiology of carnosine. Physiological Reviews
93: 1803-1845.

Effect of white striping myopathy on breast muscle protein turnover and
gene expression in broilers
K. Vignale1,2, J.V. Caldas1,3, J.A. England1, N. Boonsinchai1, A. Magnuson1, E.D. Pollock1, S. Dridi1,
C.M. Owens1 and C.N. Coon1*
1University of Arkansas, Center of Excellence for Poultry Science, Fayetteville, AR 72701, USA;
2current address: Kemin Industries, Des Moines, IA 50317, USA; 3current address: Cobb –Vantress,
P.O. Box 1030 Siloam Springs, AR 72761-1030, USA; ccoon@uark.edu
Abstract
A study was conducted to evaluate the effect of white striping (WS) on protein turnover and gene
expression of genes related to protein degradation and fatty acid synthesis. A total of 560 1-day old
male broiler chicks Cobb 500 were allocated in a total of 16 pens, 35 chicks per pen. A CRD was
conducted with a 2×3 factorial arrangement (two scores: severe and normal, and 3 breast meat samples
sites). At day 60, 20 birds were randomly selected, euthanized, and scored for white striping. Scoring
was either normal (NORM, no WS) or severe (SEV). Also, the same day, 17 birds (16 infused, one
control) were randomly selected and infused with a solution of 15N Phen 40% APE (atom percent
excess). Breast muscle tissue was taken for gene expression analysis of the following genes: MuRF1,
atrogin-1, IGF-1, insulin receptor (IR), fatty acid synthetase, and acetyl CoA carboxylase. Each
bird was humanely euthanized after 10 minutes of infusion and scored for WS (NORM or SEV).
Samples of the breast muscle (Pectoralis major) were taken at different layers (3 samples per bird:
ventral, medial, dorsal), along with a sample of excreta for 3-methylhistidine analysis. Out of the
16 breast samples taken, only 10 were selected for analysis based on the WS score (5 NORM and
5 SEV). No significant differences (P>0.05) were found in fractional synthesis rate (FSR) between
SEV WS, NORM and samples sites for breast meat. However, FBR was significantly higher in birds
with SEV WS compared to NORM (8.2 and 4.28, respectively, P<0.0001) (Table 1). Birds with
SEV WS showed significantly higher (P<0.05) relative expression of MuRF1 and slightly higher
(P=0.07) in relative expression of atrogin-1 than the NORM birds. These birds also showed lower
(P<0.05) relative expression of IGF-1 than NORM birds. The IR relative expression was higher
(P<0.05) for birds with SEV WS when compared to the NORM. Furthermore, birds with SEV
WS had significantly lower (P<0.05) relative expression of fatty acid synthetase than the normal.
However, birds with severe white striping showed higher, but not significant, relative expression of
LPL (P=0.053). Further studies are needed to better understand why birds with severe white striping
are degrading more muscular protein and mobilizing more fat.
Keywords: white striping, protein turnover, gene expression, insulin
Introduction
White striping (WS) is the white striation observed parallel to the direction of muscle fibres in
broiler breast fillets and thighs at the processing plant (Kuttappan et al., 2012, 2013). Broiler breast
fillets can be categorized as normal (NORM, no stripes), moderate (MOD), or severe (SEV) based
on the degree of WS. It is known that WS could be a potential reason for the rejection of raw breast
fillets in the market (Kuttappan et al., 2012). However, it is still not clear what the origin of the WS
problem is, or what the mechanism is for its appearance.

A CRD was conducted with a 2×3 factorial arrangement (two scores: severe and normal, and 3
breast meat samples sites). At day 60, 20 birds were randomly selected, euthanized, and scored for
white striping. Scoring was either normal (NORM, no WS) or severe (SEV). Also, the same day,

17 birds (16 infused, one control) were randomly selected and infused with a solution of 15N Phen
40% APE (atom percent excess). Breast muscle tissue was taken for gene expression analysis of
the following genes: MuRF1, atrogin-1, IGF-1, insulin receptor (IR), fatty acid synthetase, and
acetyl CoA carboxylase (ACC). Each bird was humanely euthanized after 10 minutes of infusion
and scored for WS (NORM or SEV). Samples of the breast muscle (Pectoralis major) were taken
at different layers (3 samples per bird: ventral, medial, dorsal), along with a sample of excreta for
3-methylhistidine analysis (3-MH). 3-MH ratio in muscle and excreta was utilised for fractional
breakdown rate (FBR) determination. Out of the 16 breast samples taken, only 10 were selected for
analysis based on the WS score (5 NORM and 5 SEV). Fractional synthesis rate (FSR) and FBR
were determined using GC-MS.

FBR was significantly higher in birds with SEV WS compared to NORM (8.2 and 4.28, respectively,
P<0.0001) (Table 1). Birds with SEV WS showed significantly higher (P<0.05) relative expression of
MuRF1 and slightly higher (P=0.07) in relative expression of atrogin-1 than the NORM birds. These
birds also showed lower (P<0.05) relative expression of IGF-1 than NORM birds. The IR relative
expression was higher (P<0.05) for birds with SEV WS when compared to the NORM. Furthermore,
birds with SEV WS had significantly lower (P<0.05) relative expression of fatty acid synthetase than
the normal. This is supported by studies conducted by Kuttappan et al. (2012, 2013) who evaluated
histology and proximate composition of fillets with WS. Kuttappan et al. (2012) reported a significant
increase in scores for degenerative or necrotic lesions, fibrosis, and lipidosis as the degree of white
striping increased from NORM to SEV along with supporting muscle composition data that showed
increased fat and decreased protein content in severe WS fillets relative to normal fillets.
Table 1. Fractional synthesis rate (FSR) and fractional breakdown rate (FBR) by score and sample
site of breast muscle (Pectoralis major) for 60 d old broilers.1
Score Sample site FBR % FSR %
NORM1 ventral 4.07a 14.01

NORM1 medial 4.7a 7.20
NORM1 dorsal 4.03a 10.03
SEV2 ventral 9.24b 8.45
SEV2 medial 7.54b 8.08
SEV2 dorsal 7.82b 12.04
P-value <0.001 0.725
SEM 0.38 2.00
1 a-b Means within column with no common script differ (P<0.05); n=5 per mean.
2 NORM = normal fillets with no white striping; SEV = severe white striping.
References
Kuttappan, V.A., V.B. Brewer, A. Mauromoustakos, S.R. McKee, J.L. Emmert, J.F. Meullenet and C.M. Owens, 2013.
Estimation of factors associated with the occurrence of white striping in broiler breast fillets. Poultry Science 92:
811-819.
Kuttappan, V.A., V.B. Brewer, J.K. Apple, P.W. Waldroup and C.M. Owens, 2012. Influence of growth rate on the
occurrence of white striping in broiler breast fillets. Poultry Science 91: 2677-2685.

Performance and fatty acid status in dairy cows fed a diet with reduced
essential fatty acid content
C. Weber1, A. Tröscher2, H. Kienberger3, M. Rychlik4 and H.M. Hammon1*
1Institute of Nutritional Physiology ‘Oskar Kellner’, Leibniz Institute for Farm Animal Biology
(FBN), Dummerstorf, Germany; 2BASF SE, Limburgerhof, Germany; 3Bavarian Biomolecular Mass
Spectrometry Center and 4Chair of Analytical Food Chemistry, Technical University of Munich,
Freising, Germany; hammon@fbn-dummerstorf.de
Abstract
Diets in dairy production are primary based on corn silage and corn, but fresh grass is rarely
provided to cows. Corn based diets may deliver insufficient amounts of essential fatty acids (EFA), in
particular α-linolenic acid (ALA), and could impair the availability of ALA and its more desaturated
metabolic products as eicosapentaenoic acid (EPA), as well as conjugated linolenic acid (CLA) and its
intermediates as vaccenic acid (VA). To verify this hypothesis five duodenal fistulated dairy cows in
2nd lactation were changed from a grass and corn silage-based diet to a solely corn silage-based diet,
and were investigated for five month. Performance data were collected, and fatty acid composition
was measured frequently in feed, duodenal digesta, blood plasma and milk by a GC-based method.
After the diet change, ALA and c9, t11 CLA decreased, but linoleic acid (LA) increased in milk, in
milk fat and in duodenal digesta. EPA decreased in milk fat, but was not found in digesta. In milk
and milk fat VA concentration decreased, but VA was unaltered in duodenal digesta. ALA and EPA
concentrations and their relative amount in the neutral lipid, phospholipid and free fatty acid fraction
in plasma decreased during the study, whereas LA and VA concentrations were inconsistent among
plasma lipid fractions. The diet change to a corn silage-based ration results in considerable changes
of n3-EFA and CLA availability and point to a reduced ALA and EPA status in dairy cows.
Keywords: dairy cow, nutrition, essential fatty acids
Introduction
Common diets in dairy production are mainly based on corn and corn silage, and provide less fresh
or conserved grass that delivers high amounts of essential fatty acids (EFA), especially α-linolenic
acid (ALA). Due to rapid ruminal biohydrogenation especially of silage based diets, very low
amounts of ALA are available for intestinal absorption. Therefore, all the corn-based diets may
deliver insufficient ALA to cows. An EFA deficient diet may also result in less rumen production
of conjugated linoleic acid (CLA) and trans-fatty acids, leading to reduced CLA in tissue and milk,
which both are unique to food derived from ruminants. In this study we fed a corn silage-based total
mixed ration (TMR) with low fat content and negligible amounts of ALA to investigate the influence
of an EFA-deficient diet on performance and EFA status in dairy cows.

Five duodenal fistulated German Holstein cows in 2nd lactation (57±12 days in milk at start of the
study) were investigated for 26 week, therefrom 24 week after the change from a grass and corn
silage-based (GS) TMR (fed from parturition until the feed change) to a solely corn silage-based
TMR (CS). Ingredients of GS were grass and corn silage, straw, hay, wheat grain, soybean and rape
seed meal, corn grain, and concentrate. Components of CS were corn silage, straw, soybean meal,
dried sugar beet pulp and rye grain. Diets were isoenergetic (6.8 MJ NEL/kg of dry matter (DM))
and isonitrogenous (crude protein 155 g/kg DM), but crude fat content was lower in CS than in GS
(22.4 versus 31.0 g/kg DM for CS and GS). Diets were fed ad libitum. Content of linoleic acid (LA)
was quite similar (10.8 and 11.7 g/kg DM in CS and GS), but ALA content was much lower in CS

than GS diet (1.0 and 6.2 g/kg in CS and GS). DM intake (DMI), milk yield and milk composition
were measured weekly. Fatty acid (FA) composition in feed samples and in milk fat were measured
in week -1, 2, 4, 6, 8, 16, 20, 24, in duodenal ingesta and plasma lipid fractions in week -1, 8, 16,
24 by a GC-based method. Statistical analyses were done with the MIXED procedure of SAS with
time as fixed effect.

DMI and energy intake (EI) increased, but total fat intake decreased with CS diet (Table 1). Intake
of LA increased with time, but ALA intake dropped down with the CS diet. Milk yield, energy-
corrected milk (ECM) and fat and protein yield declined with time, but protein and fat content in
milk increased at the same time (Table 1).
Concentration of ALA, c9, t11 CLA and VA decreased (P<0.001) in milk and in milk fat, whereas
concentration of LA increased in milk fat (P<0.001) after CS feeding. In duodenal ingesta, relative
and absolute mass of ALA was reduced with CS feeding (P<0.001). Concentration of ALA in blood
plasma and in fat fractions of the plasma and of EPA in blood plasma decreased after CS feeding
(P<0.001). LA increased in the neutral lipid fraction, but plasma concentration of LA decreased in
the free fatty acid fraction (P<0.001). Our results show that the CS diet leads to a reduced ALA and
EPA status in dairy cows, whereas the LA status was not impaired with a corn silage-based TMR.
However, the n3/n6 status clearly decreased with such a diet. If these changes in EFA status result in
an impaired metabolic and immune function in dairy cows will be investigated in ongoing studies.
Table 1. Zootechnical data.
Variable1 Diet type SE P-value
GS CS Time
DMI, kg/d 17.3 19.9 1.20 <0.001

EI, MJ NEL/d 118 136 8.25 <0.001
Total fat intake, g/d 536 455 21.3 <0.001
ALA intake, g/d 107 20.4 2.52 <0.001
LA intake, g/d 202 219 9.63 <0.0001
Milk yield, kg/d 34.8 28.9 1.09 <0.001
ECM, kg/d 34.8 30.4 2.01 <0.001
Milk fat, g/kg 40.1 42.3 4.00 0.01
Milk fat, kg/d 1.40 1.22 0.12 <0.001
Milk protein, g/kg 31.8 36.0 1.33 <0.001
Milk protein, kg/d 1.11 1.04 0.06 <0.001
1Values are least square means (LSM) with pooled SE, and LSM were pooled for GS from week -2 to -1, and for CS
pooled from week 1 to 24 relative to diet change.
Acknowledgements
The study was funded by BASF SE, Limburgerhof, Germany.

Part 4. E
nvironmental and animal welfare aspects of protein
and energy nutrition
Agriculture without animals? The environmental and economic role of
livestock in food production
R.R. White1* and M.B. Hall2
1Department of Dairy Science, Virginia Tech, Blacksburg, VA 24061, USA; 2USDA-ARS, US Dairy
Forage Research Center, Madison, WI 53706, USA; rrwhite@vt.edu
Abstract
This study evaluated the ability of USA food production to adequately meet the nutrient requirements
of the USA population in systems with or without food derived from animal products, and determined
diet costs, environmental impact, and exported nutrient quantities in these systems. The current
USA food production system provision of human-edible nutrients was quantified in terms of human
nutrient requirement year equivalents. The current USA diet was evaluated in comparison to 4 least-
cost diet scenarios for nutritive value, environmental impact, diet cost, and nutrient export potential.
The results suggest substantial environmental and economic gains can be achieved by eating less
food, rather than eating a different diet.
Keywords: food security, sustainability, livestock, environmental impact
Introduction
Interest in improving global food security has pushed the question of whether we should feed animals
to feed humans. On an energy efficiency basis, this concern is logical. Inevitably, feeding people
directly will result in greater caloric availability because animals are significantly less than 100%
efficient at converting feed energy to product energy. This study evaluated the ability of USA food
production to adequately meet the nutrient requirements of the current USA population in systems
with or without food derived from animal products. We hypothesized that when other nutrients
are considered (amino acids, fatty acids, vitamins and minerals), the role of livestock in the food
production system would be more important.

The current USA food production system provision of human-edible nutrients was quantified.
National average nutrient requirements were calculated and the current USA diet was evaluated for
its ability to meet nutrient requirement standards. Least-cost rations representing different scenarios
were optimized and compared. Diet cost, environmental impact, nutritive value, and resulting excess
food available for export were quantified in each scenario. Four scenarios were tested. All scenarios
minimized diet cost. Two scenarios assumed all nutrient requirements must be met and two scenarios
assumed that vitamins A, D, E, B12, choline and Ca requirements could be met with supplements.
All scenarios were constrained so that the USA population’s nutrient requirements were met with
domestically produced food.
Data from USDA/ERS (USDA/ERS, 2012) was used to identify domestic production of 93 non-
animal and 24 animal-derived food products. Data from USDA/ARS (USDA/ARS, 2014) was
sourced to identify the chemical composition of each food product. Production of 41 nutrients (energy,
protein, carbohydrates, vitamins, minerals, amino acids, and fatty acids) were calculated for each
food product included in the study. From those 41 nutrients, requirements for 36 were identified for
humans based on age and gender. Distribution of the USA population by analogous age and gender
groups (Howden and Meyer, 2011) was used to identify a weighted average nutrient requirement of
the USA population. The food production and human nutrient requirement data were compared to
identify the number of human nutrient requirement years satisfied for each nutrient. Carbon footprints

(CF) for all products were sourced from International Standards Organization compliant life cycle
assessments. Effects of removing livestock from USA agriculture were simulated assuming: (1) grain
previously consumed by animals will be available for human consumption; (2) land previously used
for forage production will be used for human food production; (3) nutrients from animal products
previously provided to humans will no longer be available; (4) emissions from livestock production
will no longer occur; and (5) human-edible grain and oilseed processing byproducts will be available
for human consumption but human-inedible byproducts will be disposed of by alternative avenues
(composting, burning, etc.).

Current food consumption patterns suggest that the USA diet is comprised of 40% animal products,
25% vegetables, 24% fruits, 10% grains, and 1% nuts and legumes. This diet is consumed at a rate
of 1,477 g/d and costs $3.91/d. After domestic consumption, exported food provided 85.9×106
yearly human energy and 59.9×106 yearly human protein requirement equivalents. The CF was 3.3
kg CO2-equivalents/d.
A least-cost diet was balanced assuming all nutrients must be greater than the minimum requirement
and that total consumption of any food had to be less than total domestic production of that food.
This cost $1.42/d, had very similar consumption patterns to the current diet but required less food per
day (1,200 g/d) and resulted in notably lower CF (0.92 kg CO2-equivalents/d) than the current USA
diet. By consuming less food domestically, greater quantities of energy (418×106 yearly requirement
equivalents) and protein (546×106) were available for export. The same optimization exercise was
conducted on a simulated system without animals and despite greater total availability of energy, a
feasible solution could not be identified meaning that the yearly nutrient requirements of the USA
population could not be met under the defined constraints. Supplies of vitamins A, D, E, B12, choline
and Ca were deficient. In the scenarios assuming these nutrients could be met with supplements, the
least-cost ration optimized in the system without animal products yielded an optimal diet comprised
of 65% grain, 31% legumes, and 4% vegetables. The predicted intake on the diet was low (788 g/
person/d) and the CF represented only 15% of the current USA diet’s CF (0.49 kg CO2E/d). In an
analogous optimization that include animal-derived foods, the diet was very similar to the current
USA diet (34% animal products, 21% vegetables, 6% fruits, 37% grains, and 2% nuts). Intake was
lower than current USA intakes (1,046 g/person/d) and the CF was 22% of the current USA diet CF.
Lysine was the most-limiting amino acid in both scenarios. Although an additional 6% reduction in
CO2 emissions was projected from the system without animals, this marginal environmental impact
reduction came at the cost of nearly 300×106 exportable human lysine requirement years. Given the
tradeoffs between environmental impact and food sufficiency, more thorough analyses of methods
to reduce environmental impact while feeding the growing global population are needed.
Acknowledgements
The authors acknowledge their respective institutions for supporting this effort.
References
Howden, L.M. and J.A. Meyer, 2011. Age and sex composition: 2010. 2010 Census Briefs. Department of Commerce,
Economics and Statistics Administration, US Census Bureau, Washington, DC, USA.
USDA/ERS, 2012. Data and statistics. Available at: http://quickstats.nass.usda.gov.
USDA/ARS, 2014. National nutrient database for standard reference, release 27. Available at: http://ndb.nal.usda.
gov/ndb.

Soybean meal supplementation in automatic milking systems
M.R. Weisbjerg, M. Johansen, M. Larsen and P. Lund
Aarhus University, AU Foulum, Department of Animal Science, P.O. Box 50, 8830 Tjele, Denmark;
martin.weisbjerg@anis.au.dk
Abstract
In automatic milking systems, high milking frequencies are obtained using concentrate offer in the
automatic milking unit (AMU) as a reward for visits. The effect of supplementing or replacing part
or all of a standard pelleted concentrate mixture (CON) with soybean meal (SBM) was examined
using 48 Holstein dairy cows. The four treatments were daily offer of (1) 3 kg of CON; (2) 3 kg CON
+ 0.5 kg SBM; (3) 1.5 kg CON + 1 kg SBM; and (4) 1.5 kg SBM. Opposite to expectations, SBM
treatments affected milking frequency and concentrate leftovers negatively but not dramatically, and
feeding SBM in the AMU is a possible tool for individual supply of cows with protein.
Keywords: milking robot, concentrate, palatability, dairy cows, Holstein
Introduction
In automatic milking systems (AMS), a satisfactory milking frequency is obtained using concentrate
offer in the AMS as a reward for visits. However, high palatability of the offered concentrate is
required to obtain the wanted effect on cows’ voluntary visits in the AMS (Madsen et al., 2010). The
concentrate feeder in the AMS could be used for a cost effective strategic protein feeding in dairy
herds, based on e.g. individual cows’ lactation stage or milk yield, if protein concentrates do not affect
milking frequency negatively. The study aimed to investigate the effect of increasing substitution of
a ‘normal’ pelleted concentrate with soybean meal (SBM) on milking frequency and performance.

The experiment was conducted in a loose housing barn with a DeLaval AMS with free cow access.
Forty-eight Holstein cows, 20 primiparous and 28 multiparous, 99±82 (mean ± SD) days from
calving were used for the experiment. The cows were blocked according to parity and days from
calving and randomly assigned to one of four treatments in a 4×4 Latin square design with one week
periods. The four treatments were daily offer of (1) 3 kg of a standard pelleted concentrate (3CON);
(2) 3 kg CON + 0.5 kg SBM (3CON+0.5SBM); (3) 1.5 kg CON + 1 kg SBM (1.5CON+1SBM);
or (4) 1.5 kg SBM (1.5SBM). Concentrate allocation was divided over daily AMS visits according
to time interval from last milking. Both concentrates were fed out separately at a rate of 0.4 kg/
minute. The partly mixed ration (PMR) was fed ad libitum in Insentic feed troughs with automatic
registration of PMR intake. The PMR was composed of 31.4% grass silage, 26.0% maize silage,
17.5% rapeseed cake, 8.1% dried sugar beet pulp, 8.0% barley, 8.0% NaOH treated wheat grain,
0.13% urea and 0.9% minerals and vitamins on dry matter basis. Milking frequency, visits without
milking permission, amounts of concentrates offered in the AMS, concentrate orts, milk yield and
PMR intake were registered daily at cow level. Milk composition was analysed at each milking
during the last 48 h of each period.
All statistical analyses were performed using the MIXED procedure of SAS (SAS® version 9.3;
SAS, 2010). The model included cow nested in parity, treatment, parity (primi- or multiparous),
day within period (day), and the two-way interactions between treatment and parity and between
treatment and day. Measures within cow by treatment were handled as repeated measures assuming
a compound symmetry covariance structure.

Milking frequency was decreased by inclusion of SBM in the concentrate offered in the AMS
(P=0.01) but not AMS visits without milking permission (P=0.6) (Table 1). Concentrate leftovers
increased when SBM was offered (P<0.01), although total concentrate offer and thereby consequently
concentrate uptake was less for the high SBM offer. Energy corrected milk yield (3.14 MJ/kg) was
not affected (P=0.3), neither was milk protein (P=0.09) or lactose concentration (P=0.6), but milk
fat concentration (P<0.01) and PMR intake (P=0.01) were affected, and seemed to increase with
reduced total concentrate offer.
Earlier intensive palatability experiments with cows in tie stalls had shown a high preference for
SBM compared to the CON (Primdal et al., 2014). Therefore it was expected that milking frequency,
etc. could be sustained using lower amounts of SBM than CON, which was tested by first adding
SBM, and then substituting CON with SBM, however with a concomitant lowering total allocation
to balance for the higher protein concentration in SBM. However, no positive effects were seen
for any of the SBM treatments regarding milking frequency and concentrate leftovers. The lack of
positive effects of SBM opposite to the findings by Primdal et al. (2014) might be due to increased
dust when meal is fed in the concentrate feeder in an AMS.
Conclusion
Increasing SBM substitution reduced the milking frequency in the AMS; however, the magnitude
was limited and ECM yield was unaffected. Therefore, feeding SBM in the AMS could be used as
a tool for individual supply of cows with protein.
Table 1. Effect of treatment on intake of partly mixed ration (PMR) and total (standard pelleted
concentrate mixture with soybean meal; CON+SBM) concentrate allocated and refused, AMU visits
with and without milking permission and milk yield (per day).1
3CON 3CON+0.5SBM 1.5CON+1SBM 1.5SBM SEM P-value
Intake of PMR (kg DM) 19.2 19.0 19.5 20.3 0.28 0.01
Concentrate allocated (kg) 2.83 3.31 2.30 1.30 0.031 <0.01
Concentrate leftovers (kg) 0.089 0.164 0.155 0.234 0.027 <0.01
Milkings 2.75 2.69 2.56 2.56 0.047 0.01
Visits, no milking permission 0.28 0.32 0.27 0.23 0.046 0.6
Milk yield (kg) 39.2 39.4 38.7 38.1 0.26 <0.01
ECM yield (kg) 38.8 38.7 38.4 38.2 0.25 0.3
1 ECM = energy corrected milk.
Acknowledgements
Financial support from the Danish Milk Levy Fund (MAF) and The Danish AgriFish Agency (GUDP
#34009-12-0510).
References
Madsen, J., M.R. Weisbjerg and T. Hvelplund, 2010. Concentrate composition for automatic milking systems – effect
on milking frequency. Livestock Science 127: 45-50.
Primdal, L., M. Johansen and M.R. Weisbjerg, 2014. Do dairy cows have preferences for different concentrate feeds? In:
S. Hatcher, G.L. Krebs and B.W.B. Holman (eds.). Proceedings of the 30th Biennial Conference of the Australian
Society of Animal Production. Animal Production in Australia 30, 363 pp.

Update of protein requirements for Zebu beef cattle
L.F. Costa e Silva, S.C. Valadares Filho, P.P. Rotta*, M.I. Marcondes, M.L. Chizzotti and A.C.B.
Menezes
Department of Animal Science, Universidade Federal de Viçosa, Viçosa, 36570-900, Brazil;
polyana.rotta@gmail.com
Abstract
A meta-analysis was conducted aiming to update the protein requirements for Zebu and crossbred
beef cattle. Our database was composed by 973 animals, being 795 animals raised in feedlot and
178 animals raised on pasture. The database had 584 Nellore, 202 dairy crossbred cattle (Holstein
× Zebu), and 187 beef crossbred cattle. All studies followed the same procedures of slaughter and
data collection. The requirements of metabolisable protein (MP) for maintenance were estimated
considering the relationship between metabolisable protein intake (g/day) and average daily gain
(kg/day). We found difference for the MP for maintenance between animals raised on pasture and
feedlot, which allowed us to develop equations separately. However, no difference was observed
for different breeds and sex evaluated in the feedlot conditions. Also, the net protein requirement for
gain (NPg) was estimated considering the relationship between retained protein, empty body gain,
and retained energy. Thus, the MP for maintenance was estimated as 4.30 g MP/BW0.75 for animals
raised on pasture and 3.66 g MP/BW0.75 for animals on feedlot conditions. The last recommendation
of the BR CORTE system was 4.00 and 4.50 g MP/BW0.75 for animals raised on feedlot and pasture,
respectively. In the same way, we found differences between animals raised on pasture and feedlot
for NPg and between breeds and sex in the feedlot, allowing us to generate new equations for each
group. Therefore, this update of protein requirements will reduce the amount of crude protein in
diets of beef cattle raised in Brazil, decreasing environment impact of nitrogen excess in manure.
Keywords: gain, maintenance, BR CORTE, Nellore
Introduction
Proteins are macromolecules that have vital functions such as composition of structural tissues,
enzymes, hormones, hormone receptors and genetic material. Thus, protein nutrition is one of
the main factors that influence animal performance. According to Berends et al. (2014) feedlots
commonly adopt high-protein levels on finishing diets to increase dry matter intake, thereby leading
to a greater average daily gain and improved feed efficiency. However there is a great correlation
between crude protein intake and urinary and faecal N excretion (Sinclair et al., 2014), contributing
to environmental contamination. In addition, the appropriate formulation of diets, meeting exactly
the protein needs of animals, is one of the ways to ensure that excess N is not excreted into the
environment. Therefore, the update of protein requirements for maintenance and gain for animals
raised on pasture and feedlot systems are necessary to optimize the production cycle.

Data were collected from studies since 1991 up to 2016 totaling 795 animals evaluated in feedlot
and 178 animals raised on pasture. The database had 584 Nellore, 202 dairy crossbred cattle and 187
beef crossbred cattle. All studies followed the same procedures of slaughter and data collection. Data
were analysed using a meta-analysis which differences regarding to production system (pasture or
feedlot), genotype (Nellore, beef crossbred cattle, and dairy crossbred cattle), and sex (bulls, steers,
and heifers) were tested. The requirements of metabolisable protein (MP) for maintenance were
estimated considering the relationship between metabolisable protein intake (g/day) and average daily
gain (kg/day). We found difference for the MP for maintenance between animals raised on pasture

and feedlot, which allowed us to develop equations separately. Also, the net protein requirement for
gain (NPg) was estimated considering the relationship between retained protein, empty body gain
(EBG), and retained energy (RE).

We found difference for the MP for maintenance between animals raised on pasture and feedlot, which
allowed us to develop equations separately. However, no difference was observed for different breeds
evaluated in the feedlot conditions. Also, the MP for gain was estimated considering the relationship
between retained protein, EBG, and RE. Thus, the MP for maintenance was estimated as 4.30 g MP/
BW0.75 for animals raised on pasture and 3.66 g MP/BW0.75 for animals on feedlot conditions. The
last recommendation of the BR CORTE system was 4.00 and 4.50 g MP/BW0.75 for animals raised
on feedlot and pasture, respectively. The requirement for MP for maintenance is greater for animals
under pasture conditions, since these animals have to walk more in order to have enough feed. In
the same way, we found differences for NPg between animals raised on pasture and feedlot and
between breed and sex in the feedlot, allowing us to generate new equations for each group (Table
1). The following equation was estimated for pasture system: NPg = 185,88 × EBG – 4,69 × RE.
Table 1. Net protein requirements for gain for animals raised on pasture and on feedlot from different
genotype and sex.
Genotype Sex Equation1
Zebu Bull NPg = 210,09 × EBG – 10,01 × RE

Steer NPg = 153,13 × EBG – 2,53 × RE
Heifer NPg = 193,90 × EBG – 12,16 × RE
Beef crossbred Bull NPg = 252,38 × EBG – 27,04 × RE
Steer NPg = 204,30 × EBG – 15,49 × RE
Heifer NPg = 132,65 × EBG + 3,54 × RE
Dairy crossbred Bull NPg = 124,26 × EBG – 0,34 × RE
Heifer NPg = 173,11 × EBG – 7,52 × RE
1 NPg = net protein requirement for gain; EBG = empty body gain; RE = retained energy.
Acknowledgements
This study was funded by Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq)
through to funding field and laboratory activities (479384/2013-4) and FAPEMIG.
References
Berends, H., J.J.G.C Van der Borne, B.A. Røjen, W.H. Hendriks and W.J.J. Gerrits, 2014. Effect of protein provision
via milk replacer or solid feed on protein metabolism in veal calves. Journal of Dairy Science 98: 1119-1126.
Sinclair, K.D., P.C. Garnsworthy, G.E. Mann and L.A. Sinclair, 2014. Reducing dietary protein in dairy cow diets:
implications for nitrogen utilization, milk production, welfare and fertility. Animal – a major new International
Journal of Animal Bioscience 8: 62-74.

Intravenous lipid infusion affects methane production apart from
reducing dry matter intake in late lactation German Holstein cows
O. Lamp1,2, M. Derno1, G. Nürnberg3, C.C. Metges1 and B. Kuhla1*
1Institute of Nutritional Physiology ‘Oskar Keller’, Leibniz Institute for Farm Animal Biology,
3Institute of Genetics and Biometry, Leibniz Institute for Farm Animal Biology, Wilhelm-Stahl-Allee 2,
18196 Dummerstorf, Germany; 2Schleswig-Holstein Chamber of Agriculture, Teaching and Training
Center Futterkamp, 24327 Blekendorf, Germany; b.kuhla@fbn-dummerstorf.de
Abstract
Methane emissions of dairy cows decrease with increased dietary fat content and this effect is
attributed to decreased dry matter intake (DMI), decreased accessibility of feed for microbes, and
toxic effects on rumen microbial populations. However, it is not known whether post-absorptive
circulating triglycerides and long-chain fatty acids affects rumen methane production as well. To
address this question, four rumen-cannulated Holstein cows were equipped with two jugular catheters
and transferred to respiration chambers to measure methane production for 2 days. All cows were
either infused a lipid emulsion (LIPO) or 0.9% NaCl in a cross-over design. When normalized to
DMI, methane yield was greater in LIPO than CON cows, but decreased from day 1 to day 2 in
LIPO cows only. Results show that circulating lipids influence methane production and yield, and
that this effect cannot be exclusively attributed to the reduction in DMI. Possible reasons for the
effect are discussed.
Keywords: methane, DMI, dietary fat, cattle
Introduction
Methane emission of a dairy cow is positively related to de novo-synthesized (C6-C16) milk fatty
acids (MFA) but negatively related to C18-MFA (Van Lingen et al., 2014). Dietary supplementation
with unsaturated or saturated C18 fatty acids increases C18-MFA content in milk and reduces
methane production in the rumen due to decreased dry matter intake (DMI), decreased accessibility
of feed for microbes and direct toxic effects on microbial populations (Chilliard et al., 2009; Martin
et al., 2008). Besides dietary sources, the portion of C18-MFA is influenced by endogenous C18
fatty acids released from adipose tissue during periods of negative energy balance (Lerch et al.,
2015). This finding let us hypothesize that intravenous infusion of C18-triglycerides (TG) would
increase C18-MFA content in milk and reduce methane production from dairy cows without direct
interference of ruminal fermentation.

Four rumen-cannulated German Holstein cows in second late lactation were equipped with two
jugular catheters and transferred to respiration chambers to measure methane production in 6-min
intervals. Animals were infused (7.4 ml/min) with 0.9% NaCl (CON) or Lipofundin N, B. Braun
Melsungen AG, Melsungen, Germany (LIPO; 20% soy oil consisting of 88% C18 and 12% C16) for
48 h. A wash-out period of 6 days between infusions was assured. Animals were fed a total mixed
ration from the same batch, before, during and between infusions. In the respiration chamber, feed
intake was recorded every 6 min by feed disappearance from the feeding bin. Animals were sampled
for blood and rumen fluid hourly from 07:00 to 17:00 and for milk twice daily at 07:00 and 16:30 h.
Blood was collected as EDTA plasma to determine concentrations TG and non-esterified fatty acids
(NEFA). Plasma and rumen acetate concentrations were determined on a gas chromatography-flame
ionization detector. Milk fatty acids were analysed as methyl esters on a GC-mass spectrometry

instrument. Data were analysed by the Mixed procedure of SAS with the factor treatment (tr), the
repeated factor time (ti) and treatment × time (tr × ti) interaction.

Lipid infusion increased plasma TG and non-esterified fatty acids (NEFA) with increased infusion
time, whereas CON infusion did not affect plasma TG and NEFA (ti × tr: P<0.01). Plasma acetate
concentrations were lower in LIPO than CON cows (tr: P<0.05) with the greater difference on day
2 compared to day 1. Rumen acetate and butyrate were not significantly affected by infusion and
time, while rumen propionate increased with time (ti: P<0.05) and was greater in CON cows (tr:
P<0.05). The rumen fluid (acetate+butyrate)/propionate ratio increased from day 1 to day 2 in LIPO,
but was unchanged in CON cows (ti: P<0.01). Milk yield and energy-corrected milk yield (ECM)
were not affected by infusion, but milk yield was lower on day 2 compared to day 1 for both groups
(ti: P=0.01). Milk fat increased with progressing time of LIPO infusion. De novo-synthesized MFA
(sum of C6:0, C8:0, C10:0, C12:0, C14:0, C16:0, C16:1) were reduced, while the sum of C18 MFA
were increased in LIPO but not CON cows (tr: P<0.05). Dry matter intake (DMI) was lower in
LIPO compared to CON cows both on day 1 and day 2 of infusion (tr: P<0.01). Methane production
continuously decreased with progressing time of LIPO but not CON infusion (tr × ti: P<0.01). When
normalized to DMI, methane yield was greater in LIPO cows (tr: P<0.05), but it decreased from
day 1 (100%) to day 2 (92%) in LIPO cows only (tr × ti: P<0.05). Methane emission per unit ECM
was lower in LIPO animals during both infusion days (tr: P=0.05). Results show that circulating
lipids influence methane production and yield, and that this effect cannot be exclusively attributed to
the reduction in DMI. It seems likely that the fat metabolism of the host influences gastrointestinal
hormones and cytokines such as cholecystokinin, motilin, ghrelin and leptin, which are all known
to activate the enteric nervous system and to regulate rumen and gut motility (Kermani et al., 1993;
Yarandi et al., 2011). Hence, lipid-induced concentration changes of these hormones may affect feed
passage rate and thus rumen fermentation kinetics including methanogenesis.
References
Chilliard, Y., C. Martin, J. Rouel and M. Doreau, 2009. Milk fatty acids in dairy cows fed whole crude linseed, extruded
linseed, or linseed oil, and their relationship with methane output. J. Dairy Sci. 92: 5199-5211.
Kermani, R.Z. and A. Rezaiee, 1993. The effects of intravenous cholecystokinin, secretin and pentagastrin on
electromyographic activity of the rumen in sheep. Regul. Pept. 45: 371-377.
Lerch, S., J.A. Pires, C. Delavaud K.J. Shingfield, D. Pomiès, B. Martin, Y. Chilliard and A. Ferlay, 2015. Rapeseed or
linseed in dairy cow diets over 2 consecutive lactations: effects on adipose fatty acid profile and carry-over effects
on milk fat composition in subsequent early lactation. J. Dairy Sci. 98: 1005-1018.
Martin, C., J. Rouel, J.P. Jouany, M. Doreau and Y. Chilliard, 2008. Methane output and diet digestibility in response
to feeding dairy cows crude linseed, extruded linseed, or linseed oil. J. Anim. Sci. 86: 2642-2650.
Van Lingen, H.J., L.A. Crompton, W.H. Hendriks, C.K. Reynolds and J. Dijkstra, 2014. Meta-analysis of relationships
between enteric methane yield and milk fatty acid profile in dairy cattle. J. Dairy Sci. 97: 7115-7132.
Yarandi, S.S., G. Hebbar, C.G. Sauer, C.R. Cole and T.R. Ziegler, 2011. Diverse roles of leptin in the gastrointestinal
tract: modulation of motility, absorption, growth, and inflammation. Nutrition 27: 269-275.

Effects of dietary carbohydrate source on milk production and
environmental impact of lactating dairy cows
E.H. Cabezas-Garcia1*, S.J. Krizsan1, K.J. Shingfield2 and P. Huhtanen1
1Department of Agricultural Research for Northern Sweden, Swedish University of Agricultural
Sciences, 901 83 Umeå, Sweden; 2Institute of Biological, Environmental and Rural Sciences,
Aberystwyth University, Aberystwyth, SY23 3EB, United Kingdom; edward.cabezas.garcia@slu.se
Abstract
The present study evaluated the effects of dietary carbohydrate source on production performance
and environmental impact of lactating dairy cows. Two grass silages were prepared from the same
primary growth of timothy grass harvested two weeks apart. Four diets fed as total mixed ratio,
were formulated to meet the metabolisable energy requirement of 35 kg of energy corrected milk
(ECM) by gradually replacing late cut grass silage (LS) and barley (B) with early cut grass silage
(ES; % on dry matter basis of LS:B:ES): L = 47:53:0, LE = 37:45:18, EL = 21:37:42, E = 0:29:71.
Sixteen Swedish Red cows were used in four replicated 4×4 Latin squares. Cows were offered diets
ad libitum and milked twice daily. Each period of 28 d comprised 14 d of diet adaptation followed
by 14 d of data collection. Intake and milk yield was recorded daily, and composition analysed. Gas
emissions were measured using the GreenFeed system. Dry matter intake (DMI) linearly decreased
(P<0.01) to increased proportion of ES in the diet from 22.8 to 19.5 kg/d. Replacing LC and B with
EC in the diets linearly decreased (P=0.01) milk protein. Total methane and CO2 production or per
kg of ECM were not influenced, but per DMI linearly (P<0.01) increased with the inclusion of ES
in the diet (P<0.01). In conclusion, replacing LS and B with ES improved conversion of feed into
milk without increases in CH4 emissions or N efficiency.
Keywords: feed efficiency, grass silage, greenhouse gases
Introduction
Increasing the proportion of concentrates in the diet is one strategy to lower CH4 emissions in cattle
by promoting propionate fermentation in the rumen. However, relatively high levels of starch rich
feeds can compromise animal performance by decreasing fibre digestibility and increasing the
incidence of acidosis. This is particularly relevant on high concentrate feed-lot diets fed to growing
cattle where concentrates can contribute to >75% diet on dry matter basis (DM). It is less clear if
increases in dietary starch content influence CH4 emissions in cows fed highly digestible grass
silage grown under Nordic conditions. The present study examined the effects of replacing barley
fed with a medium-quality silage with a highly digestible grass silage on animal performance, CH4
and CO2 emissions and N efficiency.

A production trial using 16 Swedish Red cows (79±14 DIM, 635±76 kg) was conducted as a replicated
balanced 4×4 Latin Square with 28 d experimental periods. Cows were housed in an insulated loose
housing system and fed experimental diets three times daily ad libitum with free access to water.
Two grass silages referred to as early-cut (ES) and late-cut (LS) silage were harvested 2-weeks
interval with a precision-chopper from a primary growth timothy grass sward (Phleum pratense).
The crops were wilted to around 300 g of DM/kg and ensiled in bunker silos using a commercial
acid-based additive. Two grass silages of different in vitro digestibility were obtained (DOM: 685
and 607 g/kg DM). Experimental diets were formulated to meet energy and protein requirements
of 35 kg energy corrected milk (ECM) yield. Late-cut silage and crimped barley (1:1 ratio on DM
basis) was gradually replaced with ES (0, 33, 67 and 100%). The proportion of forage in the diet

increased from 42 to 64%. Rapeseed expeller was used as a protein supplement. Intake and milk
yield was recorded daily, and composition analysed from morning and evening milk composited
according to yield. Gas emissions were measured using the GreenFeed system (C-Lock Inc., Rapid
City, SD, USA). Experimental data was analysed with the MIXED procedure of SAS including the
effects of diet, period and cow.

Treatment effects on production performance and gas emissions are shown in Table 1. Feed intake
decreased linearly with increasing proportion of ES in the diet, but ECM yield was not influenced
by diet. Milk protein yield decreased linearly as the proportion of high-quality silage increased.
Total CH4 emissions or per kg ECM did not differ among the diets, but emissions per kg dry matter
intake (DMI) increased with increased proportion of ES in the diet. Diet digestibility increased for
diets containing a higher proportion of ES compensating at some extent differences in DM intake.
Increased CH4/DMI with increased proportion of ES in the diet are partly related to diet induced
differences in DMI and digestibility (Ramin and Huhtanen, 2013). The same experimental treatments
in rumen cannulated cows indicated no effects of replacing LS and barley with ES on rumen volatile
fatty acids concentrations or molar proportions (unpublished data). Efficiency of N utilisation was not
influenced by the diet, but feed efficiency improved with increasing proportion of ES in the diet. In
conclusion, the amount of concentrate can be decreased by up to 50% through improvements in forage
quality without compromising milk production, N efficiency and CH4 emissions by lactating cows.
Table 1. The effects of graded replacement of late-cut silage and barley with early-cut silage on the
performance and methane emissions in dairy cows.
Item1 Diet2 SEM P-value
L LE EL E L Q
Production performance
Total DMI, kg/d 22.8 20.9 20.4 19.5 0.57 <0.01 0.17
ECM, kg/d 30.0 29.7 29.7 29.7 1.17 0.55 0.72
Milk protein yield, g/d 1,047 1,022 1,001 996 37.2 0.01 0.45
Methane production
CH4, g/ d 440 441 446 444 14.9 0.69 0.92
CO2, g/d 13,271 13,012 13,152 12,949 297 0.24 0.85
CH4/DMI 19.4 21.1 21.9 22.7 0.67 <0.01 0.26
CH4/ECM 14.8 15.0 15.2 15.2 0.63 0.44 0.89
Feed efficiency
ECM/DMI, kg/kg 1.32 1.43 1.46 1.52 0.05 <0.01 0.40
Milk N/N intake, g/kg 293 299 288 285 9.6 0.11 0.37
N excess, g/kg ECM3 13.8 13.2 13.7 14.0 0.68 0.53 0.21
1 DMI = dry matter intake; ECM = energy corrected milk.

2 Diet LC silage: EC silage: L = 100:0, LE = 67:33; EL = 33:67, E = 0:100.
3 Calculated as [N intake (g/d) – milk N yield (g/d)] / ECM yield (kg/d).
References
Ramin, M. and P. Huhtanen, 2013. Development of equations for predicting methane emissions from ruminants. Journal
of Dairy Science 96: 2476-2493.

Delineation of relationships between feed composition, methane emission
and milk fatty acids in cows – prerequisite for the development of an
indirect methane indicator
S.W. Engelke, G. Daş, M. Derno, B. Kuhla and C.C. Metges*
Institute of Nutritional Physiology ‘Oskar Kellner’, Leibniz Institute for Farm Animal Biology (FBN),
18196 Dummerstorf, Germany; metges@fbn-dummerstorf.de
Abstract
Methane is a greenhouse gas produced by ruminants and contributes to global warming. It is assumed
that reducing CH4-emissions is possible by genetic selection. However, phenotypic characterisation
of the animals for methane emission is challenging. By using respiration chambers, it is not possible
to screen large numbers of cows for methane production. The purpose of the study is to deduce an
improved milk fatty acid marker to predict individual methane emission for identifying low-CH4-
emitting cows. Twenty lactating, half-sib Holstein cows were fed 4 diets: rich in starch (S), starch +
linseed (SL), rich in fibre (RF), fibre + linseed (RFL). In experimental week (EW) 1, the cows were
switched step-wise from standard TMR to 1 of the 4 diets which were fed until EW 6. Thereafter,
from EW 7 to 12, diet was changed from RF to RFL, and from S to SL or vice versa. Methane
emissions were recorded in EW 4 and 10 for 48 h each using open circuit respiration chambers. During
respiration measurements a milk aliquot was collected and analysed by near-infrared spectroscopy.
So far, data from 6 cows/diet were analysed using PROC MIXED of SAS. Our findings indicate a
methane lowering effect with linseed supplementation. Furthermore, the experimental diets cause a
wide variation of methane output associated with significant changes of the saturated / unsaturated
fatty acids ratio in milk fat. This suggests that the selected experimental diets are suitable to construct
a regression equation to predict individual methane emission from milk fatty acids.
Keywords: linseed supplementation, milk fatty acids, methane proxy, dairy cows
Introduction
Methane is a greenhouse gas produced in the digestive system of ruminants that contributes to global
warming. It is known that dry matter intake (DMI) and feed composition strongly affect methane
emission. It is assumed that it is possible to reduce CH4-emissions through genetic selection of cows.
However, breeding value estimations require large datasets of individual animal measurements.
Measurements of CH4 using respiration chambers are considered to be the gold standard but are
expensive and time consuming, therefore limiting the number of cows for screening purposes.
Currently, several easy to measure CH4 proxies for estimating individual CH4 emission are under
investigation, one of them being milk fatty acid profiles. The purpose of the study is to delineate the
relationship between individual methane production and milk fatty acid profile with diverse feed
compositions. The aim is to deduce an improved fatty acid marker to predict individual methane
emission.

Twenty lactating (106±28 DIM, 580±57 kg body weight), half-sib Holstein cows were fed ad
libitum 4 diets: rich in starch (S), starch + linseed (SL), rich in fibre (RF), fibre + linseed (RFL).
Diets contained grass and corn silage at levels of 17.2 and 60.8% (S and SL), and 49.1 and 26.2%
(RF and RFL), respectively. Linseed contributed 6.4% of DM in RFL and SL diets. In experimental
week (EW) 1, cows were switched step-wise from standard TMR to 1 of the 4 diets and continued
on this diet until EW 6. Thereafter, from EW 7 to 12, diet was changed from RF to RFL and from
S to SL or vice versa. Methane emissions were recorded in EW 4 and 10 for 48 h each using open

circuit respiration chambers (Derno et al., 2009). During respiration measurements, one milk aliquot
(evening and morning milking combined) was collected and analysed by near-infrared spectroscopy
for milk composition and milk fatty acid content. So far, data from 6 cows/diet were analysed using
PROC MIXED of SAS. Values are expressed as LSM.

The DMI did not differ among diets. Irrespective of diet type methane emission per kg DMI was
similar with both linseed supplemented and non-supplemented diets (Table 1). In spite of the ~3 kg
higher DMI with S diet, RF diet feeding resulted in comparable CH4-emissions on DMI basis. This
is in line with previous findings demonstrating that at comparable DMI, CH4 production decreased
when grass silage was replaced by corn silage (Van Gastelen et al., 2015). Methane output per day
and cow ranged from 337 to 708 l/d. Linseed supplementation caused ~16% lower CH4-emission
per kg DMI. Linseed decreased saturated fatty acids (SFA) by 15% and increased unsaturated fatty
acids (USFA) by 36% in milk fat. Polyunsaturated fatty acids (PUFA) were increased whereas
monounsaturated fatty acids (MUFA) were decreased by linseed in S but not in RF diets.
Our preliminary data suggest that the experimental diets cause a wide variation of methane output
associated with significant changes in the milk SFA/USFA ratio. This might indicate that the selected
experimental diets are suitable to construct a regression equation to predict individual methane
emission from milk fatty acid concentrations.
Table 1. Diet effects on methane and performance parameters (n=6 cows/diet).1
RF RFL S SL SE P-value
DMI (kg/d) 14.8 16.1 18.0 17.7 1.04 0.126

ECM (kg/d) 19.8c 20.7bc* 28.1a 26.3ab* 1.49 0.016
CH4 l/kg DMI 32.3ac* 26.9bd 31.1ab 26.7cd* 1.65 0.007
Milk
Fat (g/l) 45.8 44.8 40.1 38.2 3.04 0.327
SFA (%) 70.8a 60.5b 71.2a 60.6b 1.46 <0.001
USFA (%) 29.2b 39.6a 28.8b 39.4a 1.46 <0.001
MUFA (% USFA) 87.5ab* 86.9ab 87.1a 86.1b* 0.43 0.033
PUFA (% USFA) 12.5ab* 13.1ab 12.9b 14.0a* 0.43 0.033
1 a,b,c,d: different letters on the same line indicate differences at P<0.05; *: tendency to differ at P<0.10.
Acknowledgements
This study was supported by the German Federal Ministry of Food and Agriculture (BMEL).
References
Derno, M., H.G. Elsner, E.A. Paetow, H. Scholze and M. Schweigel, 2009. Technical note: a new facility for continuous
respiration measurements in lactating cows. Journal of Dairy Science 92(6): 2804-2808.
Van Gastelen, S., E.C. Antunes-Fernandes, K.A. Hettinga, G. Klop, S.J.J. Alferink, W.H. Hendriks and J. Dijkstra. 2015.
Enteric methane production, rumen volatile fatty acid concentrations, and milk fatty acid composition in lactating
Holstein-Friesian cows fed grass silage- or corn silage-based diets. Journal of Dairy Science 98(3): 1915-1927.

Temperature effect on fast heat production of Anglo Nubian and Saanen
goats
M.H.M.R. Fernandes*, A.R.C. Lima, C.I.S Oporto, K.T. Resende, B. Biagioli and I.A.M.A. Teixeira
Department of Animal Science, Universidade Estadual Paulista (UNESP/FCAV), SP 14884-900,
Brazil; mhmrochaf@fcav.unesp.br
Abstract
Six dry and non-lactating Saanen (52.8±4.7 kg body weight, BW) and six dry and non-lactating Anglo
Nubian (62±4.7 kg BW) goats were used in a factorial design 2×2 (2 breeds and 2 temperatures).
The experimental period consisted of two stages corresponding to the temperature to the temperature
of 20.0±0.21 °C (thermoneutral zone) and 11.5±0.15 °C (cooling). The adaptation period in each
temperature lasted 21 days. Heat production, ventilation (l/min), respiratory rate (breaths per minute,
BPM) and evaporative water loss were recorded using facemask open-circuit respirometry. Gas
measurements were performed in groups of four animals (two of each breed). Animals were subjected
to fasting (no feed only water) for 48 hours and gas measurement was performed in the third day,
during 30 min in each goat, randomly. The rectal temperature (TR) was measured using sensor. Our
results indicated no differences (P>0.05) in ventilation (average of 14.3±1.46 l/min), respiratory rate
(average of 14.0±1.27 BPM) and fast heat production (average of 182.9 kJ/kg0.75 BW) between 10
and 20 °C, irrespective of goat breed. On the other hand, evaporative loss of goats under 20 °C was
greater than that under 10 °C (10.5±0.59 and 3.24±0.59 l/h, respectively). The lack of differences in
FHP between 10 and 20 °C in this study could suggest no need of energy requirement adjustments
within this temperature range.
Keywords: facemask, energy, respirometry, ventilation
Introduction
Goats are able to be raised in widespread worldwide conditions, from mountains of temperate
areas to tropical and subtropical regions, coping with wide range of temperatures in regarding
to energy expenditure. In the context of maintenance requirements, acclimatization encloses the
adaptive responses to changes in non-extreme climatic conditions. Even though current feeding
systems have suggested corrections for net energy maintenance requirements (NEm) to account for
acclimatization, it is still not conclusive because there is a lack of data of cold and heat effects on
NEm regarding goats. In addition, it is questionable whether the appropriate midpoint termoneutral
zone should be set 20 °C for various types of goats and types. Therefore, this study was carried out
to measure fast heat production, which represents NEm by definition, of Saanen and Anglo Nubian
goats under 10 and 20 °C.

Six dry and non-lactating Saanen (50.6±4.7 kg body weight, BW) and six dry and non-lactating Anglo
Nubian (62±4.7 kg BW) goats were used in a factorial design 2×2 (2 breeds and 2 temperatures).
The experimental period consisted of two stages corresponding to the temperature of 20.0±0.21 °C
(thermoneutral zone) and 11.5±0.15 °C (cooling). The adaptation period in each temperature lasted
21 days. Heat production, ventilation (VE, l/min), respiratory rate (breaths per minute, BPM) and
evaporative water loss (EWL) were recorded using facemask open-circuit respirometry. Goats were
allocated to individual metabolic cages and fed the same total mixed ration (9.2% CP, 8.3 MJ ME/kg
DM), which consisted of 90% of corn silage and 10% of concentrate. Dry matter intake was recorded
daily. Gas measurements were performed in groups of four animals (two of each breed). Animals
were subjected to fasting (no feed only water) for 48 hours and gas exchange measurement was

performed in the third day, during 30 min in each goat, randomly. The rectal temperature (TR) was
measured using sensor (Model MLT1407, Ad Instruments Ltda., New South Wales, Australia) inserted
into the rectum of the animal. The exhaled air temperature (Texpair) sensor was placed inside the
mask near to the animal’s nose. Another sensor continuously measured ambient temperature (Tair).
These sensors were connected directly to the thermistor pods (Model ML309, Ad Instruments Ltda.,
New South Wales, AU). Data were analysed as a factorial design 2×2 using a MIXED procedure of
SAS (SAS Inst. Inc., Cary, NC. version 9.4); with the fixed effects of breed, temperature and their
interactions and the random effects of animal. Significance was declared at P≤0.05.

Our results indicated no differences in VE (average of 14.3±1.27 l/min) and BPM (average of
14.0±0.94 BPM) between 10 and 20 °C (Table 1), irrespective of goat breed. On the other hand, the
greater EWL of goats under 20 °C than those under 10 °C (10.5±0.59 and 3.24±0.59 l/h, respectively)
indicates that goats used respiratory water loss to dissipate heat. The mean rectal temperature was
higher in Saanen than in Anglo Nubian goats. The NRC (2007) have suggested corrections for energy
maintenance requirements to account for acclimatization based on the mid-point thermoneutral zone
temperature stablished for goats of 20 °C. However, the lack of differences in FHP between 10 and
20 °C in this study could suggest no need of energy requirement adjustments within this temperature
range. Future studies should address the effects of higher temperatures on FHP.
Table 1. Temperature effect on ventilatory measurements, rectal temperature and heat production
of Anglo Nubian and Saanen goats after fasting.1
Anglo Nubian Saanen SEM Effect
10 20 10 20 Breed Temp
BPM 13.8 14.2 13.3 14.8 1.27 0.98 0.43

VE, lbtps/min 15.5 16.7 12.0 13.1 1.27 0.058 0.29
T rectal, °C 38.6 38.7 39.2 39.0 0.13 0.013 0.26
Texpair, °C 20.4 25.0 18.9 23.4 0.47 0.003 <0.0001
EWL, g/min 3.5 11.7 2.9 9.4 0.83 0.16 <0.0001
VO2, lstpd/h 7.2 8.7 8.2 6.9 1.94 0.88 0.93
VCO2, lstpd/h 5.5 6.7 6.4 5.4 1.52 0.93 0.88
FHP, kJ/kg0.75 BW 156.2 184.9 215.4 171.9 48.8 0.73 0.72
1 BTPS = body temperature pressure, saturated air; STPD = standard temperature and pressure, dry air; VE = ventilation;
BPM = respiratory rate (breaths per minute); texpair = temperature of expired air; EWL = evaporative water loss; VO2
= oxygen consumption; VCO2 = carbon dioxide production; SEM = standard error of mean.
Acknowledgements
Financial support CNPq project number 479595/2012-7; FAPESP grant # 14/26556-8.
References
National Research Council (NRC), 2007. Nutrient requirements of small ruminants. Sheep, goats, cervids, and new
world camelids. National Academy Press, Washington, DC, USA, 384 pp.

Long term implications of feeding low protein diets to first lactation
dairy cows
C.K. Reynolds, L.A. Crompton, D.J. Humphries and A.K. Jones
Centre for Dairy Research, School of Agriculture, Policy and Development, University of Reading,
P.O. Box 237, Earley Gate, Reading, RG6 6AR, United Kingdom; c.k.reynolds@reading.ac.uk
Abstract
Diet nitrogen use efficiency (NUE) for milk N production is affected by multiple dietary factors. The
aim of the present study was to assess the long term implications of feeding lower protein diets to
first lactation dairy cows. First calf Holstein heifers (n=215) were assigned to one of 3 total mixed
rations formulated to contain 14, 16, and 18% crude protein (CP) and provide metabolisable protein
below, at, and above predicted requirements in a randomized block design experiment. Animals
were maintained on treatment diets from 7 days in milk until dry off. Dry matter intake (DMI) was
similar for 14 and 16%, but greater for heifers fed 18% diets, whilst milk yield was lower for 14%
but similar for 16 and 18% diets. Decreasing dietary CP to 14% was associated with decreased milk
component yield compared to 16 and 18% diets; however, reducing dietary CP increased NUE. Body
weight and condition score were highest for the 18% CP diet. Effects of feeding the 14% CP diet
on milk yield in the present study was less than expected, but the longer term effects of these diets
may differ with greater potential milk yield and DMI in subsequent lactations.
Keywords: dietary protein, long term, first lactation cows
Introduction
Dietary protein is used inefficiently for milk protein production by dairy cows, with approximately
75% or more of N intake typically excreted in manure. Diet N use efficiency (NUE) for milk N
production is affected by a variety of dietary factors that determine metabolisable protein (MP)
supply relative to requirement. In addition, reduced diet crude protein (CP) concentration consistently
increases NUE, but often at the expense of milk yield. While there may be clear benefits of increased
NUE with lower CP diets, the strategy will only be acceptable if it can be achieved without large
reductions in milk yield or detrimental effects on health and fertility. There are innumerable studies
reporting effects of varying dietary protein type and amount on NUE of lactating dairy cows, but with
few exceptions the data are from experiments that have not allowed sufficient time for expression
of long-term effects.

In the present study, 215 first calf Holstein heifers were assigned to 3 maize silage-based diets (71
or 72 heifers each) similar to those used in a previous study at our location (Reynolds et al., 2010).
The treatment total mixed rations (TMR) fed were formulated to contain 14, 16, and 18% CP and
provide MP below, at, and above predicted requirements, using incremental substitution of wheat,
wheat feed, and soy-hulls with soybean and rapeseed meal. Heifers were assigned to treatments in
blocks based on genetic merit and body weight (BWT), body condition score (BCS), and age at
calving. After calving they were fed a herd TMR until 7 days in milk (DIM) and then maintained on
treatment diets and individually fed until dry off. Measurements include daily milk yield and feed
dry matter intake (DMI) and weekly milk composition, BWT, and BCS. Cows were inseminated
between 50 and 200 DIM. Data were analysed using Mixed Models procedures testing fixed effects
of treatment, week, and their interaction and random effects of heifer, with week as a repeated effect.
Age, BWT, and BCS at calving, genetic merit, calving date, and days pregnant were included in
the model as covariates.

Feed DMI was similar for 14 and 16%, but greater for heifers fed 18% diets. In contrast, milk yield
was lower for 14%, but similar for 16 and 18% diets. Milk protein concentration was lower for 14
vs 18% diets, whilst milk protein, fat, and lactose yield were lower for 14% compared to 16 and
18% diets. Mean NUE increased as diet CP decreased, whilst energy corrected milk yield per kg
DMI was lower for 14% diets. Both BWT and BCS were highest for the 18% diet. In the present
study, greater NUE for the 14% diet was associated with reduced milk yield, but higher DMI for the
18% diet was not associated with greater milk yield compared to 16%. However, the longer term
effects of these diets may differ with greater potential DMI and milk yield in subsequent lactations.
Table 1. Milk yield, composition, and component yield in first lactation Holstein cows fed diets
containing 3 levels of dietary protein from week 2 to 40 of lactation.1,2
Diet CP % SEM P-value3
14 16 18 Diet Week Inter
DMI, kg/d 20.4a 21.0a 21.8b 0.28 0.004 0.001 0.044

Milk yield, kg/d 27.6a 29.8b 30.0b 0.47 0.001 0.001 1.000
Milk composition, g/kg
Fat 36.5 36.3 37.1 0.61 0.576 0.001 0.005
Protein 31.7a 32.6ab 33.0b 0.38 0.043 0.001 0.576
Lactose 44.1 44.9 45.0 0.43 0.271 0.001 0.679
Urea 0.165a 0.240b 0.303c 0.003 0.001 0.001 0.001
Milk component yield, g/d
Fat 1,026a 1,085b 1,107b 16.0 0.002 0.001 0.843
Protein 895a 978b 986b 13.2 0.001 0.001 1.000
Lactose 1,257a 1,348b 1,357b 21.7 0.002 0.001 1.000
Body weight 581a 595ab 609b 6.3 0.009 0.001 0.001
Condition score 2.91a 2.92a 3.06b 0.04 0.023 0.001 0.001
ECM, kg/kg DMI 1.37a 1.47b 1.43b 0.02 0.002 0.001 0.998
NUE, % 30.9a 29.4b 25.5c 0.42 0.001 0.001 0.387
n 72 72 71
1 a,b,c
least squares means with different superscripts differ at P<0.05.
2 DMI = dry matter intake; ECM = energy corrected milk; NUE = nitrogen use efficiency.
3 Probability for effects of dietary protein level, week of lactation, and their interaction (Inter).
Acknowledgements
Funded by the UK Department for Environment, Food and Rural Affairs.
References
Reynolds, C.K., L.A. Crompton, J.A.N. Mills, D.J. Humphries, P. Kirton, A.E. Relling, T.H. Misselbrook, D.R.
Chadwick and D.I. Givens, 2010. Effects of diet protein level and forage source on energy and nitrogen balance and
methane and nitrogen excretion in lactating dairy cows. In: G.M. Crovetto (ed.). Energy and protein metabolism
and nutrition. Wageningen Academic Publishers, Wageningen, the Netherlands, pp. 463-464.

Influence of rutin and rutin-containing buckwheat seeds on methane
emission in lactating dairy cows
A.-K. Stoldt1, M. Derno1, G. Daş1, J.M. Weitzel1, S. Wolffram2 and C.C. Metges1*
1Leibniz Institute for Farm Animal Biology (FBN), 18196 Dummerstorf, Germany; 2Institute of
Animal Nutrition and Physiology, Christian-Albrechts University of Kiel, 24118 Kiel, Germany;
metges@fbn-dummerstorf.de
Abstract
It has been reported that quercetin, a flavonoid found in certain plants, mitigates methane production
in vitro. The hypothesis of the present study was that rutin influences methane production, energy
metabolism, and performance in dairy cows. Seven German-Holstein dairy cows in 2nd lactation were
fed a total mixed ration supplemented with rutin trihydrate (100 mg/kg body weight) for 2 weeks in
a cross-over-design. In a second experiment 2 cows were fed the same ration but were supplemented
with ground buckwheat seeds (Fagopyrum tartaricum) providing rutin at a similar dose. Two other
cows receiving barley supplements were used as controls in a change-over mode. At the end of the
2-weeks treatment period cows were measured in respiration chambers for two days (ad libitum
and restrictive feeding). Blood samples were taken weekly to measure plasma quercetin and its
metabolites as well as variables reflecting the metabolic status. Data were evaluated with repeated
measures ANOVA using a mixed model in SAS. Supplementation of pure rutin but not of rutin
contained in buckwheat seeds increased the plasma quercetin content from the basal level of 5.4±6.3
to 35.6±6.3 nmol/l (mean ± SE) (P<0.05). Daily methane production, heat production, and milk yield
and composition were not affected by rutin treatment in either form. In conclusion, earlier reports
of in vitro studies with rutin, indicating rutin mitigation properties could not be confirmed in vivo.
Keywords: flavonoids, energy metabolism, methane production, dairy cows
Introduction
In view of the beneficial effects flavonoids, and in particular quercetin, have in humans and rodents,
there is increasing interest in their possible health benefits in dairy cows by plant bioactive compounds.
We have recently shown that quercetin can affect glucose metabolism and can be beneficial for
liver health in dairy cows (Gohlke et al., 2013; Stoldt et al., 2015). In addition, in in vitro studies
quercetin has been reported to mitigate methane production. Flavonoids have been shown to
alter the rumen microbiome and to reduce the population of protozoa and methanogenic bacteria.
Methane is a greenhouse gas produced by ruminants and contributes to global warming. Because
the systemic availability of quercetin in cows is much better after intraruminal application of rutin
(a glucorhamnoside of quercetin) when compared with quercetin aglycone (Berger et al., 2012), we
investigated effects of oral rutin supplementation.

Seven 2nd lactation cows (100 days in milk, 608±14 kg body weight) fed with a total mixed ration
based on grass and corn silage, straw and grass hay (crude protein 161, crude fat 30, neutral detergent
fibre 343 g/kg fresh mass) were supplemented with rutin trihydrate at a dose of 100 mg per kg body
weight for 2 weeks in a cross-over-design. In a second experiment 2 cows were fed the same ration
but were supplemented with ground buckwheat seeds (Fagopyrum tartaricum) providing rutin at
a similar dose. Two other cows receiving barley supplements were used as controls in a change-
over mode. At the end of each 2-weeks treatment period (Rutin or Control) cows were measured in
respiration chambers for 2 days (1st day ad libitum and 2nd day with 10 h food withdrawal). Blood
samples were taken weekly to measure plasma quercetin and its metabolites as well as variables

reflecting the metabolic status. Data were evaluated with repeated measures ANOVA using a mixed
model in SAS.

Body weight (625 kg), dry matter intake (21.7 kg/d), milk yield (33.2 kg/d) and composition were
not affected after a 2-weeks rutin supplementation (P>0.1) (Stoldt et al., 2016). Only after pure rutin
supplementation but not with rutin contained in buckwheat plasma total flavonoids were elevated as
compared to the control treatment (35.5 vs 5.4 nmol/l; P=0.003). Quercetin was the major flavonoid
in plasma and only minor levels of kaempferol and isorhamnetin were found, but no tamarixetin could
be detected. Daily methane production (26.5 vs 26.5 l/kg dry matter intake), heat production (1,163
vs 1,174 kJ/kg BW0.75) and respiratory quotient (0.82 vs 0.82) were not different between groups
(P>0.5). Plasma glucose, β-hydroxybutyrate, and albumin tended (P=0.07) or were (P=0.013) higher
under rutin supplementation, suggesting a possible metabolic effect of rutin on energy metabolism
of dairy cows (Stoldt et al., 2016).
Our data suggest that in mid-lactation cows a 2-weeks supplementation of rutin caused systemic
availability of quercetin but no effects on methane production and energy expenditure. Thus we
could not confirm in vivo the earlier reported methane mitigation effect in vitro. We also could not
observe a rutin effect on milk yield. This is in contrast to observations in a Chinese study were a
much lower dose (recalculated approximately 0.3 mg rutin/kg body weight) was added to the diet for
10 weeks and 10% increase of milk yield was found (Cui et al., 2015). A reason for this discrepancy
is not apparent but might be related to duration of supplementation.
Acknowledgements
This study was supported by the Federal Ministry of Education and Research, Germany, under the
funding initiative ‘Kompetenznetze der Agrar- und Ernährungsforschung’ (BMBF grant number
0315538B).
References
Berger, L.M., R. Blank, S. Wein, C.C. Metges and S. Wolffram, 2012. Bioavailability of the flavonol quercetin in cows
after intraruminal application of quercetin aglycone and rutin. J. Dairy Sci. 95: 5047-5055.
Cui, K., X. D. Guo, Y. Tu, N. F. Zhang, T. Ma and Q. Y. Diao, 2015. Effect of dietary supplementation of rutin on lactation
performance, ruminal fermentation and metabolism in dairy cows. J Anim. Physiol. Anim. Nutr. 99: 1065-1073.
Gohlke, A., C.J. Ingelmann, G. Nürnberg, J.M. Weitzel, H.M. Hammon, S. Görs, A. Starke, S. Wolffram and C.C.
Metges, 2013. Influence of 4 wk intraduodenal supplementation of quercetin on performance, glucose metabolism,
and mRNA abundance of genes related to glucose metabolism and antioxidative status in dairy cows. J. Dairy
Sci. 96: 6986-7000.
Stoldt, A., M. Derno, G. Nürnberg, J.M. Weitzel, W. Otten, A. Starke, S. Wolffram and C.C. Metges, 2015. Effects
of a 6 wk intraduodenal supplementation with quercetin on energy metabolism and indicators of liver damage in
periparturient dairy cows. J. Dairy Sci. 98: 4509-4520.
Stoldt, A.-K., M. Derno, G. Das, J.M. Weitzel, S. Wolffram and C.C. Metges, 2016. Effects of rutin and buckwheat
seeds on energy metabolism and methane production in dairy cows. J. Dairy Sci. 99: 2161-2168.

Effect of genetic group and feeding level on methane production in
lactating crossbred and Zebu cows in Brazil
A.L.C.C. Borges1*, P.A.D. Vivenza1, R.R. Silva1, E.O.S. Saliba1, P.H.A. Carvalho1, H.F. Lage1, I.
Borges1, A.U. Carvalho1 and J.R.M. Ruas2
1Department of Animal Science of Veterinary School of Federal University of Minas Gerais, Belo
Horizonte, Brazil; 2Agricultural Research Company of Minas Gerais – EPAMIG, Felixlândia-MG,
Brazil; analuizavetufmg@gmail.com
Abstract
Methane gas (CH4) is resulting from the digestive process, and it´s is an important energy loss for
the animal. The aim of the study is evaluate methane emissions by dairy cows from different genetic
groups and different nutritional plans. 12 lactating cows were used, divided in two genetic groups
(Gyr and F1 Holstein × Gyr). Diet was corn silage and concentrate based on corn and soybean meal.
The nutritional plans consisted in fed ad libitum period, followed by dietary restrictions of 15 and
30% in relation to the dry matter intake in the previous period (ad libitum). Digestibility trials were
performed in all periods, and production of methane was measured in respirometry chamber. F1 cows
had increased production of methane as well as loss of energy in the form of this gas in relation to Gyr
cows in all nutritional plans. However, when expressed in percentage of gross energy consumed, the
values were similar for both genotypes. Cows F1 Holstein × Gyr was more productive and efficient
than Gyr cows, diluting the methane emissions in the production of milk. The nutritional plans
interfere with the efficiency of production and energy losses related of the methane.
Keywords: crossing, efficiency, energy, tropical conditions
Introduction
The use of metabolisable energy (ME) for description the diets of ruminants depends on the accurate
measurement of methane production. Furthermore, it is known that the level of consumption and
diet quality influence the emission of this gas. There are few studies evaluating methane emissions
by dairy cows in Latin America, especially in respirometric chambers. The aim of the study is
evaluate methane emissions by lactating dairy cows from different genetic groups and different
nutritional plans.

Twelve lactating cows were used, six of genetic group Gyr (Bos taurus indicus) and six of genetic
group F1 Holstein × Gyr. Cows were kept in individual stalls receiving corn silage and concentrate
based on corn and soybean meal, according to the recommendations of NRC (2001). The nutritional
plans was ad libitum in the 1st to 15th week of lactation (period 1), energy restriction of 15% over
the previous consumption from the 16th to 18th week of lactation (period 2), and energy restriction
of 30% from the 19th to 21th week of lactation (period 3). Methane production was determined using
respirometric chamber in which the animals were evaluated in a 24 hours period. The experimental
design was completely randomized with a split plot scheme.

In the period 1 F1 cows had higher body weight (BW, 526.2 kg), dry matter intake (DMI, 16.96 kg,
3.22% BW), gross energy intake (GEI, 75.32 Mcal/day), ME intake (44, 60 Mcal/day), production
of methane (527.63 liters/day) and energy loss as methane (4.99 Mcal/day), compared to Gyr cows
(Table 1). Relationship between methane production and dry matter intake was positive, as Kurihara et

Table 1. Dry matter intake (DMI, kg/day), gross energy intake (GEI), methane production (liters/day,
Mcal/day, % of gross energy intake, g methane/kg energy corrected milk (ECM) and metabolisability
of diet (q).1
Variables Genetic group
F1 SEM Gyr SEM
P1 P2 P3 P1 P2 P3
DMI 16.96 Aa 14.84 Ab 12.79 Ac 1.41 11.22 Ba 9.22 Bb 7.69 Bc 0.85

GEI 75.32 Aa 65.74 Ab 57.78 Ab 6.36 51.29 Ba 41.61 Bb 36.53 Bc 4.02
CH4 527.63 Aa 379.52 Ab 375.52 Ab 50.70 337.69 Ba 250.54 Bb 212.92 Bb 28.74
CH4 4.99 Aa 3.63 Aab 3.55 Ab 0.52 3.51 Ba 2.24 Bb 2.99 Bb 0.37
CH4 6.40% 5.52% 6.14% 0.01 6.86% 5.38% 5.57% 0.01
CH4/ECM 19.62 Ba 14.71 Bb 18.05 Bab 2.89 28.97 Aa 24.92 Ab 25.95 Ab 4.15
q 0.58 0.59 0.55 0.02 0.58 0.59 0.56 0.02
1 Means followed by different lowercase letters in the same row differ of nutritional plans in the same genetic group. Means
followed by different capital letters in the same row differ between genetic groups in the same nutritional plan (P<0.05).
al. (1999) found. However, evaluating the production of methane in relationship with the production
of energy corrected milk (g CH4/energy corrected milk, ECM) the F1 cows had lower values (19.62
g CH4/kg ECM) in relation to Gyr cows (28.97 g CH4/kg ECM).
Methane loss was equal between the genotypes (6.63%), when expressed as percentage of GEI, as
well as the q of diet (q=0.58). In periods 2 and 3 GEI, MEI and methane production were lower
in both groups, but F1 cows had superior values. In the first nutritional restriction (15%), both
genotypes had the same proportional loss of methane relative to GEI (5.45%) and the q of diet was
similar (q=0.59). There was improvement in the relationship g CH4/kg ECM. Methane production
in periods 2 and 3 was lower for both genetic groups (F1 14.70 / Gyr 24.92 g CH4/kg ECM). F1
Holstein × Gyr cows was more productive and efficient than Gyr cows, diluting methane emissions
in the production of milk. The nutritional plans imposed interfered with the efficiency of production
and energy losses related to methane in cows of both genotypes.
Acknowledgements
We would like to thank CNPq, CNPq-INCT, FAPEMIG, CAPES and EPAMIG for their cooperation
in carrying out this work.
References
Kurihara, M., T. Magner, R.A. Hunter and G.J. Mc Crabb, 1999. Methane production and energy partition of cattle in
the tropics. British Journal of Nutrition 81: 227-234.
National Research Council (NRC), 2001. Nutrient requirements of dairy cattle (7th Ed.). National Academic of Sciences,

Part 5.
Feed sources and feed processing related to energy and
protein digestion and metabolism
Rumen degradation kinetics of protein rich feedstuffs in dairy cows and
intensively fed beef cattle
A. Navarro-Villa, H. van Laar*, J. Doorenbos and J. Martín-Tereso
Trouw Nutrition R&D, Veerstraat 38, P.O. Box 220, 5830 AE, Boxmeer, the Netherlands;
harmen.van.laar@trouwnutrition.com
Abstract
Rumen fermentation can be influenced by the physiological stage of the animal as well as by the
type of diet offered. Quantification of how these factors affect ruminal degradation rates (kd) and
undegradable fractions (U) of protein and other components of feedstuffs in intensively fed beef
cattle is scarce. The nylon bag methodology was used to compare kd and U of dry matter (DM),
crude protein (CP), starch and neutral detergent fibre (NDF) in dairy cows versus intensively fed
beef animals for: soya bean meal, sunflower meal, rapeseed meal, gluten feed meal and a composite
sample. Combining all results of all feedstuffs, beef had higher U and kd than dairy for both DM
(U: 18.4 vs 11.1%; P<0.001; kd: 0.060 vs 0.044 /h; P<0.001) and CP (U: 6.8 vs 1.5%; P<0.01;
kd: 0.051 vs 0.039/h; P<0.001). For NDF, only U fraction was higher for beef compared to dairy
(36.7 vs 24.0%; P<0.01) with no difference in kd (0.038 /h). No difference in degradation kinetics
was found for starch between production systems. In general, differences found in U and kd for
all feedstuffs combined were numerically consistent in individual feedstuffs, although not always
reaching statistical significance individually. When calculating effective degradability (ED) of DM
and CP at an assumed passage rate of 0.06/h the higher U and kd compensate each other leading to
relatively modest differences in ED between beef and dairy.
Keywords: rumen fermentation, dairy cows, beef cattle, nylon bag method
Introduction
Modern nutritional models for ruminants are based on passage and degradation kinetics of feed
components in the rumen. Ad libitum compound feed and straw is an increasingly common approach
to feed beef cattle in Europe, however there is a lack of data for ruminal degradation kinetics in these
specific conditions. Literature indicates that effective rumen degradation (ED) of protein decreases at
higher concentrate intake levels in a beef feeding system (Swanek et al., 2001; Zinn and Owens, 1983).

In order to model specific nutritional value of feedstuffs in such feeding conditions, ruminal
degradation kinetics of dry matter (DM), crude protein (CP), neutral detergent fibre (NDF) and starch
of soya bean meal, sunflower meal, rapeseed meal, maize gluten feed meal and a composite sample
containing, corn, soya bean meal and grass meal (1:1:1) were studied in both lactating dairy cows
(dairy) and in intensively fed growing dairy heifers (beef). Dairy animals consisted of 4 lactating
fistuled Holstein Frisian cows fed a total mixed ration (TMR) ad libitum with a forage to concentrate
ratio of 64:36. Beef animals consisted of 4 growing Holstein heifers (age 9±0.5 months), fistulated at
7 months of age, fed compound feed and barley straw (separately) on ad libitum basis. Nylon bags
(53±10 um pore size, Bar Diamond, Inc) with approximately 5 g DM of each experimental feedstuff
were incubated in the rumen for 2, 4, 8, 12, 24, 48 and 72 hours in dairy and beef. Non incubated (0
h) and incubated bags were washed in a front loader washing machine and dried at 60 °C for 48 h to
determine washable fraction (W%). Before analyses, bags were pooled by incubation time, feedstuff
and cow, resulting in 4 replicates (cows) per time point. For each component, undegradable fraction
(U, %) and degradation rate (kd, /h) were estimated with a first-order exponential model using the

Newrap method in the NLMIXED of SAS. Differences in U and kd for dairy vs beef were tested
using orthogonal contrasts.

Total dry matter intake (DMI) was 17.4±1.67 kg/d TMR for dairy whereas for beef DMI was
6.8±0.51 kg/d compound feed and an estimated 1.4 kg barley straw. When results for all feedstuffs
were combined (Table 1), beef had higher U and kd than dairy for both DM and CP. For NDF, only
U fraction was higher for beef compared to dairy with no difference in kd. For starch no difference
in degradation kinetics was found. In general, the differences found in U and kd across feedstuffs
were numerically consistent, although not always reaching statistical significance for each individual
feedstuff. When calculating ED of DM and CP at an assumed passage rate of 0.06/h (Daniel et al.,
2014), the higher U and kd compensate each other leading to relatively modest differences in ED
between beef and dairy (across feedstuffs beef vs dairy: ED DM 55.3 vs 54.3, ED CP 55.2 vs 53.6).
Contrary to expectations, beef at high concentrate intakes had numerically higher ED compared
to dairy. The discrepancy of these results with results from literature could be caused by potential
specific conditions in the rumen environment of the beef feeding system used (ad libitum concentrate
and barley straw offered separately) compared to alternative TMR and corn silage based beef feeding
systems.
Table 1. Nylon bag degradation kinetics; washable fraction (W, % of component), undegradable
fraction (U, % of component) and degradation rate (kd) of dry matter (DM), crude protein (CP),
neutral detergent fibre (NDF) and starch averaged across soya bean meal, sunflower seed meal,
rapeseed meal, gluten feed meal and a composite sample in beef cattle and dairy cows.1
W U kd
Beef Dairy SEM P-value Beef Dairy SEM P-value
DM 28.9 18.4 11.1 0.83 *** 0.063 0.044 0.0026 ***

CP 22.9 6.8 1.5 0.92 ** 0.051 0.039 0.0021 ***
NDF 3.6 36.7 24.0 2.42 ** 0.042 0.035 0.0040 ns
Starch 54.2 0.5 0.3 0.35 ns 0.132 0.102 0.0345 ns
1 ns: P>0.15; *P<0.05; **P<0.01; ***P<0.001.
References
Daniel, J.B., H. van Laar, D. Warner, J. Dijkstra, A. Navarro-Villa and W.F. Pellikaan, 2014. Passage kinetics of dry
matter and neutral detergent fibre through the gastro-intestinal tract of growing beef heifers fed a high-concentrate
diet measured with internal ð13C and external markers. Animal Production Science 54: 1471-1475.
Swanek, S.S., C.R. Krehbiel, D.R. Gill and B.A. Gardner, 2001. Extent and rate of in situ ruminal degradation of protein
byproduct feeds on a high concentrate diet. 2001 Animal Science Research Reports, Publication P986, Oklahoma
State University, Stillwater, OK, USA. Available at: http://tinyurl.com/gu59xl2.
Zinn, R.A. and F.N Owens, 1983. Influence of feed intake level on site of digestion in steers fed a high concentrate
diet. Journal of Animal Science 56: 471-475.

Effect of starch content of milk replacer on energy metabolism in
growing-finishing veal calves
E. Labussière1,2*, J.N. Thibault1,2, T. Lefèbvre3 and C. Martineau3
1INRA, UMR 1348 PEGASE, Domaine de la Prise, 35590 Saint-Gilles, France; 2Agrocampus
Ouest, Domaine de la Prise, UMR 1348 PEGASE, 35000 Rennes, France; 3Institut de l’Elevage,
Monvoisin, 35650 Le Rheu, France; etienne.labussiere@rennes.inra.fr
Abstract
Veal calves are traditionally reared with large amounts of milk replacer with low content of starch.
The objectives of the experiment were to test the effects of three levels of substituting fat by starch
on energy and N utilisation during the growing (mean body weight (BW): 118 kg; starch content
from 4 to 13%) and the finishing stage (mean BW: 208 kg; starch content from 4 to 14%). At each
stage, four treatments were implemented, exclusively composed of one milk replacer, or one milk
replacer, supplemented with solid feed. Each treatment was fed to four calves per stage at 630 and
545 kJ/kg BW0.85/d of metabolisable energy (ME) intake during the growing and the finishing
stage respectively to measure N and energy balances in respiration chambers. Starch content of the
milk replacer did not affect digestive and metabolic utilisation of energy during the growing stage
whereas both decreased during the finishing stage, because of higher CH4 production. Components
of heat production (HP), total HP and total energy retention were never affected by treatment during
both stages, but respiratory quotient corrected for protein oxidation significantly increased when
starch inclusion level increased. Finally, net energy to ME ratio of all milk replacers averaged 79%.
Keywords: energy digestibility, net energy, efficiency of energy utilisation
Introduction
Veal calves are reared with large amounts of milk replacer, formulated with fat of animal and
vegetable origins that forces producers to dissolve milk replacer in hot water (65 °C). The latter
increases economic and environmental impacts. Alternatively, starch can be used as a substitute
for fat to reduce these impacts. Nevertheless, enzyme activity to digest starch in the small intestine
may be limiting (Gilbert et al., 2015). The objectives of the experiment were to test the effects of
substituting fat by starch on the energy and N utilisation during the growing (mean body weight
(BW): 118 kg) and the finishing stage (mean BW: 208 kg).

Three milk replacers were formulated, with fat content from 18 to 15 and 12% during the growing
stage (G18, G15 and G12, respectively), and from 19 to 16 and 12% during the finishing stage
(F19, F16 and F12, respectively). Starch content varied inversely from 4 to 13% and 14% during
the growing and the finishing stage, respectively. At each stage, four treatments were implemented:
the first treatment was exclusively composed of milk replacer G18 or F19 whereas the three others
were composed of one milk replacer supplemented with solid feed (at 12 and 18% of dry matter
intake during the growing and the finishing stage, respectively). Each treatment was fed at 630 and
545 kJ/kg BW0.85/d of metabolisable energy (ME) intake during the growing and the finishing stage,
respectively. At each stage, N and energy balances with calculation of heat production (HP) from
O2 consumption and CO2 production were achieved on four calves per treatment that were housed
individually in an open-circuit respiration chamber during 6 days. At the end, calves remained in
the respiration chamber during one supplementary day of feed deprivation to calculate fasting heat
production and net energy (NE) intake. The data were analysed per stage for the effect of treatment
using the GLM procedure of SAS.

During the growing stage, starch inclusion level did not affect digestive utilisation of energy (92%)
nor increase CH4 production (5 l/d). During the finishing stage, digestive utilisation of energy (Table
1) and CH4 production (from 18 to 28 l/d) increased when starch inclusion level increased. The
latter decreased the metabolisability of the digestible energy from 95.3 to 93.6%. During both stages,
digestive utilisation of N significantly decreased when starch content increased (from 88.5 to 86.0%,
and from 85.6 to 79.7% during the growing and the finishing stage, respectively), that may indicate
higher excretion of microbial biomass because of fermentation of starch. Components of HP, total
HP and total energy retention were never affected by treatment, but respiratory quotient corrected
for protein oxidation significantly increased when starch inclusion level increased. This indicates a
decrease in fat oxidation whereas lipogenesis was limited (Van den Borne et al., 2007). Finally, NE
to ME ratio of each milk replacer did not differ between starch inclusion levels and averaged 79%.
Table 1. Effect of starch content of milk replacer on energy metabolism of veal calves.1
Growing stage (mean body weight: 118 kg)
Milk replacer G18 G18 G15 G12 rsd P-value

Solid feed – with with with
DE/GE (%) 95.2 92.4 92.1 92.0 1.2 0.06

ME/DE (%) 96.6 96.3 95.7 96.0 0.5 0.26
NE/ME (%) 80.0 78.9 76.8 76.8 2.6 0.41
RQnp (l CO2/l O2) 0.94a 0.95a 0.97ab 0.99b 0.02 0.04
Finishing stage (mean body weight: 208 kg)
Milk replacer F19 F19 F16 F12 rsd P-value

Solid feed – with with with
DE/GE (%) 93.7a 92.2a 90.5b 89.5b 0.9 <0.01

ME/DE (%) 96.4a 95.3ab 93.7bc 93.6c 0.9 0.01
NE/ME (%) 80.8 79.5 76.6 78.2 2.8 0.30
RQnp (l CO2/l O2) 0.95a 0.96ab 0.98b 1.01c 0.02 <0.01
1rsd = residual standard deviation; P-value = treatment effect; GE = gross energy; DE = digestible energy; ME =
metabolisable energy; NE = net energy; RQnp = respiratory quotient corrected for protein oxidation.
Acknowledgements
The project was funded by Britany and Pays de la Loire French regions and partners from the veal
calf industry (KENAVEAU).
References
Gilbert, M.S., J.J.G.C. van den Borne, H. Berends, A.J. Pantophlet, H.A. Schols and W.J.J. Gerrits, 2015. A titration
approach to identify the capacity for starch digestion in milk-fed calves. Animal 9: 249-257.
Van den Borne, J.J.G.C., G.E. Lobley, M.W.A. Verstegen, J.M. Muijlaert, S.J.J. Alferink and W.J.J. Gerrits, 2007. Body
fat deposition does not originate from carbohydrates in milk-fed calves. Journal of Nutrition 137: 2234-2241.

Effects of dietary fatty acid chain length on performance of early
lactation dairy cows
D.E. Rico*, P.Y. Chouinard, C. Cohou, J.E. Parales, M. Plante-Dubé and R. Gervais
Université Laval, Québec, 2425 Rue de l’Agriculture, QC, G1V 0A6, Canada; dnlrico@gmail.com
Abstract
This experiment evaluated the impact of feeding saturated fatty acids (FA) differing in chain length
on milk production and composition using 21 dairy cows at the onset of lactation (6±2 days in milk;
mean ± standard deviation). Fat supplements were fed at 3% of ration dry matter for 28 days and
were: (1) free FA enriched in palmitic acid (16:0; PA); (2) free FA enriched in stearic acid (18:0;
SA); or (3) medium-chain triglycerides (25% of a 50:50 8:0 and 10:0 mix; MCT) protected in a
saturated FA matrix. Dry matter intake tended to be higher in SA than in PA on day 7. Milk yield
was higher in SA relative to PA on day 7, 21 and 28, and not different between PA and MCT. Milk
fat concentration was increased by PA relative to both SA and MCT on day 7, 21 and 28, whereas
milk fat yield did not vary among treatments. The chain length of dietary fat supplements impacted
milk production and composition during early lactation.
Keywords: dairy cows, chain length, fat supplements, medium-chain triglycerides
Introduction
Fatty acid (FA) supplements are commonly fed to dairy cows to support the high energy demands
of lactation. However, metabolism of these FA may depend on their chain length, as shorter FA are
more readily oxidized in the liver relative to longer ones. Therefore, medium-chain triglycerides
(MCT) could prevent excess hepatic fat accumulation, while providing a source of available energy
during periods of negative energy balance, such as the onset of lactation. Given the potential negative
effects of MCT on microorganisms (Desbois and Smith, 2010), their use requires ruminal bypass.
Other saturated FA, such as palmitic and stearic acids are considered to be ruminally inert, and
are fed to dairy cows to increase dietary energy density and support lactation. The objective of the
current study was to investigate the effect of fat supplements differing in chain length on animal
performance and milk composition during the early postpartum period.

Twenty one multiparous Holstein dairy cows were randomly assigned at the onset of lactation (6±2
days in milk; mean ± SD) to treatment in a complete randomized block design. Fat supplements were
fed at 3% of ration dry matter and were: (1) free FA enriched in palmitic acid (>85% 16:0; PA); (2)
free FA enriched in stearic acid (35% 16:0; 45% 18:0; SA); or (3) medium-chain triglycerides (25%
of a 50:50 8:0 and 10:0 mix; MCT) protected in a saturated FA matrix (57% 16:0 and 43% 18:0).
Dry matter intake, milk yield and milk composition were monitored on day 0, 7, 14, 21 and 28 of
treatment. Data were analysed as repeated measures using the MIXED procedure of SAS including
the random effects of cow and block, and the fixed effects of treatment, time and their interaction.
Cow was the subject of the repeated statement. Day 0 values were used as a covariate. Preplanned
contrasts tested at each time point were PA vs SA, and PA vs MCT.

Dry matter intake tended to be lower in PA than in SA on day 7 (P=0.08), but was not affected by
treatment at any other time point (Figure 1). Milk yield was higher in SA relative to PA on day 7,
21 and 28 (P<0.05), and not different between PA and MCT. No effect of treatment or treatment

Figure 1. Effect of dietary fat supplements differing in chain length on milk yield, dry matter intake,
and milk fat concentration and yield of dairy cows at the onset of lactation. Preplanned contrasts
tested the difference between palmitic acid (PA) and stearic (SA; S = P<0.05 and s = P<0.10,
respectively), and between PA and medium-chain triglycerides (MCT; M = P<0.05).
by time interaction was detected for energy corrected feed efficiency. However, PA increased milk
fat concentration relative to both SA and MCT on day 7, 21 and 28 (P<0.05), but had no effect on
milk fat yield. Palmitic acid has been shown to increase milk fat synthesis relative to 18:0 (Rico et
al., 2014). Although no reports exist on the effect of protected 8:0 and 10:0 on milk fat synthesis
or feed efficiency, unprotected 8:0 and 10:0 were previously reported to decrease dry matter intake
while having no effects on milk fat synthesis (Grummer and Socha, 1989).
Conclusions
The chain length of dietary FA affected milk yield and milk fat concentration with minor effects
on dry matter intake. However, energy-corrected feed efficiency was not different between dietary
treatments.
References
Desbois, A.P. and V.J. Smith, 2010. Antibacterial free fatty acids: activities, mechanisms of action and biotechnological
potential. Applied Microbiology and Biotechnology 85: 1629-1642.
Grummer, R.R. and M.T. Socha, 1989. Milk fatty acid composition and plasma energy metabolite concentrations in
lactating cows fed medium-chain triglycerides. Journal of Dairy Science 72: 1996-2001.
Rico, J.E., M.S. Allen, and A.L. Lock, 2014. Compared with stearic acid, palmitic acid increased the yield of milk fat
and improved feed efficiency across production level of cows. Journal of Dairy Science 97: 1057-1066.

Altering the starch and fat content of the peri-conception diet has no
effect on fertility in breeding ewe lambs
D.W. Miller*, E.J. Bowen and C.L. Jacobson
School of Veterinary and Life Sciences, Murdoch University, South St, Murdoch, Western Australia;
d.miller@murdoch.edu.au
Abstract
This study aimed to determine whether dietary manipulation of starch and fat could affect fertility
in breeding ewe lambs. The hypothesis tested was that a high starch (insulin promoting) diet
would increase the rate of onset of oestrous activity and conception compared to a high fat (insulin
suppressing) diet. Two diets, a high starch and a high fat diet, were fed to ewe lambs (n=102 per
treatment) for a continuous 60-day peri-conception period, (30 days prior to and after joining with
the ram), and a sub-sample of 39 ewes per treatment were blood sampled for insulin analysis.
Joining body mass and pre-joining growth rate did not differ between the treatment groups. There
was no overall effect nutrition on plasma insulin concentrations, though insulin concentrations in
the high starch group increased by day 18 compared to day 0 (P<0.005). Irrespective of nutritional
treatment, joining plasma insulin concentration had no effect on conception rates. There was no
effect of nutrition on the proportion of ewes cycling or the number of twin pregnancies. There was
an increase in the conception rate of the cycling ewes in the high starch group (P<0.05). Higher
body mass at joining, irrespective of diet, increased cycling rate (P<0.01), conception rate (P<0.05)
and twinning rate (P<0.01). These findings indicate that manipulation of starch and fat in the diet of
breeding ewe lambs does not affect their fertility, and that achieving a target body mass, pre-joining,
is a more effective strategy to increase conception rates.
Keywords: nutrition, insulin, fecundity, conception
Introduction
The indirect role of nutrition on reproduction in ewe lambs, through effects on metabolic hormones, is
largely unknown. The mechanism is postulated to involve increased circulating insulin concentrations
which stimulate the secretion of gonadotrophins (Miller et al., 1998). In dairy cattle, high starch
diets have been associated with increased plasma insulin concentrations, and this resulted in a
faster return to oestrus post-calving (Garnsworthy et al., 2009). High fat diets, on the other hand,
have been associated with reduced insulin concentrations in dairy cattle and decreased conception
rates (Garnsworthy et al., 2009). The main objective of this study was to determine whether dietary
manipulation of starch and fat could be used to increase fertility in breeding ewe lambs through effects
on insulin. The hypothesis was that a high starch diet that increases circulating insulin concentration
would increase the rate of onset of oestrous activity and conception rates in breeding ewe lambs,
compared to those fed a high fat diet that suppresses circulating insulin concentrations.

Two hundred and four Merino × Afrino ewe lambs with an initial live weight (mean ± SEM) of
44.1±0.3 kg were randomly assigned to two dietary treatment groups, one high in starch (n=102)
and the other high in fat (n=102). To the base ration of 700 g/head/day of a commercial pellet, the
high starch group were supplemented with 300 g/head/day barley grain and the high fat group
were supplemented with 100 g/head/day cracked lupin grain and 80 g/head/day Megalac® (Volac
International Ltd, Royston, UK). The nutritional treatments lasted 60 days and contained similar
levels of crude protein (high starch = 109 g/kg vs high fat = 108 g/kg) and metabolisable energy (high
starch = 9.7 MJ/kg vs high fat = 10.1 MJ/kg). Teaser wethers fitted with marking harnesses were
introduced to the ewe lambs to detect oestrous activity. On day 17 after the start of the nutritional

treatments, the wethers were removed and were replaced with rams fitted with marking harnesses
that remained with the ewe lambs for 30 days. Blood samples were taken from a subset of 40 ewes
on days 0, 18 and 60 and analysed for plasma insulin concentration. All of the ewes were scanned
by ultrasonography for pregnancy detection on days 55 and 85. Body mass and insulin data were
analysed by repeated measures ANOVA and fertility data was analysed by chi-square tests.

Joining body mass and pre-joining growth rate did not differ between the treatment groups, however
the final body mass of the ewes in the high starch group was significantly heavier than the high fat
group (P<0.001; Table 1). There was no overall effect of nutrition on plasma insulin concentrations,
though insulin concentrations in the high starch group increased by day 18 compared to day 0
(P<0.005). Irrespective of nutritional treatment, joining plasma insulin concentration had no effect
on conception rates. There was no effect of nutrition on the proportion of ewes cycling (high starch
= 78% vs high fat = 75%) or the number of twin pregnancies (high starch = 10% vs high fat = 5%).
There was an effect of nutrition on the conception rate of the cycling ewes (high starch = 96% vs
high fat = 85%; P<0.05), though this was most likely due to the increased body mass gain of the
high starch group (high fat = 101.8 g/head/day vs high fat = 78.6 g/head/day: P<0.001). Higher
body mass at joining, irrespective of diet, increased cycling rate (P<0.01), conception rate (P<0.05)
and twinning rate (P<0.01). These findings indicate that, unlike in dairy cattle (Garnesworthy et al.,
2009), altering insulin concentrations through manipulation of starch and fat in the diet of breeding
ewe lambs does not affect their fertility, and that providing the necessary nutrition to achieve a pre-
joining target body mass (Kenyon et al., 2005), is a more effective strategy.
Table 1. Body mass (kg) and insulin concentration (µg/ml) of breeding ewes fed the high starch or
high fat diet.1,2
Day High starch High fat SED P-values
Nutrition Time N×T
Body mass 0 44.5 43.4 0.61 NS

18 47.4 46.4 0.64 NS
60 50.2 47.8 0.67 <0.001 <0.001 NS
Insulin 0 0.32 0.32 0.04 NS
18 0.50 0.40 0.07 <0.005
60 0.41 0.32 0.05 NS <0.005 NS
1 Measurements taken on days 0, 18 and 60 after the start of the nutritional treatments.
2 SED = standard error of the difference; NS = not significant.
Acknowledgements
Wellard Agri Ltd for the ewe lambs and Lienert Australia Pty Ltd for the Megalac®.
References
Garnsworthy, P.C., A.A. Fouladi-Nashta, G.E. Mann, K.D. Sinclair and R. Webb, 2009. Effect of dietary-induced
changes in plasma insulin concentrations during the early post partum period on pregnancy rate in dairy cows.
Reproduction 137: 759-768.
Kenyon, P.R., P.C.H. Morel, S.T. Morris and D.M. West, 2005. The effect of individual liveweight and use of teaser rams
prior to mating on the reproductive performance of ewe hoggets. New Zealand Veterinary Journal 53: 340-343.
Miller, D.W., D. Blache, R. Boukhliq, J.D. Curlewis and G.B. Martin, 1998. Central metabolic messengers and the
effects of nutrition on gonadotrophin secretion in sheep. Journal of Reproduction and Fertility 112: 347-356.

Effect of toasting time on proteolysis of soluble and insoluble protein
fractions of rapeseed meal
S. Salazar-Villanea1*, E.M.A.M. Bruininx2, P. Carré3, A. Quinsac4 and A.F.B. van der Poel1
Netherlands; 2Agrifirm Innovation Centre, Royal Dutch Agrifirm Group, P.O. Box 20018, 7302
HA, Apeldoorn, the Netherlands; 3CREOL, 11 rue Monge, Parc Industriel, 33600 Pessac, France;
4Terres Inovia, 11 rue Monge, Parc Industriel, 33600 Pessac, France; sergio.salazarvillanea@wur.nl
Abstract
The aim was to analyse the effects of toasting time (TT) on the extent and kinetics of proteolysis of
the rapeseed meal (RSM) and the soluble and insoluble protein fractions separated from the RSM.
In addition, the effects of TT on the size distribution of the peptides resulting after proteolysis were
studied. Increasing TT linearly decreased the rate but not the degree of hydrolysis of the RSM and
the insoluble proteins. Rate of hydrolysis of soluble proteins was 2-3-fold higher compared to that
of insoluble proteins. Increasing TT increased peptide size in the hydrolysates of RSM and insoluble
protein fraction, which was highly correlated to their rates of hydrolysis. In conclusion, the change
in the insoluble protein fraction during toasting controls the rate of hydrolysis and the peptide size
after hydrolysis.
Keywords: rapeseed meal, hydrolysis, soluble proteins, insoluble proteins
Introduction
Protein damage during toasting of rapeseed meal (RSM) could impair the accessibility of enzymes
for proteolysis. Thermal treatments can induce protein aggregation, which reduce protein solubility,
and facilitate the formation of Maillard reaction products (Gerrard et al., 2012). These changes to the
proteins could determine the extent and speed for protein hydrolysis, which finally impact protein
digestibility and nutrient synchronisation.

The RSM was produced using cold-pressing and desolventization without direct-steam. The resulting
untoasted RSM was divided in 2 batches for replication. Each batch was toasted for 120 min at
approximately 112 °C, with samples taken every 20 min. Water soluble and insoluble proteins were
separated by centrifugation (25,500×g). Degree of hydrolysis at pH 8 was measured for 120 min
using the pH-STAT method (Pedersen and Eggum, 1983) after the addition of trypsin, chymotrypsin
and intestinal peptidase. Rate of hydrolysis was calculated using the model described by Butré et
al. (2012). High performance size exclusion chromatography was performed on the hydrolysates.
Relative areas under the curve (AUC) were calculated after integration of the profiles. Regressions
were fitted to determine linear or quadratic effects of toasting time (TT) on degree and rate of
hydrolysis and relative AUC of the hydrolysates.

Toasting time did not influence the degree of hydrolysis of the RSM and the insoluble protein fractions
(Table 1). However, the rate of hydrolysis of the RSM (P<0.001) and the insoluble protein fraction
(P=0.02) were linearly decreased with increasing TT. The rate of hydrolysis of the insoluble protein
fraction seems to control that of the RSM, as both decrease with increasing TT. The decrease of the
rate of hydrolysis of insoluble proteins is possibly mediated by structural or chemical modifications
of these proteins (Gerrard et al., 2012), which limit the access of enzymes during hydrolysis. Toasting
time had a quadratic effect on the degree and rate of hydrolysis of the soluble protein fraction. Both

Table 1. Degree and rate of hydrolysis of rapeseed meals (RSM) toasted for different times and the
soluble (SPF) and insoluble (IPF) protein fractions separated from the rapeseed meals.
Toasting time Degree of hydrolysis (%) Rate of hydrolysis (s-1)
RSM SPF IPF RSM SPF IPF
0 min 18.6 14.9 16.0 0.029 0.048 0.020

20 min 17.3 15.6 19.1 0.032 0.053 0.020
40 min 17.8 16.9 19.2 0.027 0.053 0.016
60 min 17.5 17.4 17.9 0.024 0.060 0.014
80 min 18.3 15.8 18.5 0.018 0.058 0.012
100 min 18.0 16.4 18.0 0.017 0.061 0.012
120 min 18.5 15.9 18.5 0.013 0.043 0.010
SEM 0.2 0.2 0.3 0.002 0.002 0.001
P-value
Linear 0.23 0.46 0.78 <0.001 0.52 0.02
Quadratic 0.18 0.02 0.27 0.48 0.04 0.11
the degree and rate of hydrolysis of the soluble protein fraction increase up until 60 min of toasting
and decrease thereafter. It is possible that denaturation of the native structure of protein during the
initial 60 min of toasting facilitates enzyme access for hydrolysis. After that toasting time, protein
damage may become limiting for enzyme accessibility. The rate of hydrolysis of the soluble protein
fraction was 2-3-fold higher compared to that of the insoluble protein fraction.
Increasing TT causes the proportion of peptides >5 kDa to linearly increase in the hydrolysates from
RSM and the insoluble protein fraction. At the same time, there was a decrease in the proportion of
peptides <1 kDa in these materials with increasing TT. The size distribution of the peptides in the
hydrolysates of the RSM and insoluble protein fraction were correlated to their rates of hydrolysis.
There were no effects of TT on the size distribution of peptides in hydrolysates from the soluble
protein fraction.
In conclusion, increasing TT causes a decrease in the rate of hydrolysis of the insoluble protein
fraction, which determines the rate of hydrolysis of the RSM, and an increase of the size of the
peptides produced after hydrolysis.
Acknowledgements
The authors gratefully acknowledge the financial support from the Wageningen UR ‘IPOP Customized
Nutrition’ programme financed by Wageningen UR, the Dutch Ministry of Economic Affairs,
WIAS, Agrifirm Innovation Center, ORFFA Additives BV, Ajinomoto Eurolysine s.a.s and Stichting
VICTAM BV.
References
Butré, C.I., P.A. Wierenga and H. Gruppen, 2012. Effects of ionic strength on the enzymatic hydrolysis of diluted and
concentrated whey protein isolate. Journal of Agricultural and Food Chemistry 60: 5644-5651.
Gerrard, J.A., M. Lasse, J. Cottam, J.P. Healy, S.E. Fayle, I. Rasiah, P.K. Brown, S.M. BinYasir, K.H. Sutton and N.G.
Larsen, 2012. Aspects of physical and chemical alterations to proteins during food processing – some implications
for nutrition. British Journal of Nutrition 108, Suppl. 2: S288-S297.
Pedersen, B. and B.O. Eggum, 1983. Prediction of protein digestibility by an in vitro enzymatic pH-stat procedure.
Zeitschrift für Tierphysiologie, Tierernährung und Futtermittelkunde 49: 265-277.

Rapeseed meal, faba beans and microalga (Spirulina platensis) as protein
supplements for dairy cows on grass silage based diets
A. Halmemies-Beauchet-Filleau, M. Lamminen, T. Kokkonen, A. Vanhatalo and S. Jaakkola
Department of Agricultural Sciences, P.O. Box 28, 00014 University of Helsinki, Finland;
anni.halmemies@helsinki.fi
Abstract
Owing to the dependency of European animal production on imported soya bean, alternative protein
feeds are of interest. In this 4×4 Latin Square designed experiment, the effects of rapeseed meal, faba
beans and Spirulina platensis microalga on dry matter (DM) intake, milk production and nitrogen
utilisation of dairy cows were compared. Treatments comprised 4 total mixed ratios (TMR) based
on grass silage offered ad libitum. Concentrate (45% of DM in TMR) consisted of barley, sugar beet
pulp and isonitrogenously either rapeseed meal (R), rapeseed meal and Spirulina (RS), rolled faba
bean (F) or rolled faba bean and Spirulina (FS). In microalgal diets, Spirulina protein replaced half
of the protein from rapeseed meal or faba bean. DM intake averaged 22.9 and milk yield 29.8 kg/d.
Replacing rapeseed with faba bean (R + RS vs F + FS) had no effect on DM intake, but decreased
milk, fat, protein, and lactose yields and increased milk urea concentration. Spirulina in the diet
decreased DM intake by 0.6 kg. When replacing rapeseed protein, Spirulina inclusion decreased
milk, protein and lactose yields and increased milk urea concentration. However, mixing Spirulina
with faba bean increased milk, protein and lactose yields and decreased milk urea concentration.
Compared with rapeseed supplementation, faba bean decreased arterial concentrations of several
essential amino acids including histidine and methionine. In conclusion, the milk production responses
of rapeseed meal were superior to faba beans and Spirulina. When replacing rapeseed protein,
Spirulina decreased animal performance, but improved it when replacing faba bean.
Keywords: dairy cow, nitrogen utilisation, rapeseed, faba bean, Spirulina
Introduction
European animal production is largely dependent on imported soya bean. Therefore finding alternative
protein feeds is of interest. Faba bean (Vicia faba) is an ancient grain legume with N fixing capacity
and crop rotational benefits. Microalgae are rich in high quality protein, their demands for growing
conditions are modest and they have higher productivity per land area than terrestrial crops (Mata
et al., 2010). The aim of the present experiment was to compare the effects of rapeseed meal, faba
beans and Spirulina platensis microalga on dry matter (DM) intake, plasma amino acids, nitrogen
utilisation and milk production of dairy cows.

Eight multiparous Finnish Ayrshire cows averaging 113 d in milk were used in replicated 4×4 Latin
squares with 21 d periods. Treatments comprised 4 total mixed ratios (TMR) based on grass silage
offered ad libitum. Concentrate (45% of DM in TMR) consisted of barley, sugar beet pulp, minerals,
vitamins and isonitrogenously either rapeseed meal (R, 9.9% of DM in TMR), rapeseed meal and
Spirulina (RS), rolled faba beans (F, 12% DM in TMR) or rolled faba beans and Spirulina (FS). In
microalgal diets, Spirulina protein replaced half of the protein from rapeseed or faba beans. Data
were analysed by analysis of variance.

Replacing rapeseed with faba bean (R + RS vs F + FS) had no effect (P>0.10) on DM intake, whereas
Spirulina decreased (P<0.05) DM intake (Table 1). Milk production responses were opposite, when
Spirulina replaced protein from rapeseed than from faba bean (P<0.06 for interactions). When
replacing the half of rapeseed protein, Spirulina inclusion decreased milk, protein and lactose yields
and increased milk urea concentration. However, mixing Spirulina with faba bean increased milk,
protein and lactose yields and decreased milk urea concentration. Furthermore, mixing Spirulina
with faba bean improved the conversion of feed to energy corrected milk to the same level as in
rapeseed containing diets. Overall, milk, fat, protein and lactose yields were higher and milk urea
concentration lower for rapeseed than for faba bean diets. Compared with rapeseed supplementation,
faba bean decreased (P<0.05) arterial concentrations of essential amino acids including methionine,
which is inherently low in faba bean protein. Replacing rapeseed with faba bean increased (P<0.05)
crude protein digestibility and decreased the proportion of N eaten excreted in faeces and in milk. In
conclusion, the milk production responses of rapeseed meal were superior to other protein sources
used especially to those of rolled faba bean. The utilisation of faba bean protein to milk production
improved markedly, when half of it was replaced with protein in Spirulina.
Table 1. Effect of various protein supplements on dry matter intake, nitrogen metabolism and milk
production.
Treatment SEM Significance
Rapeseed vs
Substitution
Rapeseed +
+ Spirulina
Faba beans
Faba beans
Faba beans
Interaction
Rapeseed
Spirulina
Spirulina
Dry matter (DM) intake, kg/d 23.3 22.8 23.1 22.3 0.57 0.198 0.037 0.500
Crude protein (CP) intake, kg/d 3.86 3.88 3.84 3.77 0.098 0.246 0.581 0.370
Digestibility of CP, g/kg 671 674 691 680 5.6 0.034 0.505 0.210
Digestibility of neutral detergent 696 705 696 705 11.6 0.982 0.438 0.982
fibre, g/kg
Arterial concentrations, µmol/l
Histidine 36.7 39.2 32.3 33.0 3.29 0.048 0.522 0.733
Lysine 95.9 91.9 86.8 86.3 4.92 0.014 0.395 0.515
Methionine 23.9 23.3 20.8 20.7 1.19 0.007 0.655 0.732
∑ Essential 894 873 803 815 27.7 <0.001 0.794 0.357
N partitioning
N, in milk, % 29.0 28.1 26.8 27.9 0.82 0.007 0.756 0.023
N, in faeces, % 32.9 32.6 30.9 32.0 0.58 0.028 0.429 0.221
N, urine, % 31.4 34.1 33.0 35.5 1.59 0.284 0.071 0.928
Milk production
Milk, kg/d 31.0 30.0 28.5 29.7 0.53 <0.001 0.857 0.002
Energy corrected milk, kg/d 34.1 33.8 32.3 32.9 0.86 0.006 0.696 0.303
Fat, g/d 1,469 1,483 1,414 1,433 51.9 0.074 0.552 0.942
Protein, g/d 1,127 1,106 1,050 1,070 23.4 <0.001 0.959 0.058
Lactose, g/d 1,383 1,328 1,276 1,320 27.0 0.002 0.718 0.005
Urea, mg/dl 27.0 28.9 30.2 29.6 1.52 <0.001 0.117 0.008
Energy corrected milk (kg/d)/DM 1.42 1.44 1.35 1.43 0.004 0.026 0.011 0.050
intake (kg/d)

Acknowledgements
Financial support to Sustainable Intensification project from The Development Fund for Agriculture
and Forestry in Finland (Makera) and from Raisioagro Ltd.
References
Mata, T.M., A.A. Martins and N.S. Caetano, 2010. Microalgae for biodiesel production and other applications: a review.
Renewable and Sustainable Energy Reviews 14: 217-232.

Microalgae as a substitute for soya bean meal in the grass silage based
dairy cow diets
M. Lamminen*, A. Halmemies-Beauchet-Filleau, T. Kokkonen, S. Jaakkola and A. Vanhatalo
Department of Agricultural Sciences, University of Helsinki, P.O. Box 28, 00014 Helsinki, Finland;
marjukka.lamminen@helsinki.fi
Abstract
Microalgae are attractive alternative to conventional protein feeds because of their rapid growth
and high protein content. In this experiment, the effects of different microalgae species and soya
bean meal on dry matter (DM) intake, milk production and nitrogen utilisation of dairy cows were
compared. Four multiparous Finnish Ayrshire cows averaging 112 d in milk were used in a 4×4
Latin square study. Treatments consisted of cereal and molassed sugar beet pulp-based concentrate
supplemented isonitrogenously with soya bean meal, Spirulina platensis, Chlorella vulgaris, or
mixture of C. vulgaris and Nannochloropsis gaditana microalgae. Feed intake averaged 21.5 kg/d
and milk yield 30.6 kg/d. Silage intake increased when soya bean meal was replaced by microalgae.
However, no differences were found in the total DM intake reflecting decreased concentrate intake
on microalgae diets. Replacing soya bean meal with microalgae had no effect on milk, protein, fat
and lactose yields but increased the fat content of milk. However, energy corrected milk yield was
not affected by microalgae supplementation. No differences were found in the nitrogen utilisation
of cows between soya bean meal and microalgae, and between microalgae species. In conclusion,
replacing soya bean meal with different microalgae species maintained animal performance and no
statistically significant differences were found between microalgae species.
Keywords: dairy cow, milk production, protein, soya bean meal, microalgae, N utilisation
Introduction
The high protein content (up to 700 g/kg), rapid growth and high productivity per land area of
microalgae make them a very attractive alternative to conventional protein feeds. The amino acid
composition of microalgal protein closely resembles that of soya beans, although histidine content
is usually lower in microalgae (Becker, 2007). So far, few microalgal protein feeding studies with
ruminants have been conducted. The objective of this study was to compare the effects of different
microalgae species and soya bean meal on dry matter (DM) intake, milk production and nitrogen
(N) utilisation of dairy cows.

Four multiparous Finnish Ayrshire cows averaging 112 d in milk were used in a 4×4 Latin square
study. Treatments consisted of cereal and molassed sugar beet pulp-based concentrate supplemented
isonitrogenously with soya bean meal (2.11 kg/d), Spirulina platensis (1.19 kg/d), Chlorella vulgaris
(1.42 kg/d), or mixture of C. vulgaris and Nannochloropsis gaditana (0.85 + 0.85 kg/d) microalgae.
Cows were offered 12.5 kg/d of concentrates and grass silage ad libitum. Data were analysed by
analysis of variance.

Feed intake averaged 21.5 kg/d and milk yield 30.6 kg/d (Table 1). Replacing soya bean meal by
microalgae increased (P<0.05) silage intake (+1.6 kg DM/d). The same was true for replacing part
of chlorella by nannochloropsis (P<0.05) (+1.9 kg DM/d). However, there were no differences in
the total DM intake, which reflects decreased concentrate intake on microalgae diets. Replacing

Table 1. Effect of various protein supplements on dry matter intake, nitrogen metabolism and milk
production.
Treatment SEM Significance
microalgae
microalgae
Soya bean
Soya bean
chloropsis
chloropsis
vs nanno-
Chlorella
Chlorella
Chlorella
Spirulina
Spirulina
+ nanno-
vs other
meal vs
meal
Intake, kg/d
Silage dry matter (DM) 10.6 12.9 10.9 12.8 0.51 0.034 0.156 0.045
Total DM 21.5 22.0 20.9 21.6 1.29 0.973 0.383 0.501
Nitrogen (N), g/d 530 539 516 517 33.8 0.718 0.247 0.966
Arterial concentrations, µmol/l
Histidine 63.4 52.3 56.9 45.5 7.12 0.137 0.901 0.249
Lysine 107 116 109 102 9.1 0.889 0.397 0.590
Methionine 24.5 25.9 20.9 21.7 2.76 0.574 0.232 0.830
∑ Essential 1,131 1,251 1,160 1,113 90.9 0.634 0.319 0.689
Microbial N, g/d 225 241 216 230 21.9 0.760 0.270 0.440
N partitioning
N, in milk, % 28.2 30.8 29.5 29.5 1.83 0.188 0.362 0.997
N, in faeces, % 38.3 39.8 39.1 39.4 1.61 0.499 0.776 0.886
N, in urine, % 29.6 27.4 30.1 29.9 2.92 0.781 0.261 0.950
Milk production
Milk, kg/d 29.7 32.1 29.9 30.8 1.86 0.517 0.459 0.715
Energy corrected milk, kg/d 29.3 33.9 30.0 30.5 2.02 0.290 0.165 0.822
Fat, g/d 1,215 1,484 1,261 1,287 98.2 0.186 0.098 0.818
Protein, g/d 952 1,043 957 969 61.5 0.468 0.227 0.849
Lactose, g/d 1,320 1,427 1,324 1,360 95.5 0.614 0.487 0.776
Urea, mg/dl 23.9 20.0 24.1 22.0 2.90 0.484 0.357 0.542
Energy corrected milk (kg/d)/ 1.37 1.55 1.48 1.43 0.130 0.188 0.320 0.624
DM intake (kg/d)
soya bean meal with microalgae had no significant effect on milk, protein, fat and lactose yields.
Numerically, the highest milk yield was achieved on Spirulina diet (32.1 kg/d). The fat content of
milk was increased (P<0.10) when soya bean meal was replaced by microalgae (41.0 vs 42.9 g/kg,
SEM=1,57), and Spirulina diet resulted in higher fat content of milk than chlorella diets (P<0.05)
(45.0 vs 41.8 g/kg, SEM=1,57). However, energy corrected milk (ECM) yield and feed conversion to
ECM were not affected by microalgae supplementation or different microalgae species. Numerically,
Spirulina diet resulted in highest ECM yield and feed conversion to ECM. No differences were
found in the N utilisation of cows between soya bean meal and microalgae, and between microalgae
species. Neither the production of microbial protein was affected by the protein source. In terms of
N content of milk, the N utilisation was nearly 0.30 at all treatments, and no differences were found
between the feed protein sources. Microalgae supplementation or different microalgae species had
no major effect on arterial amino acid profile of animals.
In conclusion, replacing soya bean meal with different microalgae species maintained animal
performance despite the reduced concentrate dry matter intake of microalgae diets. No statistically
significant differences were found in milk production between different microalgae species. The N
utilisation of animals was not affected by the feed protein source.

Acknowledgements
Financial support to Algae Foods project from The European Regional Development Fund.
References
Becker, E.W., 2007. Micro-algae as a source of protein. Biotechnology Advances 25: 207-210.

Impact of grain source and distillers fat level on ruminal enzymes, pH
and methane production
F. Keomanivong1*, M. Rodenhuis1, M. Ruch1, M. Crouse1, J. Kirsch1, M. Bauer1, M. Borhan2, S.
Rahman2 and K. Swanson1
1North Dakota State University, 1300 Albrecht Blvd, Fargo, ND 58102, USA; 2North Dakota State
University, 1221 Albrecht Blvd, Fargo, ND 58102, USA; faithe.doscher@ndsu.edu
Abstract
The aim of this study was to examine the effect of grain source (corn vs barley) and corn distillers
grains plus solubles fat concentration (4.53 vs 7.92%) on ruminal digestive enzyme activity, pH
and in vitro enteric methane production. The experiment was designed as a 4×4 Latin square with
eight cannulated Holstein steers (body weight: 715±61.4 kg) randomly assigned to four dietary
treatments consisting of: (1) rolled corn and low-fat dried distillers grain with solubles (DDGS); (2)
rolled corn and moderate-fat DDGS; (3) rolled barley and low-fat DDGS; and (4) rolled barley and
moderate-fat DDGS in a 2×2 factorial arrangement. Daily feed consumption was recorded. Ruminal
fluid was collected and analysed for α-amylase, trypsin and maltase activity along with pH and in
vitro microbial methane production. Steers consuming diets containing low fat DDGS had greater
(P=0.01) intake. Steers fed barley diets with moderate fat DDGS had the lowest α-amylase activity
(U/l rumen fluid; interaction P=0.03) while barley with low fat had the greatest α-amylase U/kg
starch intake (interaction P=0.02). Steers fed corn-based diets had greater (P=0.01) α-amylase U/
kg starch disappearance. Diets of corn with low fat DDGS grains had greater (interaction P≤0.03)
trypsin activity (U/l rumen fluid, U/kg crude protein (CP) intake, U/kg CP disappearance). Corn
based diets and low fat DDGS revealed greater maltase activity (U/l rumen fluid; P<0.01). Rumen
pH and enteric methane production were unaffected by dietary treatment.
Keywords: grain, distillers, rumen, enzyme, methane
Introduction
Corn and barley are commonly used in cattle rations throughout North America. Distillers grains
are also typically incorporated into rations to reduce feed costs while maintaining an energy value
similar to, or greater than grain. In recent years ethanol plants have begun extracting greater levels
of oil to be used in biodiesel production, consequently reducing the fat content of distillers grains.
These changes in distillers by-products have the potential to impact cattle production through altered
nutrient composition thereby affecting microbial and enzymatic action on feed.

Eight cannulated Holstein steers (body weight: 715±61.4 kg) were randomly assigned to four
dietary treatments in a 2×2 factorial arrangement consisting of: (1) rolled corn and low-fat dried
distillers grain with solubles (DDGS); (2) rolled corn and moderate-fat DDGS; (3) rolled barley
and low-fat DDGS; and (4) rolled barley and moderate-fat DDGS. Diets were formulated to meet
or exceed National Research Council (NRC, 2000) recommendations and were offered for ad
libitum intake. The experiment was designed as a 4×4 Latin square with 24-day periods allowing
for 10 days of intermediate dietary transition, 7 days of dietary treatment acclimation and 7 days of
sample collection. Daily feed consumption was recorded, and ort samples were composited from
each collection period to determine nutrient content. Approximately 200 ml of ruminal fluid were
sampled from d 3 to 5 in a manner to represent every other hour in a 24-h cycle and was analysed
for α-amylase, trypsin and maltase activity as well as pH and in vitro microbial methane production.

Feed intake was greater (P=0.01) in steers consuming diets containing low fat DDGS (Table 1). An
interaction (P=0.03) between grain and distillers was also observed as barley diets with moderate
fat DDGS had the lowest level of α-amylase activity (U/l rumen fluid) while barley with low fat
DDGS had the greatest α-amylase (U/kg starch intake; P=0.02). Activity of α-amylase (U/kg starch
disappearance) was greater (P=0.01) in corn-based rations. Corn with low fat DDGS treatments
had the greatest (interaction P≤0.03) trypsin activity (U/kg crude protein (CP) intake and U/kg CP
disappearance). Steers fed corn-based diets and low fat DDGS (P<0.01) had greater maltase activity
(U/l rumen fluid) while no interactions were observed. Rumen pH and enteric methane production
were unaffected by dietary treatment (P≥0.13). In conclusion, new processing methods of DDGS
may be beneficial in improving rumen enzymatic function which could lead to improved digestion
and absorption. The lack of change on ruminal pH and methane emissions may also be considered
beneficial as new DDGS processing creates no negative influence on the ruminal environment.
Table 1. Rumen enzyme activity.1
Barley Corn SEM P-value
Low Moderate Low Moderate Grain DDGS Grain ×

DDGS
Feed intake, kg/d 21.9 21.5 21.7 21.2 0.84 0.19 0.01 0.73
Amylase, 144a 106b 132a 131a 13.5 0.45 0.02 0.03
U/l rumen fluid
Amylase, 116a 86.0b 98.2ab 102ab 12.16 0.94 0.08 0.02
U/kg starch intake
Amylase, 9,094 7,258 9,969 10,030 767.1 0.01 0.20 0.17
U/kg starch
disappearance
Trypsin, 207a 211a 275b 218a 14.4 0.002 0.02 0.01
U/l rumen fluid
Trypsin, 633a 644a 875b 705a 52.2 <0.001 0.05 0.03
U/kg CP intake
Trypsin, 4,835a 4,541a 6,106b 4,565a 399.2 0.01 0.001 0.02
U/kg CP
disappearance
Maltase, 280 222 334 258 28.0 0.009 <0.001 0.56
U/l rumen fluid
1 CP = crude protein; DDGS = dried distillers grain with solubles.
Acknowledgements
The authors thank the employees of the Animal Nutrition and Physiology Center for their assistance
in data collection and animal care and handling. This project was funded by a grant from the North
Dakota Corn Council.
References
National Research Council (NRC), 2000. Nutritional requirements of beef cattle (7th rev. Ed.). National Academies
Press, Washington, DC, USA.

Oilseed meal processing affects whole body amino acid retention and
composition in growing pigs
T.G. Hulshof1,2*, A.F.B. van der Poel1 and P. Bikker2
1Animal Nutrition Group, Wageningen University, De Elst 1, 6708 WD Wageningen, the Netherlands;
2Wageningen UR Livestock Research, De Elst 1, 6708 WD, Wageningen, the Netherlands;
tetske.hulshof@wur.nl
Abstract
An experiment was conducted to determine the effects of processing of soybean meal (SBM) and
rapeseed meal (RSM) on protein and amino acid (AA) retention and whole body AA pattern in
growing pigs. Five pigs were slaughtered at the start of the experiment to determine initial body
composition. Fifty-four growing pigs were fed 1 of 6 experimental diets containing SBM, processed
SBM (pSBM), RSM, processed RSM (pRSM), or pSBM and pRSM supplemented with crystalline
AA. Pigs fed the experimental diets were slaughtered at 40 kg and the organ fractions and empty
carcasses were analysed for crude protein (CP) and AA profile. Protein and AA retentions were
calculated. Processing decreased CP and AA retention and resulted in changes in whole body AA
pattern. Supplementation with crystalline AA ameliorated the effect of processing on CP and AA
retention.
Keywords: nutrient retention, slaughter trial, whole body amino acid pattern
Introduction
The (over)processing of oilseed by-products such as soybean meal (SBM) and rapeseed meal (RSM)
may reduce the standardised ileal digestible (SID) crude protein (CP) and amino acid (AA) content
and growth performance of pigs (Hulshof et al., 2016). This can be attributed to the formation of
Maillard reaction products (Mauron, 1981). However, the effects of (over)processing on subsequent
AA utilisation for body protein retention have not been studied in great detail. This study aimed
to determine the effects of processing on whole body AA pattern, CP and AA retention and post-
absorptive utilisation of CP and AA in growing pigs.

The study consisted of a 2×3 factorial randomized complete block design with body weight (BW) as
blocking factor. Of a total of 59 growing pigs (initial BW 15.6±0.7 kg), 5 pigs were slaughtered at the
start of the experiment to determine initial body composition. The remaining 54 pigs were allocated
to 1 of 6 experimental diets based on BW. The pigs were individually housed and fed twice per day;
feeding level was 3.0 times maintenance energy requirement. The 6 isoenergetic experimental diets
consisted of a basal CP-free diet with commercial SBM or RSM as sole protein source. The SBM
or RSM were used as such (SBM or RSM diet) or first processed by toasting (95 °C for 30 minutes)
in the presence of lignosulfonate and then added to the diet (pSBM or pRSM diet). The remaining
2 experimental diets contained pSBM and pRSM and were supplemented with crystalline AA (all
essential AA, Cys, Tyr, and Gln) to meet the SID AA content in the SBM and RSM diet, respectively.
Pigs fed the experimental diets were slaughtered at 40±2 kg. The organ fractions and the empty
carcasses were analysed for CP and AA profile. The CP and AA retention in the empty body were
calculated. The post-absorptive utilisation of CP and AA was calculated by correcting CP and AA
retention for the ileal digestible CP and AA intake.

Diet type, comprising effects of processing and supplementing crystalline AA, significantly affected
CP and AA retention (P<0.001 for all) in the empty body (Table 1). Processing of SBM and RSM
decreased CP and AA retention in the empty body for SBM and RSM. This can be explained by a
lower AA availability due to a lower AA digestibility. Supplementation of pSBM and pRSM fully
restored CP and AA retention (Table 1). The utilisation of ileal digestible CP for retention was
decreased for both protein sources and the utilisation of all ileal digestible AA for retention was
decreased for pSBM. Supplementation with crystalline AA restored the utilisation for all essential
AA for both protein sources and the CP utilisation for pRSM but not for pSBM. This suggests that
some AA from pSBM cannot be used post-absorption to the same extent as AA from SBM. Feeding
processed protein sources reduced the Lys content in the empty body, especially for the pSBM diet.
In conclusion, CP and AA retention were drastically reduced when feeding pSBM or pRSM to
growing pigs but the extent of the reduction was dependent on protein source. Supplementation with
crystalline AA ameliorated the negative effect of processing on retention for CP and all AA. Changes
in whole body AA pattern suggest a metabolic flexibility of the pigs by adapting the whole body AA
pattern to the dietary available AA. The post-absorptive utilisation of CP and AA was dependent on
whether the protein source was (over)processed and/or supplemented with crystalline AA.
Table 1. Nutrient retention (g/d) in the empty body of pigs fed 1 of 6 diets containing soybean meal
(SBM), processed SBM (pSBM), rapeseed meal (RSM), processed RSM (pRSM), or pSBM and pRSM
supplemented with crystalline AA (pSBM+AA and pRSM+AA, respectively).1,2
Item Diet P-value
SBM pSBM pSBM + RSM pRSM pRSM + PS DT PS×DT

AA AA
CP 104a 52c 98a 75b 58c 77b <0.001 <0.001 <0.001

Lys 7.5a 3.3c 6.8a 4.9b 3.9c 5.5b <0.001 <0.001 <0.001
Met 2.2a 1.0d 1.9a 1.4bc 1.1cd 1.6b <0.001 <0.001 <0.001
Thr 4.0a 1.9d 3.7a 2.7bc 2.2cd 3.0b <0.001 <0.001 <0.001
Trp 1.0a 0.5d 1.0a 0.7bc 0.5cd 0.7b <0.001 <0.001 <0.001
1 Least squares means within a row lacking a common superscript letter differ significantly (P<0.05).
2 PS = protein source; DT = diet type.
Acknowledgements
The authors acknowledge the financial support from the Wageningen UR ‘IPOP Customised Nutrition’
programme financed by Wageningen UR, the Dutch Ministry of Economic Affairs, WIAS, Agrifirm
Innovation Center, ORFFA Additives BV, Ajinomoto Eurolysine s.a.s., and Stichting VICTAM BV.
References
Hulshof, T.G., P. Bikker, A.F.B. van der Poel and W.H. Hendriks, 2016. Assessment of protein quality of soybean meal
and 00-rapeseed meal toasted in the presence of lignosulfonate by amino acid digestibility in growing pigs and
Maillard reaction products. Journal of Animal Science 94: 1020-1030.
Mauron, J., 1981. The Maillard reaction in food: a critical review from the nutritional standpoint. Progress in Food
and Nutrition Science 5: 5-35.

Oilseed meal processing affects protein digestion kinetics and metabolic
organ load of growing pigs
T.G. Hulshof1,2*, A.F.B. van der Poel1 and P. Bikker2
2Wageningen UR Livestock Research, De Elst 1, 6708 WD, Wageningen, the Netherlands;
tetske.hulshof@wur.nl
Abstract
An experiment was conducted to determine the effects of processing of soybean meal (SBM) and
rapeseed meal (RSM) on crude protein (CP) digestibility along the small intestine, N solubility in
chyme, and the effect of post-absorptive amino acid (AA) availability on metabolic organ load. Fifty-
four growing pigs were fed 1 of 6 experimental diets containing SBM, processed SBM (pSBM),
RSM, processed RSM (pRSM), or pSBM and pRSM supplemented with crystalline AA. Pigs were
slaughtered at 40 kg, organ weight was recorded and chyme was collected at 3 locations along the
small intestine. The CP digestibility was reduced due to processing, which could not be explained
by an increased amount of insoluble N as fraction of total N. The weight of the kidneys, pancreas,
liver, and small intestine was affected by processing and supplementation with crystalline AA.
Keywords: nitrogen solubilisation, apparent digestibility, organ weight
Introduction
Previous studies showed that processing of protein sources, e.g. soybean meal (SBM) and rapeseed
meal (RSM), decreased the standardised ileal digestible (SID) crude protein (CP) and amino acid (AA)
content for growing pigs (Almeida et al., 2014; González-Vega et al., 2011; Hulshof et al., 2016).
This study aimed to determine the effects of processing of SBM and RSM on CP digestibility along
the small intestine and N solubilisation in chyme and the effect of post-absorptive AA availability
on metabolic organ load as determined by organ weight.

The study consisted of a 2×3 factorial randomized complete block design with body weight (BW)
as blocking factor. Fifty-four pigs (initial BW 15.6±0.7 kg) were allocated to 1 of 6 experimental
diets based on BW. Pigs were individually housed and fed twice per day; feeding level was 3.0
times maintenance energy requirement. The 6 isoenergetic experimental diets consisted of a basal
CP-free diet with commercial SBM or RSM as sole protein source. The SBM or RSM were used
as such (SBM or RSM diet) or first processed by toasting (95 °C for 30 minutes) in the presence
of lignosulfonate and then added to the diet (pSBM or pRSM diet). The remaining 2 experimental
diets contained pSBM and pRSM and were supplemented with crystalline AA (all essential AA,
Cys, Tyr, and Gln) to meet the SID AA content in the SBM diet and RSM diet, respectively. Pigs
were slaughtered at 40±2 kg after which the weight of each organ was recorded. The small intestine
was divided in 3 segments and chyme was collected from the last meter of each segment. Chyme
of pigs fed the SBM, pSBM, RSM, and pRSM diets was analysed for N and titanium dioxide to
determine apparent CP digestibility and separated in a soluble and insoluble phase by centrifugation.
Subsequently, the N in the insoluble phase was determined.

The amount of N in the insoluble phase as fraction of total N was not affected by protein source or
processing and was about 30% in the first and second segment and increased to 50% in the third

segment. Processing decreased (P<0.001) apparent CP digestibility in the third segment (i.e. ileal
digestibility) from 81 to 63% for SBM and from 62 to 43% for RSM. Diet type, comprising effects of
processing and supplementing crystalline AA, affected the weight of the kidneys (P=0.013), pancreas
(P=0.003), liver (P=0.013), and small intestine (P=0.002; Table 1). Processing lowered the weight
of the kidneys, pancreas, and small intestine while supplementation with crystalline AA increased
the weights of these organs to similar levels as for SBM or RSM. Supplementation with crystalline
AA of pSBM and pRSM lowered the weight of the liver which could be related to a more balanced
AA profile in the supplemented diets compared with the pSBM and pRSM diets.
In conclusion, the total amount of (undigested and endogenous) N in chyme was greater for pSBM
and pRSM compared with SBM and RSM which was not caused by an increased amount of insoluble
N as fraction of total N. The subsequent change in post-absorptive available N and AA altered the
weights of organs involved in nutrient digestion and metabolism, presumably as a result of the
metabolic load.
Table 1. Organ weight (g/kg empty BW) of pigs fed 1 of 6 diets containing soybean meal (SBM),
processed SBM (pSBM), rapeseed meal (RSM), processed RSM (pRSM), or pSBM and pRSM
supplemented with crystalline AA (pSBM + AA and pRSM + AA, respectively).1,2
Organ Diet P-value
SBM pSBM pSBM + RSM pRSM pRSM + PS DT PS×DT

AA AA
Kidneys 4.6ab 4.4ab 4.7a 4.7ab 4.2b 4.5ab 0.139 0.013 0.302
Pancreas 1.9a 1.5bc 1.8ab 1.5bc 1.4c 1.6abc 0.002 0.003 0.311
Liver 23.1bc 25.8a 24.3abc 25.3ab 24.8abc 22.9c 0.783 0.013 0.004
Small 33.7abc 31.3c 36.3ab 36.8a 32.3bc 34.4abc 0.382 0.002 0.075
intestine
1 Least squares means within a row lacking a common superscript letter differ significantly (P<0.05).
2 PS = protein source; DT = diet type.
Acknowledgements
The authors acknowledge the financial support from the Wageningen UR ‘IPOP Customised Nutrition’
programme financed by Wageningen UR, the Dutch Ministry of Economic Affairs, WIAS, Agrifirm
Innovation Center, ORFFA Additives BV, Ajinomoto Eurolysine s.a.s., and Stichting VICTAM BV.
References
Almeida, F.N., J.K. Htoo, J. Thomson and H.H. Stein, 2014. Effects of heat treatment on the apparent and standardized
ileal digestibility of amino acids in canola meal fed to growing pigs. Animal Feed Science and Technology 187:
44-52.
González-Vega, J.C., B.G. Kim, J.K. Htoo, A. Lemme and H.H. Stein, 2011. Amino acid digestibility in heated soybean
meal fed to growing pigs. Journal of Animal Science 89: 3617-3625.
Hulshof, T.G., P. Bikker, A.F.B. van der Poel and W.H. Hendriks, 2016. Assessment of protein quality of soybean meal
and 00-rapeseed meal toasted in the presence of lignosulfonate by amino acid digestibility in growing pigs and
Maillard reaction products. Journal of Animal Science 94: 1020-1030.

Increasing level of full-fat rapeseeds in broiler chicken diets changes the
plasma nontargeted metabolic profile
E. Ivarsson1*, K. Hanhineva2 and H. Wall1
1Departement of Animal Nutrition and Management, Swedish University of Agricultural Sciences.
P.O. Box 7024, 75007 Uppsala, Sweden; 2Deptartment of Clinical Nutrition, University of Eastern
Finland. P.O. Box 1627, 70211 Kuopio, Finland; emma.ivarsson@slu.se
Abstract
Non-targeted metabolite profiling analysis of plasma samples from 35 day old chickens (Ross 308)
was conducted to elucidate metabolic events related to decreased growth performance observed in
chickens fed diets with 24% rapeseed (RS) as non-pelleted as compared to chickens fed 24% RS
in pelleted (P) diet or a non-pelleted control (C) without RS. The results showed that both RS24
and RS24P had a higher plasma abundance of α-linolenic acid and docosapentaentanoic acid than
C. Moreover the abundance of goitrin and sinapine was higher in both RS diets but no difference
between RS24 and RS24P was observed. The RS24 group had lower abundance of several fatty
acids and lysine, methionine and DL-homoserine than RS24P and C, which indicates that most likely
mainly a low nutrient availability caused the observed growth depression in RS24.
Keywords: rapeseed, chicken, metabolites
Introduction
Rapeseed (RS) can successfully be grown in Nordic countries and potentially be used in a higher
extent in broiler chicken diets, especially in home-mixed. Today, mainly RS meal is used but full-fat
RS can also be considered as a valuable source of both energy and protein. A study by Ivarsson et al.
(2014) showed large differences in RS tolerance dependent on if the feed was pelleted or not. When
fed non-pelleted growth depression was observed already at 8% inclusion, whereas if pelleted (P)
24% could be included without affecting growth. In the previous study it could not be concluded
whether the growth depression was due to an effect of glucosinolates or a result of oil encapsulating
effect of the hull fraction described in previous studies (Józefiak et al., 2010). The aim of this study
was to evaluate plasma metabolic profile of chickens fed diets with high levels of RS, pelleted or
non-pelleted, to find the potential cause of the observed phenomenon.

At day 35 in the growth experiment (Ivarsson et al., 2014) two chickens per group – a hen and a
rooster – were selected and blood samples were taken from wing vein. The blood was collected in
EDTA tubes centrifuged 1,300×g for 10 minutes. Plasma was harvested and stored in -80 °C until
analysis. The samples were analysed by liquid chromatography quadrupole time-of-flight mass
spectrometry (LC-qTOF-MS). The analyses and the raw data were processed in accordance with
Hanhineva et al. (2015). Further data processing and selection for the most discriminating compounds
between diets control (C), RS24 and RS24P, were performed in Excel (Microsoft 2010). A total of
5,784 compounds were used in the data analysis. Student’s t-test were used to find compounds that
differed (P<0.05) between groups. Identification of metabolites was based on the MS/MS spectral
comparison of the pure standard compounds and on the search of the candidate compounds in
published databases such as Metlin.

Selected metabolites are shown in Table 1. Inclusion of 24% RS increased the abundance of
α-linolenic acid and docosapentaentanoic acid. The fold change was higher in the pelleted compared
to the non-pelleted diets, indicating a higher fat availability on the pelleted diets. Moreover the
abundance of goitrin and sinapine was higher in the RS diets than in the control. Goitrin is a
degradation product from the glucosinolate progoitrin and has been reported to give a bitter taste to
RS, and might also decrease the production of thyroid hormones (Fenwick et al., 1982). However,
there was no difference in the fold change between RS24 and RS24P when compared to C, suggesting
that goitrin was not responsible for the decreased growth performance. Moreover, lysine, methionine
and DL-homoserine were lower in RS24 compared to C but there was no difference (P>0.05) between
C and RS24P. In conclusion, the lower abundance of several important dietary components in the
RS24 compared to the RS24P diet indicates that the lower nutrient availability caused the decreased
growth performance in the RS24 group.
Table 1. Selected metabolites with putative identity that differed (P<0.05) between C, RS24 and/or
RS24P. FC indicates the fold change with d = down regulated in C higher abundance in treatments,
u = upregulated in C lower abundance in treatment.1
Column Mode MW m/z Rt, min Putative annotation RS24 RS24P
P-value FC P-value FC
RP ESI– 278.22 277.22 10.23 α-linolenic acid 0.003 1.70 d 6.36×10-12 5.41 d
RP ESI– 330.26 329.25 10.81 Docosapentaentanoic acid 1.67×10-4 2.20 d 9.57×10-5 4.03 d
RP ESI+ 129.025 130.03 2.29 Goitrin – 16.0 d – 16.0 d
RP ESI+ 113.05 114.05 1.99 Sinapine – 16.0 d – 16.0 d
HILIC5 ESI– 119.06 118.05 5.70 DL-Homoserine 0.003 2.05 u 0.54 1.19 u
HILIC ESI– 149.05 148.04 4.44 Methionine 1.31×10-4 2.23 u 0.32 1.16 u
HILIC ESI+ 146.10 147.11 7.11 Lysine – 3.72 u 0.11 1.53 d
1 MW = molecule weight; m/z = identified ion; rt = retention time; RP = reversed phase; HILIC = hydrophilic interaction.
References
Fenwick, G.R., N.M. Griffiths and R.K. Heaney, 1982. Bitterness in Brussels sprouts (Brassica oleracea L. var.
gemmifera): the role of glucosinolates and their breakdown products. Journal of Science of Food and Agriculture
34: 73-80.
Hanhineva, K., M.A. Lankinen, A. Pedret, U. Schwab, M. Kolhemainen, J. Paananen, V. de Mello, R. Sola, M. Lehtonen,
K. Poutanen, M. Uusitupa and H. Mykkänen, 2015. Nontargeted metabolite profiling discriminates diet-specific
biomarkers for consumption of whole grains, fatty fish and bilberries in a randomized controlled trail. The Journal
of Nutrition 145: 7-17.
Ivarsson E., H. Wall, R. Tauson, L. Jönsson and K. Elwinger, 2014. Slutrapport: ökat utnyttjande av raps och åkerböna
i slaktkycklingfoder. Institutionen för husdjurens utfodring och vård, SLU, Uppsala, Sweden. Available at: http://
tinyurl.com/zod7m6p.
Józefiak, D., A. Ptak, S. Kaczmarek, P. Maćkowiak, M. Sassek and B.A. Slominski, 2010. Multi-carbohydrase and
phytase supplementation improves growth performance and liver insulin receptor sensitivity in broiler chickens
fed diets containing full-fat rapeseed. Poultry Science 89: 1939-1946.

Protein digestion kinetics of different protein sources in broilers
H. Chen1,4*, S. de Vries1,2, J. de los Mozos3 and A.J.M. Jansman4
2Trouw Nutrition R&D, Veerstraat 38, 5831 JN Boxmeer, the Netherlands; 3Trouw Nutrition R&D,
Ctra. CM-4004, km 10.5, 45950, Casarrubios del Monte, Spain; 4Wageningen UR Livestock Research,
De Elst 1, 6708 WD Wageningen, the Netherlands; hsuan.chen@wur.nl
Abstract
The present study evaluated protein digestion kinetics of diets with different dietary protein sources
in broilers. The apparent digestibility of crude protein (CP) and the mean retention time of digesta in
different compartments of the small intestine were measured in birds fed diets containing soybean
meal (SBM), rapeseed meal (RSM) or potato protein (PP) as main dietary protein source. Calculated
maximum small intestinal CP digestibility did not differ among diets. SBM, however, showed a
higher digestion rate for CP compared to RSM and PP. In conclusion, protein sources differ in protein
digestion kinetics in broilers, with SBM being more quickly digested compared to RSM and PP in
the small intestine.
Keywords: dietary protein, digestion kinetics, broilers
Introduction
In current feed evaluation systems, the nutritive value of a protein source in diets for broilers is based
on the concentrations of indispensable amino acids, and their digestibility up to the end of the ileum
or over the total gastrointestinal tract (GIT). These values, however, do not provide information
on the digestion kinetics in the GIT. Digestion kinetics can affect the postprandial appearance of
absorbed amino acids and peptides in blood and their post-absorptive utilisation. The objective of
the present study was to evaluate the small intestinal protein digestion kinetics in broilers containing
three different protein sources.

A total of 96 28-day-old female broilers (Ross 308) were randomly allocated to one of four
experimental diets containing soybean meal (SBM), rapeseed meal (RSM) or potato protein (PP)
as the main dietary protein source (70% of total dietary protein). TiO2 (2.5 g/kg feed) was included
as an indigestible marker. Broilers were housed individually in the metabolic cages. On day 38 and
39 of age, birds were euthanized and digesta samples from duodenum, proximal jejunum, distal
jejunum, and ileum were collected quantitatively. 24-hour feed intake before dissection was recorded.
Digesta samples were analysed for crude protein (CP) and TiO2 to determine the apparent digestibility
of CP (ADCP) and the mean retention time (MRT) in the different small intestinal sections was
calculated (Weurding et al., 2003).
The protein digestion kinetics curve of the different diets were calculated by relating the apparent
digestibility of CP in each compartment of the GIT with the sum of the MRT of digesta up to that
compartment. The digestion kinetics curve was then described by the exponential modal developed
by Ørskov and McDonald (1979). The potential digestibility of CP (Dmax) and the digestion rate
constant (K) were estimated.
The effect of protein source was analysed by analysis of variance using the GLM procedure of SAS
(version 9.3; SAS Inst. Inc., Cary, NC, USA). A probability level of less than 5% was considered
to be significantly different.

Up to the proximal jejunum, SBM had a higher ADCP compared to RSM (47 and 27%, respectively)
(P<0.001) and the ADCP of PP did not differ from SBM and RSM. Up to the terminal ileum, PP and
SBM had a higher ADCP compared to RSM (82, 80 and 75, respectively) (P<0.001). The difference
of ADCP between SBM and RSM was larger in the proximal jejunum compared to the ileum. MRT
did not differ among diets in either of the small intestine compartments. MRT in the duodenum,
proximal jejunum, distal jejunum and ileum were 9, 33, 47 and 125 min, respectively, with an
estimated MRT in the entire small intestine of 213 min. Dmax for CP in the small intestine did not
differ among diets. However, the diet with SBM had a higher digestion rate for CP compared to diets
with RSM and PP (2.68×10-2, 1.15×10-2 and 1.55×10-2 unit/min, respectively) (P<0.001; Figure 1).
In conclusion, protein sources differ in protein digestion kinetics in broilers, with SBM being more
quickly digested compared to RSM and PP in the small intestine.
A SBM B RSM
100 100
Digestibility of CP (%)
80 Digestibility of CP (%) 80
60 60
40 40
20 20
0 0
0 50 100 150 200 250 0 50 100 150 200 250 300
Time (min) Time (min)
Figure 1. Relationship between mean retention time and crude protein (CP) digestibility coefficient
of diets with (A) soybean meal (SBM), and (B) rapeseed meal (RSM). Observed values (♦) and
predicted values (grey line) from the equation: Dt = Dmax(1-e-kt).
Acknowledgements
Nutrition’ programme financed by Wageningen UR, the Dutch Ministry of Economic Affairs, WIAS
graduate school and Nutreco and Darling Ingredients International.
References
Ørskov E.R. and I. McDonald, 1979. The estimation of protein degradability in the rumen from incubation measurements
weighted according to rate of passage. Journal of Agricultural Science 92: 499-503.
Weurding, R.E., H. Enting, M.W.A. Verstegen, 2003. The relation between starch digestion rate and amino acid level
for broiler chickens. Poultry Science 82: 279-284.

Maintenance energy requirements in modern broilers fed exogenous
enzymes
J.V. Caldas1,2, K.M. Hilton1, N. Boonsinchai1,3, G. Mullenix1, J.A. England1 and C.N. Coon1*
1Center of Excellence for Poultry Science, University of Arkansas, Fayetteville, AR 72701, USA;
2current address: Cobb –Vantress, P.O. Box 1030, Siloam Springs, AR 72761-1030, USA; 3current
address: CP group, C.P. Tower 14/F, 313 Silom Road, Bangkok, Thailand; ccoon@uark.edu
Abstract
Maintenance energy requirement is a large component of the energy needs for poultry. Two experiments
with 100 and 360 Cobb male broilers were conducted. Feeding levels (10-50% experiment 1 from
16-22 d, and 30-100% trial 2 from 16-27 d). In experiment 2, a negative control (NC) and NC +
Multi-enzyme (glucanase + xylanase + protease + phytase) were studied. The enzymes were added
toa basal corn-soybean mash diet. Metabolisable energy (ME) for maintenance was evaluated by a
linear regression fitting retention energy in the body kcal/kg0.70 as the response variable and ME intake
kcal/kg0.70 as the independent variable; MEm was determined as ME intake at zero energy retention.
Net energy (NE) for maintenance was evaluated fitting a logarithmic model (HP kcal/kg0.70 = a ×
e (b × MEI)), where the constant ‘a’ corresponds to NEm. The MEm was 152±8 kcal/kg0.70 (R2=0.91)
and 128±6 kcal/kg0.70 (R2=0.98) for trial 1 and 2, respectively (P<0.01). The NEm was 97.2±8 kcal/
kg0.70 (R2=0.87) and 97.9±6 kcal/kg0.70 (R2=0.95) for trial 1 and 2, respectively (P<0.01). The NC
+ multi-enzymes showed lower MEm in 8.5 kcal/kg0.70 which represents 6.6% of the maintenance
energy requirement (P<0.01). When this value is expressed as kg of feed intake, the energy savings
ranged from 75 kcal at ad libitum up to 236 kcal at maintenance intake. Further investigation is
needed to understand the mechanism by which enzymes are decreasing the maintenance energy
requirement for broilers.
Keywords: tissue retention, metabolisable energy, net energy, broilers
Introduction
The lower heat production (HP) kcal/kg feed shown when exogenous enzymes are added to poultry
corn-soybean diets (Caldas, 2015) lead to believe enzymes saving energy at the maintenance level
since HP includes maintenance and heat increment. Maintenance energy requirement for a growing or
producing animal is a dynamic equilibrium of protein and fat turnover to maintain body temperature
Chwalibog (1985). The maintenance energy needs are fulfilled first before growth can occur, so a
lower energy needs for maintenance means the bird using the feed more efficiently towards growth.

A linear regression to analyse metabolisable energy for maintenance (MEm), and a logarithmic model
to determine net energy for maintenance (NEm) were fitted in two experiments. Increasing feeding
ME levels (10-50%, for 16-22 d in experiment 1) and (30-100% for 16-27 d in experiment 2) were
considered as the independent variable X. Energy retention in the body was the response variable ‘Y’
to determine MEm and HP was the response variable to determine NEm. In the second experiment,
a diet effect was added to the model to account differences in MEm when the enzymes were added.
The ME was determine by total collection in the ad libitum birds, the RE (retained energy) was
calculated to be: protein gain (g) × 5.66 kcal/g + fat gain (g) × 9.35 kcal/g (Okumura, 1979), both
normalized by metabolic body weight (BW0.70). The HP was calculated to be: ME intake – RE.

Maintenance energy accounted for 34% of the ME intake in broilers from 16-27 d of age. This
value is smaller of the 42-44% reported by Lopez and Leeson (2005) for 0-49 d, may be due to
age difference in the study. Maintenance energy is higher in older animals because a bigger tissue
is needed to maintain. The MEm (152 in experiment 1, and 128 kcal/kg0.70 in experiment 2) differ
in 16%, while NEm (net energy for maintenance) was almost the same, (97 in experiment 1 and 98
kcal/kg0.70 in experiment 2). The difference between MEm and NEm is the heat increment which is
discounted in the NEm. These results suggest that broilers in the first experiment spent more energy
as heat increment probably for thermoregulation Sakomura (2004) since the feeding levels were
lower than experiment 2. When a multi-enzyme blend (NC+Enz) was added to NC in experiment
2, the NC+Enz decreased the MEm in 8.5 kcal (128.1 NC vs 119.6 kcal/kg0.70/d NC+Enz.) (Figure
1) which corresponds to 6.6% of the energy for maintenance, taking 8.5 kcal over kg feed intake,
the energy savings from the enzymes varies from 75 kcal at ad libitum intake up to 265 kcal at
30% of feeding level, so the more feed restricted is the chick, the more energy for maintenance the
enzymes can spare. To the author’s knowledge there is no research that exogenous enzymes have
been reported to decrease the maintenance energy, so no information is available for comparison.
180
Lineair (NC) Lineair (NC+Enz)
160
140
120
RE kcal/kg0.70
100
80
60 R2 0.98
RMSE 5.72
40 Intercept <0.0001**
20 MEI <0.0001**
Diet 0.0002**
0
148 152 158 186 193 202 218 222 237 250 263 267 283 288 293 302 308 323 333 336 364 368 384
ME intake kcal/kg0.70
Figure 1. Linear regression: retained energy (RE) regressed on metabolisable energy (ME) intake
(kcal/kg0.70) with and without enzymes in the diets.
References
Caldas, J.V., 2015. Calorimetry and body composition research in broilers and broiler breeders. PhD thesis, University
of Arkansas, AR, USA.
Chwalibog, A., 1985. Studies on energy metabolism in laying hens. Report 578. Institute of Animal Science, Copenhagen,
Denmark, 190 pp.
Lopez, G. and S. Lesson, 2005. Utilization of metabolizable energy by young broilers and birds of intermediate growth
rate. Poult. Sci. 84: 1069-1076.
Okumura, J.I. and M. Setsutaka, 1979. Effect of deficiencies of single essential amino acids on nitrogen and energy
utilisation in chicks. British Poultry Science 20: 421-429.
Sakomura, N.K., 2004. Modeling energy utilization in broiler breeders, laying hens and broilers. Braz. J. Poult. Sci.
6: 1-11.

Laying performance of layer vs dual purpose genotypes under low
methionine supply
S. Mueller*, R.E. Messikommer, M. Kreuzer and I.D.M. Gangnat
ETH Zurich, Institute of Agricultural Sciences, Universitaetstrasse 2, 8092 Zurich, Switzerland;
sabine.mueller@usys.ethz.ch
Abstract
With dual purpose genotypes the females are used for egg and the males for meat production.
However, a lower performance and feed efficiency are to be expected with such genotypes compared
to specialised genotypes. The aim of the present study was to investigate whether the omission of
synthetic methionine (MET) supplementation would be better tolerated by layers of dual purpose
genotypes than by a specialised layer genotype. Ten individually kept hens each of three dual
purpose genotypes (Lohmann Dual, Mechelner, Schweizer Huhn) were compared with a layer hybrid
(Lohmann Brown plus). In a cross-over design, each hen received for 3 weeks a control diet (+1.6
g DL-MET/kg feed) or an unsupplemented diet, both containing 11.5 MJ metabolisable energy and
168 g crude protein per kg. There were significant genotype differences in most performance and
egg quality traits measured, but no diet effect and no interaction occurred. In conclusion, none of the
genotypes seems to have really required the extra MET supplementation during the 3 weeks even
though the dietary MET content, when omitting the supplementation, was lower than recommended
for layer genotypes at this stage of lay. From the dual purpose genotypes, only Lohmann Dual was
approaching performance and MET use efficiency of the layer genotype with, however, still a lower
laying performance and lighter eggs.
Keywords: methionine, dual purpose poultry, layer, egg quality
Introduction
The practice of culling 1-day old male chicks from layer genotypes is controversially discussed in
the public and is anticipated to result in legal changes in some countries soon. Instead of fattening
the males with the result of unfavourable carcass appearance and low feed efficiency, production
systems based on dual purpose genotypes could be established. This approach is facilitated by new
genotypes provided by breeding companies like Lohmann Dual (LD) and Walesby Special (Damme
et al., 2015). In addition there are less specialised but long established poultry breeds which may
serve a dual purpose. However, a limited performance in both laying and fattening is to be expected
with dual purpose genotypes (Damme et al., 2015), which is unfavourable from a feed efficiency
perspective unless a diet of lesser quality can be fed. The objective of the present study was to test
whether the omission of synthetic methionine (MET) supplementation, an obligatory demand by
organic farming rules, would be better tolerated by dual purpose hens compared to layer hybrids which
were shown to be susceptible to that (Koreleski and Świątkiewicz, 2009; Van Krimpen et al., 2015).

An experiment was conducted with 4×10 individually kept hens of Lohmann Brown plus (LB),
Lohmann Dual (LD), Mechelner (ME) and Schweizer Huhn (CH). The hens had ad libitum access to
feed and water. The room temperature was maintained at around 20 °C and 14 h light were provided
per day. An enriched environment was provided. The experiment was approved by the Cantonal
Veterinary Office. In a cross-over design, each animal received a control diet (+1.6 g DL-MET/kg
feed) and an unsupplemented diet for 3 weeks in different order (laying stage from 4 to 6 months). The
analysed total MET contents were 4.2 vs 3.1 g/kg as fed (4.0 vs 2.4 g digestible MET as calculated),
respectively. Both diets were calculated to contain, per kg as fed, 11.5 MJ metabolisable energy

and 168 g crude protein. Data were subjected to analysis of variance considering genotype, diet,
interaction and sequence, and hens as subject in the repeated statement.

There were significant genotype differences (P<0.001) in all performance and egg quality traits
measured. Body weights (kg) of the ME hens were highest (3.4) followed by CH (2.6) and, similarly,
LD (1.9) and LB (2.0), whereas laying performance was 66, 60, 87 and 98%, respectively. The order
in feed consumption was ME, LB, CH and LD (131, 120, 116 and 104 g/day). Average egg weights
(g) were lower with ME and CH (57-58), slightly higher with LD (61) and highest with LB (65)
but all were in the normal range (>53 g). Across diets, the MET use efficiency (g egg mass/g MET)
was favourably high in LB (147) and LD (142) and clearly lower in CH (94) and ME (81). The LB
and LD hens laid rounder eggs (shape index 79) whereas ME and CH laid more ovoid eggs (shape
index 75). The shell strength (N) was best in LB (47.6 N) compared to ME and CH (35.8 and 35.5
N, respectively) with intermediate values for LD (40.6 N). With Haugh Units of above 70, the egg
white of all eggs was of a good quality (Grashorn, 2008). The ME hens were superior to the other
genotypes in egg yolk proportion (32%), followed by CH (30%), LD (27%) and LB (25%). With
the omission of synthetic MET, small numerical declines occurred in laying performance and egg
mass (g/day) which were not significant. All other traits were even numerically very similar between
groups. There was no significant interaction between genotype and diet. Other studies found clear
responses in performance and egg quality of layer genotypes due to the omission of MET (e.g.
Koreleski and Świątkiewicz, 2009; Van Krimpen et al., 2015), but in both studies the experimental
period was longer than in the present study.
In conclusion, none of the genotypes seems to have really required the extra MET supplementation
during the 3 weeks even though the dietary MET content when omitting the supplementation was
lower than recommended for layer genotypes at this stage of lay (about 4 g/kg as fed; depending
on the feed intake). From the dual purpose genotypes, only LD was approaching performance and
MET use efficiency of the layer genotype, but still their laying performance and egg size were not
fully competitive. An unexpected finding was given by the large genotype differences found in yolk
proportion.
Acknowledgements
The project was funded by the Coop Research Program of the ETH Zurich World Food System
Center, Zurich, and by the Swiss Federal Office of Agriculture, Berne, Switzerland.
References
Damme, K., S. Urselmans and E. Schmidt, 2015. Der Eierpreis muss es richten. DGS Magazin 6: 30-38.
Grashorn, M.A, 2008. Eiqualität. Landbauforschung – vTI Agriculture and Forestry Research, Sonderheft 322: 18-33
(W. Brade, G. Flachowsky, L. Schrader, Hrsg., Legehuhnzucht und Eiererzeugung – Empfehlungen für die Praxis).
Koreleski, J. and S. Świątkiewicz, 2009. Laying performance and nitrogen balance in hens fed organic diets with
different energy and methionine levels. Journal of Animal and Feed Sciences 18: 305-312.
Van Krimpen, M.M., G.P. Binnendijk, M.A. Ogun and R.P. Kwakkel, 2015. Responses of organic housed laying
hens to dietary methionine and energy during a summer and winter season. British Poultry Science 56: 121-131.

Possible mechanism of down-regulation of atrogin-1 mRNA level in
butoxybutyl alcohol-fed chicken
T. Kamizono1, M. Kikusato1, K. Hayashi2 and M. Toyomizu1*
1Animal Nutrition, Life Sciences, Graduate School of Agricultural Science, Tohoku University, 1-1
Tsutsumidori-Amamiyamachi, Aoba-ku, Sendai 981-8555, Japan; 2Faculty of Agriculture, Kagoshima
University, 1-21-24 Korimoto, Kagoshima 890-0065, Japan; toyomizu@bios.tohoku.ac.jp
Abstract
We found that feeding butoxybutyl alcohol (BBA) to broiler chickens depresses skeletal muscle
proteolysis and thereby increases skeletal muscle mass, and that one of the causes of this decrease
in proteolysis might be down-regulation of the gene expression of an ubiquitin ligase atrogin-1. The
aim of the present study was to clarify the endocrine and molecular mechanisms responsible for
decreasing the level of atrogin-1 gene transcription in BBA-fed broiler chickens. Twelve 0-day-old
male broiler chicks were divided into two groups (n=6 per group), and fed either basal (control)
or BBA-supplemented diets (30 mg BBA/kg basal diet) for 4 weeks. While the mRNA and total
protein levels of forkhead box O1 (FoxO1), which regulates atrogin-1 mRNA transcription, were
significantly lower, relative to controls, in the muscle of birds given BBA, the phosphorylation level
at Thr 24 of the FoxO1 protein (p-FoxO1), which inactivates transcription, tended to be increased
(P=0.07), resulting in a significant increase in the p-FoxO1/total FoxO1 ratio. The birds given BBA
showed a significant decrease in plasma corticosterone concentration, while they did not exhibit any
changes in the levels of muscle insulin-like growth factor-1 (IGF-1) or its receptor (IGF1R) mRNA;
or in the total or phosphorylated Akt protein levels, which could affect the phosphorylation level of
FoxO1. These results suggest that BBA-induced down-regulation of atrogin-1 mRNA expression
may be induced by inactivation (phosphorylation) of FoxO1, possibly via decreasing the plasma
corticosterone concentration.
Keywords: growth promotion, skeletal muscle proteolysis, FoxO1
Introduction
Butoxybutyl alcohol (BBA) is a by-product of the fermentation and distillation processes involved
in the production of Shochu, a traditional Japanese spirit. Our previous study demonstrated that the
feeding of BBA increases the skeletal muscle mass of broiler chickens, probably due to a suppression
of skeletal muscle proteolysis (Kamizono et al., 2015). The study also showed that in BBA-fed
chickens, there was a decrease in the muscular mRNA level of atrogin-1, which is an ubiquitin
ligase and a rate-limiting enzyme within the ubiquitin-proteasome protein degradation system. This
decrease in the atrogin-1 mRNA level was relatively larger than the observed decreases in mRNA
expression of other proteases such as calpains, cathepsin B and caspase-3. We therefore focused
our attention on the signal transduction pathway that down-regulates atrogin-1 gene expression
to further understand the mechanism responsible for the increased skeletal muscle mass of birds
given BBA. The present study investigated the effect of feeding BBA on the gene expression and
phosphorylation level of forkhead box O1 (FoxO1), which regulates atrogin-1 gene transcription.
The study also characterized endocrine and intramolecular changes in factors affecting FoxO1 gene
expression and phosphorylation, plasma corticosterone concentration, and the expression of signal
transduction proteins IGF-1, IGF1R and Akt.

Twelve 0-d-old male broiler chicks (Ross strain) were randomly divided into two groups, and fed
either basal (control) diet (22% crude protein and 3.0 Mcal metabolisable energy/kg) or BBA-

supplemented diet (30 mg BBA/kg basal diet) for 4 weeks. Birds were euthanized by decapitation
under carbon dioxide anesthesia, and plasma and skeletal muscle (pectoralis profundus muscle) were
taken. Real time RT-PCR analysis was conducted to measure muscle mRNA expression levels of
atrogin-1, FoxO1, IGF-1 and IGF1R. Western blot analysis was conducted to determine the levels of
the FoxO1 protein and the degree of phosphorylation at its Thr 24 residue. Phosphorylation of FoxO1
inactivates its transcription activity, as the protein translocates from the nuclei. The protein level of
Akt and its phosphorylation level were determined as phosphorylated Akt induces the phosphorylation
of FoxO1. Plasma corticosterone concentration was determined using a commercially-available EIA
kit. Statistical differences were determined using a one-sided Student’s t-test.

The level of atrogin-1 mRNA and the mRNA and total protein levels of FoxO1 were significantly
decreased in the muscles of birds given BBA, relative to untreated controls. The p-FoxO1 protein
level tended to be increased by BBA supplementation (P=0.07), leading to a significant increase
in the p-FoxO1/total FoxO1 ratio. The birds given BBA showed a significant decrease in plasma
corticosterone concentration, while they did not show any changes in IGF-1 or IGF1R mRNA levels;
or changes in the total amount of Akt protein or its phosphorylation level. From the results, the present
study demonstrates that BBA-induced down-regulation of the muscle atrogin-1 mRNA level may
be caused by inactivation of FoxO1, possibly via decreasing plasma corticosterone concentration.
This leads to the observed decrease in skeletal muscle proteolysis in the BBA-fed broiler chickens.
References
Kamizono, T., D. Saputra, I. Miura, M. Kikusato, K. Hayashi and M. Toyomizu, 2015. Effect of feeding butoxybutyl
alcohol on the growth performance and status of skeletal muscle proteolysis in broiler chickens. Journal of
Agricultural Science 153: 920-928.

Effects of increased diet density through increased dietary fat level on
energy balance characteristics of broilers during the first week of life
D.M. Lamot1,2*, D. Sapkota1, P.J.A. Wijtten2, I. van den Anker1, M.J.W. Heetkamp1, B. Kemp1 and
H. van den Brand1
1Adaptation Physiology Group, Wageningen University, P.O. Box 338, 6700 AH Wageningen,
the Netherlands; 2Cargill Animal Nutrition Innovation Center Velddriel, Veilingweg 23, 5334 LD
Velddriel, the Netherlands; david_lamot@cargill.com
Abstract
The current study aimed to determine the effect of increased diet density through increasing dietary
fat level on growth performance and energy balance characteristics of broiler chickens during the
first week of life. The effects of diet density on energy and nitrogen metabolism were studied using
a dose response design that comprised 5 dietary fat inclusion levels (3.5, 7.0, 10.5, 14.0, and 17.0%)
while maintaining a constant digestible amino acid to energy ratio. Chickens were housed in open
circuit climate respiration chambers. Preplanned contrasts were used to determine significant linear
and quadratic relationships with diet density. Feed intake and BW gain linearly decreased and gain
to feed ratio increased (P<0.001) with increasing dietary density. Nutrient efficiencies (calculated as
gain per unit of nutrient consumed) for fat, nitrogen and gross energy linearly decreased (P<0.001).
A linear decrease in heat production and the respiratory exchange ratio (CO2/O2) were found with
increasing diet density (P<0.001). Protein intake and total energy, fat and protein retention were not
affected by diet density. To conclude: increased diet density during the first week of age resulted
in improved feed efficiency, but not nutrient efficiency. Protein and fat deposition in the body of
broilers was similar.
Keywords: diet density, dietary fat, metabolism, energy balance, broiler chickens
Introduction
To date, studies to determine growth and maintenance requirements of broiler chickens often base
these requirements on a prolonged growth trajectory. They discount that these requirements may
be age related and depending on the physiological status of the chicken. Especially requirements
during the first week of life remain obsolete. Also, there is little insight in how metabolic processes
might be affected through diet composition during this first week period. The aim of the current
study was to determine the effect of increased diet density through increased dietary fat level on
growth performance, energy and nitrogen metabolism of broiler chickens during the first week of life.

A dose response design comprising 5 dietary fat inclusion levels (3.5, 7.0, 10.5, 14.0, and 17.0%,
respectively) was used to study the effects of increased diet density through increased dietary fat
level. Increased dietary fat levels were obtained through increased soybean oil inclusion, while
maintaining a constant digestible amino acid to energy ratio. As such, diets were not kept isocaloric
nor isonitrogenous. Per treatment, 108 broiers were divided over 6 replicates. Chickens were housed
in open circuit climate respiration chambers. BW was measured on day 0 and 7, while feed intake
was measured daily. For calculation of energy and nitrogen balances, the oxygen and carbon dioxide
exchange were measured combined with feed and excreta collection and analysis.
Preplanned contrasts were used to determine significant relationships for linear and quadratic effects
with diet density.

Body weight gain and feed intake decreased linearly as the diet density increased (P<0.05; Table 1).
Total gross energy and metabolisable energy intake were not affected by diet density. This corresponds
with earlier research that showed broiler chickens are able to control their feed intake in order to
maintain a constant energy intake (Leeson et al., 1996), where previous research suggested that
this capability only occurs from 2 weeks of age onwards (Brickett et al., 2007). The gain to feed
ratio from 0 to 7 days increased linearly due to increased diet density (P<0.001; Table 1). However,
nutrient efficiencies (calculated as gain per unit of nutrient consumed) for fat, nitrogen and gross
energy linearly decreased (P<0.05; Table 1). A linear decrease in heat production and the respiratory
exchange ratio (CO2/O2) were found with increasing diet density (P<0.001; Table 1). Respiratory
exchange rates (RER) are known to be dependent on macro nutrient availability, where increased
dietary fat levels resulted in increased fatty acid oxidation, and increased O2 usage relative to CO2
production. A lowered heat production suggests a more efficient transition from metabolisable into
retained energy. However, energy retention for protein and fat was not affected by diet density.
Concluding, increased diet density resulted in increased feed efficiency, but not nutrient efficiency.
Despite a significant shift from carbohydrates to fat as energy source, increased diet density through
increased dietary fat level did not change protein and fat deposition rates in broiler chickens during
the first week of life.
Table 1. Effects of increased diet density through dietary fat level on growth performance, nutrient
efficiency, energy intake, heat production (HP), respiratory exchange ratio, and energy retention
(as protein, ERp; as fat, ERf) of broiler chickens of 0 to 7 days of age.
Dietary fat level (%) SEM P-value
3.5 7.0 10.5 14.0 17.5 Linear Quadratic
BW gain 0-7 d (g/d) 19.3 18.6 18.7 18.8 18.0 0.4 0.199 0.047 0.877
Feed intake 0-7 d (g/d) 18.2a 17.2b 16.4c 16.7bc 14.8d 0.3 <0.001 <0.001 0.570
Gain to feed ratio 0-7 d 1.059a 1.081a 1.140b 1.128b 1.212c 0.017 <0.001 <0.001 0.348
Fat efficiency (g gain/g nutrient) 25.8d 14.2c 10.4b 8.0a 7.4a 0.3 <0.001 <0.001 <0.001
Nitrogen efficiency (g gain/g nutrient) 29.6b 29.1b 29.3b 27.8a 28.6ab 0.5 0.044 0.019 0.568
Gross energy efficiency (g gain/MJ) 63.2c 61.1bc 62.1bc 58.4a 60.2ab 1.0 0.008 0.004 0.319
Gross energy intake (kJ/kg0.75/d) 1,686.5b 1,705.1b 1,682.4b 1,794.2a 1,700.2b 25.2 0.015 0.121 0.296
Metabolisable energy intake (kJ/kg0.75/d) 1,287.3 1,314.3 1,250.5 1,331.1 1,252.1 25.7 0.131 0.506 0.476
HP (kJ/kg0.75/d) 557.5a 535.4bc 527.7c 540.0b 531.7bc 0.00 <0.001 <0.001 0.001
Respiratory exchange ratio 1.05a 1.02b 0.98c 0.94d 0.91e 4.6 <0.001 <0.001 0.511
Energy retention (kJ/kg0.75/d) 730.5 777.5 724.0 792.0 719.5 24.6 0.148 0.921 0.200
ERp (kJ/kg0.75/d) 349.6 355.6 346.1 370.7 354.0 6.9 0.139 0.272 0.656
ERf (kJ/kg0.75/d) 381.1 421.7 377.9 421.1 365.8 18.3 0.114 0.578 0.123
Acknowledgements
The authors gratefully thank all staff of Carus, research facility of the Department of Animal Sciences
of Wageningen University (the Netherlands) for their assistance during the study.
References
Brickett, K.E., J.P. Dahiya, H.L. Classen and S. Gomis, 2007. Influence of dietary nutrient density, feed form, and
lighting on growth and meat yield of broiler chickens. Poultry Science 86: 2172-2181.
Leeson, S., L. Caston and J.D. Summers, 1996. Broiler response to diet energy. Poultry Science 75: 529-535.

Effects of metabolisable energy and crude protein levels in balanced
digestible essential amino acids on body weight gain and carcass
composition of Brown laying hens in the late phase of production
N. Chauychuwong* and K. Soisuwan
Department of Animal Science, Faculty of Agriculture, Rajamangala University of Technology
Srivijaya, Thungsong, Nakhon Si Thammarat 80110, Thailand; nantanachu@yahoo.com
Abstract
This study was conducted to evaluate the effect of metabolisable energy (ME) and crude protein (CP)
in balanced digestible essential amino acids on body weight gain (BWG) and carcass composition
of brown laying hens in the late phase of production. A 4×3 factorial experiment with 4 dietary ME
levels (2,500, 2,600, 2,700 and 2,800 kcal ME/kg) and 3 CP levels (16, 17 and 18%) was used. A
total of 720 commercial Lohmann Brown laying hens, 50 wk of age, were randomly assigned into
12 treatments (5 replicates with 12 birds per replicate). Feed and water were provided ad libitum
throughout the experiment (16 wk). There was no significant interaction between dietary energy
and protein levels; however it was found that BWG, carcass yield (CY) and breast yield (BY) were
affected (P<0.05) by the ME and CP levels. At low and moderate CP levels (16.0 and 17.0%) and
moderate and high ME levels (2,700 and 2,800 kcal/kg) improved (P<0.05) BWG, CY and BY while
abdominal fat pad (AFP) decreased (P<0.05) when reduced ME from 2,700 to 2,600 and 2,500
kcal/kg. The ME and CP levels had no effect on thigh yield, drumstick yield, wing yield and gibbet
yield. Based on the data under this experimental condition it was concluded that 2,700 kcal ME/
kg feed and 17% CP are the optimum levels for improving BWG and edible meat yield of brown
laying hens in the late phase of production in the balanced dietary digestible essential amino acids.
Keywords: metabolisable energy, crude protein, digestible essential amino acids, body weight gain,
carcass composition
Introduction
Dietary metabolisable energy (ME) and crude protein (CP) are the major nutritional parameters of
the diets of laying hens (Lesson et al., 2001). The objective of this study was to evaluate the effect
of dietary metabolisable energy and crude protein levels in balanced digestible essential amino acids
on body weight gain and carcass composition of brown laying hens in the late phase of production.

720 Lohmann Brown laying hens in five replicates per treatment (12 birds/replicate) were used.
Twelve experimental diets which contained of 4 levels of dietary energy levels (2,500, 2,600, 2,700
and 2,800 kcal ME/kg feed) and 3 levels of crude protein (16, 17 and 18%). All dietary treatments
were formulated base on ideal protein concept to balance digestible essential amino acids while
another nutrient contents of the recommendation were met by of NRC (1994). Birds were raised in
laying cage throughout the experimental period from 50 to 66 weeks of age and received feed in mash
form with feed were provided for ad libitum consumption. At the end of 66 weeks of age, 2 birds per
replicate will be randomly selected for processing. Carcasses and abdominal fat pad weights will be
recorded. Carcass weight will be determined without neck, giblets, and abdominal fat. All data were
analysed by the GLM procedures as suggested by Khunthum (2006) as 4×3 factorial arrangement
of dietary treatments. Two-way interaction between ME and CP were not significant (P>0.05); thus,
data were pooled and analysed for main effects.

Results and discussions
There were no interaction between main treatments. It was found that body weight gain (BWG),
carcass yield (CY) and breast yield (BY) were affected (P<0.05) by the ME and CP levels (Table
1). At low and moderate CP levels (16.0 and 17.0%) and moderate and high ME levels (2,700 and
2,800 kcal/kg) improved (P<0.05) BWG, CY and BY while abdominal fat pad decreased (P<0.05)
when reduced ME from 2,700 to 2,600 and 2,500 kcal/kg. Based on the results of this excessive ME
and CP concentrations in diets lead to substantial negative effects on body weight gain and carcass
composition of the layer. It was also found that ME and CP levels had no effect on thigh yield,
drumstick yield, wing yield and gibbet yield which included of heart and gizzard.
Conclusions
The present study has shown that it can be used the diets composed of 2,700 kcal of ME/kg feed
and 17% crude protein in balanced digestible essential amino acids to optimize body weight gain
and carcass composition of laying hens in the late phase of production.
Table 1. Dietary metabolisable energy (ME) and crude protein (CP) levels on body weight gain
(BWG) and carcass composition of laying hens in the late phase of production in balanced digestible
essential amino acids diets.1,2
Item Parameters
BWG CY BY TY DY WY GY AFPY
(g) (%) (%) (%) (%) (%) (%) (%)
Metabolisable energy (kcal/kg)

2,500 612.7b 72.46b 18.10b 10.39 9.71 5.34 4.36 1.52b
2,600 621.4b 71.01b 18.47b 10.21 9.21 5.65 4.24 1.56b
2,700 662.9a 79.28a 21.46a 10.06 9.10 5.47 4.32 3.01a
2,800 671.3a 77.13a 20.62ab 10.71 9.03 5.09 4.27 3.10a
Crude protein (%)
16 641.1a 76.94a 20.49ab 10.92 9.07 5.21 4.28 1.47
17 658.7a 77.94a 21.81a 11.01 9.01 5.10 4.32 1.76
18 628.1b 74.21ab 18.88b 10.96 9.66 5.22 4.31 1.69
Pooled SEM 11.21 0.76 0.24 0.18 0.05 0.04 0.33 0.04
Main effect and interaction
ME 0.042 0.046 0.047 NS NS NS NS 0.039
CP 0.048 0.048 0.046 NS NS NS NS NS
ME×CP NS2/ NS NS NS NS NS NS NS
1 a,b means within comparisons with different superscripts differ (P<0.05); NS means not significant at P>0.05.
2 CY = carcass yield; BY = breast yield; TY = thigh yield; DY = drumstick yield; WY = wing yield; GY = gibbet yield;
AFPY = abdominal fat pad yield.
References
Khunthum, A., 2006. The principle of experimental design. Department of Statistic, Faculty of Science, Kasetsart
University, Thailand 343 pp.
Lesson, S., J.D. Summers and L.J. Caston, 2001. Response of layers to low nutrient density diets. J. Appl. Res. 10: 46-52.

Protein digestion kinetics of different protein sources in pigs
H. Chen1,3*, P.A Wierenga2 and A.J.M. Jansman3
2Laboratory of Food Chemistry, Wageningen University, Bornse Weilanden 9, 6708 WG Wageningen,
the Netherlands 3Wageningen UR Livestock Research, De Elst 1, 6708 WD Wageningen, the
Netherlands; hsuan.chen@wur.nl
Abstract
The objective of the present study is to evaluate protein digestion kinetics for various protein sources
in pigs. Postprandial concentration of total free amino acids in the plasma was determined using the
ninhydrin method in growing pigs fed diets containing soybean mean, rapeseed meal, wheat gluten,
plasma meal and insect protein meal as the only dietary protein source. Wheat gluten and plasma
protein treatments, which are fast digested, showed a peak like curve whereas treatments soybean
meal, rapeseed meal and insect protein meal, which are slowly digested, showed a more plateau
like postprandial response.
Keywords: dietary protein, digestion kinetics, pigs
Introduction
Currently, the nutritive value of a protein source in diets for pigs is based on the composition and
concentration of indispensable amino acids, and their digestibility up to the end of ileum. These
values, however, do not provide information on the digestion kinetics in the gastrointestinal tract. The
kinetics of protein digestion may affect the postprandial appearance of amino acids and peptides in
blood and their post-absorptive metabolism (Boirie et al., 1997). Fast digested dietary proteins such
as whey protein show an faster postprandial appearance of amino acids in blood compared to more
slowly digestible sources such as casein (Boirie et al., 1997). The objective of the present study is
to evaluate protein digestion kinetics of various protein sources in pigs in terms of the postprandial
appearance of amino acids in blood.

Twenty boars (initial weight 32±3.5 kg) were suited with an ear-vein catheter and housed individually
in metabolic cages. Five experimental diets containing soybean mean (SBM), rapeseed meal (RSM),
wheat gluten (WG), plasma protein (PP) and insect protein meal (IPM) as the only protein source
were used. Diets were provided twice a day at a level of 2.5 times the maintenance requirement
for energy. On day 10,11 or 12, blood samples were collected at 30 and 60 min before feeding and
30, 60, 90, 120, 150, 180, 240, 360 and 480 min after feeding. Blood samples (1 ml per time) were
collected in tubes containing lithium-heparin and immediately centrifuged at 3,000×g for 10 min at
4 °C. Postprandial concentrations of free amino acids (FAA) in the plasma were determined using
the ninhydrin method. As a measure of quantitative postprandial appearance of FAA in blood, the
area under (AUC) the curve was calculated using the trapezoidal method. The effect of protein source
on AUC was analysed by ANOVA using the GLM procedure of SAS (Release 9.3. SAS Inst. Inc.,
Cary, NC, USA). A probability level of less than 5% was considered to be significantly different.

The postprandial concentrations of FAA showed two types of response among treatments: a peak-
like appearance (WG and PP) or a plateau-like appearance (SBM, RSM and IPM). Peak-like
curves showed a relatively fast increase of FAA in the plasma after feeding while the depletion of

FAA from the plasma was fast as well (Figure 1). Alternatively, plateau-like responses showed a
slower increase of concentration of FAA in plasma after feeding and the concentration stayed at its
maximum for a longer period of time compared to the peak-like response curves. The faster increase
of FAA in the plasma of pigs fed WG and PP might due to the high solubility protein in WG and PP,
which affects both enzymatic hydrolysis and passage rate trough the stomach and small intestine.
Soluble proteins are emptied faster from the stomach and hydrolysed faster enzymatically in vivo
than insoluble proteins (Low, 1990; Tonheim et al., 2007). Up to 120 min after feeding, WG had
the highest AUC compared to other protein sources (P<0.001). This could due to a higher quantity
of available cleavage sites for pepsin, trypsin and chymotrypsin in WG, compared to proteins in
the other protein sources.
In conclusion, digestion kinetics of protein sources differ in pigs. WG and PM could be regarded
as fast digestible while SBM, RSM and IPM could be considered as more slowly digestible protein
sources.
Figure 1. Postprandial concentration of free amino acids in the plasma of pigs fed diets containing
soybean mean (SBM), rapeseed meal (RSM), wheat gluten (WG), plasma protein (PP) and insect
protein meal (IPM) as the only dietary protein source.
Acknowledgements
Nutrition’ programme financed by Wageningen UR, the Dutch Ministry of Economic Affairs, WIAS
graduate school and Nutreco and Darling Ingredients International.
References
Boirie, Y., M. Dangin, P. Gachon, M.P. Vasson, J.L. Maubois and B. Beaufrère, 1997. Slow and fast dietary proteins
differently modulate postprandial protein accretion. Proceedings of the National Academy of Sciences 94: 14930-
14935.
Low, A., 1990. Nutritional regulation of gastric secretion, digestion and emptying. Nutrition Research Reviews 3:
229-252.
Tonheim, S., A. Nordgreen, I. Høgøy, K. Hamre and I. Rønnestad, 2007. In vitro digestibility of water-soluble and
water-insoluble protein fractions of some common fish larval feeds and feed ingredients. Aquaculture 262: 426-435.

Impact of silages with bioactive compounds on ruminal fermentation
and microbial protein synthesis
A. Grosse Brinkhaus1,2, G. Bee1, F. Dohme-Meier1*, M. Kreuzer2 and J.O. Zeitz2,3
1Agroscope, Institute for Livestock Sciences ILS, 1725 Posieux, Switzerland; 2ETH Zurich,
Institute of Agricultural Science, 8092 Zurich, Switzerland; 3Justus-Liebig-University
Gießen, Institute of Animal Nutrition and Nutritional Physiology, 35392 Gießen, Germany;
frigga.dohme-meier@agroscope.admin.ch
Abstract
The objective of the present study was to determine the impact of two legumes (sainfoin (SF) and
birdsfoot trefoil (BT, cultivars Polom (BTP) and Bull (BTB))) containing condensed tannins (CT)
and their 1:1 mixtures with red clover (RC) containing polyphenol oxidase compared to lucerne (LU)
and RC alone on ruminal fermentation and microbial protein synthesis using the Rumen Simulation
Technique. Total volatile fatty acid concentration was lower (P<0.05) when SF was present either
alone or as RC-SF compared to RC-BTP, whereas the other treatments took an intermediate position.
Degradability of crude protein (CP) and ruminal ammonia (NH3) concentration were lowest (P<0.05)
with SF and RC-SF compared to all other treatments. Compared to RC and LU, the microbial protein
recovered in particle associates microbes was highest (P<0.05) with BTP, followed by SF, whereas
the microbial protein recovered in liquid associates microbes was highest (P<0.05) with SF, followed
by BTP. The content of utilisable protein at the duodenum (uCP) related to kg dry matter was highest
(P<0.05) for RC and RC-SF and lowest for LU whereas SF, BTP, BTB and the other mixtures took
an intermediate position. In conclusion, these results demonstrate several advantages of adding CT
legumes strategically to ruminant diets.
Keywords: condensed tannins, birdsfoof trefoil, polyphenol oxidase, sainfoin
Introduction
Microbial protein is synthesized during ruminal degradation of nutrients. Together with this microbial
protein, bypass protein makes up the utilisable crude protein (uCP) being the second most limiting
factor after energy for the productivity of animals. Bioactive compounds like condensed tannins
(CT) and polyphenol oxidase (PPO) may interfere in the ruminal degradation process by building
complexes with protein and carbohydrates. The objective of the present study was to determine the
impact of two CT-containing legumes (sainfoin (SF) and birdsfoot trefoil (BT)) and their mixtures
with red clover (RC) containing PPO compared to lucerne (LU) and RC alone on ruminal fermentation
and microbial protein synthesis in vitro.

Approximately 12 g silage dry matter (DM) were incubated in six runs (n=6) using the RUmen
SImulation TEChnique with eight 1-liter fermenters in a 39 °C water bath. Test plants were LU
(196 g CP/kg DM), RC (196 g CP/kg DM)), SF (139 g CP and 136 g CT/kg DM), BT (cultivars
Polom (BTP) (198 g CP and 23 g CT/ kg DM) and Bull (BTB) (196 g CP and 35 g CT/kg DM)) as
well as 1:1 mixtures of each CT-containing legume with RC. Incubations lasted for 10 days. The
last 5 days were used for data and sample collection. Concentration of NH3 and volatile fatty acid
(VFA), apparent degradability of CP, and purine content in the liquid associates microbes (LAM)
and particle associates microbes (PAM) were determined and used to calculate microbial protein
synthesis by LAM and PAM. For the calculation of uCP, the nitrogen content of the silages and
the NH3 concentrations of blanks and incubation fluids were taken into account (Scharenberg et
al., 2007). Data were analysed with the MIXED Procedure of SAS considering treatment as fixed

effect and run as random effect. In addition, orthogonal contrasts of the mixtures compared to the
respective pure legumes were calculated.

Total VFA concentration was lower (P<0.05) when SF was present either alone (92.5 mmol/l) or in
RC-SF (90.5 mmol/l) compared to the RC-BTP mixture (110 mmol/l), whereas the other treatments
took an intermediate position. The degradability of CP and concomitantly, the molar percentage of
valerate and the concentration of NH3 in the incubation fluid were lowest (P<0.05) with SF compared
to all other treatments indicating a reduction in ruminal proteolysis as a consequence of dietary CT
(Waghorn, 2008). However, in contrast to SF, no or just minor effects on ruminal CP degradation
were observed with the CT-containing BT cultivars and the PPO containing RC compared to LU. In
case of BT, this might be the result of a lower CT content (Azuhnwi et al., 2012) or differences in
CT structure (Mueller-Harvey, 2006) compared to SF. The calculated content of uCP (g/kg DM) was
highest (P<0.05) for RC (201) and RC-SF (196) and lowest for LU (142) whereas RC-BTB (183),
SF (181), BTB (175), BTP (174) and RC-BTP (171) took intermediate positions. The microbial
protein recovered (mg N/d) in PAM was higher (P<0.05) with BTP (76) and that recovered in
LAM was higher (P<0.05) with SF (122) compared to RC, RC-BTB, RC-BTP and LU (42 to 49
with PAM and 52 to 62 with LAM, respectively). These findings are of particular interest because
Min et al. (2003) reported that the amount of undegraded feed protein flowing out of the rumen
increases with increasing CT content whereas the amount of microbial protein synthesized in the
rumen remains unaffected. In addition, compared to the pure legumes there were some additive
effects of mixing SF and RC, which can be regarded positive in terms of ruminal CP degradation
and calculated uCP content.
Acknowledgements
This study was supported by the EU Marie Curie Initial Training Network (‘LegumePlus’; PITN-
GA-2011-289377).
References
Azuhnwi, B.N., B. Thomann, Y. Arrigo, B. Boller, H.D. Hess, M. Kreuzer and F. Dohme-Meier, 2012. Ruminal dry
matter and crude protein degradation kinetics of five sainfoin (Onobrychis viciifolia Scop) accessions differing in
condensed tannin content and obtained from different harvest. Animal Feed Science and Technology 177: 135-143.
Min, B.R., T.N. Barry, G.T. Attwood and W.C. McNabbb, 2003. The effect of condensed tannins on the nutrition
and health of ruminants fed fresh temperate forages: a review. Animal Feed Science and Technology 106: 3-19.
Mueller-Harvey, I., 2006. Unravelling the conundrum of tannins in animal nutrition and health. Journal of the Science
of Food and Agriculture 86: 2010-2037.
Scharenberg, A., Y. Arrigo, A. Gutzwiller, C.R. Soliva, U. Wyss, M. Kreuzer and F. Dohme, 2007. Palatability in
sheep and in vitro nutritional value of dried and ensiled sainfoin (Onobrychis viciifolia) birdsfoot trefoil (Lotus
corniculatus), and chicory (Cichorium intybus). Archive of Animal Nutrition 61: 481-496.
Waghorn, G., 2008. Beneficial and detrimental effects of dietary condensed tannins for sustainable sheep and goat
production – Progress and challenges. Animal Feed Science and Technology 147: 116-139.

Postprandial net portal and liver fluxes of essential amino acids in dairy
cows fed rumen escape protein
M. Larsen* and A.C. Storm
Department of Animal Science, Aarhus University, Foulum, 8830 Tjele, Denmark;
mogens.larsen@anis.au.dk
Abstract
The effect of allocating rumen escape protein concentrates in a total mixed ration (TMR) or separately
either as pellets, or in an ‘as is’ mix on postprandial patterns of net portal-drained visceral (PDV)
and net total splanchnic (TSP) release of essential amino acids (EAA) was investigated. Six lactating
multiparous Danish Holstein cows catheterised in major splanchnic vessels were used in a replicated
3×3 Latin square design. At milking, a concentrate mixture of Soypass, maize gluten, barley and
molasses was allocated as pellets in a TMR or separately, or ‘as is’ separately. The net PDV and TSP
release of EAA did not increase immediately after feeding high amounts of rumen escape protein as
the EAA release decreased after feeding with the nadir appearing around 3 hours after feeding. The
study indicated that separate allocation of rumen escape protein may result in greater PDV release
of EAA; however, the liver uptake appeared to be of an equivalent magnitude.
Keywords: cattle, splanchnic metabolism, concentrate, processing
Introduction
Recent published research results show that the normal protein deficiency in postpartum transition
cows negatively affect milk production and potentially also basal maternal body functions. Milk
production was increased with around 20% by increasing the protein supply just after calving.
However, initial on-farm tests of increasing the protein supply with rumen escape protein in pelletized
concentrates have indicated difficulties in transferring the research results to practical farming. The
aim of the experiment was to investigate the effect of different allocation strategies and processing
of rumen escape protein concentrates on postprandial patterns of net portal-drained visceral (PDV)
release and liver metabolism of essential amino acids (EAA) in lactating dairy cows.

Six multiparous Danish Holstein cows with indwelling catheters in major splanchnic blood vessels
were used in a replicated 3×3 Latin square design with 14 day periods. Treatments were allocation
of 3.1 kg DM of a protein compound concentrate at each of two daily milking’s in conventional
5 mm pellets in a total mixed ration (TMR-PEL) or separately (SEP-PEL), or in an ‘as is’ form
separately fed (SEP-MÜS). The protein compound concentrate was composed of (g/kg DM) 387
Soypass, 322 rolled barley, 194 maize gluten, and 97 sugar beet molasses. To ease palatability of
the ‘as is’ form, a müsli was prepared by mixing with rolled barley and sugar beet molasses. Cows
were fed to 95% of pre-experimental ad libitum intake with a basal diet (kg DM/day): 6.0 maize
silage, 4.0 grass silage, 2.0 dried sugar beet pulp, 0.5 mineral/vitamin premix, and 0.3 vegetable fat.
Ten hourly simultaneous arterial, portal vein, and hepatic vein samples were collected beginning
½ h prior to feeding at 08.00 h on day 14 of each period. Venous plasma flows were measured by
down-stream dilution of para-aminohippuric acid infused into an upstream vein. The portal catheter
was misplaced in three cows; hence n=3 for PDV and liver data. Statistical analysis was conducted
using the MIXED procedure of SAS with fixed effects of period, treatment, time, and treatment
× time. Square and cow × square were considered as random, and time within cow × period as a
repeated measurement. Treatment was tested with predefined contrasts: TMR versus SEP and PEL
versus MÜS. Significance was declared at P≤0.05 and tendencies at 0.05<P≤0.10.

Dry matter intake averaged (±SE) 19.1±0.1 kg/d. Milk yield averaged 30.6±1.3 kg/d and protein
yield 1,015±15 g/d; neither differed among treatments (P≥0.32). The arterial concentration of group
1 and 2 EAA tended to or were greater with SEP allocation as compared to TMR (P=0.09 and
P=0.02, respectively), but did not differ between PEL and MÜS (P≥0.12). For splanchnic plasma
flows and EAA fluxes (Table 1), no clear interactions between treatment and time were observed
(0.09≤P≤0.97). The net PDV and total splanchnic release of G1 and G2 EAA did not increase after
feeding high amounts of rumen escape protein; in fact the release decreased after feeding with a nadir
appearing around 3 hours after feeding (Figure 1). However, a postprandial increase net PDV release
of EAA of rumen escape feed may have been masked by an even greater postprandial decrease in
net PDV release of EAA from microbial protein. The study indicates that separate allocation rumen
escape protein may result in greater PDV release of EAA (Table 1); however, an equivalent liver
uptake appears.
Table 1. Plasma flows (l/h) and net fluxes (mmol/h) of essential amino acids (EAA) in portal-drained
visceral (PDV), liver (LIV), and total splanchnic (TSP) tissues.
Item Site Treatments3 SEM1 P-values
TMR-PEL SEP-PEL SEP-MÜS TMR vs SEP PEL vs MÜS Time
Plasma flows PDV 1,201 1,105 1,291 125 0.98 0.08 0.26
TSP 1,275 1,327 1,422 84 <0.01 <0.01 <0.01
EAA PDV 195 238 236 40 0.02 0.16 <0.01
LIV -39 -62 -47 36 0.40 0.77 0.34
TSP 167 168 174 8.5 0.63 0.46 <0.01
G12 EAA PDV 68 80 82 12 0.02 0.04 <0.01
LIV -32 -46 -41 13 0.01 0.34 <0.01
TSP 34 33 33 3.1 0.63 0.89 <0.01
G22 EAA PDV 124 152 148 26 0.04 0.23 <0.01
LIV -7.2 -15 -7.0 24 0.80 0.70 0.66
TSP 127 129 134 5.7 0.48 0.37 <0.01
1 Standard error of the mean; n=3 for PDV and LIV, and n=6 for TSP.
2 Group 1 EAA: His, Met, Phe, Trp, and Tyr; Group 2 EAA: Ile, Leu, Lys, and Val.
3 TMR = total mixed ration; PEL = pellets; MÜS = ‘as is’.
A Group 1 EAA B Group 2 EAA
Figure 1. Postprandial net fluxes of essential amino acids (EAA) in group 1 (A; His, Met, Phe, Trp,
and Tyr) and group 2 (B; Ile, Leu, Lys, and Val) for portal-drained visceral (PDV), liver (LIV), and
total splanchnic (TSP) tissues (n=3 for PDV and LIV, and n=6 for TSP).

Effect of the type of dietary fibre in the feed on digestibility and
fermentation parameters in dogs
O. Lasek
Department of Animal Nutrition and Dietetics, University of Agriculture in Krakow, 32-120 Krakow,
Poland; o.lasek@ur.krakow.pl
Abstract
The aim of the current study was to investigate how the type of dietary fibre (from easily fermentable
to non-fermentable) in feed can influence on digestibility and microbiological activity as well as
fermentation parameters in the intestines in dogs. Experiment was divided into four stages using
Graeco – Latin square and was conducted on 8 dogs (mixed breed). Digestibility trial was performed
using indicatory method (with AIA as a marker). Gas production was estimated using in vitro method
(gas test). In conclusion, digestibility of nutrients, kinetics of fermentation gases, total gas production,
maximum gas production, pH value of feaces, in vitro dOM and content of short chain fatty acids
(branched chain fatty acids included) were changing depending on the type of dietary fibre in feed.
Keywords: dogs, fibre, digestibility, fermentation
Introduction
Nowadays, pet foods for dogs are mainly composed of plant components what makes them a
significant source of a dietary fibre for these animals. It is a well known fact that the influence of
dietary fibre on the coefficient of digestibility and the activity of the gut microflora in dogs depends
of its type (Flickinger et al., 2003). Pet food may contain ingredients that cause excess production of
fermentation gases. It has been proven that food with low digestibility, content of high protein, dietary
fibre (readily fermentable) cause an increase in gas production (Sunvold et al., 1995). Therefore the
aim of the study was to determine digestibility of nutrients, gas production, fermentation parameters
(pH, volatile fatty acids (VFA), lactic acids and ammonia) for dogs fed a diet differ in dietary fibre.

8 dogs (mixed breed), with an average body weight of about 30 kg, were in the experiment for 45 days.
Experiment was divided into four stages using Graeco – Latin square. Dogs were randomly allocated
into four dietary groups: (1) without fibre addition; (2) with easily fermentable fibre addition; (3) with
moderately fermentable fibre addition; and (4) with non-fermentable fibre addition. Digestibility trial
was performed using indicatory method (with AIA as a marker), with 7 d. of adaptation and 4 d. of
sampling collection. Gas production was estimated using gas test (RF-Gas Production System) and
the fermentation parameters such as: pH, short chain fatty acids (SCFA), branched chain fatty acids
(BCFA), lactic acid and ammonia concentration, and degradability of organic matter dOM were
determined using the methods described by Flickinger et al. (2003). Chemical composition of dog
diets and faeces were evaluated by standard method (AOAC, 2005). Obtained data were analysed
using Variance Components and Mixed Model ANOVA/ANCOVA (StatSoft, Inc, Version 7.1). The
differences were considered significant at P<0.05. In addition, Pearson’s correlation coefficient was
calculated.
Result and discussion

Chemical analysis showed that pet food varied content of the nutrient primarily crude fibre (P<0.001)
and NDF (P<0.02), ADF (P<0.001), ADL(P<0.001). Digestibility coefficient of nutrients were
significantly higher for dogs fed diet without dietary fibre. The lowest digestibility coefficient of crude

protein (P<0.09) was found in a group fed a diet with addition of moderately fermentable dietary
fibre whereas the lowest digestibility coefficient of crude fat (P<0.001) and energy (P<0.001) was
observed in the group fed a diet with addition of non-fermentable dietary fibre. Maximum production
of gases characterized by the faecal samples of dogs fed diet with non-fermentable fibre (P<0.002).
Similarly, content of fermentation parameters such as the VFA, SCFA, BCFA and ammonia were
the highest in faeces of dogs fed a diet with non-fermentable fibre. Gas production was positively
correlated (P<0.05) with the content of crude fibre (r=0.58), cellulose (r=0.62) and crude protein
(r=0.60) in diet. Furthermore GPmax was correlated with content of butyric acids (r=0.37) and
ammonia (r=0.46) in faeces. Some fermentation product such as SCFA are important energy source
for the colonic mucosa but others like ammonia can have deleterious effect (Flickinger et al., 2003).
In conclusion, digestibility of nutrients, kinetics of fermentation gases, total gas production, maximum
gas production, pH value of feaces, and content of SCFA (BCFA included), ammonia was changing
depending on the type of dietary fibre in diet.
Table 1. Chemical composition of diet for dogs and fermentation parameters.1
Items WF EF MF NF SEM P-value
Chemical composition of dog’s diet

Crude protein, g/kg DM 383.28 391.43 393.03 398.84 2.93 0.34
ME, MJ/kg DM 20.38 19.65 19.62 19.24 0.09 <0.001
Crude fibre g/kg DM 5.02 22.25 18.39 25.25 2.36 <0.001
NDF g/kg DM 41.20 77.22 71.92 89.17 5.39 <0.001
ADF g/kg DM 16.50 47.75 27.60 47.38 4.40 <0.001
ADL g/kg DM 3.18 13.35 3.66 3.97 1.29 <0.001
Fermentation parameters
pH0 6.33 6.22 6.05 6.22 0.05 0.37
pH24 6.34 5.9 5.64 6.13 0.06 <0.001
GPmax, ml/kg DM 7.56 11.52 10.34 14.38 0.69 0.002
VFA0, mmol/kg DM 10.99 15.28 12.51 15.51 0.49 <0.001
ammonia0, mg/kg DM 10.83 13.01 10.09 16.96 0.61 <0.001
VFA24, mmol/kg DM 29.13 28.17 30.74 40.89 1.42 0.014
ammonia24, mg/kg DM 24.04 28.79 22.45 35.18 1.41 0.008
1 WF = diet without fibre; EF = diet with easily fermentable fibre; MF = diet with moderately fermentable fibre; NF =
diet with non-fermentable fibre; ME = metabolisable energy; NDF = neutral detergent fibre; ADF = acid detergent fibre;
ADL = acid detergent lignin; GPmax = maximum gas production; VFA = volatile fatty acids.
References
Association of Official Analytical Chemists (AOAC), 2005. Official methods of analysis (18th Ed.). AOAC, Washington,
DC, USA.
Flickinger, E.A., E.M.W.C. Schreijen, A.R. Patil, H.S. Hussein, C.M. Grieshop, N.R. Merchen and G.C. Fahey Jr.,
2003. Nutrient digestibilities, microbial populations, and protein catabolites as affected by fructan supplementation
of dog diets. Journal of Animal Science 81: 2008-2018.
Sunvold, G.D., G.C. Fahey, N.R. Merchen, E.C. Titgemeyer, L.D. Bourquin, L.L. Bauer and G.A. Reinhart, 1995.
Dietary fiber for dogs: IV. In vitro fermentation of selected fiber sources by dog fecal inoculum and in vivo digestion
and metabolism of fiber-supplemented diets. J. Anim. Sci. 73: 1099-1109.

Digestion pattern in horses of fibre in grass haylage cut at different
stages of maturity
J.E. Lindberg1* and S. Ragnarsson2
1Department of Animal Nutrition and Management, Swedish University of Agricultural Sciences,
P.O. Box 7024, 750 07 Uppsala, Sweden; 2Hólar University Collage, 551 Saudárkrókur, Iceland;
jan.erik.lindberg@slu.se
Abstract
Mature Icelandic geldings were used to study the impact of cutting time of grass haylage on the
digestion pattern of dietary fibre, based on analysis of neutral detergent fibre (NDF) and monomeric
neutral sugars. There were strong correlations between the digestibility of total neutral sugars and
NDF (R2=0.97), of glucose and xylose (R2=0.94), of glucose and arabinose (R2=0.89), and of xylose
and arabinose (R2=0.98). The results indicate that the digestion of cellulose (glucose) and hemi-
cellulose (xylose and arabinose) in grass haylage is linked, and that the digestion follows the same
pattern irrespective of the stage of maturity.
Keywords: Icelandic horses, haylage, grass, fibre, digestibility
Introduction
In practice and in nutrition studies, the fibre content in feeds is often assessed by analyzing for neutral
detergent fibre (NDF). However, the NDF fraction will only comprise the insoluble fibre components
while the water soluble fibre components will be lost during the analysis (Bach Knudsen, 2001).
In contrast, enzymatic-chemical procedures used to assess the dietary fibre (DF) content of feeds
provide information on the monomeric composition of the non-starch polysaccharide (NSP) fraction
of the DF (Bach Knudsen, 2001). Therefore, it is of interest to examine if the digestions pattern of
total and individual neutral sugars in the NSP fraction of the feed differs from that of NDF.

Mature Icelandic geldings, fed at maintenance level of energy intake, were used to study the influence
of cutting time of timothy (Pleum pratence L.) haylage (Ragnarsson and Lindberg, 2008) and of mixed
grass haylage (Ragnarsson and Lindberg, 2010) on the coefficient of total tract apparent digestibility
(CTTAD) of dietary fibre. The grass species composition of the mixed grass haylage differed but was
dominated by Poa pratensis, Agrostis capillaris, Festuca richardsonii and Deschampsia caespitosa.
The two experiments were arranged as 4×4 balanced change-over designs.
All forages were plastic wrapped and wilted, and baled in large (250-450 kg) round or square bales
and fed straight out of the bales in long form. The forage-only diets were fed for a total of 20 days,
comprising 14 days of adaptation and 6 days for total collection of faeces. During adaptation periods
horses had access to salt lick stones and were fed minerals during non-collection periods.
NDF was analysed according to Chai and Udén (1998) using undiluted ND solution, sodium sulphate
and amylase. Individual monomeric neutral sugars were analysed as described by Bach Knudsen
(1997).

The content of fibre increased and crude protein content decreased with advancing stage of maturity.
The total neutral sugars (TNS) made up 410 to 489 g/kg dry matter (DM) in timothy haylage and

408 to 437 g/kg DM in mixed grass haylage. Bach Knudsen (1997) reported 366 to 426 g/kg DM
of TNS in grass meal from 1st, 2nd and 3rd cut. The major neutral sugars comprised 95.2 to 97.1% of
TNS in timothy haylage and 94.1 to 94.7% of TNS in mixed grass haylage, which was comparable
to grass meal (Bach Knudsen, 1997). The major individual neutral sugars in the fibre fraction of both
timothy and mixed grass haylage were glucose (53-57%), xylose (30-34%) and arabinose (7-9%),
which was in good agreement with grass meal (Bach Knudsen, 1997).
The CTTAD of NDF, TNS, glucose, xylose and arabinose declined (P<0.001) with advancing
maturity in both timothy haylage and mixed grass haylage. The fibre digestibility data from the two
experiments were pooled and correlations were calculated to reveal any potential common digestion
pattern. Interestingly, there were very strong correlations between the CTTAD of TNS and NDF
(R2=0.97), of glucose and xylose (R2=0.94), of glucose and arabinose (R2=0.89), and of xylose and
arabinose (R2=0.98) for the pooled data (n=8).
Thus, the results indicate that the digestion of cellulose (glucose) and hemi-cellulose (xylose and
arabinose) in grass is linked, and that the digestion follows the same pattern irrespective of the stage
of maturity. Moreover, the amount of individual sugars released during digestion from the fibre
fraction in grass will be determined by their respective proportion of the TNS.
Conclusion
NDF digestibility in grass haylage in horses will reflect the digestion pattern of total and individual
monomeric neutral sugars in the fibre fraction.
References
Bach Knudsen, K.E., 1997. Carbohydrate and lignin contents of plant materials used in animal feeding. Animal Feed
Science and Technology 67: 319-338.
Bach Knudsen, K.E., 2001. The nutritional significance of ‘dietary fibre’ analysis. Animal Feed Science and Technology
90: 3-20.
Chai, W. and P. Udén, 1998. An alternative oven method combined with different detergent strengths in the analysis
of neutral detergent fiber. Animal Feed Science and Technology 74: 281-288.
Ragnarsson, S. and J.E. Lindberg, 2008. Nutritional value of timothy haylage in Icelandic horses. Livestock Science
113: 202-208.
Ragnarsson, S. and J.E. Lindberg, 2010. Nutritional value of mixed grass haylage in Icelandic horses. Livestock
Science 131: 83-87.

Effect of the site of starch infusion on urea kinetics in dairy cows
D.R. Ouellet*, F. Hassanat and H. Lapierre
Agriculture and Agri-Food Canada, 2000 College St., Sherbrooke, QC, J1M 0C8, Canada;
daniel.ouellet@agr.gc.ca
Abstract
Our objective was to determine the impact of the site of starch infusion on urea kinetics in dairy
cows. Four cows were used in a randomized complete block design with 3 periods of 28 d. To the
total mixed ration providing 2.59±0.21 kg/d of starch was added continuous starch infusion (1.80
kg/d) either in the rumen, the abomasum, or equally partitioned in both sites (Mix). Cows were
continuously infused in a jugular vein [15N15N]urea for 72 h (d13-16): on day 16, urine and faeces
were collected to determine 15N enrichment to estimate urea kinetics. From d 13-19, total collection
of urine and faeces was performed and milk was sampled. Milk yield (30.3b, 26.9a, 32.0b±1.6 kg/d)
was lowest for abomasal infusion whereas milk composition was not affected by treatment. Total
tract starch (99.5a, 94.1b, 99.1a±1.1%) and N (75.9a, 70.5b, 71.7b±0.5%) apparent digestibilities were,
respectively, reduced with abomasal and abomasal and Mix starch infusion. Urea-N production,
urea-N gut entry rate, urinary urea-N excretion, urea-N loss to faeces, and urea-N re-use for anabolism
were not affected by infusion site. Return of urea-N through the ornithine cycle (114a, 84b, 83b±5
g/d) was highest when starch was totally infused into the rumen. Important amount of starch to be
digested in the small intestine penalized milk yield, reduced apparent digestibility of starch and N but
had limited effect on urea kinetics except a reduction of the return of urea gut entry to ornithine cycle.
Keywords: dairy cattle, urea kinetics, starch infusion
Introduction
High producing dairy cows require important amount of glucose to support maintenance and
productive functions. Starch is the major source of glucose precursors via rumen fermentation into
propionate or via direct intestinal absorption of glucose. However, small intestine capacity to digest
starch is limited and excess supply would either be fermented in the hindgut or excreted in faeces
(Owen et al., 1986). Extensive hindgut fermentation can impact N metabolism (Reynolds, 2006).
Thus, the objective of this study was to determine the impact of the site of starch infusion on urea
kinetics in dairy cows.

Four mid-lactation cows fitted with ruminal fistula were used in a randomized complete block design
with 3 periods of 28 d. Cows were fed a total mixed ration providing 2.59±0.21 kg/d of starch at
16% crude protein. Treatments were continuous starch infusion (1.80 kg/d) either in the rumen,
the abomasum, or equally partitioned in both sites (Mix). From d 13-16, cows were continuously
infused in a jugular vein [15N15N]urea (0.5 mmol/h). On d 16, urine and faeces spot samples were
collected after 72 h of infusion; enrichment of [15N15N]- and [15N14N]urea in urine and of total 15N in
faeces was determined to estimate urea kinetics (Lobley et al., 2000). From d 13-19, total collection
of urine and faeces was performed and milk was sampled. Data were analysed using the MIXED
procedure of SAS. Differences between treatments were determined using adjusted Tukey-Kramer
multiple comparisons test.

Data are presented as LSMeans for rumen, abomasal, Mix, respectively ± SEM. Milk yield (30.3ab,
26.9a, 32.0b±1.6 kg/d) was lowest for abomasal treatment. Milk composition was not affected by

treatment (data not shown) and milk lactose yield decreased with abomasal infusion (1,339ab, 1,205b,
1,442a±74 g/d). Total tract starch apparent digestibility (99.5a, 94.1b, 99.1a±1.1%) and apparent
digestibility of N (75.9a, 70.5b, 71.7b±0.5%) were reduced with abomasal and abomasal and Mix
starch infusion, respectively. Urea-N production, gut entry rate, urinary excretion, loss to faeces,
and re-use for anabolism were not affected by infusion site (Table 1). Return of urea-N through
the ornithine cycle (114a, 84b, 83b±5 g/d) was highest when starch was infused into the rumen;
however, the proportion of urea-N that return to ornithine cycle over gut urea-N entry rate (r) was
not affected by treatments.
Table 1. Effect of site of starch infusion on urinary and faecal N excretion, milk N, and urea kinetics
in lactating dairy cows.
Parameter, g N/d Site of starch infusion SEM
Rumen Abomasal Mix
N intake 448 444 432 26

Urinary N 155 135 132 15
Faecal N 108 131 122 6
Milk N 158 145 161 14
UER1 260 243 219 15
UUN2 60 67 55 6
GER3 200 176 165 14
ROC4 114a 84b 83b 5
UFE5 9 13 13 2
ANABOL6 77 79 68 10
r, % GER7 57 48 50 2
f, % GER8 5 7 8 1
a, % GER9 38 44 41 3
1 Urea-N production.
2 Urinary urea-N excretion.
3 Gut urea-N entry rate.
4 N from urea returning to the ornithine cycle.
5 N from urea excreted in the faeces.
6 N from the urea used for anabolic purposes.
7 ROC/GE.
8 UFE/GER.
9 ANABOL/GER.
Conclusion
Important amount of starch to be digested in the small intestine penalized milk yield, reduced
apparent digestibility of N and had limited effect on urea recycling although return of gut entry to
ornithine cycle was reduced.
References
Lobley, G.E., B.M. Bremner and G. Zuur, 2000. Effects of diet quality on urea fates in sheep as assessed by refined,
non-invasive [15N15N]urea kinetics. British Journal of Nutrition 84: 459-468.
Owen, F.N., R.A. Zinn and Y.K. Kim, 1986. Limits to starch digestion in the ruminant small intestines. Journal of
Reynolds, C.K., 2006. Production and metabolic effects of site of starch digestion in dairy cattle. Animal Feed Science
and Technology 130: 78-94.

Effect of pelleted feed use for Addax nasomaculatus on feed intake and
nutrient digestibility
M. Przybyło1, P. Tyl1, J. Kański1, A. Kloska2 and P. Górka1*
1Department of Animal Nutrition and Dietetics, University of Agriculture in Krakow, 30-059 Krakow,
Poland; 2Silesian Zoological Garden, Promenada gen. Jerzego Zietka 7, 41-500 Chorzow, Poland;
p.gorka@ur.krakow.pl
Abstract
The aim of this study was to determine the effect of pelleted cereal-based feed use in the diet for
addax antelope (Addax nasomaculatus) on feed intake and nutrient digestibility. The animals were
allocated to 1 of 3 treatments according to 3×3 Latin square and fed a basal diet consisting of a
mixture of ground concentrates, chopped dehydrated lucerne, vegetables and fruits (DIET A) or a
basal diet where 50% (DIET B) or 100% (DIET C) of dry matter (DM) provided with concentrates
and chopped dehydrated lucerne was replaced with a pelleted cereal-based feed. Dry matter intake
of basal diet was not different between treatments whereas hay intake and total DM intake linearly
decreased with increasing pellet inclusion in the basal diet. Cell wall digestibility tended to linearly
decrease and hemicellulose digestibility linearly decreased with increasing pellet inclusion in the
diet. This study indicates that pelleted cereal-based feed use in the diet for addax may negatively
affect feed intake and decrease nutrient digestibility.
Keywords: wild ruminant, concentrate, zoo, health
Introduction
Pelleted cereal-based supplements are commonly used in the diets for wild captive ruminants.
However, it has been shown that pelleting of feeds affect feeding behaviour and gastrointestinal tract
function in livestock ruminants (Bertipaglia et al., 2010; Waghorn and Reid, 1983). Nevertheless,
little is known about effects of pelleted cereal-based feed use in the diets for wild captive ruminants,
such as addax antylope (Addax nasomaculatus). The aim of this study was to determine the effect of
pelleted cereal-based feed use in the diet for addax antelope on feed intake and nutrient digestibility.

Four females of addax were allocated to 1 of 3 treatments according to 3×3 Latin square design with
experimental period lasted 28 days. Thorough whole study period two females were kept together
and thus pen was used as an experimental unit in this study. Animals were fed a typical diet used
for addax in Silesia Zoological Garden (Chorzów, Poland) that consisted of basal diet (a mixture of
ground concentrates, chopped dehydrated lucerne, vegetables and fruits; DIET A) fed at 2.25 kg/day/
animal (as fed) with additional ad libitum access to grass hay. Dietary treatments were applied by
replacing 50% (DIET B) or 100% (DIET C) of concentrates and chopped dehydrated lucerne in the
basal diet with pelleted cereal-based feed. Feed intake was controlled daily. The diets were formulated
to be similar for crude protein (CP) and crude fibre content. Apparent total tract digestibility was
calculated using acid insoluble ash as a digesta marker. Data were analysed as Latin square design
using the MIXED procedure of SAS (version 9.2).

Dry matter intake of basal diet was not different between treatments (Table 1). However, hay
intake and total DM intake linearly decreased with increasing pellet inclusion in the diet, leading
to linear decrease of CP, cellulose and non-fibrous carbohydrates (NFC) intake. Apparent total tract

digestibility of organic matter, CP and NFC was no different between treatments. However, cell
wall constituents digestibility tended to linearly decrease and hemicellulose digestibility linearly
decreased with increasing pellet inclusion in the diet. In livestock ruminants pelleted feed use in the
diet increases feeding rate and decreases rumination time as well as decreases digesta retention time
in the rumen and ruminal and total tract digestibility of fibre (Waghorn and Reid, 1983). Furthermore,
pelleting of cereal-based feeds affect starch fermentability in the rumen and in consequence may
affect rumen environment and nutrient digestion (Bertipaglia et al., 2010). This study indicates that
pelleted cereal-based feed use in the diet for addax, large Sahara desert grazer with high proportion
of monocots in the natural diet (80%), may negatively affect feed intake and nutrient digestibility.
Table 1. The effect of pelleted cereal-based feed inclusion in the diet for Addax nasomaculatus on
feed intake and nutrient digestibility.
Item Treatment1 SEM Contrasts
DIET A DIET B DIET C Linear Quadratic
Dry matter intake, kg/day

Basal diet 1.20 1.22 1.16 0.01 0.64 0.52
Hay 1.33 1.14 1.08 0.03 0.05 0.47
Total 2.53 2.36 2.25 0.03 0.03 0.76
Apparent digestibility, %
Organic matter 60.6 56.4 53.6 3.6 0.17 0.90
Crude protein 60.7 56.9 54.8 2.9 0.12 0.77
Crude fat 67.5 54.7 42.9 8.6 0.24 0.98
Cell wall constituents 40.3 31.8 28.3 5.9 0.09 0.58
Hemicelluloses 59.3 51.1 44.7 5.9 0.05 0.99
Cellulose 40.0 31.8 31.9 5.7 0.12 0.43
Non-fibrous carbohydrates 84.0 84.9 84.3 1.1 0.84 0.63
1 Treatment: DIET A = basal diet without pelleted cereal-based feed inclusion; DIET B = 50% of concentrates and chopped
dehydrated lucerne in the basal diet DM replaced with pelleted cereal-based feed; DIET C = 100% of concentrates and
chopped dehydrated lucerne in the basal diet DM replaced with pelleted cereal-based feed.
References
Bertipaglia, L.M.A., M. Fondevila, H. van Laar and C. Castrillo, 2010. Effect of pelleting and pellet size of a concentrate
for intensively reared beef cattle on in vitro fermentation by two different approaches. Anim. Feed Sci. Tech.
159: 88-95.
Waghorn, G.C. and C.S.W. Reid, 1983. Rumen motility in sheep and cattle given different diets. New Zealand J.
Agirc. Res. 26: 289-295.

Lactobacillus buchneri on sugarcane silage fermentation and
performance of cattle in Brazil: a meta-analysis
C.H.S. Rabelo*, C.J. Härter and R.A. Reis
UNESP, São Paulo State University, Department of Animal Science, Jaboticabal, São Paulo, 14884-
900, Brazil; carlos.zoo@hotmail.com
Abstract
Lactobacillus buchneri is a heterofermenter lactic-acid bacterium widely used to enhance aerobic
stability of silages, which has been related with improvements on animal performance in some
cases. The objective of this study was to investigate the impact of L. buchneri on sugarcane silage
fermentation and performance of cattle in Brazil through a meta-analysis. Two data base were
used to investigate the effect of L. buchneri on the silage fermentation and cattle performance. L.
buchneri altered the fermentation patterns in sugarcane silages leading an increase on body gain in
cattle fed inoculated silage.
Keywords: digestibility, intake, lactic-acid bacteria, sugarcane
Introduction
Lactobacillus buchneri is a heterofermenter lactic-acid bacterium used in order to enhance the
aerobic stability of silages (Kleinschmit and Kung, 2006), which also has been related to increases
on the microbial protein supply and body gain in ruminants (Basso et al., 2014). Our objective was
to investigate the impact of L. buchneri on sugarcane silage fermentation and performance of cattle
in Brazil through a meta-analysis.

A database compiled of 75 treatments means from 35 studies that evaluated sugarcane silage treated
with L. buchneri was used to investigate the silage fermentation. The application rate of L. buchneri
ranged from 0 to 2.5×1010 cfu/g of fresh sugarcane, and the treatments were classified into the
following categories: (1) sugarcane silage with no inoculant added (untreated); (2) sugarcane silage
treated with L. buchneri at <1×105 cfu/g of fresh forage (low inoculant); and (3) sugarcane silage
treated with L. buchneri at ≥1×105 cfu/g of fresh forage (high inoculant). In addition, a second
database compiled of 19 treatments means from 6 studies that evaluated sugarcane silage treated with
L. buchneri was used to investigate the cattle performance. The treatments were classified only into
two categories: (1) sugarcane silage without inoculant (untreated); and (2) sugarcane silage treated
with L. buchneri (inoculated). A minimum of two studies per each treatment was the prerequisite
for keeping the dependent variables in the final database. Data were analysed using the MIXED
procedure of SAS (v. 9.4).

Concentrations of lactic, propionic, butyric, and total acids were unaffected by L. buchneri (P>0.05),
as well as ammonia-N (Table 1). Acetic acid concentration increased (P<0.05) in L. buchneri treated-
silages because this bacterium anaerobically converts lactic acid to acetic acid. The greater dose of
L. buchneri reduced (P<0.01) the ethanol concentration by 28.47% compared to untreated silage.
In vitro dry matter digestibility of sugarcane silages increased 8.94% (P<0.01) when the lower dose
of L. buchneri was used.

Table 1. The effects of Lactobacillus buchneri on fermentation and in vitro dry matter digestibility
(IVDMD) of sugarcane silage (data are given in g/kg of DM, unless otherwise stated).1
Item Untreated Low inoculant High inoculant P-value
n Mean SEM n Mean SEM n Mean SEM
Lactic acid 20 29.4a 4.73 11 28.3a 4.96 15 32.5a 4.92 0.3131

Acetic acid 20 33.7b 3.97 10 45.9a 5.11 17 42.2a 4.45 0.0304
Propionic acid 18 4.09a 2.01 9 4.15a 2.45 16 7.62a 2.18 0.1775
Butyric acid 12 0.91a 0.25 7 0.89a 0.31 9 0.51a 0.29 0.4272
Ethanol 20 82.2a 14.27 12 76.8ab 14.78 16 58.8b 14.72 0.0034
Ammonia-N2 10 60.5a 15.12 6 65.4a 15.69 5 55.2a 16.56 0.6660
IVDMD 18 472.2b 21.72 12 514.4a 22.73 7 484.3ab 24.53 0.0074
1 a-b means in the same row with different superscripts differed (P<0.05).
2 g/kg of total N.
Dry matter (DM) and crude protein intake were unaffected (P>0.05) by inoculation (Table 2).
Likewise, inoculation did not affect (P>0.05) DM and organic matter digestibility, and also the feed
efficiency. However, inoculation increased (P<0.05) the average daily gain of cattle likely because
the alterations in fermentation profile led by L. buchneri. Data of this meta-analysis suggest that
cattle fed inoculated silage likely uses protein and energy most efficiently than cattle fed untreated
silage. Improvements on the microbial protein yield and body gain has been reported in ruminants
fed silages inoculated with L. buchneri (Basso et al., 2014).
Table 2. The effects of Lactobacillus buchneri on cattle performance.1,2
Item Untreated Inoculated P-value
n Mean SEM n Mean SEM
DM intake, kg/d 8 9.81 0.86 10 9.93 0.86 0.7244

CP intake, kg/d 3 1.62 0.26 3 1.57 0.26 0.4669
DM digestibility, % 3 57.2 7.36 3 54.1 7.36 0.4291
OM digestibility, % 3 59.4 7.29 3 57.3 7.29 0.5052
Feed efficiency 2 7.54 1.37 2 7.09 1.37 0.7698
ADG, kg/d 3 1.04b 0.18 4 1.28a 0.17 0.0107
1 a-b means in the same row with different superscripts differed (P<0.05).
2 DM = dry matter; CP = crude protein; OM = organic matter; ADG = average daily gain.
References
Basso, F.C., A.T. Adesogan, E.C. Lara, C.H.S. Rabelo, T.T. Berchielli, I.A.M.A. Teixeira, G.R. Siqueira and R.A. Reis,
2014. Effects of feeding corn silage inoculated with microbial additives on the ruminal fermentation, microbial
protein yield, and growth performance of lambs. Journal of Animal Science 92: 5640-5650.
Kleinschimit, D.H. and L. Kung Jr., 2006. A meta-analysis of the effects of Lactobacillus buchneri on the fermentation
and aerobic stability of corn and grass and small-grain silages. Journal of Dairy Science 89: 4005-4013.

Lactobacillus buchneri applied in corn silage or directly into the rumen
of wethers: effect on apparent digestibility and ruminal fermentation
C.H.S. Rabelo*, L.G.O. Jorge, F.C. Basso, E.C. Lara, C.J. Härter, L.M. Delevatti and R.A. Reis
UNESP, São Paulo State University, Department of Animal Science, Jaboticabal, São Paulo, 14884-
900, Brazil; carlos.zoo@hotmail.com
Abstract
Bacterial inoculants are used in order to improve the fermentation or aerobic stability of silage, or
both. Enhanced performance has been reported in animals feeding inoculated silage. The purpose of
this study was to investigate the impact of Lactobacillus buchneri applied in corn silage or directly
into the rumen of wethers on the apparent digestibility and ruminal fermentation. Three treatments
were evaluated: (1) wethers fed untreated silage (control); (2) wethers fed the inoculated silage
(inoculated); and (3) wethers fed untreated silage and receiving a daily dose of L. buchneri directly
into the rumen (1×107 cfu of L. buchneri/g of provided silage [LB rumen]). L. buchneri applied
in corn silage or directly into the rumen unaffected apparent digestibility and production of total
volatile fatty acids.
Keywords: bacterial inoculation, protein, volatile fatty acid
Introduction
Improvements on the microbial protein yield and body gain has been reported in ruminants fed silages
inoculated with Lactobacillus buchneri, a heterofermenter lactic-acid bacterium able in enhancing
the aerobic stability of silages (Basso et al., 2014). However, few studies were carried out to evaluate
the influence of L. buchneri on the digestion and metabolism in the rumen. Our objective was to
investigate the impact of L. buchneri applied in corn silage or directly into the rumen of wethers on
the apparent digestibility and ruminal fermentation.

Corn silage was treated with water (2 l/t; control) or with L. buchneri CNCM I-4323 at 1×105 cfu/g
of fresh forage (Lallemand Animal Nutrition, Milwaukee, WI, USA; inoculated). Four silos per each
treatment (control and inoculated) were filled with 350 kg of corn forage and remained closed for
229 d. We evaluated three treatments: (1) wethers fed untreated silage (control); (2) wethers fed the
inoculated silage (inoculated); and (3) wethers fed untreated silage and receiving a daily dose of L.
buchneri directly into the rumen (1×107 cfu of L. buchneri/g of provided silage [LB rumen]). For
this, six Dorper × Santa Ines crossbred ruminally-cannulated wethers, each fitted with a silicone,
2.5-inch ruminal cannula were used. Wethers were fed 70% of corn silage and 30% of concentrate
composed of soybean meal and ground corn, on a dry matter (DM) basis. Digestibility measurements
were taken over 3 experimental 17-d periods, each consisting of 10 d for diet adaptation and 5 d for
the digestibility assay. In the 17th d of each period we collected the ruminal fluid prior to feeding
animals (time 0 h) and after 6, 12, and 18 h. Apparent digestibility was analysed as a double 3×3
Latin square design, whereas ruminal fermentation was analysed as repeated measurements over
time, both using the MIXED procedure of SAS (v. 9.4). Differences between means were determined
using the PDIFF, which differentiates means based on Fisher´s F-protected least significant difference
test. Significance was declared at P ≤ 0.05.

Apparent digestibility of DM, organic matter, crude protein, and neutral detergent fibre was unaffected
(P>0.05) by application of L. buchneri in corn silage or directly into the rumen (Table 1).
Except butyrate, all variables regarding ruminal fermentation were unaffected (P>0.05) by treatments
(Table 2). Positive results from inoculation of corn silage with L. buchneri were not observed in this
study, likely because the similar characteristics of untreated and inoculated silages, and also by lack
of differences in ruminal community of bacteria (unpublished data).
Table 1. Impact of Lactobacillus buchneri (LB) applied in corn silage or directly into the rumen of
wethers on the apparent digestibility (g/kg).1
Item Control Inoculated LB rumen SEM P-value
DM 0.879 0.885 0.887 0.03 0.9792

OM 0.883 0.890 0.898 0.03 0.9387
CP 0.835 0.840 0.852 0.03 0.9469
NDF 0.653 0.681 0.655 0.09 0.9668
1 DM = dry matter; OM = organic matter; CP = crude protein; NDF = neutral detergent fibre.
Table 2. Impact of Lactobacillus buchneri (LB) applied in corn silage or directly into the rumen of
wethers on the ruminal fermentation profile (mMol/l).1
Item Treatments (T) SEM P-value
Control Inoculated LB rumen T Hour (H) T×H
pH 6.21 6.16 6.15 0.196 0.5254 <0.0001 0.9885

Total VFA2 58.82 60.00 56.50 6.966 0.4214 0.0053 0.3913
Acetate 44.10 42.96 40.67 4.935 0.4122 0.0087 0.3927
Propionate 8.28 8.83 7.91 2.867 0.6605 0.0014 0.5517
Butyrate 7.88a 6.44b 5.90b 0.666 0.0113 0.0323 <0.001
AC:P ratio3 5.91 5.53 6.31 1.251 0.3476 0.0165 0.2407
1 a-b
means in the same row with different superscripts differed (P<0.05).
2 VFA = volatile fatty acids.
3 Acetate:propionate ratio.
Acknowledgements
The authors are grateful to São Paulo Research Foundation – FAPESP for its financial support
(Project no. 2012/25463-0).
References
Basso, F.C., A.T. Adesogan, E.C. Lara, C.H.S. Rabelo, T.T. Berchielli, I.A.M.A. Teixeira, G.R. Siqueira and R.A. Reis,
2014. Effects of feeding corn silage inoculated with microbial additives on the ruminal fermentation, microbial
protein yield, and growth performance of lambs. Journal of Animal Science 92: 5640-5650.

Effects of the level of soybean oil on the kinetics of fibre digestion in
dairy cows fed sugarcane based diets
J.P.P. Rodrigues1, R.M. de Paula1, L.N. Rennó1, M.M.S. Fontes1, P.P. Rotta1*, S.C. Valadares Filho1,
P. Huhtanen2 and M.I. Marcondes1
1Department of Animal Science, Universidade Federal de Viçosa, Viçosa, 36570-900, Brazil;
2Department of Animal Science, Swedish University of Agricultural Sciences, 90183 Umeå, Sweden;
polyana.rotta@ufv.br
Abstract
Our objective was to quantify the effects of soybean oil (SBO) inclusion in high concentrate diets with
sugarcane as main forage for dairy cows. Duplicated 4×4 Latin square design was conducted with
8 rumen-cannulated Holstein cows averaging 122±6.9 days in milk. Animals were fed sugarcane-
based diets with a roughage to concentrate ratio of 40:60. Four levels of refined SBO were used
to increase dietary ether extract (EE) concentrations [g/kg dry matter (DM)]: baseline (without
SBO), low level (40 g EE/kg DM), medium level (70 g EE/kg DM), and high level (100 g EE/kg
DM). Rumen evacuations were conducted and the omasal flow of nutrients was estimated by using
the double marker system (Co-EDTA and indigestible neutral detergent fibre (iNDF)). The SBO
inclusion did not affected the passage rate of potential digestible neutral detergent fibre (pdNDF).
The digestion rate of pdNDF decreased linearly with SBO level. The passage rate of iNDF varied
quadratically with the SBO inclusion rate with the highest rate at medium level. The reduction in fibre
digestibility was more associated with the reduction of digestion rate than effects of passage rate.
Keywords: digestion rate, fat supplementation, passage rate, rumen pool
Introduction
The sugarcane fibre have a high proportion of lignin and low pdNDF, causing low dry matter
(DM) intake when compared to corn silage. When formulating diets for high-producing dairy cows
using sugarcane as main roughage it is necessary to increase the amount of concentrate in the diet.
In this way, diets with sugar cane present a high proportion of non-fibre carbohydrates (NFC) in
the diet and thus, rumen acidosis. Although the knowledge regarding to the negative effects of fat
supplementation on fibre digestibility and DM intake, the inclusion of fat in sugarcane diets for high
yield dairy cows can be an option to diminish the high NFC content and improve energy intake. In
these diets, the replacement of NFC by fat supplements can be an option to reduce rumen problems
such as acidosis and low fibre digestion. Plant oils are one of the most considerable rumen-active fat
supplements, because of its high rumen release when compared to seeds or rumen protected fats and
high concentration of unsaturated FA. We hypothesized that the soybean oil (SBO) supplementation
when used in low levels can improve the fibre digestibility, whereas high levels can decrease the
fibre digestion. Based on such hypothesis, our objective was to quantify the effects of SBO inclusion
in fibre digestion kinetics in dairy cows fed sugarcane-based diets.

We used a duplicated 4×4 Latin squares design with 8 rumen-cannulated Holstein cows in mid-
lactation (574±19.1 kg BW, 122±6.9 days in milk), averaging 22.5±1.22 kg/d of milk production.
The experimental period had 21 days, with 14 days for adaptation followed by a sampling period
from day 15 to 21. Animals were fed sugarcane based diets with roughage to concentrate ratio of
40:60. Four levels of refined SBO were used to increase dietary ether extract (EE) concentrations
[g/kg DM]: baseline (without SBO), low level (40 g EE/kg DM), medium level (ML; 70 g EE/kg
DM), and high level (HL; 100 g EE/kg DM). In each period, we conducted rumen evacuations on
day 19, 4 hours after feeding, and 2 hours before feeding on day 21. To estimate the omasal flow

we collected omasal digesta samples. The omasal flow was estimated by using the double marker
(Co-EDTA and iNDF) system. Digestion kinetics were calculated as: ingestion rate (ki; %/h) =
(100/24) × (intake/rumen pool), passage rate (kp; %/h) = (100/24) × (omasal flow/rumen pool), rate
of digestion (kd; %/h) = ki – kp. The statistical analysis were conducted considering the random
effects of square, period within square, cow within square, and the model error and the fixed effect
of treatment. The effects of EE levels were tested with polynomial linear and quadratic contrasts. The
PROC IML’s ORPOL function was used to obtain the appropriate coefficients to the CONTRAST
statements. Significance of the effect was declared at P<0.05 and P-values from 0.05 to 0.10 were
considered as trend.

Increasing SBO levels did not affect (P>0.10) the kp of pdNDF. The kd of pdNDF decreased (P<0.01)
55% when comparing the BS and HL diets (Table 1). The rate of passage of iNDF was quadratically
affected by the SBO inclusion (P<0.05), with highest values in ML diet and the lowest kp was verified
in the HL diet (Table 1). The reduction in digestion rate of pdNDF indicates that the reduction in
fibre digestibility is more associated with the effectiveness of fibre digestion by microbes than
the passage rate in the rumen. The reduction in cellulolytic bacteria and protozoa count in cows
fed 40 g/kg of soybean and linseed oil mixture (1:1 g/g) by direct inhibition and/or coating action
was reported by Yang et al. (2009). Increasing SBO supplementation reduces the digestion rate of
pdNDF. The reduction in fibre digestibility is more associated with the reduction in digestion rate
than effects on passage rate.
Table 1. Rumen pool and kinetics of fibre fractions in dairy cows fed sugarcane based diets with
different levels of soybean oil.
Treatment1 SEM Contrast
BL LL ML HL Lin. Quad.
Pool, kg
Total, as fed 93.6 87.9 85.7 77.6 3.32 <0.01 0.87
DM 10.9 10.6 9.9 9.4 0.59 <0.01 0.92
pdNDF
ki, %/h 5.43 5.20 5.21 3.74 0.466 <0.01 0.13
kp, %/h 2.30 2.16 2.68 2.35 0.307 0.56 0.52
kd, %/h 3.12 3.04 2.56 1.40 0.427 <0.01 0.12
iNDF
kp, %/h 2.41 2.36 2.61 2.25 0.170 0.41 0.02
1Treatment: BL = baseline level – without soybean oil; LL = low level – soybean oil added to reach 40 g EE/kg DM;
ML = medium level – soybean oil added to reach 70 g EE/kg DM; HL = high level – soybean oil added to reach 100
g EE/kg DM.
Acknowledgements
This study was funded by Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq)
through to funding field and laboratory activities (479384/2013-4) and FAPEMIG.
References
Yang, S.L., D.P. Bu, J.Q. Wang, Z.Y. Hu, D. Li, H.Y. Wei, L.Y. Zhou and J.J. Loor, 2009. Soybean oil and linseed oil
supplementation affect profiles of ruminal microorganisms in dairy cows. Animal 3: 1562-1569.

Effects of metabolisable energy and crude protein levels in balanced
digestible essential amino acids on production performances and egg
quality of Brown laying hens in the late phase of production
K. Soisuwan* and N. Chauychuwong
Department of Animal Science, Faculty of Agriculture, Rajamangala University of Technology
Srivijaya, Thungsong, Nakhon Si Thammarat 80110, Thailand; ksoisuwan52@gmail.com
Abstract
A 4×3 factorial experiment with 4 dietary metabolisable energy (ME) levels (2,500, 2,600, 2,700
and 2,800 kcal ME/kg) and 3 crude protein (CP) levels (16, 17 and 18%). A total of 720 commercial
Lohmann Brown laying hens, 50 weeks of age, were randomly assigned into 12 treatments (5
replicates with 12 birds per replicate). Feed and water were provided ad libitum throughout the
experiment (16 weeks). The result shown that ME had no effect on EW whereas EW increased
(P<0.05) with CP levels increased from 16.0 to 18.0% CP. The egg quality parameters in this
experiment, only YC increased (P<0.05) with dietary ME increased while YP decreased and AP
increased with dietary CP levels increased (P<0.05). Based on the data under this experimental
condition it was concluded that 2,700 kcal ME/kg feed and 17% CP are the optimum levels for
improving production performances and egg quality of brown laying hens in the late phase of
production in the balanced dietary digestible essential amino acids.
Keywords: metabolisable energy, crude protein, digestible essential amino acids, production
performance, egg quality
Introduction
Dietary metabolisable energy (ME) and crude protein (CP) are the major nutritional parameters of
the diets of laying hens (Lesson and Summers, 2005). The objective of this study was to evaluate
the effect of differing dietary metabolisable energy and crude protein levels in balanced digestible
essential amino acids on production performances and egg quality of brown laying hens in the late
phase of production.

720 Lohmann Brown laying hens in five replicates per treatment (12 birds/replicate) were used.
Twelve experimental diets which contained of 4 levels of dietary energy levels (2,500, 2,600, 2,700
and 2,800 kcal ME/kg feed) and 3 levels of crude protein (16, 17 and 18%). All dietary treatments
were formulated base on ideal protein concept to balance digestible essential amino acids while
another nutrient content of the recommendation were met by of NRC (1994). Birds were raised in
laying cage and received 16-h constant lighting regime throughout the experimental period from
50 to 66 weeks of age and received feed in mash form with feed and water were provided for ad
libitum consumption.
Data were subjected to ANOVA using GLM procedures as suggested by Khunthum (2006) as 4×3
factorial arrangement of dietary treatments with ME, CP as main effects. Two-way interaction
between ME and CP and time periods were not significant (P>0.05); thus, data were pooled across
period and analysed for main effects. Least significant difference comparison were made between
treatment means for main effects when there was a significantly different at P<0.05.

Results and discussions
It was found that EP, EM and FCR were affected (P<0.05) by the ME and CP levels. At medium
and high CP levels (17.0 and 18.0%) and ME levels (2,700 and 2,800 Kcal/kg) improved (P<0.05)
EP, EM and FCR while FI decreased (P<0.05) when increased ME from 2,500 to 2,700 Kcal/kg.
However, a further increase of ME up to 2,800 Kcal/kg had no effect on FI and FCR (P>0.05). The
egg quality parameters in this study, only YC increased (P<0.05) with dietary ME increased. YP
decreased and AP increased with dietary CP levels increased (P<0.05). Lesson et al. (2001) have
observed that birds can tolerate low-density CP and ME diets as long as the nutrients are balanced.
Dietary ME and CP in this study had no adverse effect (P>0.05) on mortality (data had not shown).
Conclusions
The present study has shown that it can be used the diets composed of 2,700 kcal of ME/kg feed and
17% crude protein in balanced digestible essential amino acids to optimize production performance
and egg quality of laying hens in the late phase of production.
Table 1. Dietary metabolisable energy (ME) and crude protein (CP) levels on performances and
egg quality of laying hens in the late phase of production in balanced digestible essential amino
acids diets.1
Item EP EW EM FI FCR YC AP YP
(%) (g) (g/d) (g/d) (g/g) (%) (%)
ME (kcal/kg)
2,500 86.16b 57.90 52.61b 127.64 a 2.60 a 7.03b 64.30 24.02
2,600 90.14a 58.22 54.21 a 126.32 a 2.31b 7.67 a 64.17 23.99
2,700 90.16 a 58.07 53.62 a 123.42b 2.28b 7.87 a 63.97 24.12
2,800 90.04 a 58.65 53.86 a 123.10b 2.26b 7.96 a 63.88 24.01
CP (%)
16 90.14 a 57.49b 52.64b 125.90 2.34b 7.52 63.14b 24.61 a
17 90.75 a 58.14 a 54.99 a 125.31 2.31b 7.56 63.42b 25.09b
18 86.14b 58.41 a 52.14b 125.19 2.59 a 7.54 64.49 a 23.64b
Pooled SEM
Main effect and interaction
ME 0.036 NS 0.042 0.039 0.039 NS NS NS
CP 0.047 0.041 0.041 NS 0.043 0.044 0.042 0.039
ME×CP NS NS NS NS NS NS NS NS
1 Mean within a column and under each main effect with no common superscripts differ significant (P<0.05); NS =
mean not significant at P>0.05.
References
Khunthum, A., 2006. The principle of experimental design. Department of Statistic, Faculty of Science, Kasetsart
University, Bangkhen Campus, Thailand, 343 pp.
Lesson, S., J.D. Summers and L.J. Caston, 2001. Response of layers to low nutrient density diets. J. Appl. Res. 10: 46-52.
Lesson, S. and J.D. Summers, 2005. Commercial poultry nutrition. Nottingham University Press, Nottingham, UK.

Effect of level and micronizing of lupin seeds on microbial activity in the
large intestine of pigs
A. Tuśnio, M. Barszcz, E. Święch, J. Skomiał and M. Taciak*
Department of Monogastric Nutrition, The Kielanowski Institute of Animal Physiology and Nutrition,
Polish Academy of Sciences, Instytucka 3, 05-110 Jabłonna, Poland; m.taciak@ifzz.pl
Abstract
The study aimed at determining the effects of dietary level and micronizing of blue sweet lupin on
microbial activity in the large intestine of growing pigs. Two-factorial experiment was performed on
32 castrated male pigs fed cereal-based diets with low (LL) and high (HL) level of raw or micronized
lupin seeds. After three weeks of feeding, pigs were slaughtered and short-chain fatty acids and
ammonium concentration were analysed in the digesta from the caecum and colon. Feeding HL diets
increased valerate concentration in all parts of the large intestine, whereas acetate, propionate and
butyrate concentrations only in the distal colon. Ammonium level was decreased in the caecum and
proximal colon and increased in the distal colon by feeding HL diets. Pigs fed diets with micronized
lupin had greater concentration of valerate in the caecum and proximal colon. In conclusion, dietary
lupin level has greater impact on microbial activity in the large intestine of growing pigs than process
of micronizing.
Keywords: lupin, micronizing, fermentation, large intestine
Introduction
Blue sweet lupin is considered as an alternative protein source to soybean meal in diets for pigs.
However, its use may be limited due to high content of non-starch polysaccharides (NSP) and
other antinutritional factors. Micronizing is a technological process involving infrared treatment of
feeds that may improve the nutritional value of lupin seeds. Application of this process may allow
formulation of diets for young pigs with greater content of this protein source. The study aimed at
determining the effect of dietary level and micronizing process of blue sweet lupin on microbial
activity in the large intestine of growing pigs.

The experiment was performed on 32 castrated male pigs (DanBred × Duroc) of initial body weight
8 kg, divided into 4 groups (n=8). The animals were fed cereal-based diets in which 50% (low level,
LL) or 100% (high level, HL) of soybean meal protein was replaced by protein of either raw or
micronized blue sweet lupin seeds of Baron variety. After 21 days of feeding, pigs were slaughtered
and digesta samples were taken from the caecum and proximal, middle and distal colon for analyses
of short-chain fatty acids (SCFA) and ammonium concentration. SCFA were analysed by a gas
chromatography method and ammonium was determined spectrophotometrically. Data were analysed
by two-way analysis of variance and Tukey’s test.

Feeding HL diets increased acetate, propionate and butyrate concentrations in the distal colon, and
valerate concentration in all parts of the large intestine (Table 1). This may indicate that replacing
soybean meal by lupin seeds changed the composition of dietary fibre, mainly NSP, being an energy
source for microflora. Ammonium concentration was also influenced by lupin level. In the caecum
and proximal colon it was decreased while in the distal colon increased by feeding HL diets. As the
HL diets provided greater amount of protein of lower ileal digestibility compared to soybean meal

protein (Rubio, 2005), greater ammonia level in the distal colon may be explained by an altered
carbohydrates to nitrogen ratio and intensified deamination of amino acids (Windey et al., 2012).
Micronizing process influenced only valerate concentration in the caecum and proximal colon. Neither
lupin level nor micronizing affected isoacids concentration in the colon (data not shown). It can be
concluded that dietary lupin level has greater impact on microbial activity in the large intestine of
growing pigs than process of micronizing.
Table 1. Concentration of SCFA and ammonium (µmol/g) in large intestine of pigs fed diets with low
(L) and high (H) content of raw (R) or micronized (M) blue sweet lupin (BSL) seeds.
BSL R BSL M P-value
L H L H Level Process L×P
Caecum
Acetate 27.15 28.10 28.58 25.32 0.46 0.66 0.18
Propionate 19.37 19.25 17.79 21.13 0.16 0.89 0.13
Butyrate 7.88 10.34 9.83 10.19 0.23 0.44 0.36
Valerate 1.78 3.36 2.80 4.98 0.00 0.02 0.58
Ammonium 8.21 3.92 4.09 3.88 0.04 0.06 0.06
Proximal colon
Acetate 30.34 29.29 28.73 27.77 0.46 0.25 0.97
Propionate 19.61 19.07 17.50 22.04 0.11 0.72 0.04
Butyrate 8.55 10.45 10.54 10.32 0.41 0.36 0.30
Valerate 2.18 3.43 3.22 5.17 0.00 0.01 0.45
Ammonium 13.96 3.94 6.92 4.86 0.00 0.08 0.03
Middle colon
Acetate 27.52 29.13 26.41 24.77 0.99 0.09 0.30
Propionate 15.34 17.09 14.37 17.44 0.15 0.85 0.69
Butyrate 6.91 9.08 7.19 7.22 0.28 0.43 0.29
Valerate 1.70 2.47 2.15 3.01 0.04 0.21 0.90
Ammonium 11.17 7.90 7.30 10.53 0.99 0.72 0.08
Distal colon
Acetate 12.99 15.18 12.78 14.97 0.01 0.79 1.00
Propionate 6.51 7.82 6.46 8.78 0.03 0.55 0.52
Butyrate 2.59 3.95 2.84 3.73 0.01 0.97 0.55
Valerate 0.69 1.30 0.87 1.50 0.00 0.17 0.93
Ammonium 10.45 13.79 9.38 15.17 0.06 0.91 0.43
Acknowledgements
Financial support Project RM HOR No 3/2015/OB4 and 4/2015/OB4.
References
Rubio, L.A., 2005. Ileal digestibility of defatted soybean, lupin and chickpea seed meals in cannulated Iberian pigs: I.
Proteins. Journal of the Science of Food and Agriculture 85: 1313-1321.
Windey, K., V. de Preter and K. Verbeke, 2012. Relevance of protein fermentation to gut health. Molecular Nutrition
and Food Research 56: 184-196.

Influence of silver nanoparticles on growth and health of broiler
chickens challenged with Campylobacter jejuni
K.P. Vadalasetty1, C. Lauridsen2, R.G. Engberg2, E. Sawosz3 and A. Chwalibog1*
1Department of Veterinary Clinical and Animal Sciences, University of Copenhagen, 1870
Frederiksberg, Denmark; 2Department of Animal Science, Aarhus University, 8830 Tjele, Denmark;
3Department of Animal Nutrition and Biotechnology, Warsaw University of Life Sciences, 02-786
Warsaw, Poland; ach@sund.ku.dk
Abstract
Silver nanoparticles (AgNPs) have gained much attention in recent years due to their biomedical
applications, especially as an antimicrobial agent. The present study was to investigate the effects
of AgNPs oral administration via drinking water on growth, haematological, immunological and
microbial profile of ileum, caecum, and faeces in broilers with Campylobacter jejuni challenge. One
hundred 1-day-old Ross broiler chickens were randomly assigned into two groups: control (0 mg/kg;
n=45) and AgNPs (50 mg/kg; n=45). At day 11, all birds were orally challenged with an overnight
C. jejuni culture. The body weight gain, relative weights of bursa and spleen, the concentration of
plasma IgG and IgM of AgNPs birds was significantly lower. Expression of TNF-α and NF-kB at
mRNA level was significantly higher in AgNPs group. Taken together, the negative influence on
growth performance and impaired immune responses may suggest that the 50 mg/kg AgNPs have
harmful effects on broiler chickens.
Keywords: broiler chickens, silver nanoparticles, microflora, immunoglobulins, gene expression
Introduction
In broiler production, different kinds of antimicrobial agents are used for preventing and controlling
diseases. The overuse of antimicrobial agents (antibiotics) promotes the emergence of resistance
in microorganisms, being harmful to animal and human health. The use of all antibiotics has been
prohibited in the European Union since 2006. When the conventional therapies are ineffective
in curing poultry infections, it is necessary to explore novel drug compounds. Recent studies on
antibacterial nanomaterials have received increasing attention. Among metal nanoparticles, silver
is one of the most promising components in several nanotechnology products. Currently, there are
several consumer products containing various silver nanoparticles (AgNPs) because of antimicrobial
properties. Ag+ binds and denatures the bacterial DNA and RNA, thus inhibiting cell replication
(Dunn et al., 2004). Furthermore, evidence has been obtained suggesting that silver nanoparticles
may modulate the phosphotyrosine profile of putative bacterial peptides that could affect cellular
signalling and, therefore, inhibit the growth of bacteria (Shrivastava et al., 2007). However, little
is known about their toxicity, antibacterial mechanisms, biocompatibility and the intolerance of the
immune system to AgNPs. The objective of this study was to investigate the effect of AgNPs on the
growth, microbial profile of digestive tract and immune status of chickens exposed to Campylobacter
jejuni infection.

Ninety-day-old male broiler chickens of Ross 308 breed were randomly assigned into two groups:
control (0 mg/kg; n=45) and AgNPs 50 mg/kg (50 mg/kg; n=45) obtained from Nano-Tech (Warsaw,
Poland). At day 11, all birds were orally challenged with an overnight C. jejuni culture. On days 15,
22 and 30 chickens were weighed, and blood and organ samples were collected.

We demonstrated that the efficacy of AgNPs against C. jejuni infection in broiler chickens has shown
that orally-administered AgNPs in drinking water (50 mg/kg) did not influence the feed intake, water
intake, feed conversion ratio, microbial profile and red blood cell count. However, AgNPs reduced
body weight gain (Table 1), lowered concentrations of plasma immunoglobulins (Figure 1A and
1B) and upregulated mRNA expression of TNF-α and NF-Kb (Figure 1C and 1D), indicating their
toxicity and impaired immune response.
Table 1. Body weight of broiler chickens challenged with Campylobacter jejuni and treated with
silver nanoparticles (AgNPs).
Age Control AgNPs P-value
Age 11 213±3.43 167±2.26 0.0040

Age 15 380±0.25 318±1.43 0.0003
Age 22 788±7.80 722±1.73 0.0001
Age 30 1,424±11.88 1,346±3.67 0.0123
A Treatment×Age: B Treatment×Age: C Treatment×Age: D Treatment×Age:

P=0.0047 P=0.0014 2.5 P=0.0007 P=0.0001
1.4 0.18
IgG IgM NF-κB * 1.8 TNF-α
TNF-α mRNA/ACTB
1.2 2.0 *
NF-kB mRNA/ACTB
0.16 1.6
ELISA O.D values
ELISA O.D values
1.0 * 0.14 1.4

0.12 1.5 1.2
0.8 0.10 1.0
0.6 0.08 1.0 0.8
0.4 0.06 0.6
0.04 0.5 0.4
0.2 0.02 0.2
0.0 0.00 0.0 0.0
15 22 30 15 22 30 15 22 30 15 22 30
Age of chickens (days) Age of chickens (days) Age of chickens (days) Age of chickens (days)
Control AgNPs
Figure 1. Influence of silver nanoparticles (AgNPs) on immunoglobulins concentrations (A) IgG

and (B) IgM, mRNA expression of (C) NF-κB and (D) TNF-α in broiler chickens infected with
Campylobacter jejuni. Error bars represent the mean ± standard errors of 6 isolators, each with 15
birds. * indicates significant difference between control and AgNP (P<0.05).
Acknowledgements
This research work was supported by the Danish Agency for Science Technology and Innovation
# 2106-08/0025.
References
Dunn, K. and V. Edwards-Jones, 2004. The role of Acticoat with nanocrystalline silver in the management of burns.
Burns 30, Suppl. 1: S1-S9.
Shrivastava, S., T. Bera, A. Roy, G. Singh, P Ramachandrarao and D. Dash, 2007. Characterization of enhanced
antibacterial effects of novel silver nanoparticles. Nanotechnology 18: 225103.

The effects of dried leaves of Manihot esculenta and Artemisia annua on
coccidiosis in organically reared pullets in Brazil
G.F.D. Almeida1*, S.M. Thamsborg2, D.M.B. Campos1, K. Horsted3, P.M. de Magalhães4, J.F.S.
Ferreira5 and J.E. Hermansen3
1Federal University of Sao Carlos, CCN, Lauri S. de Barros, km 12, 18290-000, Buri, SP, Brazil;
2University of Copenhagen, Danish Centre for Exp. Parasitology, Dyrlægevej 100, 1870 Frederiksberg
C, Denmark; 3Aarhus University, Department of Agroecology, Blichers allé, 20, P.O. Box 50, Tjele
8830, Denmark; 4University of Campinas, Centro P. P. Quím, Biol e Ag – CPQBA, 13140-000
Paulínia, SP, Brazil; 5USDA-ARS, Sal. Laboratory, 450 W. B. Springs Rd, Riverside, CA 92507-
4617, USA; gufoal@gmail.com
Abstract
The effects of Manihot esculenta and Artemisia annua as natural coccidiostats were investigated
as compared to a vaccinated group. The inclusion of Artemisia annua showed poorer performance
compared to the vaccinated group whereas dried leaves of M. esculenta presented similar results
of a commercial vaccine in performance and smaller oocyst shedding at 21 days of age. Manihot
Esculenta might be an option as natural coccidiostat for organic and slow input poultry systems and
deserves further investigation.
Keywords: natural anti-protozoa drug, cassava, organic systems, layers
Introduction
Coccidiosis in poultry systems has been prevented through the addition of anti-coccidial drugs
in feed. However, the risk of drug residues in poultry products has drawn attention to food with
minimal drug use. In this respect, new methods to control the disease through food supplements
described as ‘natural’ are likely to play an increasing role since they have been well accepted by
consumers. The use of live attenuated vaccines is a useful option available in the market, although
not always affordable to the small scale farmer. Thus, we investigate possible benefits of Manihot
esculenta and Artemisia annua as potential coccidiostats provided with daily feed on performance
and oocyst excretion.

The study was performed in an organic commercial farm located in São Paulo state, Brazil (22°38′
S, 47°00′ W). Eight groups were randomly allocated to pens (4×8 m) with concrete floor and access
to an outdoor chicken run/corridor (during the experimental period birds did not access outdoor
corridors). 1,280 one-day Isa Brown pullets were distributed in a completely randomized design of
four treatments with two replicates of 160 birds per pen. Treatment 1 (Vac), birds were vaccinated
at 4 days of age with a commercial vaccine (Livacox Merial®). Treatment 2 (Aad1), dried powder of
A. annua was supplied in a concentration of 3% of the diet since one day old. Treatment 3 (Aad30),
A. annua was supplied in a concentration 3% of the diet from 30 days of age. Treatment 4 (Cad8),
M. esculenta dried leaves were supplied in the first 8 days of birds life at 3% through diet. From
first week to seven weeks old, 10% of animals per group were weighed and feaces was collected
for oocysts counting. An ANOVA test was performed to assess the differences considering time and
treatment effects including interactions on body weights (BW) and mean oocyst excretion (OE). Box-
Cox transformations were used in order to stabilize variances due to the small number of replicates.
The Gompertz equation was also fitted to the means in order to estimate the growth parameters.

Supplementation of A. annua at 3% at daily feed influenced negatively BW in the 49 days trial.
Gompertz equation presented different parameters for mature body mass, age of inflection point and
growth rate for the experimental treatments (Figure 1). The mature body mass of Vac and Cad8 were
similar, suggesting that M. esculenta dried leaves might not affect growth rate of the Isa Brown pullets.
An interaction among treatment and time was found for OE for the group supplemented with cassava
at 21 days (Table 1) suggesting an infection delay attributed to cassava active ingredients. A negative
effect of Aad1 over bird’s growth was observed. Artemisinin, the main bioactive component in A.
annua leaves is bitter. It influenced negatively feed consumption in agreement with a previous study
with broilers in a free range system (Almeida et al., 2012). For Cad8, bioactive components are
believed to be condensed tannins. Seng and Rodriguez (2001) showed its potential coccidiostatic
effect in parasitized goats. In our study with pullets, dried leaves of Manihot esculenta presented
similar results of a commercial vaccine in performance and smaller OE at 21 days of age. It would be
a cheap strategy to control coccidiosis in organic slow input poultry systems and the plant deserves
further investigation.
Weight gain (g)
Days
Figure 1. Growth parameters curves for Isa Brown pullets when 49 days old.
Table 1. Log transformed means of oocyst excretion of Isa Brow pullets of 21 to 49 days old.1
Treatments Time (days)
21 28 35 42 49
Vac 9.32 Aa 11.18 Aa 8.97 Aa 7.20 Aa 6.49 Aa

Aad1 7.49 Aa 7.80 Aa 10.14 Aa 9.23 Aa 10.52 Aa
Aad30 6.68 Aa 9.15 Aa 8.79 Aa 9.14 Aa 11.32 Aa
Cad8 -0.69 Ab 7.50 Aa 9.05 Aa 9.97 Aa 11.63 Aa
1 Log means followed by different letters, uppercase in columns and lowercase in rows, differ statistically from each
other (Tukey<0.05).
References
Almeida, G.F.D., K. Horsted, S.M. Thamsborg, N.C. Kyvsgaard, J.F.S. Ferreira and J.E. Hermansen, 2012. Use
of Artemisia annua as a natural coccidiostat in free-range broilers and its effects on infection dynamics and
performance. Vet. Parasitol. 186: 178-187.
Seng, S. and L. Rodriguez, 2001. Foliage from cassava, Flemingia macrophylla and bananas compared with grasses
as forage sources for goats: effects on growth rate and intestinal nematodes. Livestock Research for Rural
Development 13: 1-6.

Effects of rich in docosahexaenoic acid algae supplementation on
performance of calves
J. Flaga*, Ł. Korytkowski, P. Górka and Z.M. Kowalski
University of Agriculture in Krakow, Department of Animal Nutrition and Dietetics, al. Mickiewicza
24/28, 30-059 Krakow, Poland; j.flaga@ur.krakow.pl
Abstract
There are indications showing that dietary decosahexaenoic acid (DHA) supplementation may have
beneficial effect on health and performance of dairy cattle. Its supplementation may be especially
justified in the diet of milk replacer (MR) fed calves, due to the negligible content of DHA in MR.
The aim of this study was to determine the effect of DHA-rich algae (All-G Rich, Alltech Inc.)
supplementation in MR on performance of newborn dairy calves. Forty female Holstein-Friesian
calves (8.6±0.8 day-old and 41.1±4.3 kg; mean ± SD) were blocked by age of birth and allocated into
four experimental groups (10 animals/group): (1) not supplemented with DHA-rich algae (DHA0);
(2) supplemented with 9 g of DHA-rich algae/day in MR (DHA1); (3) supplemented with 18 g of
DHA-rich algae/day in MR (DHA2); and (4) supplemented with 27 g of DHA-rich algae/day in MR
(DHA3). Data were analysed as randomised complete block design using PROC MIXED of the SAS
and polynomial contrasts were used to determine linear, quadratic, or cubic changes that occurred
with increasing supplemental dose of DHA-rich algae fed in MR. When offered at the low dose (9 g/
day), DHA-rich algae supplementation in MR had positive effect on average daily gain, gain-to-feed
ratio and faecal score of calves between 1 to 14 day of the study; however, negative effect of DHA-
rich algae supplementation in MR on MR and starter mixture intake, and in consequence average
daily gain was observed between day 15 to 49 of the study as well as in the whole study period.
Keywords: growth, health, feed efficiency
Introduction
There are indications showing that dietary decosahexaenoic acid (DHA) supplementation may have
beneficial effect on health and performance of dairy cattle (Silvestre et al., 2011a,b). Moreover,
the dietary omega-3 fatty acids supplementation seems to influence not only on pre-weaning
growth of calves but also on heifers productivity during first lactation (Santos et al., 2013). DHA
supplementation may be especially justified in the diet of milk replacer (MR) fed calves due to the
negligible content of DHA in the MR. Therefore, the aim of this study was to determine the effect of
DHA-rich algae (All-G Rich, Alltech Inc.) supplementation in the MR on performance of newborn
dairy calves.

Forty female Holstein-Friesian calves (8.6±0.8 day-old and 41.1±4.3 kg; mean ± SD) were blocked
by age of birth and allocated into four experimental groups (10 animals/group): (1) not supplemented
with DHA-rich algae (DHA0); (2) supplemented with 9 g of DHA-rich algae/day in MR (DHA1);
(3) supplemented with 18 g of DHA-rich algae/day in MR (DHA2); and (4) supplemented with 27
g of DHA-rich algae/day in MR (DHA3). The dose of DHA-rich algae corresponded to 0, 1, 2 and
3% of MR powder fed/day for DHA0, DHA1, DHA2 and DHA3, respectively. MR was fed in the
amount equal to 900 g/day, two times a day, for 49 days. Starter mixture (SM) was fed ad libitum
beginning on 15 day of the study. MR, SM intake and faecal score were recorded daily and body
weight was recorded weekly. Data were analysed as randomised complete block design using PROC
MIXED of the SAS and polynomial contrasts were used to determine linear, quadratic, or cubic
changes that occurred with increasing supplemental dose of DHA-rich algae fed in MR.

Initial age, body weight and growth of heifers was not statistically different among the experimental
groups. Average daily gain increased between 1 to 14 day of the study from 409 for DHA0 to 476
g/day for DHA1 and then decreased to 326 g/day for DHA3 (quadratic, P=0.03); however, average
daily gain in the whole study period linearly decreased (from 609 to 492 g/day; P=0.01) as well
as body weight gain during study period linearly decreased (from 29.8 to 24.8 kg; P=0.05) with
increasing supplemental dose of DHA-rich algae. There was also a group × time interaction, P=0.04,
for mean body weight.
MR intake decreased linearly between 1 to 14 day of the study (from 5.10 to 4.68 l/day; P<0.01)
and in the whole study period (from 5.74 to 5.57; P=0.05) with increasing dose of supplemental
DHA-rich algae. Starter mixture intake decreased from 302 for DHA0 to 171 g/day for DHA2
and then increased to 205 g/day for DHA3 (quadratic, P=0.05). However, the significant group ×
time interaction (P=0.01) was detected for this parameter. In the beginning of the study the biggest
consumption of starter mixture was observed in groups DHA1 and DHA3, whereas at the end, the
highest intake was observed in control group.
Gain-to-feed ratio increased in first 14 days of the study from 572 for DHA0 to 705 for DHA1 and
then decreased to 511 g/day for DHA3 (quadratic, P=0.03) and was no different between treatments
in the latter stage of the study as well as in the whole study period (group × time interaction, P=0.04).
In first 14 days of the study faecal fluidity decreased from 2.41 for DHA0 to 2.03 for DHA1 and
2.06 for DHA2, and than increased to 2.23 for DHA3 (quadratic, P=0.03).
In conclusion, when offered at the low dose (9 g/day) DHA-rich algae supplementation in MR had
positive effect on average daily gain, gain-to-feed ratio and faecal score of calves between 1 to 14
day of the study; however, negative effect of DHA-rich algae supplementation in MR on MR and
SM intake, and in consequence average daily gain was observed between day 15 to 49 of the study
as well as in the whole study period.
References
Santos, J.E.P., L.F. Greco, M. Garcia, W.W. Thatcher and C.R. Staples, 2013. The role of specific fatty acids on dairy
cattle performance and fertility. In: Proceedings of the 24th Annual Ruminant Nutrition Symposium, Gainesville,
FL, USA, February 5-6, pp. 74-88.
Silvestre, F.T., T.S. Carvalho, N. Francisco, J.E.P. Santos, C.R. Staples, T.C. Jenkins and W.W. Thatcher, 2011a. Effects
of differential supplementation of fatty acids during the peripartum and breeding periods of Holstein cows: I.
Uterine and metabolic responses, reproduction, and lactation. J. Dairy Sci. 94: 189-204.
Silvestre, F.T., T.S. Carvalho, N. Francisco, J.E.P. Santos, C.R. Staples, T.C. Jenkins and W.W. Thatcher, 2011b. Effects
of differential supplementation of fatty acids during the peripartum and breeding periods of Holstein cows: II.
Neutrophil fatty acids and function, and acute phase proteins. J. Dairy Sci. 94: 2285-2301.

Determining the optimal essential amino acid ratios in juvenile of Nile
tilapia
F.H.F. Rodrigues1, J.C.P. Dorigam2, N.K. Sakomura2, T.M.T. Nascimento1, C.F.M. Mansano1, E.P.
Silva2 and J.B.K. Fernandes1*
1Aquaculture Center, Universidade Estadual Paulista ‘Julio de Mesquita Filho, Jaboticabal, São
Paulo 14884-900, Brazil; 2Department of Animal Science, Faculdade de Ciências Agrárias e
Veterinárias, Universidade Estadual Paulista ‘Julio de Mesquita Filho,’ Jaboticabal, São Paulo
14884-900, Brazil; jbatista@caunesp.unesp.br
Abstract
The deletion method has been used to determine the optimal essential amino acid (AA) ratio for
different animal species, including fishes. Thus, the purpose of this study was to determine the ideal
essential amino acid ratio (IAAR) for Nile tilapia (Oreochromis niloticus) juveniles using the deletion
method. The fishes were fed with 11 experimental diets and four replications. One balanced diet (BD)
was formulated to meet all the tilapia’s requirements. Ten other diets were formulated, in which the
BD diet was diluted with cornstarch to meet 45% of the requirements and refilled to 100%, except for
the test amino acid. Groups of fishes were euthanized for further determination of the body nitrogen
(N) composition at the beginning and at the end of the experiment to calculate nitrogen deposition
(ND). The individual responses (ND) for each amino acid deleted diet was compared to the BD
responses and were used to calculate the AA requirements. The AA requirements obtained in this
study were Lys 1.33, Met 0.51, Thr 1.50, Trp 0.38, Arg 1.27, His 0.64, Ile 0.85, Leu 1.37, Phe 1.03
and Val 0.91 g/kg. The requirement for each AA was divided by the requirements of lysine resulting
in the following IAAR: Lys 100, Met 38, Thr 113, Trp 29, Arg 96, His 48, Ile 64, Leu 103, Phe 78
and Val 68%. The deletion method appear to be a successful approach.
Keywords: balanced diet, deletion method, fish, nitrogen deposition, protein
Introduction
One strategy used to improve fish production is reducing dietary protein content, which is possible
by using the ideal protein concept. A number of techniques have been employed to estimate the
quantitative amino acid (AA) requirement for fish. The dose-response experiments can be performed
to determine the ideal essential amino acid ratio (IAAR) (Furuya, 2010), but they are time-consuming
and expensive (Rollin et al., 2003). The deletion method has been used as an alternative to determine
the IAAR. This method relies on the assumption that the reduction of a non-limiting AA has no
effect on nitrogen deposition (ND). However, if a single AA is limiting in the diet, the rate of ND is
directly related to the reduction in levels of that individual AA. The changes in ND are a function
of reduction of each essential amino acid (EAA), which in turn is used to calculate the IAAR where
all EAA are limiting. Several studies have been conducted in order to determine the requirements
for lysine in each creation phase of Nile tilapia. However, little information is available on other AA
requirements and on their relationship to lysine, which is used as reference (Furuya, 2010). Using
the deletion method, we aimed to determine the IAAR for juvenile Nile tilapia.

This study was conducted at the Aquaculture Center of the Universidade Estadual Paulista
(CAUNESP), located in Jaboticabal, São Paulo, Brazil. The experimental design consisted of 11
experimental diets with four replications, distributed in a randomized block design. For this trial,
one balanced diet (BD) was formulated to meet all the nutritional requirement for tilapia (Furuya,
2010) and the other 10 diets were obtained by the deletion technique, i.e. the BD diet was reduced

to 45% of the requirements with the inclusion of cornstarch and then the amino acids were refilled
again to 100% using synthetic amino acids, except for the AA tested, which remained at 45% of the
requirement. Groups of fish at the beginning and at the end of the experiment were euthanized to
determine body nitrogen N composition by Kjeldahl method. The ND was calculated as the difference
between final and initial body N composition. The EAA requirement was calculated by the following
equation proposed by Rollin et al. (2003): requirement = (EAA)BD × (2 – DEL – (NDEAA/NDBD)),
were (EAA)BD is the EAA concentration in the BD (g/kg DM), DEL is the deletion rate, NDEAA
and NDBD are the N deposition (mg/BWkg0.75 per day) corresponding to the limiting diets and BD,
respectively. The Dunnett test at 5% of probability was used to compare the ND in control diet (BD)
against each other limiting diet for each AA tested.

The fishes fed the balanced diet presented a higher ND (P<0.05) in comparison to the limiting diets,
validating the calculation for the AA requirements. The most limiting AA for ND was Leucine, which
resulted in lower ND (Table 1). A higher requirement for threonine was observed and this contributed
for a higher Thr:Lys ratio, probably because Thr was the second most limiting AA in the deleted diets.
In conclusion, the amino acid profile found in this study can successfully be used by nutritionists to
make a proper balanced diet, meeting the IAAR for juvenile Nile tilapia while improving performance
and reducing the environment pollution due to the excess in nitrogen excretion.
Table 1. Responses of Nile tilapia juveniles (Oreochromis niloticus) fed diets with individual amino
acid deletions and the calculated requirement and ideal amino acid ratios (IAAR).1
Diets Body nitrogen composition (%) Nitrogen deposition Requirement IAAR

(mg/BWkg0.75 per (g/kg) (%)
Initial Final day)
Balanced diet 8.01±0.5 7.63±0.1 222.0±35.7 a

Lysine 8.01±0.5 7.24±0.6 138.1±22.4 b 1.33 100
Methionine 8.01±0.5 7.45±0.3 108.3±6.8 bc 0.51 38
Threonine 8.01±0.5 6.30±0.9 34.5±23.1 de 1.50 113
Tryptophan 8.01±0.5 7.36±0,0 33.0±13.3 de 0.38 29
Arginine 8.01±0.5 7.60±0,4 102.3±26.3 bc 1.27 96
Histidine 8.01±0.5 7.11±0,4 46.8±29.8 de 0.64 48
Isoleucine 8.01±0.5 7.61±0.3 127.7±25.4 bc 0.85 64
Leucine 8.01±0.5 7.31±0.2 14.1±17.5 e 1.37 103
Phenylalanine 8.01±0.5 6.27±0.7 40.4±12.3 de 1.03 78
Valine 8.01±0.5 7.58±0.1 67.1±27.9 cd 0.91 68
1 Different superscript letters refer to a significant difference at 5% probability by the Dunnett’s test.
References
Furuya, W.M., 2010. Tabelas Brasileiras para a Nutrição de Tilápias. GFM, Toledo, Brazil.
Rollin, X., M. Mambrini, T. Abboudi, Y. Larondelle and S.J. Kaushik, 2003. The optimum dietary indispensable amino
acid pattern for growing Atlantic salmon (Salmo salar L.) fry. British Journal of Nutrition 90: 865-876.

Part 6.
Methodological aspects of research on protein and energy
A new modelling system to estimate lactation requirements and the
efficiency of utilising metabolisable protein to synthesise milk protein
L.E. Moraes1*, E. Kebreab1, L. Doepel2 and H. Lapierre3
1Department of Animal Science, University of California, Davis, CA 95616, USA; 2University of
Calgary, 2500 University Dr. NW, Calgary, AB, T2N 1N4, Canada; 3Agriculture and Agrifood
Canada, 2000 College Street, Sherbrooke, QC, J1M 0C8, Canada; lemoraes@ucdavis.edu
Abstract
The efficiency of utilising metabolisable protein decreases as a function of protein supply so the
requirement of an additional unit of milk produced is expected to increase with increasing protein
supply. In this study we used four nonlinear models to estimate protein requirements with variable
efficiency of synthesising milk protein. The ability of the four models in describing the data was
similar and the requirement for metabolisable protein was similar for all models up to 0.85 kg/d of
milk protein yield. At higher true protein yields, the requirements determined by the monomolecular
and logistic models were larger than the ones from the quadratic plateau and linear plateau models.
The latter determined the lowest protein requirement. In conclusion, we propose a new system to
calculate metabolisable protein requirements for lactation based on nonlinear models which respect
the biological principles underlying milk protein synthesis.
Keywords: lactation, metabolisable protein, requirement, efficiency, nonlinear models
Introduction
The efficiency of utilising metabolisable protein to synthesise milk decreases as a function of protein
supply. However, current requirement models assume constant efficiency and often overestimate the
allowable metabolisable protein for milk protein yield. The objective of this study was to develop a
system to estimate the requirements of metabolisable protein for lactation with variable efficiency.

Four nonlinear response functions were used to represent the relationship between true protein yield
in milk and metabolisable protein net supply (Table 1). The instantaneous, or marginal, efficiency
of utilising metabolisable protein to yield true milk protein was defined as the first derivative of the
curve. The lactation requirement of metabolisable protein net supply, for a given level of milk protein
yield, was defined as the inverse of the response function. Models were fitted with 217 records from
lactating dairy cows gathered from 40 scientific publications and were compared with the Akaike
Information Criteria (AIC). The metabolisable protein supply and all protein outputs were computed
as described in Lapierre et al. (2014).

The ability of the four models in describing the data was similar as suggested by the similar AIC
(Table 1). The biological principles underlying each model were revealed with the determination of
the marginal efficiencies and the lactation requirements. For instance, in the linear plateau model, the
instantaneous efficiency is constant (β1=0.39, SE=0.02) up to the breakpoint (b=1.73 kg/d, SE=0.07)
at which it becomes null. In the quadratic plateau model, the instantaneous efficiency decreases
linearly as a function of protein supply up to the breakpoint (b=2.34 kg/d with SE=0.20) where it
becomes null. In the monomolecular model, the instantaneous efficiency decreases monotonically
as a nonlinear function of the protein supply. Conversely, in the logistic model the instantaneous
efficiency increases until the curve inflection point (θ2=0.60 kg/d with SE=0.04) where it starts its

Table 1. Parameter estimates, standard errors (SE) and Akaike Information Criteria (AIC).
Parameter Functional form Estimate SE AIC
Linear plateau1 𝑦𝑦𝑦𝑦 = 𝛽𝛽𝛽𝛽0 + 𝛽𝛽𝛽𝛽1 𝑥𝑥𝑥𝑥 if 𝑥𝑥𝑥𝑥 < 𝑏𝑏𝑏𝑏 -358.0
�
𝑦𝑦𝑦𝑦 = 𝑝𝑝𝑝𝑝 if 𝑥𝑥𝑥𝑥 ≥ 𝑏𝑏𝑏𝑏
β0 0.298 0.030
β1 0.387 0.027
Quadratic plateau2 𝑦𝑦𝑦𝑦 = 𝛽𝛽𝛽𝛽0 + 𝛽𝛽𝛽𝛽1 𝑥𝑥𝑥𝑥 + 𝛽𝛽𝛽𝛽1 𝑥𝑥𝑥𝑥 2 if 𝑥𝑥𝑥𝑥 < 𝑏𝑏𝑏𝑏 -356.0
�
𝑦𝑦𝑦𝑦 = 𝑝𝑝𝑝𝑝 if 𝑥𝑥𝑥𝑥 ≥ 𝑏𝑏𝑏𝑏
β0 0.121 0.059
β1 0.732 0.090
β2 -0.156 0.031
Monomolecular 𝑦𝑦𝑦𝑦 = 𝜃𝜃𝜃𝜃1 − (𝜃𝜃𝜃𝜃1 − 𝜃𝜃𝜃𝜃2 )exp(−𝜃𝜃𝜃𝜃3 𝑥𝑥𝑥𝑥) -352.0
θ1 1.071 0.055
θ2 -0.034 0.121
θ3 1.094 0.205
Logistic 𝜃𝜃𝜃𝜃1 -354.6
𝑦𝑦𝑦𝑦 =
1 + exp[(𝜃𝜃𝜃𝜃2 − 𝑥𝑥𝑥𝑥)/𝜃𝜃𝜃𝜃3 ]
θ1 1.015 0.035
θ2 0.602 0.039
θ3 0.510 0.067
1 b=1.725 with SE=0.073 and P=0.967 with SE=0.021.

2 b=2.341 with SE=0.198 and P=0.978 with SE=0.029.
nonlinear decrease as a function of protein supply. The requirements for the metabolisable protein
were similar for all models up to 0.85 kg/d of milk true protein yield. At higher true protein yields, the
requirements determined by the monomolecular and logistic models were larger than the ones from the
quadratic plateau and linear plateau models. The latter determined the lowest protein requirement. In
conclusion, we propose a new system to calculate the metabolisable protein requirements for lactation
based on nonlinear models which respect the biological principles underlying milk protein synthesis.
References
Lapierre, H., L. Doepel, D. Pacheco and D.R. Ouellet, 2014. Amino acid requirement and post-absorptive metabolism
in cattle: implications for ration formulation. In: Proceedings of the Florida Ruminant Nutrition Symposium,
University of Florida, Gainesville, FL, USA, pp. 167-178.

Incorporating Theil-Sen regression method into Bartlett’s 3-group Type
II unknown variances model to improve its robustness
M.S. Dhanoa1* and R. Sanderson2
1Rothamsted Research, North Wyke, Okehampton, EX20 2SB, United Kingdom; 2Institute of
Biological, Environmental and Rural Sciences, Aberystwyth University, Gogerddan, Aberystwyth,
SY23 3EB, United Kingdom; dan.dhanoa@rothamsted.ac.uk
Abstract
In cases where measurement error variances are unknown robustness of Bartlett’s type II 3-group
linear regression model may be improved by using group medians. Incorporating an incomplete
Theil-Sen procedure and using the median-median estimate gives a more robust estimate of slope
than Bartlett’s original method.
Keywords: type II regression, unknown variances, 3-group, robustness, Bartlett, Theil-Sen
Introduction
In cases of linear regression when both variables are subject to measurement errors, choice of a type
II model depends whether these error variances are known or unknown. For known variances we
may use the maximum likelihood (ML) solution to estimate the slope (β)
(
βˆML =σˆ y2 − λMLσˆ x2 + (σˆ y2 − λMLσˆ x2 ) 2 + 4λMLσˆ xy2 ) 2σˆ xy (1)
where λML = σˆε2 σˆδ2 with σˆ ε2 and σˆ δ2 representing the measurement error variances of the y- and
x-variables respectively, σˆ x2 and σˆ y2 are the sample variances of the y- and x-variables and σˆ xy2 is
the sample covariance between the x- and y-variables. Major axis (MA) regression may be used
when error variances of the y- and x-variables are considered equal and reduced major axis (RMA)
regression is relevant when the error variances are proportional to their underlying true variances.
However, error variances are not always available. For these cases Bartlett (1949) proposed a 3-group
method to estimate the slope. Here x-variable data are ranked in ascending order and divided into
non-overlapping lower, middle and upper subgroups with the lower (Group 1) and upper (Group 3)
groups of equal size (N≈⅓∑N). Mean y-values and x-values are calculated for Groups 1 and 3 and
the slope estimate is calculated as (mean Y3 – mean Y1) / (mean X3 – mean X1). Replacing means
with medians will guard against undue influence of extreme or outlier observations. The aim of
this study is to further improve the robustness of slope estimates from Bartlett’s 3-group method.
Methods
In Theil’s nonparametric regression (Theil, 1950) slope is calculated for all pairs of data points and
then the median of these slopes is taken as the slope estimate. Sen (1968) pointed out that replicate
y-values should be either omitted or averaged out to avoid any bias. This provides us an opportunity
to improve robustness of the 3-group method by incorporating an incomplete Theil-Sen procedure.
For each data point in Group 1 we calculate slope up to each point in Group 3 giving N samples
of slopes. The N means or medians of these samples are further summarised by their overall mean
or median giving a mean-mean or median-median Theil-Sen Bartlett slope estimate. This provides
2-stage protection against the influence of extreme values or outliers. To illustrate the effects of this
modification we used metabolic rate (y; ml O2/h) and mass (x; g) data for 469 mammal species (White
et al., 2006) compressed into 40 groups by non-hierarchical clustering. The allometric index, i.e.
b in y = axb, was estimated by linear regression of loge-scaled data using mean-mean and median-
median estimates obtained from the combined Theil-Sen-Bartlett method. These estimates were

compared with those from ordinary least squares, major axis (MA) and reduced major axis (RMA)
regression and Bartlett’s 3-group method. A linear functional relationship estimate (FREML) was
obtained by the method of Ripley and Thompson (1987) (as implemented in an Excel Add-In (RSC,
2002)). Sensitivity of the each estimate to outliers was examined by increasing the loge y-value for
a randomly selected cluster in Group 1 and Group 3, either the 8th (y8) or 32nd (y32) ranked group,
by a factor of 1.5.

The estimate of the allometric index, b, varied between regression methods (Table 1). The Theil-
Sen-Bartlett mean-mean estimate was sensitive to outliers as was Bartlett’s original method. The
Theil-Sen-Bartlett median-median estimate was more robust but showed slope attenuation relative
to type II parametric methods (MA, RMA and FREML).
Table 1. Allometric indices (b) estimated for mammal data of White et al (2006).1
Method b s.e. y8 × 1.5 y32 × 1.5
b s.e. b s.e.
OLS2 0.6789 0.0242 0.6569 0.0295 0.7071 0.0343

Bartlett’s 3-group2 0.6523 0.0267 0.6191 0.0386 0.7052
T-S-B mean-mean 0.6378 0.0225 0.5993 0.0401 0.7049 0.0217
T-S-B median-median3 0.6642 0.0272 0.6552 0.0490 0.6668 0.0291
MA2 0.6881 0.0231 0.6747 0.0249 0.7406 0.0559
RMA2 0.6932 0.0220 0.6852 0.0220 0.7543 0.0595
FREML 0.6788 0.0183 0.6525 0.0181 0.7340 0.0186
1 OLS = ordinary least squares; T-S-B = Theil-Sen Bartlett; MA = major axis; RMA = reduced major axis; FREML =
linear functional relationship estimate.
2 Bootstrapped s.e.
3 s.e. = sqrt(π/2) × σ/sqrt(N).
Conclusion
This test data on log-scale did not suffer from outliers, even so the Theil-Sen Bartlett mean-mean
slope estimate does not seem to be sufficiently robust whereas the median-median slope estimate
is more in line with other methods although still shows slope attenuation compared to type II
parametric methods.
References
Bartlett, M.S., 1949. Fitting a straight line when both variables are subject to error. Biometrics 5: 207-212.
Ripley, B.D. and M. Thompson, 1987. Regression techniques for the detection of analytical bias. Analyst 112: 377-383.
Royal Society of Chemistry (RSC), 2002. Fitting a linear functional relationship to data with error on both variables.
Technical Brief Number 10.
Sen, P.K., 1968. Estimates of the regression coefficient based on Kendall’s tau. Journal of the American Statistical
Association 63: 1379-1389.
Theil, H., 1950. A rank-invariant method of linear and polynomial regression analysis. II. Proceedings of the Royal
Netherlands Academy of Sciences 53: 521-525.
White, C.R., N.F. Phillips and R.S. Seymour, 2006. The scaling and temperature dependence of vertebrate metabolism.
Biology Letters 2: 125-127.

Use of the pig to determine digestible indispensable amino acid scores
(DIAAS) in human foods
H.H. Stein* and J.K. Mathai
Division of Nutritional Science and Department of Animal Sciences, University of Illinois, 1207
West Gregory Dr., Urbana, IL 61801, USA; hstein@illinois.edu
Abstract
Values for the digestible indispensable amino acid score (DIAAS) were determined in 21 foods in
four different experiments using the pig as the model for humans. Results indicated that DIAAS
values are greatest in milk proteins (125 to 139), intermediate in plant protein concentrates (73 to
105), and least in cereal grains and cereal grain protein concentrates (29 to 77). The first limiting
amino acid in whey proteins was histidine, but the sulfur containing amino acids were first limiting
in other milk proteins and in soy and pea proteins, whereas lysine was first limiting in cereal grains
and in oat protein concentrate. Results indicate that DIAAS values can be used to distinguish foods
with high protein values from foods with lower protein value and as such, DIAAS values will be
valuable in evaluating food proteins. Results also confirmed that the pig can serve as a model to
generate DIAAS values for foods.
Keywords: amino acid digestibility, digestible indispensable amino acid score
Introduction
In 2 recent FAO reports it was suggested that the pig is the preferred model to assess amino acid
quality of foods for humans (FAO, 2013, 2014). The term ‘digestible indispensable amino acid scores’
(DIAAS) was introduced to evaluate amino acid quality in human foods (FAO, 2013). Values for
DIAAS are calculated from values for true or standardised ileal digestibility of amino acids, and
unless these values can be determined in humans, values obtained in growing pigs are preferred,
although values may also be determined in rats. Indeed, specified details on how to determine
DIAAS values using the pig as the model have also been published (FAO, 2014). The objective of
the present work was to determine DIAAS values in 21 different foods using the pig as the model
for human digestion of amino acids.

Four experiments were conducted to determine DIAAS values in 21 different foods. Each experiment
followed the same protocol and used ileal cannulated pigs (Stein et al., 1998). Diets containing the
21 foods were fed and ileal digesta were collected following standard procedures (Stein et al., 1998).
The standardised ileal digestibility of each amino acid was calculated (Stein et al., 2007). These
values were then multiplied by the concentration of each amino acid in each food and expressed
relative to the crude protein in the food (mg/g protein) and divided by the reference standards
for foods for children older than 3 years to calculate dispensable amino acid reference values for
children older than 3 years, adolescents, and adults (FAO, 2013). The amino acid that had the least
reference value determined the DIAAS value of each food and this amino acid was considered the
first limiting amino acid.

In the first experiment, DIAAS values in 8 cereal grains were determined and it was concluded that
lysine is the first liming amino acid in all 8 cereal grains (Cervantes-Pahm et al., 2014). However,
dehulled oats had a DIAAS value of 77 whereas sorghum had a DIAAS value of 29, and wheat, rye,

pearled barley, two varieties of maize, and polished rice were intermediate having DIAAS values of
43, 47, 51, 48, 54, and 64, respectively. In subsequent experiments, it was determined that DIAAS
values in casein, milk protein concentrate, skim milk powder, whey protein concentrate, and whey
protein isolate are between 125 and 139 with the first limiting amino acid in casein, skim milk powder
and milk protein concentrate being the sulfur containing amino acids, whereas the first limiting amino
acid in whey protein isolate and whey protein concentrate is histidine. Digestibility of amino acids
in soy flour and soy protein isolates were determined in 2 experiments and DIAAS values close to
100% were obtained for these proteins with the sulfur containing amino acids being first limiting.
The DIAAS values in oat protein concentrate and in pea protein concentrate were determined to be
56 and 73, respectively, with lysine and the sulfur containing amino acids being first limiting in oat
protein concentrate and pea protein concentrate, respectively.
Overall, results of the four experiments confirm that the pig can be used as a model to generate
DIAAS values for human foods and results are repeatable among experiments. To the best of our
knowledge, this dataset represents the first data for DIAAS in human foods that have been generated
using the pig as a model. However, DIAAS values based on the rat as a model have also been reported
(Rutherfurd et al., 2015). Results confirmed that differences among food proteins in terms of protein
quality exist and that DIAAS values generated from pigs can differentiate among protein qualities.
There is, however, a need to determine DIAAS values in a large number of food ingredients to
make it possible to formulate diets for humans based on values for digestible indispensable amino
acids and thereby make sure that amino acids are provided in sufficient quantities to support growth
and development in children, adolescents, and adults. There is also a need to determine effects of
processing on DIAAS values because most foods are consumed after some kind of processing. It is
also important to establish the additivity of DIAAS values calculated in individual foods when they
are mixed together in balanced diets consumed by humans. In such mixed diets, complementary
effects of individual food proteins may overcome limitations in DIAAS values in an individual food
and thus make the best use of all amino acids provided in the combined diet.
References
Cervantes-Pahm, S.K., Y. Liu and H.H. Stein, 2014. Digestible indispensable amino acid score and digestible amino
acids in eight cereal grains. British Journal of Nutrition 111: 1663-1672.
Food and Agriculture Organisation (FAO), 2013. Report of an FAO Expert Consultation. FAO of the United Nations.
Dietary protein quality evaluation in human nutrition.
Food and Agriculture Organisation (FAO), 2014. Report of an FAO Expert Consultation. FAO of the United Nations.
Research approaches and methods for evaluating the protein quality of human food.
Rutherfurd, S.M., A.C. Fanning, B.J. Miller and P.J. Moughan, 2015. Protein digestibility-corrected amino acid scores
and digestible indispensable amino acids scores differentially describe protein quality in growing male rats. Journal
of Nutrition 145: 372-379.
Stein, H.H., C.F. Shipley and R.A. Easter, 1998. Technical note: a technique for inserting a T-cannula into the distal
ileum of pregnant sows. Journal of Animal Science 76: 1433-1436.
Stein, H.H., B. Sève, M.F. Fuller, P.J. Moughan and C.F.M. de Lange, 2007. Invited review: amino acid bioavailability
and digestibility in pig feed ingredients: terminology and application. Journal of Animal Science 85: 172-180.

Blood biomarkers in short-term studies on amino acid requirement in
pigs
J.V. Nørgaard1*, E.A. Soumeh1, M. Curtasu1, H.D. Poulsen1, E. Corrent2, J. van Milgen3 and M.S.
Hedemann1
1Department of Animal Science, Aarhus University, Foulum, 8830 Tjele, Denmark; 2Ajinomoto
Eurolysine S.A.S., 75817 Paris Cedex 17, France; 3INRA, UMR1348 PEGASE, 35590 Rennes,
France; janvnoergaard@anis.au.dk
Abstract
The objective was to evaluate a short-term approach with a low number of pigs based on blood
samples as a method to determine the requirement for Ile, Leu, and Val. Three independent 6×6
Latin square experiments with six diets containing increasing concentrations of Ile, Leu or Val
fed to six 8-9 kg pigs for two d each were conducted during a period of 12 d. Blood samples were
collected and analysed for amino acids (AA) and other metabolites. The results on plasma AA and
other metabolites indicated that the items, which were possible to model to obtain an optimum,
provided estimations of Ile, Leu and Val requirements close to those obtained in previous studies,
and that two days of adaptation may be sufficient to reflect relevant biological changes in blood to
different levels of dietary AA.
Keywords: branched chain amino acid, growth performance, metabolomics, pigs
Introduction
In our previous studies on Ile (Soumeh et al., 2014), Leu (Soumeh et al., 2015a), and Val (Soumeh et
al., 2015b), the requirement of these amino acids (AA) was estimated for 8-18 kg pigs by evaluating
feed intake and weight gain. Blood plasma and urine samples from these studies were analysed by
a metabolomics approach to identify metabolites which could be related to growth performance
(Soumeh et al., 2016). As an alternative to evaluate animal performance to determine AA requirement,
Pedersen and Boisen (2001) developed an approach which was based on plasma urea nitrogen as
the sole response trait in dose-response studies with pigs fed the experimental diets for only two
consecutive days. It was hypothesized that biomarkers could be used in short-term studies using
blood metabolites as response criteria instead of feed intake and weight gain. The objective of the
current study was to evaluate a short-term approach with a low number of animals based on blood
samples as a method to determine the requirement for Ile, Leu, and Val. The diets were previously
used in dose-response studies, thus having a documented effect on growth performance.

Six diets on standardised ileal digestible (SID) Ile:Lys (0.42, 0.46, 0.50, 0.54, 0.58 and 0.62, SID
Leu:Lys (0.70, 0.80, 0.90, 1.00, 1.10 and 1.20), and SID Val:Lys (0.58, 0.62, 0.66, 0.70, 0.74 and
0.78) from the three previous studies (diets stored at -20 °C) were used in three new 6×6 Latin squares
experiments, where six female 8-9 kg pigs were fed one of the six diets for two d, i.e. 12 d in total.
After two d and an overnight fasting, pigs were offered 25 g feed/kg0.75 and jugular vein blood was
obtained 180 min. later. Blood plasma was analysed for AA and discriminating metabolites by a
metabolomics approach using LC-MS/MS. The mixed model included AA level as a fixed effect and
pig and sampling day as random effects. Orthogonal polynomial contrast coefficients were used to
determine linear and quadratic effects. The PROC NLIN procedure of SAS was used to estimate the
optimum SID AA:Lys by subjecting the least squares means of the plasma components to broken-line
(BL) and curvilinear-plateau (CLP) models. PROC GLM was used for fitting quadratic (Q) models.

Of the 18 plasma AA analysed, 10, 16 and 3 AA showed significant linear or quadratic responses
to increasing dietary levels of Ile, Leu or Val, respectively. This is in agreement with our previous
study (Soumeh et al., 2016) showing more discriminating metabolites caused by different levels
of dietary Leu compared to Val. Of the metabolites we identified to describe optimum dietary Ile
and Leu (Soumeh et al., 2016), we found 7 and 9 showing significant linear or quadratic responses
to the increasing levels of Ile and Leu, respectively. However, the pattern of plasma AA and other
metabolites only allowed fitting a few of them to BL, CLP and Q models (Table 1). The average
of optimums found across the three types of models reflected well the optimums found in previous
standard-design dose-response studies using the same batches of feed (Table 1). Only metabolites
showing biologically meaningful patterns are reasonable to model, and thus it may be difficult to
model response curves for AA where the response is unknown. Plasma Ile, Leu and Val concentrations
showed close to identical relation to AA levels compared to our previous three studies. This indicates
that two d of adaptation may be sufficient. In conclusion, the results on plasma AA and other
metabolites indicated these traits, which were possible to fit to BL, CLP and Q models, provided
estimations close to those obtained with ADG as response criterion in previous studies using the
same diets.
Table 1. Optimum SID Ile:Lys, Leu:Lys and Val:Lys of broken-line (BL), curvilinear-plateau (CLP)
and quadratic (Q) models fitted to blood metabolites, and the number of metabolites.
Fitting model Mean/n metab.1 Former study
BL CLP Q Reference Conclusion
Ile:Lys 0.53 0.54 0.54 0.53 Soumeh et al., 2014 0.52

Leu:Lys 0.96 1.06 1.10 1.04 Soumeh et al., 2015a 0.93
Val:Lys 0.66 0.68 0.69 0.68 Soumeh et al., 2015b 0.70
Ile:Lys, n metab.2 4 4 5 6
Leu:Lys, n metab. 15 12 4 16
Val:Lys, n metab. 2 2 1 2
1 Mean of the three models /number of unique amino acids and other metabolites used for modelling.
2 Number of amino acids and other metabolites used for modelling.
References
Pedersen, C. and S. Boisen, 2001. Studies on the response time for plasma urea nitrogen as a rapid measure for dietary
protein quality in pigs. Acta Agriculturae Scandinavica, Section A – Animal Sciences 51: 209-216.
Soumeh, E.A., J. van Milgen, N.M. Sloth, E. Corrent, H.D. Poulsen and J.V. Nørgaard, 2014. The optimum ratio of
standardized ileal digestible isoleucine to lysine for 8-15 kg pigs. Animal Feed Science and Technology 198:
158-165.
Soumeh, E.A., J. Van Milgen, N.M. Sloth, E. Corrent and H.D. Poulsen, 2015a. The optimum ratio of standardized
ileal digestible leucine to lysine for 8 to 12 kg female pigs. Journal of Animal Science 93: 2218-2224.
Soumeh, E.A., J. van Milgen, N.M. Sloth, E. Corrent, H.D. Poulsen and J.V. Nørgaard, 2015b. Requirement of
standardized ileal digestible valine to lysine ratio for 8- to 14-kg pigs. Animal 9: 1312-1318.
Soumeh, E.A., M.S. Hedemann, H.D. Poulsen, E. Corrent, J. van Milgen and J.V. Nørgaard, 2016. Biomarkers of
optimum dietary branched chain amino acids for the best growth performance in pigs. In: J. Skomial and H. Lapiere
(eds.). Energy and protein metabolism and nutrition. EAAP Scientific Series No. 137. Wageningen Academic
Publishers, Wageningen, the Netherlands, pp. 195-196.

Variation in protein content and efficiency of lysine utilisation in
growing-finishing pigs
S. Ghimire1*, C. Pomar1 and A. Remus1,2
1Agriculture and Agri-Food Canada, Sherbrooke, QC, J1M 0C8, Canada; 2Department of Animal
Science, Univ. of São Paulo State, Jaboticabal, São Paulo, Brazil; sandip.ghimire@agr.gc.ca
Abstract
Amino acids affect growth performance and environmental impacts from pig production and are
therefore important component in their diet. In this study, individual performance data from two
separate experiments were analysed to assess the variation in protein as a percentage of body weight
gain (fPD) and efficiency of available lysine utilisation (kLys) in growing-finishing pigs. Data
from 137 growing and 132 finishing barrows of either maternal cross line or terminal cross line fed
various levels of lysine were used. The body protein content was measured at the start and end of
each growth phase. Lines of the pig did not affect fPD or kLys (P>0.05). However, the fPD was
greater in growing pigs (18±1.2%) than in finishing pigs (13.8±1.4%). The level of available lysine
did not affect fPD (P>0.05), but higher kLys was observed at lower levels of the intake (P<0.05).
Keywords: lysine efficiency, protein fraction
Introduction
Optimizing amino acids (AA) supplied to the pigs has favourable effects both on the production and
on the environmental cost. Most widely used feeding programs involve feeding growing pigs to meet
some set AA requirements for two or more feeding phases. The requirements are usually established
using a factorial approach (NRC, 2012). One of the limitations of the factorial approach is that they
have to overestimate requirements to ensure optimal response of the population. For example, in the
case of lysine, this is achieved by lowering efficiency of available lysine utilisation (kLys) (NRC,
2012) or by using the 82nd centile pig of the population (Hauschild et al., 2010). Furthermore, the
variation in protein as a percentage of body weight (BW) gain (fPD) is usually taken constant as
16% (De Lange et al., 2003) across all stages of growth. We hypothesized that kLys and fPD are
different across the level of available lysine intake and growth phases. The objective of this study was
to assess growing pigs’ kLys and fPD at different growth stages and available lysine intake levels.

Individual animal data from two separate studies, Zhang et al. (2012) and Cloutier et al. (2015),
designed to assess performance of growing-finishing pigs at different levels of lysine intake was used
for the study. In Zhang et al. (2012) study growing (n=57; 25 to 55 kg BW) and finishing (n=59; 70
to 100 kg BW) barrows of terminal cross line were used. Whereas, in Cloutier et al. (2015) study
barrows of either terminal cross line (n=75, 37 growing and 38 finishing) or maternal cross line (n=72,
37 growing and 35 finishing) barrows were used. The mean starting BW of growing pigs was 25.8 kg
and finishing pigs was 73.3 kg and both stages lasted for 28 days. The standardised ileal digestible
(SID) lysine intake ranged from 6.9 to 26.3 g/d across the experiments. The body protein content
was measured at the start and end of each growth phase by dual energy absorptiometry (DXA). The
kLys was calculated by assuming lysine content as 7% of the body protein, basal endogenous loss
of 313 mg/kg DM intake, integument loss as 4.5 mg/kg BW0.75, and losses due to protein turnover
as 23.9 mg/kg BW0.75.

Results of the analysis indicated that the SID lysine intake did not affect fPD (P>0.05). However,
fPD was significantly higher (18±1.2%) in growing (P<0.05) than in finishing (13.8±1.4%) pigs
(Figure 1A), which explains why more finishing pigs were fed above requirements than growing
pigs (Figure 1B). The AA requirements in the experiments were supplied with an assumption of
constant fPD of 16% across all ages of the pigs. Therefore, the overestimation of protein in the body
led to overfeeding of SID lysine to the finishing pigs. The fPD and the kLys was similar in both
maternal and terminal cross lines of the pigs. Similarly, the kLys was not affected by growth stage
of the pigs (P>0.05). However, kLys increased with decreasing level of available lysine (P<0.05),
indicating that pigs were more efficient in utilising lysine when they were fed below requirements.
However, increased efficiency is not a reasonable biological explanation for efficiency close to and
above 1 considering the length of growth period assessed in this study. Possible explanation for such
high efficiency could be that during lysine restriction: (1) growing animals prioritize growth over
maintenance; and (2) the AA composition of protein can be different.
Results of this study suggest the fPD can be lower at the finishing stage and the kLys is higher at
lower levels of lysine. Therefore, it might be cost effective and environmentally beneficial to reduce
the lysine supply to animals both in group and individually fed animals.
A B
Protein as % of growth
Efficiency of lysine
utilization (kLYS)
(fPD)
Available SID lysine (g/d) Available SID lysine (g/d)
Figure 1. Protein as a percentage of (A) body weight gain and (B) efficiency of lysine utilisation in
growing finishing pigs fed various levels of standardised ileal digestible (SID) lysine.
References
Cloutier, L., C. Pomar, M.L. Montminy, J.F. Bernier and J. Pomar, 2015. Evaluation of a method estimating real-time
individual lysine requirements in two lines of growing-finishing pigs. Animal 9: 561-568.
De Lange, C.F.M., P.C.H. Morel and S.H. Birkett, 2003. Modeling chemical and physical body composition of the
growing pig. Journal of Animal Science 81: E159-E165.
Hauschild, L., C. Pomar and P.A. Lovatto, 2010. Systematic comparison of the empirical and factorial methods used
to estimate the nutrient requirements of growing pigs. Animal 4: 714-723.
Zhang G.H., C. Pomar, J. Pomar and J.R.E. Del Castillo, 2012. Precision feeding in growing–finishing pigs: estimating
the dynamic requirements of lysine supporting maximal daily gain. Journées de la Recherche Porcine 44: 171-176.

Evaluation of compound-specific N isotope analysis in key amino acids to
predict feed efficiency in growing lambs
G. Cantalapiedra-Hijar1,2*, S. Prache1,2, I. Téa3, S. Faure1,2, C. Chantelauze1,2 and I. Ortigues-
Marty1,2
1INRA, UMR 1213 INRA-VetAgro Sup, Unité Mixte de Recherches sur les Herbivores, 63122 St
Genès Champanelle, France; 2Clermont Université, VetAgro Sup, UMR 1213 Herbivores, BP
10448, 63000 Clermont-Ferrand, France; 3Elucidation of Biosynthesis by Isotopic Spectrometry
Group, CEISAM, UMR6230, CNRS, University of Nantes, BP92208, 44322 Nantes, France;
gonzalo.cantalapiedra@clermont.inra.fr
Abstract
To measure feed efficiency (FE) in growing ruminants we proposed an improved isotopic method
based on natural 15N abundance (δ15N). Individual FE was measured for 75 days in 48 growing
lambs fed diets based on Lucerne and supplemented with either a high or low amount of barley. The
δ15N analysis was conducted in bulk N from feed ingredients and animal muscle to calculate the
isotopic N fractionation (Δ15Nanimal-diet, δ15Nanimal – δ15Ndiet) and by compound-specific isotopic
analysis on amino acids to obtain the δ15N of transaminating amino acids (δ15NTAAs). Isotopic values
obtained with both approaches (Δ15Nanimal-diet and δ15NTAAs) were regressed against the observed
FE. We show an improvement in FE prediction was achieved for analysis of 15N natural abundances
on transaminating amino acids rather than on bulk N.
Keywords: feed efficiency, amino acids, natural abundance, 15N
Introduction
Feed efficiency (FE) is a key factor in profitability of livestock farm systems. However, FE is costly,
laborious and most times not possible to measure in field conditions. The difference in 15N natural
abundance (δ15N) between an animal and its diet (Δ15Nanimal-diet = δ15Nanimal – δ15Ndiet) has been
recently correlated to FE in ruminants (Cantalapiedra-Hijar et al., 2015). However, bulk N isotopic
values lack specificity, representing integrated isotopic values of various N-containing molecules in
animal tissues. In this regard, the δ15N in animals differs among individual amino acids according
to their ability to be transaminated (Braun et al., 2014). To refine the use of 15N natural abundance
to predict FE in ruminants we have conducted amino acid compound-specific N isotope analysis
(AA-CSIA). The objective of this study was to assess in ruminants the improvement in FE prediction
when shifting the isotopic analysis from bulk N to transaminating AAs.

Daily individual feed intake and fortnightly body weight gain were recorded for 48 growing Romane
lambs fed for 75 days diets based on alfalfa dehydrated pellets supplemented with either 400 or 100
g of barley and with straw offered ad libitum. Muscle samples (longissimus thoracis) were obtained
for all animals at the slaughterhouse while feeds were sampled weekly and pooled throughout the
experiment. Muscle (n=48) and feed (n=3) samples were analysed by EA-IRMS to obtain the 15N
natural abundance (δ15N) in bulk N. Eight muscle samples were randomly chosen within each dietary
treatment (n=16) for AA-CSIA by GC-C-IRMS. Average daily gain was determined for each lamb
as the slope of the regression of live body weight against time. The FE (%) was calculated as the
average daily gain divided by dry matter intake (kg/d) and multiplied by 100. Linear regressions
(XLStat v2015.2.02) were conducted to model FE from isotopic natural abundances values in either
bulk N or from the sum of transaminating AAs (alanine + valine + isoleucine + leucine + serine +
aspartate + glutamate + glutamine; Braun et al., 2014).

As expected, a negative and significant (P<0.001) relationship was found between FE and Δ15Nanimal-
diet in agreement with previous results in growing beef cattle (Cantalapiedra-Hijar et al., 2015).
This high correlation between FE and N utilisation (N retained/N intake) supportsthe prediction of
FE from this isotopic biomarker. Both diets were similar in isotopic N composition (δ15N = -0.55
and -0.30) and thus relationships between FE and natural 15N abundances were not different when
using either Δ15N (Figure 1A; r2=0.53) or δ15N (r2=0.44; P<0.001). This highlights the potential
of this isotopic biomarker to predict between-animal variation of FE fed the same diet. When the
isotopic analysis was shifted from bulk N (Figure 1A) to AA-CSIA (Figure 1B) the δ15N values
were a better fit to FE (r2=0.80 vs 0.53) and prediction error was less (RSE = 1.85 vs 2.74). The δ15N
values on transaminating AAs are more relatedto FE compared to bulk N isotopic analysis since
(1) isotopic N fractionation occurs during transamination reactions (Macko et al., 1986); and (2)
higher AA catabolism andintensity of transamination reactions are usually associated with lower feed
(N) efficiency. In conclusion, our results show that an improvement in the prediction of FE can be
achieved in ruminants when the analysis of δ15N is conducted on specific AAs rather than on bulk N.
A B
30.0 30.0
Feed conversion efficiency (%)
Feed conversion efficiency (%)
27.5 27.5
25.0 25.0
22.5 22.5
20.0 20.0
Y = 39.7 -4.63X Y = 45.7 -0.56X
17.5 r2 = 0.53 17.5 r2 = 0.80
RSE = 2.74 RSE = 1.85
15.0 n = 48 15.0 n = 16
3.0 3.5 4.0 4.5 5.0 30 35 40 45 50 55

Δ15Nanimal-diet (‰) δ15N transaminating AAs (‰)
Figure 1. Relationship between feed conversion efficiency (%) and (A) Δ15Nanimal-diet (bulk N) and
(B) δ15NTAAs (AA-CSIA) in growing lambs fed diets based on luzerne supplemented with either low
(white circles) or high (black circles) amounts of barley.
References
Braun, A., A. Vikari, W. Windisch and K. Auerswald, 2014. Transamination governs nitrogen isotope heterogeneity
of amino acids in rats. Journal of Agricultural and Food Chemistry 62: 8008-8013.
Cantalapiedra-Hijar, G., I. Ortigues-Marty, B. Sepchat, J. Agabriel, J.F. Huneau and H. Fouillet, 2015. Diet-animal
fractionation of nitrogen stable isotopes reflects the efficiency of nitrogen assimilation in ruminants. British Journal
of Nutrition 113: 1158-1169.
Macko, S.A., M.L.F. Estep, M.H. Engel and P.E. Hare, 1986. Kinetic fractionation of stable nitrogen isotopes during
amino acid transamination. Geochimica et Cosmochimica Acta 50: 2143-2146.

Using computed tomography to assess fat distribution in lamb carcasses
F. Anderson, A. Williams, L. Pannier, D.W. Pethick and G.E. Gardner
Australian Cooperative Research Centre for Sheep Industry Innovation, University of New England,
Armidale, NSW 2351, Australia; School of Veterinary and Life Sciences, Murdoch University,
Murdoch, WA 6150, Australia; f.anderson@murdoch.edu.au
Abstract
This study used computed tomography to assess the body composition of Australian lambs. Reducing
sire post weaning c-site fat depth and increasing c-site muscle depth breeding values decreased carcass
fat weight uniformly across the carcass by 24.7% and as much as 16.3%. When lambs are compared
at the same carcass weight selection for increased growth has minimal impact on carcass fat weight.
Overall, the genetic effects had a far greater impact on carcass fat weight than the environmental
or production factors.
Keywords: breeding values, computed tomography, leanness
Introduction
A high proportion of saleable meat in the carcass is an important determinant of carcass value, as it
reduces the processing costs associated with fat trim (Gardner et al., 2010). Producers can select for
these carcass types indirectly using three Australian Sheep Breeding Values (ASBVs): increased post
weaning weight (PWWT), eye muscle depth (PEMD), and reduced post weaning fat depth (PFAT).
Previous studies have shown the impact of these ASBVs on carcass fatness, relying on fat depths,
or the weight of fat depots (Gardner et al., 2010), but are likely to be biased due to the effect of
ASBVs on muscle and fat distribution. This study used computed tomography (CT) to investigate
the carcass composition of lambs and report the impact of ASBVs and production factors on the
weight of fat and its distribution within the lamb carcass.

Lamb carcasses (n=1,665) from the Sheep CRC Information Nucleus Flock were collected from 6
sites over 5 years. These lambs were the progeny of sires divergent for numerous production traits,
including PFAT, PEMD and PWWT ASBVs. Carcases were scanned in 3 sections, fore, saddle
and hind, using CT to determine fat, lean and bone weights. Data was analysed using an allometric
equation fitted in its log-linearised form logy = log a + b.logx. Fixed effects were site-year, sex,
sire type, birth-type rear-type and kill group within site-year, with random terms sire and dam
by year. Fixed and random effects act as adjustments to log a and are interpretable as percentage
differences. To assess whole carcass composition of fat, log CT weight of carcass fat was included
as the dependent variable (log y), with log CT carcase weight the independent variable (log x). To
assess fat distribution, log section fat weight was log y with log CT carcass fat weight as log x.

The impact of the ASBVs on carcass fat weight was far greater than that of the non-genetic effects,
however there was minimal impact on fat distribution within the carcass. Decreasing sire PFAT had
a marked impact on whole carcass fat weight (P<0.01) which was reduced by 24.7% across the 5.1
mm sire PFAT range. This indicates that the use of this breeding value, which is based on fat depth
measurement at the c-site (12 mm from the midline at the 12th rib), effectively decreases whole
carcass fat and that its impact is not solely localised around its point of measurement – an effect
previously identified in pigs (Trezona-Murray, 2008). Increasing sire PEMD reduced carcass fat in

all carcass sections, with this effect greatest in the Terminal sired lambs (16.3%, P<0.01), with a
smaller effect in the Maternal sired lambs (15.34%, P<0.01), and no effect in Merino lambs. The
PFAT and PEMD ASBVs have the potential to significantly improve carcass value by decreasing
fat trimming requirements, in combination with known impacts on increasing carcass lean weight
(Anderson et al., 2015). The impact of increasing sire PWWT ASBV was small and inconsistent
across the experimental sites.
Relative to Merino and Terminal sired lambs, the Maternal sired lambs contained the most carcass fat
when compared at the same carcass weight, aligning well with their improved reproductive capacity.
The Terminal sired lambs had the least carcass fat and the highest portion of lean (Anderson et al.,
2015). This lean/fat profile is consistent with selection focus for fast-lean-growth, which is likely
to impact on mature size. Therefore at a given carcass weight, Terminal sired lambs would be less
mature and have less carcass fat.
The weight of carcass fat varied between site-years and sexes (P<0.01), with fat distribution varying
between site-years, sex, and birth-type rear-types (P<0.01). Between the site-years carcass fat varied
by as much as 15%, compared to the smaller impact of sex, where the ewe lambs had on average
6.9% more fat in the carcass than wether lambs. Among the production and management effects, site
and kill group had the largest impact on carcass fat and its distribution between sections. Site had
more than double the impact of the sex and birth-type rear-type effects, demonstrating the potential
for nutrition, and other environmental factors to impact on carcass fat and its distribution.
Conclusion
The impact of the ASBVs on carcass fat was far greater than that of the non-genetic effects. The
PFAT and PEMD breeding values reduced carcass fatness across all regions of the carcass, and the
PWWT breeding value had little effect on carcass fatness.
Acknowledgements
The CRC for Sheep Industry Innovation is supported by the Australian Government’s Cooperative
Research Centre Program, Australian Wool Innovation Ltd. and Meat & Livestock Australia.
References
Anderson, F., A. Williams, L. Pannier, D.W. Pethick and G.E. Gardner, 2015. Sire carcass breeding values affect body
composition in lamb – 1. Effects on lean weight and its distribution within the carcass as measured by computed
tomography. Meat Science 108: 145-154.
Gardner, G.E., A. Williams, J. Siddell, A.J. Ball, S. Mortimer, R.H. Jacob, K.L. Pearce, J.E. Hocking Edwards, J.B.
Rowe and D.W. Pethick, 2010. Using Australian sheep breeding values to increase lean meat yield percentage.
Animal Production Science 50(12): 1098-1106.
Trezona-Murray, M., 2008. Conventional and deep-litter pig production systems: the effects on fat deposition and
distribution in growing female large white × landrace pigs. PhD Thesis, Murdoch University, Murdoch, Australia.

Dynamics of nutrient utilisation, heat production and body composition
in broiler breeder hens during egg production
J.V. Caldas1, K. Hilton2, M. Schlumbohm2, N. Boonsinchai3, J.A. England2 and C.N. Coon2
1Cobb – Vantress, P.O. Box 1030, Siloam Springs, AR 72761-1030, USA; 2Center of Excellence for
Poultry Science, University of Arkansas, Fayetteville, AR 72701, USA; 3CP group, C.P Tower 14/F,
313 Silom Road, Bangkok, Thailand; ccoon@uark.edu
Abstract
Changes in heat production and body composition in modern broiler breeders can provide means to
understand nutrient utilisation and an opportunity to improve feeding strategies. Twelve Cobb 500
fast feather breeders were evaluated every 3 weeks from 26 to 59 weeks of age. Heat production,
and respiratory exchange ratio were determined by an indirect calorimetry and body lean and fat
composition using a dual X-ray absorptiometry. Feed allocation was 123 g/d (352 kcal) at 26.3 wk.
and changed to 136 g/d (390 kcal) at 29.6 wk until the end of production. Light program was 16L:8D
from 29.6 wk. HP increased with age (d) in 0.28 kcal/d. During the light period, hens consumed more
VO2 (+17.5 l/d) (P<0.01) than in the dark. HP during the dark period was 83 kcal/kg0.75 which could
be considered resting metabolic rate and during the light period was 115 kcal/kg0.70. RER decreased
with age in -0.1×10-3 per day suggesting more fat being used later in production. Lean body mass
changed from 642-783 g/kg reaching the lowest at 37 and 50 wk and the highest at the beginning
26-33 wk (P<0.001). Fat body mass changed from 168-261 g/kg with the lowest at the beginning
of production 26-33 wk and the highest at 50 wk of age (P<0.001). Broiler breeders may be using
body fat reserves for energy from 50 wk because lean mass increases when the egg production has
reduced below 50%. Broiler breeders change nutrient fuel use along egg production.
Keywords: calorimetry, DEXA, lean mass, fat mass
Introduction
Meat-type hens or broiler breeders have been intensively selected for growth rate, feed efficiency, and
breast meat yield traits, but not necessarily for reproductive traits; in fact, these hens have less eggs
than table-egg producing hens (Robinson et al., 2003). Therefore, management and nutrition of the
broiler breeder is the most complex piece of the poultry production (Kleyn, 2013). Body composition
has changed over time resulting in leaner breeders being lean protein very important at the onset of
sexual maturity (De Beer and Coon, 2007). Calorimetry can explain the nutrient oxidation, and dual
X-ray absorptiometry (DEXA) body synthesis. The objective of the present study is to follow the
same breeder during production to evaluate calorimetry parameters: VO2, VCO2, RER, HP, along
with body lean and fat mass.

A mixed model was used to evaluate calorimetry parameters HP kcal/d, VO2, VCO2 l/d and RER
by age (10 points of evaluation), time of day (2 levels: light and dark), and hen as random because
of repeated measurements. Heat production was calculated after determining oxygen consumption
and carbon dioxide production (HP kcal/d = 3.866 VO2 l/d + 1.233 VCO2 l/d) (Brouwer, 1965). A
complete randomized design, CRD – one way ANOVA (age) with hen as random effect was used for
body composition, lean and fat gain g/d. Means were separated by Tukey-HSD test. Egg production
was recorded daily and averaged at every week of evaluation. Hens were scanned alive one day
before calorimetry. Hens were scanned using a dual energy X-ray absorptiometry, DEXA scanner
(GE, Madison, WI, USA) with small animal body software module (Lunar Prodigy from GE encore
version 12.2). No chemicals or anesthesia were used during the scan.

Heat production increased 0.28 l/d because the hen kept increasing body weigh during production.
Past research reports 14.6 l/kg0.75 for oxygen consumption (Waring and Brown, 1965) which is lower
than the value reported in the present experiment, 20 l/kg0.75. This may be due to modern breeders
having more lean tissue than birds in 1965 in the same basis (l/kg0.75). This increase represents 37%
more oxygen in 2015 compared to 1965. RER reached the lowest point at 40-43 wk of age which
could mean fat oxidation is higher compared to beginning of production. Salas (2012) reported hens
using glucose for egg production at the beginning of production and fat at the end of production.
Lean tissue mass reached the lowest point at 37 and 50 wk (Figure 1) which is in full agreement with
data found by Salas (2012). Protein degradation rate was found to be the highest at peak production
(30-37 wk) Vignale (2014), suggesting the hen using breast protein for egg synthesis. After 50 wk,
egg production decreased and the hen started increasing lean tissue at the expense of fat oxidation.
Lean mass Fat mass

850
a
ab abc
750 abcd cde
def ef
cde bcde f
650
550
g/kg
450
350
ab a
ab ab
250 b b ab
b b b
150
26 30 33 37 40 43 45 50 54 59
Age (week)
Figure 1. Lean and fat body mass (AS IS) from 26-59 wk of age. Levels (a-f) not connected by same
letter are significantly different.
References
Brouwer, E., 1965. Report of sub-committee on constants and factors. In: K.L. Blaxter (ed.). Energy metabolism.
Academic Press, London, UK, 441 pp.
De Beer, M. and C.N. Coon, 2007. The effect of different feed restriction programs on reproductive performance,
efficiency, frame size, and uniformity in broiler breeder hens. Poultry Science 86: 1927-1939.
Kleyn, R., 2013. Chicken nutrition. A guide for nutritionists and poultry professionals. British Library Press, London,
UK, pp. 21-42.
Robinson, F.E., G.M. Fasenko and R.A. Renema, 2003. Optimizing chick production in broiler breeders. Vol. 1. Broiler
Breeder Production, Alberta, Canada.
Salas, C., R.D. Ekmay, J. England, S. Cerrate and C.N. Coon, 2012. Determination of chicken body composition
measured by dual energy X-ray absorptiometry. International Journal of Poultry Science 11: 462-468.
Vignale, K., 2014. Protein turnover in broiler, layers, and broiler breeder. PhD thesis, University of Arkansas,
Fayetteville, USA.
Waring, J.J. and W.O. Brown, 1965. A respiration chamber for the study of energy utilization for maintenance and
production in the laying hen. Journal of Agricultural Science 65: 139.

Use of artificial neural networks to improve estimation of energy
requirements of cattle
M.P. Gionbelli1, D.D. Ferreira2, L.K. Ferreira1, M.L. Chizzotti3 and S.C. Valadares Filho3
1Department of Animal Science, Federal University of Lavras, MG, Brazil; 2Department of
Engineering, Federal University of Lavras, MG, Brazil; 3Department of Animal Science, Federal
University of Viçosa, MG, Brazil; mateus.pg@dzo.ufla.br
Abstract
An artificial neural network (ANN) was compared to multiple linear and nonlinear regression models
(MRM) to predict energy requirements (metabolisable energy intake) of beef cattle. Construction of
models (MRM), network (ANN) and validation was made using a dataset containing data from 31
comparative slaughter experiments (n=840, 60% for training and 40% for validation). Comparisons
were made based on accuracy and precision estimations. Both systems were accurate to predict energy
requirements (accuracy = 0.924 and 0.995 for MRM and ANN, respectively), but the precision of
estimates from ANN was better than MRM (random proportion of mean square error of prediction
was 13.5 for MRM and 7.68 for ANN). These results shown, for the first time, the great potential
of use of ANN to predict nutritional requirements of beef cattle based on large dataset training.
Keywords: accuracy, precision, metabolisable energy, multilayer perceptrons
Introduction
Heuristic (expert systems and adaptive filtering) was suggested to be a future approach to improve the
evolution of net energy systems in predicting nutritional energetics of cattle (Ferrell and Oltjen, 2008).
Heuristic principles are the base to design artificial neural networks (ANNs). The traditional view of
an ANN is of a program that emulates biological neural networks and ‘learns’ to recognize patterns
or categorize input data by being trained on a set of sample data from the domain (Walczak and
Cerpa, 1999). In cattle, ANNs have been successfully applied in several areas, but not in bioenergetics
until the present date. Nutrient requirements of cattle have been predicted using multiple linear and
nonlinear regression models (MRM). One of the most common ANN system used is the multilayer
perceptrons (MLP), which is a feedforward ANN and consists of multiple layers of nodes, one or
more hidden layers, an input layer and an output layer (Haykin, 2009). We hypothesized that ANNs
could predict energy requirements of cattle better than multiple linear regression (MLR) systems.
Thus, the main objective of this work was to compare the accuracy and precision of ANNs and MLR
models to predict energy requirements for maintenance and gain of beef cattle.

A dataset containing data from 31 comparative slaughter experiments, with individual information of
840 animals was used. Dataset was composed by 558 bulls, 175 steers and 107 heifers; 544 purebred
Zebu and 296 crossbred and; 672 animals from feedlot and 168 from pasture studies. Dataset used
was a slice of BR-CORTE System (Valadares Filho et al., 2010). Dataset was divided at random into
training (60%, n=504) and validation (40%, n=336). Same data was used to train and validate ANN
and MRM. Linear and non-linear multiple regression models were estimated similar BR-CORTE
System (Valadares Filho et al., 2010). The MLP used was composed of three layers (only one
hidden layer). The weights and biases were initialized according to the Nguyen and Widrow (1990)
algorithm. The activation function used was the hyperbolic tangent, except for the output layer where
a pure linear neuron was used for obtaining the network response. The training algorithm used was
the scaled conjugate gradient backpropagation. The input vector of the neural network comprises
5 collected variables: empty body weight, rate of gain, gender, genetic group and feeding system.

Models generated by both methods were used to estimate MEI using validation dataset. Estimated
data was regressed on observed data. Comparisons between ANNs and MRM were performed using
mean square error of prediction (MSEP) and its partition, accuracy (Cb) and other statistics based
on the Model Evaluation System (MES, http://nutritionmodels.tamu.edu) (Tedeschi, 2006).

Statistics of comparisons between MRM and ANN are summarized in Table 1. Both methods were
considered accurate do estimate energy requirements of beef cattle. However, ANN estimations
were most precise than MRM estimations, probably due to the ability of this system to account for
multiple interactions between input variables that are very difficult to account when using MRM.
Both systems presented a great proportion of MSEP (>98%) as random errors, but the gross error
was 76% higher in MLR than in ANN. These results had shown a great potential of use of ANN
to improve the precision to predict nutrient requirements of cattle, especially using large datasets.
Mathematical operations performed by ANN are not easy to synthesize and describe in equations,
but can be easily performed using computers, making it easy to use in beef cattle production systems.
Table 1. Coefficient of determination (r2), accuracy (Cb), mean square error of prediction (MSEP)
and its composition of multiple linear and nonlinear regression models (MRM) and artificial neural
networks (ANN) estimations of MEI in function of observed values.1
Method Statistic (n=336)
r2 Cb MSEP MSEP decomposition
Mean bias Systematic bias Random errors
MLR 0.630 0.924 13.7 0.05 (0.39%) 0.12 (0.90%) 13.5 (98.7%)
ANN 0.789 0.995 7.74 0.01 (0.18%) 0.05 (0.68%) 7.68 (99.2%)
1 MLR = multiple linear regression.
References
Ferrell, C.L. and J.W. Oltjen, 2008. Centennial paper: net energy systems for beef cattle-concepts, application, and
future models. Journal of Animal Science 86: 2779-2794.
Haykin, S.S., 2009. Neural networks and learning machines. Pearson Education, Upper Saddle River, USA.
Ngugen, D. and B. Widraw, 1990. Improving the learning speed of 2-layer neural networks by choosing initial values
of the adaptive weights. In: Procceedings of the International Joint Conference on Neural Networks, pp. 21-26.
Tedeschi, L.O., 2006. Assessment of the adequacy of mathematical models. Agricultural Systems 89: 225-247.
Valadares Filho, S.C., M.I. Marcondes, M.L. Chizzotti and P.V.R. Paulino, 2010. Nutrient requirements of Zebu Beef
cattle – BR-CORTE. Suprema Gráfica e Editora, Visconde do Rio Branco, MG, Brazil.
Walczak, S. and N. Cerpa, 1999. Heuristic principles for the design of artificial neural networks. Information and
Software Technology 41: 107-117.

Mature weight of male and female Saanen goats
A.K. Almeida1*, K.T. Resende1, L.O. Tedeschi2, M.H.M.R. Fernandes1 and I.A.M.A. Teixeira1
1Department of Animal Science, Universidade Estadual Paulista, Jaboticabal, SP 14884-900,
Brazil; 2Department of Animal Science, Texas A&M University, College Station, TX 77843-2471,
USA; almeida.amelia@gmail.com
Abstract
The objective of this work was to provide ways to determine mature weight for female and male
(intact and castrated) Saanen goats, using body composition data. The used database combined
seven comparative slaughter studies: 244 individual records of body composition of Saanen goats
weighing from 4.6 to 51.0 kg BW. Nonlinear regressions were fitted to predict empty body water,
fat (EBF), protein (EBP), and ash, expressed as amounts and percentages of the empty BW (EBW)
and water-free EBW. The selected nonlinear functions were the allometric function to describe the
EBF content and the exponential function to describe EBP content in the water-free matter basis. We
estimated that at maturity, castrate males and females had 21.9 and 22.8% EBF while intact males
presented 36.8% EBF. Considering that an animal is mature when the EBF percentage approaches
22%, one can backward estimate mature EBW of 42.9, 34.1, and 25.8 kg for intact and castrated males,
and females. This work indicated that fat percentage in the body may be used to describe maturity.
Keywords: body fat, body protein, dairy goats, growth, maturity
Introduction
Mature size is used to scale animals’ growth curve because animals of distinct mature weights
exhibit different patterns of growth and body composition (Owens et al., 1995), affecting their daily
requirements of energy and nutrients. Many ruminant feeding systems have adopted it approach
(CSIRO, 2007; NRC, 2007). The objective of this study was to provide ways to determine mature
weight for female and males (intact and castrated) Saanen goats, using body composition data.

Database combined seven comparative slaughter studies and was comprised of 244 individual records
of body composition of intact male (n=94), female (n=71) and castrated male (n=79) Saanen goats
weighing from 4.6 to 51.0 kg BW. Nonlinear regressions (Brody, Von Bertalanffy, Richards, Logistic
and Gompertz models) were fitted to predict empty body water, fat (EBF), protein (EBP), and ash,
expressed as amounts and percentages of the empty BW (EBW) and water-free EBW. Candidate
equations were selected based on preliminary graphical examination of observed body composition
and the best one to describe the data was selected based on convergence and biological interpretation
of mature weight. The selected nonlinear functions were the allometric function (Y=a×Xb) to describe
the EBF content and the exponential function (Y=a×e-b×X) to describe EBP and ash contents in the
water-free matter basis. Due to the absence of overlap among all studies, the statistical analyses was
performed in two steps. The variance due to study in the body composition and EBW was considered
using the RANDOM statement of PROC MIXED in SAS, to generate an adjusted database before
fitting the nonlinear equations. After that, PROC NLIN was used in the nonlinear parameterization
using the MARQUARDT convergence method.

None of the tested nonlinear functions was able to describe ash content possibly because of its large
variation. Mature weight was assumed to be the weight when net protein deposition (i.e. accretion

minus degradation) tended to zero. The EBP (% of water-free EBW) plotted against the EBW
using the exponential function enabled to estimate the mature weight of intact and castrated males,
and females as 84.8, 33.6, and 26.7 kg EBW, respectively Equations 1, 2 and 3). Indicating that
the decrease of protein accretion in intact male approaches zero later than in females and castrated
males, during growth.
EBPintact male = 35.8±0.758 × (1 + exp(-0.0374±0.00394×EBW)(1)
EBPcastrated male = 39.5±1.813 × (1 + exp(-0.0712±0.0138×EBW)(2)
EBPfemale = 36.7±2.51 × (1 + exp(-0.0747±0.0230×EBW) (3)
In which, EBP is % of protein in the water-free EBW, and EBW is empty body weight in kg.
Replacing these mature EBW estimates in the allometric function to describe the fat content in the
EBW (Equations 4,5 and 6), we estimate that at maturity, castrate males and females had 21.9 and
22.8% EBF while intact males presented 36.8% EBF. This value (i.e. 36.8% EBF); however, poses
some challenges, and may not be biologically acceptable.
EBFintact male = 1.27±0.196 × EBW0.759±0.0481 (4)
EBFcastrated male = 1.088±0.205 × EBW0.852±0.0582(5)
EBFfemale = 1.069±0.142 × EBW0.931±0.0394(6)
In which, EBF is % of fat in the EBW, and EBW is empty body weight in kg.
Considering that an animal is mature when the EBF percentage approaches 22% (Tedeschi et al.,
2002; Trenkle and Marple, 1983), one can backward estimate mature EBW of 42.9, 34.1, and 25.8
kg for intact and castrated males, and females. This work indicated that fat percentage in the body
may describe maturity, as long as dietary challenges are not imposed to the animals. In addition,
females Saanen goats may reach maturity earlier than males.
Acknowledgements
Financial support No. 2014/14734-9, 2014/14939-0, and 2015/22600-5; FAPESP – Sao Paulo
Research Foundation.
References
Commonwealth Scientific and Industrial Research Organization (CSIRO), 2007. Nutrient requirements of domesticated
ruminants. CSIRO Publishing, Collingwood, Australia.
National Research Council (NRC), 2007. Nutrient requirements of small ruminants: sheep, goats, cervids, and new
world camelids (1st rev. Ed.). National Academy Press, Washington, DC, USA.
Owens, F.N., D.R. Gill, D.S. Secrist and S.W. Coleman, 1995. Review of some aspects of growth and development of
feedlot cattle. Journal of Animal Science 73: 3152-3172.
Tedeschi, L.O., C. Boin, D.G. Fox, P.R. Leme, G.F. Alleoni and D.P.D. Lanna, 2002. Energy requirement for maintenance
and growth of Nellore bulls and steers fed high-forage diets. Journal of Animal Science 80: 1671-1682.
Trenkle, A. and D.N. Marple, 1983. Growth and development of meat animals. Journal of Animal Science 57: 273-283.

Dual NMR and LC-MS-ToF analysis highlights new markers of nitrogen
use in dairy cows
H. Boudra1*, M. Lagrée2, M. Doreau1, P. Nozière1 and D.P. Morgavi1
1INRA, UMRH1213 Herbivores, 63122 Saint-Genès-Champanelle, France; 2ICCF, UMR 6296,
Plateforme d’Exploration du Métabolisme, Université Blaise Pascal, 24 avenue des Landais, 63171
Aubière, France; abdelhamid.boudra@clermont.inra.fr
Abstract
The aim of this study is to investigate the effect of two levels of nitrogen (N) diet on animal
metabolome. The excess or deficiency of N in diet can alter animal’s metabolism and, in this context,
a metabolomic approach may highlight novel markers of N use efficiency. HILIC-ToF-MS and 1H
NMR spectroscopy methods were able to distinguish diets of distinct N proportions. There was a
complementary between these two method with sixteen metabolites identified that were discriminant
for high and low level of N in the diet. Most of these metabolites were implicated in amino acids
pathways. Furthermore, the urinary metabolome obtained by HILIC-ToF-MS could accurately predict
the efficiency of N utilisation in dairy cows (R2=0.82).
Keywords: metabolomics, dietary N, dairy cow, HILIC-MS-ToF, 1H NMR
Introduction
In ruminant production systems, it is important to maximize the transfer of dietary N in milk and
meat, and therefore to reduce losses contributing to water and air pollution. The use of an untargeted
metabolomic approach followed by multivariate analysis is a promising strategy for discovering
markers of N efficiency. Due to the metabolome complexity, a current trend is to use a multi-platform
approach that provides complementary biochemical information. In this context, we set up a HILIC-
MS-ToF/NMR method to measure metabolic profiles in dairy cows urine.

Four Holstein cows, used in a 4×4 Latin square design, received different diets in terms of N levels
(high vs low) and energy supply (starch vs fibre). Urinary metabolic profiles were performed on a
Metabolic ProfilerTM system which combines MS and NMR detection. MS and NMR data were
then analysed by chemometric tools (SIMCA-P+ 13, Sweden).

PLS-DA models including the level of N and the type of energy supply showed a separation that was
more important for MS data (Figure 1). When PLS-DA models included only the level of N without
considering the type of energy supply, the separation was more marked for both analytical techniques
(data not shown). A total of 16 metabolites discriminating the dietary N were identified, 13 by MS
and 4 by NMR, with only one metabolite in common. The identified biomarkers were involved in
some important metabolic pathways including amino acid metabolism (indole-3 carboxylic acid,
indole acetic acid, taurine, creatine, hippuric acid, pantothenic acid and pipecolic acid), purine
metabolism (allantoine, methylguanine), and protein digestion and absorption (L-leucyl-proline,
isoleucyl-hydroxyproline).
In addition, to investigate whether the urine profile obtained by MS technique can predict N diets
efficiency, a Partial least square regression was done with all MS variables (n=830) and the N

A B
HNF
HNS
0.4 LNF
10
LNS
0.2 5
0
T[2]
T[2]
0
-0.2 -5
-0.4 -10
-0.6 -15
-0.6 -0.4 -0.2 0 0.2 0.4 0.6 -15 -10 -5 0 5 10
T[1] T[1]
Figure 1. PLS-DA plots of MS (A) and NMR (B) data according to diet fed to dairy cattle: HNS =
high N-starch; HNF = high N-fibre; LNS = low N-starch; LNF = low N-fibre.
efficiency calculated as the percentage of the ratio of N in milk and N intake. A good correlation
was obtained (R2=0.82) (Figure 2).
It is concluded that untargeted metabolomics is a useful tool for identifying potential markers of
dietary N supply and N efficiency. These results show that the combination of the 2 analytical
techniques are complementary and offers a better coverage of the metabolome (Naz et al., 2013).
y = 1x + 9.30635e-007
32 R2=0.825719
Yvar (N-efficiency)
30
28
26
24
22
20
20 22 24 26 28 30
YPred[1] (N-efficiency)
Figure 2. PLS modelling of N efficiency: HNS = high N-starch; HNF = high N-fibre; LNS = low
N-starch; LNF = low N-fibre.
Acknowledgements
This work was partially funded by the Commission of the European Communities; project FP7,
KBBE-2007-1 (RedNex).
References
Naz, S., A. Garcia and C. Barbas, 2013. Multiplatform analytical methodology for metabolic fingerprinting of lung
tissue. Analytical Chemistry 85: 10941-10948.

Early life nutritional programming of long-term weight gain and feed
intake in the porcine model
C. Clouard1*, W.J.J. Gerrits2, B. Kemp1, D. Val-Laillet3 and J.E. Bolhuis1
1Adaptation Physiology Group, Wageningen University, P.O. Box 338, 6700 AH Wageningen, the
Netherlands; 2Animal Nutrition Group, Wageningen University, P.O. Box 338, 6700 AH Wageningen,
the Netherlands; 3INRA, UR1341 ADNC, Domaine de la Prise, 35590 Saint Gilles, France;
caroline.clouard@wur.nl
Abstract
We studied the effect of a prenatal and/or postnatal Western-like diet high in fat, refined sugar, and
cholesterol on long-term feed intake and growth of piglets. Thirty-two sows and their offspring were
allocated to 1 of 4 dietary treatments in a 2×2 factorial design, with 8-week prenatal and 8-week
postnatal exposure to a Western-like diet or control diets as factors. From weaning onwards, 4-week-
old piglets were housed in groups (n=8) of 3 littermates and fed ad libitum. From the end of the
dietary treatment onwards, all piglets were fed a standard commercial diet, and followed for 8 weeks.
Piglets exposed to the prenatal Western diet were heavier at weaning than controls. Piglets fed the
postnatal Western diet weighed less than controls at the end of the treatment and 8 weeks later. From
weaning to the end of the treatment, piglets fed the postnatal Western diet had a lower average daily
gain (ADG), gain:feed (G:F) and average daily feed intake (ADFI), but a higher energy intake than
controls. During the 8 weeks following the end of the treatment, piglets fed the postnatal Western
diet had a 20% decrease in ADFI, but only a 7% decrease in ADG, resulting in a higher G:F. Thus,
feeding a high-sugar high-fat diet for only 8-week post-partum can drastically reduce feed intake,
and improve feed efficiency in later life.
Keywords: Western-like diet, piglets, feed efficiency, growth, fat
Introduction
In the last century, modern Western societies have been subjected to a drastic shift in dietary energy
sources, from complex carbohydrates to refined sugars and saturated fats. Perinatal exposure to this
so-called ‘Western diet’ has been found to program long-term health, weight gain and cognitive
function in rodent models. Little is known, however, on the period (prenatal vs postnatal) which
is the most sensitive to dietary influences. Recently, we found beneficial effects of a late prenatal,
but not early postnatal, Western-like diet on spatial memory of pigs, a model for humans. Here, we
present the effects of prenatal and/or postnatal exposure to this Western-like diet on long-term feed
intake and growth of these piglets.

Thirty-two sows and their offspring were allocated to 1 of 4 dietary treatments in a 2×2 factorial
design, with 8-week prenatal and 8-week postnatal exposure to a Western-like diet (13.2 MJ NE/kg
dry matter (DM), sugar: 225 g/kg DM, fat: 147 g/kg DM, starch: 238 g/kg DM) or standard control
diets (gestation, lactation or post-weaning starter diets; 10.7-11 MJ NE/kg DM, sugar: 45-60 g/kg
DM, fat: 37-51 g/kg DM, starch: 420-466 g/kg DM) as factors. During gestation and lactation, sows
received iso-caloric (NE) feed rations. From weaning onwards, 4-week-old piglets were housed
in groups of 3 littermates (n=8 groups/treatment), and fed ad libitum. From the end of the dietary
intervention onwards, all piglets were fed a standard commercial diet, and followed for 8 weeks. The
effects of the diets on performance were analysed using a mixed model (SAS 9.1.3.) at group level.

The prenatal diet did not affect birth weight, but piglets exposed to the prenatal Western-like diet
were heavier at weaning than controls (Table 1), which may be caused by diet-induced increase
in milk fat levels (Laws et al., 2009). Piglets fed the postnatal Western-like diet weighed less than
controls at the end of the treatment and 8 weeks later. From weaning to the end of the treatment,
piglets fed the postnatal Western-like diet had a lower average daily feed intake (ADFI; -12%),
which might be related to their higher energy intake (6.4 vs 5.8 MJ NE/d), and also a lower average
daily gain (ADG; -30%) and gain:feed (G:F), which may be explained by the lower essential amino
acid (AA) levels in the Western-like diet (Liao et al., 2015). During the 8 weeks following the end
of the treatment, piglets fed the postnatal Western-like diet had a 20% decrease in ADFI, but only a
7% decrease in ADG, resulting in a higher G:F. These results indicate that piglets fed the postnatal
Western-like diet seemed to be able to partially compensate for the growth reduction induced by a
lower AA intake in early life. The long-term reduction in growth could be due to changes in body
composition, with more adipose tissue (due to high fat intake) and/or less muscle tissue caused by a
delayed hypertrophic muscle growth (due to low AA intake). In conclusion, our study highlights the
key role of early postnatal nutrition for long-term programming of feed intake and growth in pigs.
Table 1. Performance of piglets.1,2
Prenatal diet Control Western P-values3
Postnatal diet Control Western Control Western Pre Post Pre×Post
BW, kg
Birth 1.41±0.06 1.50±0.09 1.49±0.09 1.48±0.07 n.s. ‒ ‒
Weaning 7.94±0.34 8.43±0.16 8.71±0.29 8.60±0.15 * n.s. n.s.
End treatment 19.2±1.13 15.9±0.46 19.6±0.91 16.3±0.37 n.s. *** n.s.
8 weeks later 73.0±1.85 65.6±1.66 73.8±2.35 67.6±1.93 n.s. *** n.s.
ADG, kg/day
Weaning-end 0.36±0.03 0.26±0.02 0.39±0.03 0.27±0.01 n.s. *** n.s.
End-8 weeks later 0.96±0.03 0.89±0.02 0.97±0.03 0.92±0.03 n.s. * n.s.
ADFI, kg/d
Weaning-end 0.58±0.04 0.53±0.03 0.62±0.04 0.53±0.03 n.s. * n.s.
End-8 weeks later 2.11±0.09 1.65±0.06 2.07±0.13 1.70±0.06 n.s. *** n.s.
G:F, kg /kg
Weaning-end 0.67±0.01 0.50±0.03 0.63±0.03 0.52±0.03 n.s. *** n.s.
End-8 weeks later 0.43±0.02 0.51±0.01 0.45±0.01 0.51±0.01 n.s. *** n.s.
1 Data (means ± SEMs) are averaged per pen.

2 BW = body weight; ADG = average daily gain; ADFI = average daily feed intake; G:F = gain:feed.
3 Pre, post and pre × post represent prenatal diet, postnatal diet and their interactions. n.s. = non-significant, P>0.10;
*P≤0.05; ***P<0.001.
References
Laws, J., E. Amusquivar, A. Laws, E. Herrera, I.J. Lean, P.F. Dodds and L. Clarke, 2009. Supplementation of sow diets
with oil during gestation: sow body condition, milk yield and milk composition. Livestock Science 123: 88-96.
Liao, S.F., T. Wang and N. Regmi, 2015. Lysine nutrition in swine and the related monogastric animals: muscle protein
biosynthesis and beyond. SpringerPlus 4: 147.

Net energy requirements for maintenance of F1 – Holstein × Gyr
crossbred bulls determined by the calorimetry and comparative
slaughter technique
A.L. Ferreira1*, A.L.C.C. Borges1,2, R.R. Silva1, A.S. Souza1, A.C.A. Duque1, J.S. Silva1, J.R.M.
Ruas2, L.C. Gonçalves1 and E.O.S. Saliba1
1Department of Animal Science, Federal University of Minas Gerais, Belo Horizonte, MG,
Brazil; 2Agricultural Research Company of Minas Gerais-EPAMIG, Felixlândia, MG, Brazil;
analuizavetufmg@gmail.com
Abstract
Several countries have set nutritional standards for beef and milk cattle, taking into account the
peculiarities of their realities. These systems are based on studies involving energy metabolism using
respiration chambers or comparative slaughter techniques. There are methodological differences in the
application of the concepts of net energy for these techniques. In Brazil, the vast majority of studies
of nutritional requirements are based on comparative slaughter, but recently studies using respiration
chambers have been conducted. This study was carried out to estimate the nutritional requirements of
net energy (NEm) and metabolisable energy (MEm) for maintenance of F1 – Holstein × Gyr crossbred
bulls, non-castrated, determined by comparative slaughter and by respiration calorimetry. The NEm
estimated with comparative slaughter and respiration calorimetry was 307 and 312 kJ/kg0.75 body
weight and the efficiency of utilisation of ME for maintenance was 0.62 and 0.60, respectively.
Keywords: respirometric chamber, respirometry, Zebu

Twenty bulls were used. Five animals were slaughtered at the beginning of the experiment as a
reference group and the remaining 15 animals were divided into three groups. The animals were
fed a diet of 40% concentrate, formulated according to NRC (2000) for low, medium and high
gain treatments (510, 699 and 891 kJ ME/kg0.75 live weight/day, respectively). Digestibility and
metabolism assays for determination of energy losses from faecal, urinary and methane emission
were performed. Digestible energy, metabolisable energy (ME) and net energy (NE) of diets were
determined. In the end (128 days after the beginning of the experiment), all animals were slaughtered
and body composition and empty body weight were determined. The heat production (HP) and the
energy retained in the comparative slaughter methodology were determined from method by Lofgreen
and Garrett (1968). HP through calorimetry was determined by Brouwer (1965) equation. Regression
equations of the log of HP as a function of metabolisable energy intake (MEI) were estimated. Net
energy requirement for maintenance (NEm) was estimated as the antilog of the intercept of the
equation obtained by the linear regression between the logarithm of HP and the MEI.

There were differences in nutrient digestibility, energy partition and respirometric parameters obtained
for the low, medium and high gain groups. The NEm for non-castrated Holstein × Gyr crossbreed
bulls, estimated with comparative slaughter and respiration calorimetry was 307 and 312 kJ/kg0.75
body weight (BW) and MEm of 494 and 523 kJ/kg0.75 BW, respectively (Table 1).
In order to determine the ME of the diet and compare the traditional way (Slaughter 1) to that in
which the ME is obtained by adding the loss of urinary energy and methane (measured in respiration
chamber) determined in metabolism assays (Slaughter 2), separate analyses for each of these
procedures were performed. The traditional method resulted in a 9.24% lower value. The efficiency

Table 1. Parameters of regression of heat production for the logarithm (kJ/kg0.75 BW) due to the
metabolisable energy intake (kJ/kg0.75 BW) of F1 – Holstein × Gir bulls obtained by the technique
of comparative slaughter and respiration calorimetry.1
Item Intercept (a) Coefficient n R2 RSE NEm MEm km

(b) (×1000)
Slaughter 12 7.71±0.10 7.469±0.559 15 0.93 0.072 291 452 0.64

Slaughter 23 7.81±0.10 7.330±0.514 15 0.94 0.068 307 494 0.62
Calorimetry4 7.84±0.15 7.499±0.799 15 0.88 0.093 312 523 0.60
1 NEm = net energy requirement for maintenance; MEm metabolisable energy for maintenance.
2 Metabolisable energy intake (MEI) determined by the traditional method using ratio metabolisable energy/digestible
energy = 0.82.
3 MEI determined by subtracting the energy losses of urine and methane (respirometric chamber).
4 Regression equation obtained by the relationship between heat production and MEI obtained by measuring in
respirometric chamber.
of utilisation for maintenance (km) was slightly higher than for traditional methodology (0.64 vs
0.62). The net energy for maintenance (NEm) was similar between the slaughter 2 and calorimetry,
representing minimal differences of 1.63%. For MEm this difference was 5.96%, showing km
with slightly higher for slaughter 2. Further differences were found between NEm and MEm when
comparing slaughter 1 and calorimetry, which differed by 7.34 and 15.74%, respectively.
The estimated fasting HP for lower energy intake animals was higher (444 KJ/kg0.75 BW) than
that estimated by iterative method. The efficiency of utilisation of ME for maintenance was 0.62
and 0.60 for the comparative slaughter 2 and calorimetry, respectively. The methodologies of
respiration calorimetry and comparative slaughter proved effective to determine the nutritional
energy requirements of Zebu crossed animals in tropical conditions.
Acknowledgements
We would like to thank CNPq, CNPq-INCT, FAPEMIG, CAPES and EPAMIG for their cooperation
in carrying out this work.
References
Brouwer, E., 1965. Report of sub-committee on constants and factors. In: Proceedings of the 3rd Symposium on Energy
Metabolism. EAAP Publication No. 11, Academic Press, London, UK, pp. 441-443.
Lofgreen, G.P. and W.N. Garrett, 1968. A system for expressing net energy requirements and feed values for growing
and finishing beef cattle. Journal of Animal Science 27: 793-806.
National Research Council (NRC), 2000. Nutrient requirements of beef cattle (7th rev. Ed.). National Academy Press,

Energy requirements during pregnancy in dairy goats
C.J. Härter1*, L.D. Lima1, H.G.O. Silva1, D.S. Castagnino1, A.R. Rivera1, J.L. Ellis2,3, J. France3
and I.A.M.A. Teixeira1
1Department of Animal Science, School of Agrarian Sciences and Veterinary Medicine, Universidade
Estadual Paulista – UNESP, Campus Jaboticabal, São Paulo, 14884-900, Brazil; 2Animal Nutrition,
Wageningen UR Livestock Research, Wageningen, the Netherlands; 3Centre for Nutrition Modelling,
Department of Animal and Poultry Science, University of Guelph, Guelph, ON, N1G 2W1, Canada;
harter.carla@gmail.com
Abstract
This study determined the net and metabolisable energy requirements of pregnant dairy goats carrying
single and twin foetuses. The net energy requirements during pregnancy (NEp) were estimated
using an exponential model, while the metabolisable energy requirement during pregnancy (MEp)
was estimated by the conversion efficiency (kP) of metabolisable energy intake into Nep. The daily
MEp ranged from 15.8 to 99.5 kJ/kg BW for does carrying single foetuses and from 14.6 to 131
kJ/kg BW for twins.
Keywords: conceptus, does, energy demand, gestation
Introduction
The current feeding systems used for goats estimate pregnancy energy requirements based on data
from sheep. However, it is known that goat’s metabolism differs from that of sheep (Van Soest,
1994), which may result in different nutritional requirements by these species. Furthermore, there
is a lack of studies on nutrient requirements and on the efficiency of nutrient utilisation by pregnant
goats, especially regarding single or multiple (twin) pregnancies. Therefore, the aim of this study
was to determine the dietary and metabolisable energy requirements during pregnancy in dairy goats
carrying single and twin foetuses.

Data from two experiments on pregnant dairy goats (82 observations) were used to determine the
net energy requirements during pregnancy (NEp). The energy content of the gravid uterus and
mammary gland was determined using the comparative slaughter technique. The NEp requirements
were estimated using an exponential model, comparing litter size (single versus twin) from 50
to 140 days of pregnancy (DOP). To estimate the requirements of metabolisable energy during
pregnancy (MEp), 54 goats carrying twins were randomly distributed into 6 groups, subjected to 3
levels of feed restriction (0, 20 and 40% restriction), and slaughtered at 3 different gestational age
(80, 110 and 140 DOP), using a split plot design. The maternal body and products of conception
(gravid uterus and mammary gland) were ground and samples freeze-dried. The energy content of
the samples was determined by a bomb calorimeter (Parr Instrument Co., Moline, IL, USA). The
efficiency of metabolisable energy utilisation during pregnancy (kP) was estimated by Rattray et
al. (1974) regression equation: MEI (kcal/d) = a + b × EG + c × Preg; where, a = intercept, b and c
are the slopes, EG = Δ maternal body energy retention (kcal/d), and Preg = products of conception
energy accretion (kcal/d). The inverse of slope c was considered as kP. The metabolisable energy
intake (MEI) was estimated as the gross energy intake (GEI) minus energy losses in faeces, urine
and gases. The losses in faeces and urine were estimated by digestibility trials at 50, 80, 110 and 140
DOP; using 12 pregnant goats carrying single and twins, and fed ad libitum. The energy lost through
gas production was estimated using the equation proposed by Blaxter and Clapperton (1965). The
MEp requirements were estimated by the kP value. The MEI as a proportion of GEI was considered

as the metabolisability value (q). The parameters for the exponential model used to predict NEp
requirements were obtained by comparison between single and twin pregnancies, using %NLINMIX
macro procedure of SAS software (v 9.4; SAS Inst. Inc., Cary, NC). The equation used to estimate
kP was analysed using the %NLINMIX macro procedure of SAS and the data on MEI and q were
analysed as mixed models in SAS. Significance was considered at P<0.05.

The NEp and MEp were significantly (P<0.05) greater for does carrying twins than for those carrying
single foetuses (Table 1). The kP obtained was 20.4%, which is greater than the 16.1% reported for
sheep (Rattray et al., 1974). At 110 DOP, the does carrying twins had greater q than those carrying
a single foetus, whereas at 140 DOP the reverse was observed (P=0.049). The q observed in does
carrying twins at 110 DOP is probably due to the increase in net energy requirements by goats
carrying multiple foetuses. Conversely, the decrease of q at 140 DOP in goats carrying twins may
be related to the increase in the size of the pregnant uterus, which can impair rumen digestion and
increase energy loss in faeces.
Table 1. Daily energy requirements and metabolisability values at different days of pregnancy in
dairy goats carrying single and twin foetuses.
Singles Twins
Days of pregnancy 50 80 110 140 50 80 110 140
Daily energy requirements

Net energy, kJ/kg BW 3.22 4.02 7.82 20.3 2.97 4.89 10.5 26.6
Metabolisable energy, kJ/kg BW1 15.8 19.7 38.4 99.5 14.6 24.0 51.5 131
Metabolisability (MEI/GEI)2,3,4 0.860 0.849 0.856 0.868 0.860 0.861 0.879 0.842
1 Net energy accretion in the gravid uterus and mammary gland obtained by the models: gravid uterus (single) = 45 ×
e0.034 × days; gravid uterus (twins) = 67.8 × e0.034 × days; mammary gland (single) = 262 × e 0.021 × days; mammary gland
(twins) = 312 × e0.021 × days
2 The k of 0.204 found in this study was used to calculate the metabolisable energy requirements during pregnancy.
P
3 Standard error of mean of metabolisability was 0.879; effect of interaction between litter size and days of pregnancy
(P=0.049).
4 MEI = metabolisable energy intake; GEI = gross energy intake.
Acknowledgements
The authors would like to thank the State of São Paulo Research Foundation (FAPESP) for the
financial support (Project No. 2014/11166-0, 2013/04758-5, 2006/60480-2).
References
Blaxter, K.L. and J.L. Clapperton, 1965. Prediction of amount of methane produced by ruminants. British Journal
Nutrition 19: 511-522.
Rattray, P.V., W.N. Garrett, N.E. East and N. Hinman, 1974. Efficiency of utilization of metabolizable energy during
pregnancy and the energy requirements for pregnancy in sheep. Journal of Animal Science 38: 383-393.
Van Soest, P.J., 1994. Nutritional ecology of the ruminant (2nd Ed.). Cornell University Press, Ithaca, NY, USA.

Intake and digestibility of diets containing crude glycerin to steers
determined with markers
C.R.M. Silva, E.O.S. Saliba*, F.A. Silva, G.S.S.C. Barbosa, G.M. Rocha and H. Lopes
Department of Animal Science, Veterinary School of the Federal University of Minas Gerais, Av.
Antônio Carlos 6627, Belo Horizonte, Brazil; saliba@ufmg.br
Abstract
By providing energy and sweet flavor, glycerin is characterized as a promising material for animal
feed, and it may replace part of the energy from others feeds in a ration. The objective of this study
was to evaluate the intake and digestibility of heifers fed different levels of crude glycerin through
internal indicators (indigestible dry matter (iDM), and neutral indigestible detergent fibre (iNDF))
and external Marker (purified and enriched lignin-LIPE®). Five individually fed heifers with average
weight of 602 kg and 37 months old were used. Diets were 88% corn silage and 12% corn meal and
soybean meal, with the inclusion of 0 (control); 2.5; 5.0; 7.5 and 10% of crude glycerin in the dry
matter, replacing the corn meal. Results show that inclusion of crude glycerin up to 10% does not
affect consumption and digestibility. The intake evaluation showed that the LIPE® was similar to
the data obtained by the standard method (faeces output with total faeces collection) as opposed to
the internal markers, iDM and iNDF, that overestimated intake. Regarding apparent digestibility of
dry matter, LIPE® results were similar to the standard method, but the internal markers iNDF and
iDM resulted in lower digestibility estimates.
Keywords: by-products, dairy cattle, glycerin, internal and external markers
Introduction
Because of its high energy and sweet flavor, glycerin is characterized as a promising material for
animal feed. It may replace part of the concentrates in a ration, especially corn. Digestibility and
consumption are important in the formulation of diets for ruminants. Thus, it is necessary to measure
these variables, because they are correlated with the efficiency of absorption and utilisation of
nutrients. The apparent digestibility coefficients used in the evaluation of feed can be influenced
by a number of factors, and the forage:concentrate ratio is among the most important factor. This
work was to test different crude glycerin inclusions in the diet of crossbred heifers in relation to
intake and digestibility by the method of internal markers, indigestible dry matter (iDM) and neutral
indigestible detergent fibre (iNDF), and the external marker LIPE®.

The experiment was conducted at the Federal University of Viçosa, Forest Campus. Laboratory tests
were performed at the Animal Nutrition Laboratory of the Federal University of Minas Gerais. Five
heifers, with average live weight of 602.4 kg and 37 months old were used. The animals were kept
in confinement in individual stalls. The experiment lasted 75 days, divided into five periods of 15
days with 10 days for diet adaptation and five days of collection (faeces, leftovers and feed offered).
The diets were composed of corn silage, corn meal and soybean meal. Crude glycerin was added
to the diet by replacing corn meal, diet 1 being the control (0%), 2.5% diet 2, diet3 with 5%, diet 4
with 7.5% and diet 5 with 10% inclusion. To obtain the indigestible fractions iNDF and iDM, the
samples were incubated for 288 hours as suggested by INCT(2012).

Table 1 shows the intake of different crude glycerin inclusion in the diet. The dry matter intake of
the control diet was similar to all inclusion levels. In work of Farias et al. (2012), there were no
differences in dry matter intake of grazing heifers supplemented with crude glycerin up to 9% of
the diet. When we evaluated markers for determining intake, LIPE® was similar to actual intake.
The iDM and iNDF markers overestimated intake.
The data found in Table 2, following assessments by marginal means, show no difference due to
glycerin compared to the control group. These values possibly may be from the same source of dietary
fibre derived from corn silage. Digestibility estimates by LIPE® were similar to that measured by
faecal collection, corroborating work of Saliba et al. (2003) who found that there was no difference
for DM digestibility of Tifton hay when comparing total collection and LIPE®. But when using
iNDF and iDM internal indicators, we found lower digestibilities again demonstrating the possible
influence of glycerin on indigestible fibre fractions.
Table 1. Real intake (faeces output with total collection) in kg dry matter, and the estimated intake
by LIPE® Marker, indigestible dry matter (iDM) and neutral indigestible detergent fibre (iNDF)
markers1.
Methods 0% 2.5% 5% 7.5% 10% Medium
Total 12.92 13.11 13.39 13.55 13.98 13.39 A

LIPE® 13.50 13.12 14.24 14.09 13.71 13.73 AB
iDM 14.42 14.36 16.42 15.37 15.64 15.29 BC
iNDF 14.68 14.46 17.57 16.01 19.60 16.41 C
Medium 13.88 13.76 15.40 14.75 15.74
1 0, 2.5, 5, 7.5 and 10% = crude glycerin levels in the diet DM. Means followed by different capital letters in the column
represent differences for test SNK (P<0.05). CV(%)=17.35.
Table 2. Digestibility of drymatter (DDM) anddigestibilityestimated bydifferent markers as LIPE®,

indigestible dry matter (iDM) and neutral indigestible detergent fibre (iNDF) in %1.
Methods 0% 2.5% 5% 7.5% 10% Medium
DDM 74.50 74.90 77.01 75.51 74.77 75.33A

DLIPE® 73.39 74.99 75.43 74.98 76.09 74.97A
DiDM 69.82 72.74 71.55 72.36 72.38 71.77B
DiNDF 70.52 72.60 69.49 71.36 65.85 69.98B
Medium 72.06 73.81 73.37 73.55 72.29
1 0, 2.5, 5, 7.5 and 10% = crude glycerin levels in the diet DM. Means followed by different capital letters in the column
represent differences for test SNK (P<0.05). CV(%)=5.18.
References
Farias, M.S., I.N. Prado and M.V. Valero, 2012. Níveis de glicerina para novilhas suplementadas em pastagens:
desempenho, ingestão, eficiência alimentar e digestibilidade. Semina, Ciências Agrárias, Londrina 33: 1177-1188.
Saliba, E.O.S., N.M. Rodriguez and D. Pilo-Veloso, 2003. Purified lignin extruded from Eucalyptus grandis (PELI), used
as an external marker in digestibility trials in various animal species. In: Proceedings of the 9th World Conference
on Animal Production, Porto Alegre, Brazil.

Estimate of the dry matter intake for grazing horses
R.H.P. Silva*, A.S.C. Rezende, E.O.S. Saliba, D.F.S. Inácio, S. Maruch, J.N.S.M. Queiroz and K.M.C.
Barcelos
Escola de Veterinária, Universidade Federal de Minas Gerais, Brasil, Endereço: 6627, Avenida
Antônio Carlos, campus Pampulha, 31270-901;rafaelrhps@yahoo.com.br
Abstract
Dry matter intake (DMI) of horses on pasture can be estimated by the use of external indicators,
such as the Purified and Enriched Lignin (LIPE®). This indicator has shown good results in the
determination of DMI in several species, being important to know the digestibility coefficient of dry
matter (CDDM) of feed. However, consensus is needed on the technique to determine CDDM. The
objective was to compare the technique of mobile bags with in vitro technique and internal indicator
Klason lignin (Kl) in order to check which should be used with LIPE® in estimating the DMI in
grazing horses. We used 10 grazing mares that received concentrate. Each animal received a capsule
of LIPE® for 7 days. Stool samples collections were made for 5 days and the DMI estimated with
LIPE®, associated with techniques of mobile bags, in vitro digestibility and Kl, which constituted
treatments T1, T2 and T3, respectively, and the in vivo technique using mobile bags was the control
treatment. A block design was used, with each animal being a block. DMI was 18.8; 18.3 and 16.3
g/kg body weight for treatments T1, T2 and T3, respectively. Results of T1 and T2 were similar
(P>0.05) and very close to the estimated 20 g/kg body weight for equine adults at maintenance.
The treatment T3 differed (P<0.05) from the others, probably because of errors contained in the
technique for determination of Kl. In vitro technique can be used in association with LIPE® to
estimate CDAMS of grazing horses.
Keywords: mares, forages, purified and enriched lignin
Introduction
The estimative of dry matter intake (DMI) for grazing animals is complex and cannot be performed
directly as is done when the animals are confined. Therefore, markers must be used to estimate
the DMI of grazing horses. Purified and enriched lignin (LIPE®) has been successfully used in
determining DMI in several species (Saliba et al., 2015). But, to use LIPE®, it is important to know
the digestibility coefficient of dry matter (CDDM) of feed. The evaluation techniques of in vitro
digestibility (Tilley and Terry, 1963) and mobile bags (Araújo et al., 2000) and LIPE® has also
been associated with Klason lignin (Effand, 1977) to estimate DMI (Saliba and Rodriguez, 2011).
The objective was to compare the result of CDDM obtained with mobile bags with those calculated
using other techniques, to check which should be recommended to use LIPE® in estimating the
DMI in grazing horses.

Ten Mangalarga Marchador mares of 429.0±30.0 kg body weight (BW), in early pregnancy, were
kept in pasture with Panicum maximum cv. Tanzania grass with minerals, salt and water ad libitum.
Concentrate was offered at 5 g/kg BW at 7 a.m. in individual stalls. After a 28 day adaptation
period, a seven-day digestibility trial followed. Each animal received a 500 mg capsule of LIPE®
daily, with the first two days for adaptation to the marker. Fecal samples were collected the final
five days of the digestibility trial to assess the marker concentration and dry matter (DM) content.
The DMI estimated by LIPE® was obtained from the calculations described by Saliba et al. (2015).
DMI: FP/(1-CDDM), where FP is the faecal production (kg) estimated by LIPE®. The diet’s CDDM
was determined with the use of the techniques of mobile bags (Araújo et al., 2000) and in vitro

digestibility (Tilley and Terry, 1963), constituting the treatments T1 and T2, respectively. In T2,
the in vitro digestibility was estimated according to Tilley and Terry (1963). For T3, Klignin, the
DMI was obtained from the association between LIPE® and Klason lignin analysis (Effand, 1977)
according to Saliba and Rodriguez (2011). The experiment employed a randomized block design in
which each animal represented a block.

Mares (average weight 429 kg) had DMI of 18.8, 18.3, and 16.3 g/kg BW for treatments T1, T2
and T3, respectively. Thus, T1 and T2 were close to the estimated 20 g/kg BW (NRC, 2007) for
equines at maintenance. T1 and T2 had similar DMI (P>0.05), indicating that mobile bag and in vitro
techniques can be used to estimate horse CDDM. In others studies, CDDM obtained with in vitro
methodology with equine faeces inoculum was similar to CDDM obtained with total collection of
faeces (Earing et al., 2010). The T3 estimate was lower DMI (P<0.05) than T1 and T2. Kl analysis
might have underestimated the DMI due to errors in the analytical technique. Pereira (2010) compared
DMI estimated by LIPE® associated with the in situ technique or associated with Kl in mares and
found that the DMI by LIPE® associated with the in situ technique was lower (P<0.05) than DMI
estimated by LIPE® associated with Kl. The DMI by LIPE® associated with the technique of in
situ digestibility was the closest to the DMI described by NRC (2007). The mobile bag and in vitro
techniques can be used in association with LIPE® to estimate CDDM of grazing horses.
References
Araújo, K.V., J.A. Lima, E.T. Fialho and J.C. Teixeira, 2000. Comparison of the mobile nylon bag technique with the
total collection method to determinate the forages nutrient digestibilities in equine. Brazilian Journal of Animal
Science 29: 752-761.
Earing, J.E., B.D. Cassill, S.H. Hayes, E.S. Vanzant and L.M. Lawrence, 2010. Comparison of in vitro digestibility
estimates using the Daisy incubator with in vivo digestibility estimates in horses. Animal Science 88: 3954-3963.
Effand, M.J., 1977. Modified procedure to determinate acid-insoluble lignin in wood and pulp. TAPPI, Tillis 60: 143-144.
National Research Council (NRC), 2007. Nutrients requirements of horses (6th rev. Ed.). National Academy Press,
Pereira, R.V.G., 2010. Digestibility and intake of horses in training and raised on pasture. MSc thesis, Animal Science,
Federal University of Minas Gerais, Minas Gerais, Brazil.
Saliba, E.O.S., E.P. Faria, N.M. Rodriguez, G.R. Moreira, I.B. Sampaio, J.S. Saliba, L.C. Gonçalves, I. Borges and
A.L.C.C. Borges, 2015. Use of infrared spectroscopy to estimate fecal output with marker LIPE®. International
Journal of Food Science, Nutrition and Dietetics S004: 1-10.
Saliba, E.O.S. and N.M. Rodriguez, 2011. Use of markers in assessing the digestibility in ruminants: LIPE® lignin
purified and enriched. In: Proceedings of the International Symposium on Advances in Research Techniques of
Ruminant Nutrition, Pirassununga, Brazil, pp. 50-67.
Tilley, J.M.A. and R.A. Terry, 1963. A two stage technique for the in vitro digestion of forage crops. Journal of the
British Grassland Soceity 18: 104-111.

Use of stable isotopes as a tracer for broiler chickens
A.R. Troni1, D.M.B. Campos2, R.M. Suzuki1*, H.S. Doreto1, N.K. Sakomura1 and J.A. Bendassolli3
1São Paulo State University, Via de Acesso Prof. Paulo Donato Castellane s/n, 14884-900 Jaboticabal,
SP, Brazil; 2Federal University of São Carlos, Campus Lagoa do Sino, Rodovia Lauri de Simões
de Barros, km 12, SP 189, Brazil; 3Center for Nuclear Energy and Agriculture, Av. Centenário, no.
303, Caixa Postal 96, 13400-970 Piracicaba, SP, Brasil; rafael-msuzuki@hotmail.com
Abstract
The hypothesis that is possible to achieved substantial isotopic enrichment for broiler chicken
tissues, supplying a tracer through diet was tested. In the first trial, the treatment T1 presented the
lowest values of 16.92, 14.72 and 12.12‰ for plasm, liver and breast, respectively. The T5 presented
51.24, 45.55 and 42.66‰ for the same evaluated tissues. The other treatments showed increasing
and intermediate values between T1 and T5. In the second trial, T5 dosage was chosen. Liver had a
higher enrichment in delta values, compared to the collected tissues. The values of δ15N for feathers
and breast were similar. Therefore, these data confirm the efficacy of the method: the use of a tracer
supplied through diet on these conditions.
Keywords: threonine, metabolism, tracer, isotopic enrichment
Introduction
The protein deposition is the difference between synthesis and degradation, a process known as a
turnover, which is an important factor in nitrogen metabolism. This process could be explored with
metabolic tracers as stable isotopes by diverse methodologies: bolus injection, a constant infusion or
primed-infusion. In poultry production, these invasive methods could present practical limitations,
for example, the input of the tracer occurs by vein or artery. To test a tracer supply through diet as
the same condition of nutritional studies, this assay was outlined to investigate the enrichment in
broiler tissues with L-[15N] threonine in the diet.

In the first assay, it was used 24 broilers chicken male Cobb 500® from 7 to 14 days old, each animal
was housed individually in metabolism cages in a completely randomized design with five treatments
and four replications. Dietary treatments consisted on inclusion of L-[15N] threonine (98%; Cambridge
Isotope Laboratories®): T1 = 17.4; T2 = 26.1; T3 = 34.8; T4 = 52.2; T5 = 69.6 μmol/kg bird. On the
first day, four birds were slaughtered to establish enrichment reference of δ15N for plasm, liver and
breast, respectively. After seven days of L-[15N] threonine supplementation in diets, 20 birds were
slaughtered to verify tissue enrichment. Based on these results, another assay was conducted using
8 broiler chickens male Cobb 500® from 14 to 28 days of age. The 69.6 μmol/kg bird dosage was
supplied in the diet during the first two days. The birds were slaughtered at 14 days (as reference),
16, 21 and 28 days of age, to measure enrichment values of δ15N in feathers, liver and breast.

In the first assay, the first dose (17.4) presented the lowest enrichment values of 16.92; 14.72; 12.12‰
in plasm, liver and breast, respectively. The highest dose (69.6 μmol/kg bird) presented 51.24, 45.55
and 42.66‰ for the same evaluated tissues. The other treatments showed intermediate values between
T1 and T5 (Table 1). The enrichment tissues for first dose compared to reference provided a difference
of 13.457, 10.753 and 9.955‰ for the plasm, liver and breast, respectively. These enrichments are
higher than those found in the animals tissues fed diets formulated with ingredients commonly used

in animal nutrition. In the second assay, the maximum enrichment occurred on day 16 of age. The
liver had a higher enrichment compared to the other tissues (Table 2). The values δ15N for feathers
and breast were similar (Table 2). These results indicate a metabolic difference and/or nitrogen
incorporation between organs and tissues. For both experiments a rapidly incorporation of L-[15N]
threonine could be an outcome of a high metabolic activity of the evaluated tissues (Tieszen et al.,
1983). But also, this rapidly incorporation could be due to threonine requirement is relative higher
than other amino acids, because of great turnover rate and endogenous losses by intestinal secretions
(Fernandez et al., 1994). In addition, threonine is important in protein synthesis, collagen formation,
elastin and antibodies (López et al., 2001). In conclusion, these results confirm the efficacy of the
use of L-[15N] threonine as tracer supplied in the diet.
Table 1. Tracer supply and tissues isotopic enrichment of broilers.
Treatments Feed intake Isotopic enrichment (‰)1

(µmol/kg) (Tracer – mg)
Plasma Liver Breast
0.0 0.00±0.00 3.465±0.065 e 3.975±0.255 f 2.173±0.205 f

T1 = 17.4 3.357±0.078 16.922±0.408 d 14.728±0.318 e 12.128±0.406 e
T2 = 26.1 4.952±0.176 23.513±0.547 cd 18.835±0.478 d 18.478±0.443 d
T3 = 34.8 6.665±0.193 29.203±0.417 c 26.345±0.257 c 23.393±0.372 c
T4 = 52.2 10.178±0.291 40.995±2.225 b 36.915±0.937 b 32.108±0.676 b
T5 = 69.6 12.964±0.450 51.243±2.080 a 45.555±1.133 a 42.668±0.965 a
P-value and CV 0.0001; 9.17 0.0001; 5.42 0.0001; 5.20
1 δ‰ relationship between 15N/14N, value for IRMS. Means followed by different letters in the column differ by Tukey
test (α<0.05).
Table 2. Tracer supply and tissues isotopic enrichment of broilers.
Time Feed intake Isotopic enrichment (‰)1

(days) (Tracer2 – mg)
Feathers Liver Breast
14 2.987±0.0792 2.175±0.205 3.172±0.007 1.975±0.075

15 3.249±0.0788 – – –
16 0.00 19.715±2.545 30.975±8.965 17.320±0.310
21 0.00 14.000±0.450 12.805±0.005 12.730±0.140
28 0.00 11.145±0.535 6.575±0.035 7.045±0.085
1 δ‰ relationship between 15N/14N, value for IRMS.

2 Tracer dosage 69.6 μmol/kg bird.
References
Fernandez, R.S., S. Aoyagi and Y. Han, 1994. Limiting order of amino acid in corn and soybean cereal for growth of
the chick. Poultry Science 73: 1887-1896.
López, R.M., T.J. Méndez and E.A. González, 2001. Necesidades de treonina en pollos sometidos a dos calendários
de vacunación. Veterinária México 32: 189-194.
Tieszen, L.L., T.W. Boutton, K.G. Tesdahl and N.A. Slade, 1983. Fractionation and turnover of stable carbon isotopes
in animal tissues: implications for δ13C analysis of diet. Oecologia 57: 32-37.

Author index
A Boudra, H. 363
Adamczak, L. 239 Boutry, C. 25
Agabriel, J. 233 Bowen, E.J. 277
Agarwal, U. 157, 159 Bruckmaier, R. 105
Aguilera, J.F. 153, 213 Bruininx, E.M.A.M. 279
Agyekum, A.K. 165 Burakowska, K. 81
Ahvenjärvi, S. 87 Burrin, D.G. 157
Alferink, S.J.J. 89 Busato, K.C. 137
Al-Jammas, M. 229, 233
Almeida, A.K. 361 C
Almeida, A.M. 113 Cabezas-Garcia, E.H. 257
Almeida, G.F.D. 335 Cabrita, A.R.J. 77
Alston, C. 109 Caldas, J.V. 185, 187, 243, 299, 357
Aluwé, M. 35, 221 Camacho, L.E. 83
Anderson, F. 225, 355 Campos, D.M.B. 191, 193, 335, 375
Arguello, A. 113 Cantalapiedra-Hijar, G. 77, 95, 353
Armentano, L.E. 71 Capote, J. 113
Ashaba, S. 135 Carlson, K.B. 53
Augustyn, R. 201 Carré, P. 279
Carvalho, A.U. 267
B Carvalho, P.H.A. 267
Bailey, M. 27 Castagnino, D.S. 149, 369
Bannink, A. 67 Castex, M. 155
Baracat-Pereira, M.T. 137 Castro, J. 107
Barbosa, G.S.S.C. 371 Castro Marquez, J. 117
Barcelos, K.M.C. 373 Castro, N. 113
Barioni, L.G. 69 Caton, J.S. 91
Barros, E. 137 Chantelauze, C. 353
Barszcz, M. 331 Chapoutot, P. 133
Barteczko, J. 201 Chauychuwong, N. 307, 329
Bartelt, J. 219 Cheng, L. 77
Basso, F.C. 325 Chen, H. 297, 309
Bauer, M. 289 Chizzotti, M.L. 137, 253, 359
Bee, G. 173, 311 Chouinard, P.Y. 275
Bendassolli, J.A. 193, 375 Chwalibog, A. 215, 333
Benis, N. 175 Cliche, S. 241
Berends, H. 97 Clouard, C. 365
Berk, A. 169 Cohou, C. 275
Biagioli, B. 261 Coon, C.N. 185, 187, 243, 299, 357
Bikker, P. 291, 293 Corrent, E. 161, 195, 219, 221, 349
Błońska, A. 125 Costa e Silva, L.F. 253
Blouin, R. 241 Criscioni, P. 123
Bokkers, E.A.M. 97 Crompton, L.A. 263
Bolhuis, J.E. 365 Crosswhite, M.S. 91
Bollwein, H. 105 Crouse, M.S. 91, 289
Boonsinchai, N. 185, 187, 243, 299, 357 Cruzen, S.M. 53
Borges, A.L.C.C. 267, 367 Curtasu, M. 349
Borges, I. 267
Borhan, M. 289 D
Borowicz, P.P. 91 Dahlen, C.R. 91

Dänicke, S. 169 Fiorotto, M.L. 25
Daniel, J.B. 95, 121 Firkins, J.L. 71, 141
Daş, G. 259, 265 Flaga, J. 337
D’Astous-Pagé, J. 241 Fonseca, A.J.M. 77
Davis, T.A. 25 Fontes, M.M.S. 327
De Boever, J. 35 Fontoura, A.B.P. 83
De Campeneere, S. 35, 221 Ford, J.L. 191
De Lange, C.F.M. 165, 179, 203 Fortin, F. 241
Delevatti, L.M. 325 Fouillet, H. 77
De los Mozos, J. 297 Frahm, J. 169
De Magalhães, P.M. 335 France, J. 117, 369
Denadai, J.C. 193 Friggens, N.C. 121
De Paula, R.M. 327 Furukawa, K. 181
Derno, M. 139, 255, 259, 265
De Sutter, J. 221 G
De Vries, S. 297 Galindo, C.E. 115
Dewhurst, R.J. 77 Gangnat, I.D.M. 301
Dhanoa, M.S. 345 Gardner, G.E. 109, 111, 225, 355
Didelija, I.C. 157, 159 Gariépy, C. 241
Dijkstra, J. 67 Garnett, R. 71
Doepel, L. 343 Gerrits, W.J.J. 75, 89, 97, 145, 163, 167,
Dohme-Meier, F. 311 217, 365
Doorenbos, J. 271 Gervais, R. 275
Doreau, M. 363 Ghimire, S. 351
Doreto, H.S. 375 Giger-Reverdin, S. 93
Dorigam, J.C.P. 339 Gilbert, M.S. 75
Dridi, S. 185, 243 Gimsa, U. 105
Dubois, S. 55, 155 Gionbelli, M.P. 359
Duque, A.C.A. 367 Gomes, R.A. 137
Dykier, K.C. 101, 103 Gonçalves, L.C. 367
González-Valero, L. 197
E Górka, P. 81, 125, 321, 337
El-Kadi, S.W. 25 Grandl, F. 73
Ellis, J.L. 369 Green, M.H. 191
Elo, K. 85 Grosse Brinkhaus, A. 311
Engberg, R.G. 333 Grossmann, J. 113
Engelke, S.W. 259 Grubbs, J.K. 53
England, J.A. 185, 187, 243, 299, 357 Gruse, J. 41, 139
Enishi, O. 129
Estes, K. 107 H
Halik, G. 209
F Hall, M.B. 249
Faure, S. 353 Halmemies-Beauchet-Filleau, A. 281, 285
Fernandes, J.B.K. 339 Hammon, H.M. 41, 139, 245
Fernandes, M.H.M.R. 261, 361 Hanhineva, K. 295
Fernández, C. 123 Hanigan, M.D. 71, 107, 117, 127, 131, 177
Fernández-Fígares, I. 197 Haro, A. 153
Ferreira, A.L. 367 Härter, C.J. 149, 323, 325, 369
Ferreira, A.M. 113 Harthan, L.B. 127
Ferreira, D.D. 359 Hassanat, F. 319
Ferreira, J.F.S. 335 Hayashi, K. 303
Ferreira, L.K. 359 Hedemann, M.S. 195, 349

Heetkamp, M.J.W. 89, 305 Krizsan, S.J. 257
Hermansen, J.E. 335 Kruijt, L. 175
Hernandez-Castellano, L.E. 113 Kuhla, B. 99, 255, 259
Hidalgo-Checa, N. 213
Higuchi, K. 129 L
Hilton, K.M. 187, 299, 357 Laarveld, B. 81
Hirche, F. 219 Labussière, E. 55, 145, 155, 273
Horsted, K. 335 Lachica, M. 197
Htoo, J.K. 165, 199, 203 Ladeira, M.M. 137
Huff-Lonergan, E. 53 Lage, H.F. 267
Huhtanen, P. 87, 257, 327 Lagrée, M. 363
Hulshof, T.G. 291, 293 Lammers, A. 163
Humphries, D.J. 263 Lamminen, M. 281, 285
Lamot, D.M. 305
I Lamp, O. 99, 255
Inácio, D.F.S. 373 Lapierre, H. 115, 117, 131, 133, 135, 141,
Ingvartsen, K.L. 121 319, 343
Ivarsson, E. 295 Lara, E.C. 325
Lara, L. 153, 197, 213
J Larsen, M. 143, 251, 313
Jaakkola, S. 281, 285 Larsen, T. 123, 143
Jacobson, C.L. 277 Larsson, C. 205
Jansman, A.J.M. 161, 163, 175, 297, 309 Lasek, O. 201, 315
Jensen, L.M. 235 Lauridsen, C. 333
Johansen, M. 251 Lefèbvre, T. 273
Jones, A.K. 263 Le Gall, E. 221
Jorge, L.G.O. 325 Lima, A.R.C. 261
Junghans, P. 205 Lima, L.D. 149, 369
Júnior, P.M. 193 Lindberg, J.E. 317
Lin, X.Y. 177
K Li, S. 125
Kamizono, T. 303 Liu, G.M. 177
Kański, J. 321 Lonergan, S.M. 53
Kar, S.K. 175 Lopes, H. 371
Kebreab, E. 343 Łozicki, A. 209, 239
Kelman, K.R. 109 Lund, P. 251
Kemp, B. 163, 305, 365
Keomanivong, F.E. 83, 289 M
Kienberger, H. 245 Magnuson, A. 187, 243
Kikusato, M. 181, 183, 211, 303 Makarski, M. 209, 239
Kirsch, J. 289 Mansano, C.F.M. 339
Kloska, A. 321 Mansilla, W.D. 165, 203
Kluess, J. 169 Marcondes, M.I. 253, 327
Kluge, H. 219 Marini, J.C. 157, 159
Kobayashi, Y. 129 Martineau, C. 273
Koch, F. 99 Martineau, R. 117
Koczoń, P. 239 Martín-Tereso, J. 271
Kokkonen, T. 85, 281, 285 Maruch, S. 373
Korytkowski, Ł. 337 Mathai, J.K. 347
Kosieradzka, I. 209, 239 Matthiesen, C.F. 205
Kowalski, Z.M. 81, 125, 337 Matusiewicz, M. 209, 239
Kreuzer, M. 73, 105, 147, 301, 311 Matzapetakis, M 113

McGilchrist, P. 111 Palma, M. 113
McGill, T. 71 Pannier, L. 355
McLean, K.J. 91 Parales, J.E. 275
Medeiros, S.R. 69 Paulick, M. 169
Meese, S. 105, 147 Pellerin, D. 141
Menezes, A.C.B. 253 Penner, G.B. 81, 125
Messikommer, R.E. 301 Peruzzi, N.J. 191
Metges, C.C. 255, 259, 265 Pethick, D.W. 109, 111, 225, 355
Mielenz, M. 145 Pietrzak, D. 239
Miller, D.W. 277 Plaizier, J.C. 125
Millet, S. 35, 221 Plante-Dubé, M. 275
Mitloehner, F.M. 101, 103 Pollock, E.D. 185, 243
Montanholi, Y.R. 83 Pomar, C. 351
Moraes, L.E. 343 Poulsen, H.D. 195, 349
Morales, J. 199 Prache, S. 353
Morgavi, D.P. 363 Prezotto, L.D. 83
Mueller, S. 301 Przybyło, M. 321
Mullenix, G. 187, 299 Putsakum, M. 185
Myers, A.J. 117
Q
N Qin, N. 85
Nanni, P. 113 Queiroz, J.N.S.M. 373
Nanto, F. 211 Quinsac, A. 279
Nascimento, T.M.T. 339
Navarro-Villa, A. 271 R
Neville, B.W. 91 Rabelo, C.H.S. 323, 325
Nichols, K. 67 Ragnarsson, S. 317
Niemiec, T. 209, 239 Rahman, S. 289
Nieto, R. 153, 213 Raulhac, F. 133
Noblet, J. 55 Reichstadt, M. 229
Nonaka, I. 129 Reis, R.A. 323, 325
Nørgaard, J.V. 195, 349 Remus, A. 351
Nørgaard, P. 235 Renaudeau, D. 155
Nozière, P. 77, 93, 95, 133, 135, 363 Rennó, L.N. 327
Nürnberg, G. 255 Resende, K.T. 149, 261, 361
Nyachoti, C.M. 165 Reynolds, C.K. 263
Reynolds, L.P. 91
O Rezende, A.S.C. 373
Ohtani, F. 129 Rico, D.E. 275
Ohwada, S. 211 Rivera, A.R. 149, 369
Oksbjerg, N. 39 Robinson, J. 157
Oltjen, J.W. 69, 101, 103 Rocha, G.M. 371
Oporto, C.I.S 261 Rodenhuis, M. 289
Ortigues-Marty, I. 77, 93, 133, 135, 229, Rodrigues, F.H.F. 339
233, 353 Rodrigues, J.P.P. 327
Ouellet, D.R. 115, 131, 141, 319 Rodrigues, R.T.S. 137
Owens, C.M. 185, 243 Rodríguez-López, J.M. 197
Roman-Garcia, Y. 71
P Röntgen, M. 105, 147
Pacheco, L.G. 191, 193 Rotta, P.P. 253, 327
Palin, M.F. 241 Rovers, M. 161
Palma-Granados, P. 153, 213 Ruas, J.R.M. 267, 367

Ruch, M. 289 Suzuki, R.M. 191, 193, 375
Rudar, M. 179 Swanson, K.C. 83, 289
Ruiz-Ascacibar, I. 173 Święch, E. 331
Rychlik, M. 245 Szopa, J. 209
S T
Sainz, R.D. 69, 101, 103 Taciak, M. 331
Sakomura, N.K. 191, 193, 339, 375 Taponen, J. 85
Salazar-Villanea, S. 279 Tauson1, A.-H. 205
Saliba, E.O.S. 267, 367, 371, 373 Téa, I. 353
Salin, S. 85 Tedeschi, L.O. 361
Sanderson, R. 345 Teixeira, I.A.M.A. 149, 261, 361, 369
Sapkota, D. 305 Thamsborg, S.M. 335
Sauerwein, H. 145 Therkildsen, M. 39
Sauvant, D. 93, 95, 121, 133 Thibault, J.N. 273
Sawosz, E. 333 Toyomizu, M. 181, 183, 211, 303
Schäff, C.T. 41, 139 Tran, A.T. 169
Schatzmayr, D. 169 Troni, A.R. 191, 193, 375
Schlumbohm, M. 357 Tröscher, A. 245
Schurmann, B.L. 125 Tuśnio, A. 331
Schwarm, A. 73, 105, 147 Tyl, P. 321
Scott, A. 215
Seiquer, I. 153 U
Selim, S. 85 Ulbrich, S.E. 105, 147
Seppänen-Laakso, T. 85
Shingfield, K.J. 87, 257 V
Silva, C.R.M. 371 Vadalasetty, K.P. 215, 333
Silva, E.P. 339 Valadares Filho, S.C. 253, 327, 359
Silva, F.A. 371 Val-Laillet, D. 365
Silva, H.G.O. 149, 369 VandeHaar, M.H. 71
Silva, J.S. 367 Van den Anker, I. 305
Silva, R.H.P. 373 Van den Borne, J.J.G.C. 75, 89, 145
Silva, R.R. 267, 367 Van den Brand, H. 305
Skomiał, J. 331 Van der Meer, Y. 163
Smits, M.A. 175 Van der Poel, A.F.B. 279, 291, 293
Sodsee, P. 185 Van Diepen, H. 161
Soisuwan, K. 307, 329 Van Erp, R.J.J. 167, 217
Sok, M. 141 Vanhatalo, A. 85, 87, 281, 285
Soumeh, E.A. 195, 349 Van Hees, H.M.J. 167, 217
Souza, A.S. 367 Van Laar, H. 67, 121, 271
Spek, J.W. 115 Van Milgen, J. 55, 195, 349
Stangl, G.I. 219 Van Reenen, C.G. 75, 97
Stefański, T. 87 Vernet, J. 133, 135, 229, 233
Stein, H.H. 347 Vignale, K. 185, 243
Stewart, S.M. 111 Vital, C.E. 137
Stoldt, A.-K. 265 Vivenza, P.A.D. 267
Stoll, B. 157 Vonnahme, K.A. 83
Stoll, P. 173
Storm, A.C. 143, 313 W
Sullivan, B. 241 Wall, H. 295
Suryawan, A. 25 Walpole, M.E. 125
Sutoh, M. 129 Wang, S. 147

Wang, Z.H. 177
Webb, L.E. 97
Weber, C. 41, 245
Weisbjerg, M.R. 251
Weitzel, J. M. 265
Wellnitz, O. 105
Wessels, A.G. 219
White, R.R. 71, 117, 127, 249
Wierenga, P.A 309
Wijtten, P.J.A. 305
Williams, A. 355
Wolffram, S. 265
Y
Yayou, K. 129
Yuan, Y. 157
Z
Zeitz, J.O. 73, 311
Zhao, K. 177
Zhu, C.L. 165, 179
Zijlstra, R.T. 167, 217
Zuk, M. 209

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Energy and protein

metabolism and nutrition

EAAP publication No. 137

The Kielanowski Institute of Animal Physiology and Nutrition

The Committee of Animal Sciences and Aquaculture

University of Agriculture in Krakow

Warsaw University of Life Sciences – SGGW

5th EAAP International Symposium on Energy and Protein Metabolism

EAAP publication No. 137

This work is subject to copyright. All rights

EAN: 9789086862863 The individual contributions in this

The publisher is not responsible for possible

The symposium was chaired by:

Energy and protein metabolism and nutrition 5

Interaction between immunological system, microflora of the gastrointestinal tract and

Pork production with maximal nitrogen efficiency 35

Proteomic applications to study livestock growth efficiency and meat quality 53

Evaluation of the 2001 Dairy NRC and derivation of new equations 71

Predicting variation in feed efficiency in veal calves by early life characterization 75

Energy and protein metabolism and nutrition 7

Ruminal metabolism of rapeseed meal insoluble nitrogen 87

Determinants of feed preferences and intake in calves 97

Energy metabolism in high and low RFI beef cattle 103

8 Energy and protein metabolism and nutrition

Estimation of endogenous urinary N excretion in lactating dairy cattle 115

Milk metabolites as biomarkers of energy balance in goats during lactation 123

Effects of rumen thermodynamics on volatile fatty acid production and interconversion in

Energy and protein metabolism and nutrition 9

Diet-dependent energy metabolism of neonatal calves determined by indirect calorimetric

Amino acid composition of rumen microorganisms in cattle 141

Effect of plasma factors on in vitro activation and proliferation of leukocytes from

Effect of dietary live yeast supplementation on thermal heat acclimatization in finishing

Citrulline and arginine synthesis in perinatal and young pigs 157

Dietary citrulline supplementation is an efficient strategy to increase arginine availability 159

10  Energy and protein metabolism and nutrition

Effect of dietary proteins on immunity and metabolism in mice 175

Roles of corticosterone and superoxide in the ubiquitin proteasome system in heat-

A comparison of reactive oxygen species regulation in skeletal muscle with different

Breast muscle protein turnover in broiler breeder parent stock 185

De novo lipogenesis in broiler breeder hens 187

Application of compartmental analysis to study nitrogen kinetics in broilers 191

Energy and protein metabolism and nutrition 11

The protein requirement before and after implantation in mink 205

Transgenic flax overexpressing polyphenols and inflammation: anti-inflammatory

12  Energy and protein metabolism and nutrition

The optimal lysine requirement of modern genotype piglets 221

Effects of diet composition on carcass fat in beef cattle: a meta-analysis 233

Energy and protein metabolism and nutrition 13

Soybean meal supplementation in automatic milking systems 251

Update of protein requirements for Zebu beef cattle 253

Effects of dietary carbohydrate source on milk production and environmental impact of

Influence of rutin and rutin-containing buckwheat seeds on methane emission in lactating

14  Energy and protein metabolism and nutrition

Effect of starch content of milk replacer on energy metabolism in growing-finishing veal

Effect of toasting time on proteolysis of soluble and insoluble protein fractions of

Protein digestion kinetics of different protein sources in broilers 297

Energy and protein metabolism and nutrition 15

Possible mechanism of down-regulation of atrogin-1 mRNA level in butoxybutyl

Protein digestion kinetics of different protein sources in pigs 309

Impact of silages with bioactive compounds on ruminal fermentation and microbial

Lactobacillus buchneri on sugarcane silage fermentation and performance of cattle in

16  Energy and protein metabolism and nutrition

Effects of rich in docosahexaenoic acid algae supplementation on performance of calves 337

Energy and protein metabolism and nutrition 5

Energy and protein metabolism and nutrition 7

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10 Energy and protein metabolism and nutrition

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