LD5655.V856 1995.V39

EVALUATION OF RUMINAL ESCAPE POTENTIAL OF CRAB
MEAL AND OTHER PROTEIN SUPPLEMENTS AND INFLUENCE

OF STEAM EXPLOSION ON RUMINAL DEGRADABILITY OF
CRAB MEAL
by
T. V. Viswanathan
Dissertation submitted to the Faculty of the

Virginia Polytechnic Institute and State University
in partial fulfillment of the requirements for the degree of
DOCTOR OF PHILOSOPHY
in
Animal and Poultry Sciences
APPROVED:
bounkire
Dri PFontenot, Chairman
Ah e/ | {) Pay .
' (Dr. H. J. Gerken, Jr”/ “Dr. C. E. Polan
\
A Dr. D. E. Eversole mafpart 4 Yih
November, 1995
Blacksburg, Virginia
Ce
EVALUATION OF RUMINAL ESCAPE POTENTIAL OF CRAB MEAL
AND OTHER PROTEIN SUPPLEMENTS AND INFLUENCE OF STEAM
EXPLOSION ON RUMINAL DEGRADABILITY OF CRAB MEAL
by
T. V. Viswanathan
J. P. Fontenot, Chairman
Animal and Poultry Sciences
(ABSTRACT)
Four experiments were conducted, three to study the value of crab meal
and other protein supplements, and the other to explore the potential of steam
explosion technique to improve the nutritive value of crab meal. In Expt. 1, 48
Angus x Hereford and Angus x Simmental steers (avg. BW, 223 kg) were used in a
126-d growth study. Diets were formulated to contain 10.5% CP and 63% TDN,
DM basis. In each diet, one third of the N was supplied by the protein
supplement. Steers were randomly allotted to the following six supplements: 1)
soybean meal (SBM); 2) supplement based on industrial byproducts of both plant
and animal origin (IPA); 3) experimental supplement based on byproducts of
animal origin (ESA); 4) hydrolyzed supplement No. 3 (HESA); 5) commercial
supplement (Pro-Lak®) based on animal protein (CS) and 6) crab meal (CM).
There were no significant positive responses in performance and feed efficiency
for any of the protein supplemented groups compared to SBM. There was a trend
for a positive response in gain to feed for steers fed CS and IPA. Lower weight
gain and gain:feed were recorded for the steers fed HESA supplement. Steers fed
CM diet had numerically higher growth and gain:feed than those fed SBM.
In Expt. 2, two metabolism trials were conducted, each with 24 wether
lambs (avg. BW, 25 kg). In addition to the six diets that were used for the growth
Abstract
trial, two other diets were used, a negative control (NC) with no supplemental N,
and a diet supplemented with urea (U). The supplements supplied one third of the
total dietary N. There were no differences in DM and OM digestibilities among
the lambs fed the different protein supplements. Lower (P < .05) apparent
absorption of N was recorded for the lambs fed the HESA and NC diets. There
were no differences in ruminal fluid pH among the sheep fed different protein
supplements. Sheep fed CM tended to have higher total VFA compared to other
supplements. Highest (P < .05) ruminal NH; N and blood urea N were observed in
lambs fed the U diet.
In Expt. 3, the ruminal degradability of DM and CP of crab meal and other
protein supplements were estimated in situ, in a ruminally cannulated steer. The
highest DM degradability was for SBM. The ruminal escape of protem was lowest
(P < .05) for SBM (23.2%) and the highest (P < .05) for the ESA supplement
(79.8%). The respective values for IPA, HESA, CS and CM were, 60.6, 67.3, 69.8,
and 48.4%. The IVDMD of feather meal and blood meal combinations (ESA and
HESA) were lowest (P < .05). The IVDMD of crab meal was 67%.
In Expt. 4, the potential of steam explosion technique to enhance the
nutritive value of crab meal was explored. Crab meal was steam exploded in a
batch steam explosion reactor at two levels of severities. Steam explosion
decreased (P < .05) N content of crab meal by 20%, and did not improve DM
degradability nor increase escape of CP. A 60% increase in chitin degradability,
from 21.5 to 34.2% was observed for steam exploded CM. Steam explosion
improved IVDMD of CM from 65.9% to 75.2%.
These studies illustrated that substituting other protein supplements used in
this study for SBM did not elicit a significant positive response in steer
performance. Crab meal is comparable to SBM or other commercial products as a
Abstract
protein supplement for steers. Steam explosion does not seem to be a promising
processing method for improving utilization of crab waste for ruminants.
Abstract
Acknowledgements
I would like to extend my deepest gratitude and appreciation to Dr. J. P.

Fontenot, Committee Chairman, for his support, guidance, encouragement and
patience throughout this program.
I am grateful to the John Lee Pratt Animal Nutrition program for the
Fellowship and financial support of my research program. J thank Dr. H. J.
Gerken, Jr., Dr. C. E. Polan, Dr. D. E. Eversole, and Dr. W. G. Glasser for serving
on my advisory committee. Also, I appreciate the encouragement of Dr. V. G.
Allen who was a member of my advisory committee, until she accepted a position
at Texas Tech University.
Harmony Products Inc., Chesapeake, VA and Perdue Farms Inc., Salisbury,
MD are acknowledged for supporting, in part, the research.
My thanks go to Scott Baker and Dr. Joseph Mathew for their help,
friendship, and encouragement. Smithfield and feed mill crews, Gary Bradley,
Kenneth Hines, Tim Lucas, and Eugene Long are acknowledged for their
involvement in the feed mixing and management of experimental animals. The
technical assistance, support and encouragement of Nancy Frank is appreciated. |
thank other members of our group, Mercy Joseph, Tina Shanklin, Ann Meredith
Howes, and John Fike for their help in the conduct of the animal studies. I express
appreciation to Mrs. Kay Johnson for her assistance.
Acknowledgements Vv
Appreciation is extended to Dr. W. G. Glasser for providing the facilities
for the steam explosion experiment. Special thanks go to Dr. Rajesh Jain
(Biobased Materials Center, Dept. of Forest Products and Wood Science) and Bob
Wright (pilot plant) for their generous help and sincere friendship.
I am grateful to Dr. E. Sivaraman, retired Professor and Head of the
Department of Nutrition, and the late Dr. G. Nirmalan, Dean, College of
Veterinary and Animal Sciences, Kerala Agricultural University, Mannuthy,
Trichur, Kerala, India, for encouraging me to pursue a doctoral degree in the USA
My thanks go to Dr. C. T. Thomas, Professor and Head, and other members of the
Department of Nutrition for their encouragement. I thank the Kerala Agricultural
University for providing me the required leave for this program.
Last but not least, I am sincerely grateful to my wife, Amritha, our sons.
Gopan and Anish, and my mother who waited patiently for this day.
Acknowledgements vi
w/ wg wife, pettha
Dedication Vil
Table of Contents
page
ADSEY at oo... cece ccensceeceecesneeeeeesestaeesceseessteeeeceesenaaeeeeeeeesereeetenaneags ii
AcknowledgementtS .00.............cccccccccececseesesseeseeseeeeececeeseeeeeasneeeceseeueneessosans Vv
Dedication ........00..cccccc cc cccccccccessceeeesnececceneeeccseaneceeseneeessesaaeeesesenaaneneoeees vil
Table of Contents 00.00.00... ceceeceeeeeceeneeeeneecenctcnensesseesnsessseeeeeereceseeeeneas Vill
List of Tables 00.0.0... cee ceesecceccceeeseseeessaseaeeeeeseeeeeeeeesteesesteeeseeeeeeeees xii
List Of Figures ...0........ cece ccccceeeceecceceennaaeeeecesceneaneeseseeeaaaeesesaaueesoeeaaess XV
List of Appendix Tables 20.00.0000... cece eeeseceseesnneeceseeesaeeeseseensanaeeeeeees xvii
Chapter I. Introduction ............... ccc cccceecsceeneseeeeaeeaaaeeeeaaeeeeaaenenaneaes ]
Chapter I. Review of Literature.......00.0..000

ccc ccccccccseceneteeceaeeeeeeeeaeeeaes 3
Evaluation of Protein Supplements ..................:.cccccesesseteteceeeeetees 3

Protein Degradation in the RUMEN .........ccccccceesssesttsentntettensaes 3
Ammonia Production and Microbial Growth ............00000cc0008s 5
Factors Affecting Microbial Growth .......ccccccccccceecsccceeeesteesesens 10
Protein FractionS And De@ gradation ........cccccccccsecssesecescsstneseenees 1]
Methods of Estimation of Ruminal Degradability .........0..000000. 12
Factors Affecting Ruminal Degradation of Protein ........0...0.00.. 17
Protein Supplements ........ccccccccceessscescecseeeecessueeseecsssusnesseeeseeeens 20
Combination of Different Protein oo... ..ccccccccccccccccceesceeseststseeesees 32
Crab Processing Waste ..........0..cccccccsssccceseccssssecceensateceeseteeeesensaes 33
Steam Explosion ...........cccccccccecccsseceessesesesseeeessececessaseseceseeeeseessetess 35
ODjeCHIVES ooo... ccc cccctcscecessseseeeessseeecestaeeceesesstesesesstsateeeeseestsassesesens 39
Chapter III. Journal Paper 1. Effects of Feeding Crab Processing

Waste and other Protein Supplements on Growth and Ruminal
Table of Contents Vill

Characteristics of Steers Fed High Roughage Diet ..................0.0... 40
ADSUraCt o0.. occ cccccceccececeeencneeeeeentenaeeeeeesecanecesesseeeseecnneeeeeeeeeereees 40
TntrOduction 2.0.0.0... cccccceccccceceeeecnetenceeeeeeceseceeeeeeaaaaaaaeaaaaneaeaaaaeaaaaaas 4]
Experimental Procedures ............ccccccccceceeeseesesenennneneeeececeeeneceeeeeaeanes 43

StALISTICAT ANALYSIS oo... ccccccccccctencc cece eceeeeeeceeseeeeaaaaaanaaaaeaananaaaaas 48
Results .0.......cccccccccccccessesceececeesneeeeeeeescaneeeeeecesesuaeeeseeeeseeseseqnaaaeaeeeeees 49
DISCUSSION ......... ccc ccc ccccceeceeeceseeseseessedeeseeeseteeseteeeeeeseseeseeceeeeeeeeseeenaas 59
Tp lications 20.00.00... eeeeeeccccenceneeeeeceeeeeeeeceeeeteceeseeaeaeaasaeacaesseseseeess 71
Literature Cited ....0..0..ccccccccesscccnceceeceeeeeeeteeeseeetsaeaesassaaeaasasaaaaaas 71
Chapter IV. Journal Paper 2. Effects of Feeding Crab Meal and

Escape Protein Supplements on Nitrogen Balance, Digestibility
and Calcium and Phosphorus availability in sheep ........................... 80
ADS act 20... cece ccccccccccecessessssnceeeeeeeceeecessssssassaueeeeeeceseseeeseceseceneeess 81
Introduction ...........ceccccecccccssscsesssccesssececeseeeceesecesssseeescssseececesseeeeess 81
Experimental Procedures .0...........ccccccccccecesseeceeeeeeesenssssseceeeeseeeeeeeees 83

SLALISTICAL ANALYSIS 6... .eecccccececncccecenteceeteeneceteeesaeeeeesenesseeeeeseeseens 87
Results and Discussion ..............ccccccccsesssccceesssceeceeeesssuseseeeeesssanees 88

Apparent DigestibDility .......cccccccceccccccestcccesenseecceseensceeeseeseusnsaees 88
Ruminal Volatile Fatty ACIS .....ccccccccccccscseccesssteeeseeeeesssasesseees 98
Other Ruminal and Blood PAraMeter ...ccccccccccceccscccsesseessseseccees 102
Tmplications .........cc cc ccceccccccessscceeeeseeeecesseeeeceseseeeeccesssseeeceeeeesseneeeeees 110
Literature Cited 0... cccccceccccccssscessesseeesseccesseeesesssesesessseeeceseaaes 112
Table of Contents
Chapter V. Journal Paper 3. In Situ Dry Matter and Crude Protein
Degradability and In Vitro Dry Matter Digestibility of Crab
Processing Waste and Other Protein Supplements ........................ 118
ADStract .....c ce ccccccce ccc eeeeceneeeeeeeeeeseeeeneaaaeeeeeeeeeteeeeteeteneneeeeeeeneneneees 118
Tmtroduction 2.0.0.0... ccc eeeeecsseeceeeeeeeeceneeeeeeeeeeeeeteeseseseeseeeneneneeeseeee 119
Experimental Procedure ..............ccccccecceeeessncceceeeeeeeenenaeeeeceeeeeoeees 12]

In Situ EXPeriment 0...ccceccccsscscesecnsacccccecceseaseseesenaseseaeesaeeeeteeses 121
Tn Vitro EXPerimen ....ccccccccccccccccseeeceeecceeecaeeeeeeeeeeuneseeeeaeessenees 123
StAtiStiCA] ANALYSIS .o.c.ccccccccceeeeeeesettneteeane seaeea saa saaaeseneseseseeeeeees 125
Results o.oo... ccccccccceceeesceesseceeseesesesecseeeedeesetseseeeseescseeeeeeeeeeeenaas 125

In Situ EXPeriment ...cccccccccccccccencsneeeeeeeeeeeeececeeeeaaaeneteceeenaanneees 125
In Vitro Dry Matter Digestibility (IVDMD) ............cccccccccccceees 131
DISCUSSION ......0..cccccccescccceeesetceeecceeeseeeeeseseneteaseeseeseseeeneaaaeeseeesees 13]
Implications ..........cccecccccccsesseceeeeessecesesaceececesseecesssnsseeeceseesssatesens 142
Literature Cited 2... ccccecccscsceesseeessesessssecesetsesceseeeeseesteeeeeees 143
Chapter VI. Journal Paper 4. Effect of Steam Explosion at two levels

of Severity on Dry Matter and Chitin Digestibilities and Escape
of Protein as Measured by /n Situ and In Vitro Methods ............. 149
ADSUracCt 20.0... cccccceccccsccccceccusccceuccssnceeusetauscseseeectsestensseusecrscenscescees 149
150
Experimental Procedures .0.........0.cccccccsccessscesseceesseecessseeesseesenaes 152

In Vitro EXPeriment .........cccccccceccccece eee eeceeeenesaeeaaaeseaeaaaeaeneaaaaas 153
In Sit EXPeriment .....cccccccccccccceccectteneeeteeeeeeeeeeeceteeteeeeeeeeeeenes 154
STALISTICAL ANALYSIS oc. cccceccccccceccccescseteeeesaeeeeseeeaseeeeeeesesnsasees 156
Results and DISCUSSION .............cccccccccccccccccccesccecesccuceesencecceucencecs 156

IN Vitro EXPCViMentt .....ccccccccccccccecccccccesececseeceseeeeeaansaassesesetes 158
Tn Sit Experiment .......ccccccccceeeteccsccecccceeeeeesesteceeetessseeesnneas 160
Table of Contents
Tmplicationss 2.0.00... cceesccececeeeenneecceesesneceeeeeseteneaeeeseeeeseneestaeeeees 175
Literature Cited ......0.. 0c ccc cccccccccccceccecceccesceccecsecuececcesecencesensusesuess 175
179
186
213
219
Table of Contents xl
List of Tables
POCO Re OREO SHEET HEARTH OTH R HERETO EEH ER HOEH OEE SE REE OHE OSE SEE OTHE EERE HOT H RR OTET ETOH HSER HEE HOES OHO RHEE
Chapter III (Journal Paper 1)
Composition of protein supplements ..............:::cccccecscceeeees wee eeereneveee
Chemical composition of ingredients used for formulation

of the diets for the Steers ...............ccccccecceccecceccececcucecceseeces Cee neoccvears 45
Ingredient and chemical composition of diets fed to steers Soe enero nee 47
Performance of steers fed different protein supplements ... 50
Ruminal pH and blood urea of steers fed different

protein supplements ............c ee ccceeeeccecssssstteceeseseeeetsnaeeees 55
Ruminal volatile fatty acids of steers fed the different

protein supplements (56 d) oo........c ce cccceseesseeeceeeesesenssseaes wee ewan necan 57
Ruminal volatile fatty acids of steers fed the different

protein supplements (126 d) ...........c
cc cceeccceceeecesensssseeees 58
Chapter IV (Journal Paper 2)
Ingredient composition of experimental diets fed to sheep 84
Chemical composition of experimental diets fed to sheep . eee earewseens 85
10. Apparent digestibility by sheep fed experimental diets ..... 89
11. Nitrogen balance by sheep fed experimental diets ................0.0.....
List of Tables xii

ee eeeeeenseeeeeeenaeaeeeeeeeeeeeesaeeeseaeeesesaeeeesesaaeseseaeeeeseesececeeesuaueeeeseseaaeeeeeesd page
Calcium balance by sheep fed different protem supplements ......... 97
Phosphorus absorption by sheep fed experimental diets ................ 99
14, Ruminal volatile fatty acids of sheep fed the different

protein supplement diets........... cece eecceeececeeeeeeeee
tees teseeeeeeeeeeeeeey 100
15. Blood urea nitrogen, and rumen pH and Ammonia nitrogen

of sheep fed experimental diets .0............ccccccccsescseeeeeeeeeeseseeeennaaes 103
Chapter V (Journal Paper 3)
16. Degradability of dry matter of protein supplements in situ ............ 127
17. Degradability of crude protein of protein supplements in situ ........ 130
Chapter VI (Journal Paper 4)
18. Effect of steam explosion on crude protein and chitin

levels of crab meal and Chitin 20.0.0... cece ccceesesseeeeseeseneeees 157
19, Effect of steam explosion on fractionation and degradability

of dry matter of crab meal by in situ experiment ................0..0..0060 162
20. Effect of steam explosion on fractionation and degradability
of dry matter of chitin by in situ experiment ................::cccceceeeeeees 163

of crude protein of crab meal by in situ experiment ..................... 167

of crude protein of chitin by in situ experiment ...................0...006. 169

of chitin of crab meal by in situ experiment ........... deeeeeetseeeeeeeesaes 172
List of Tables xii

of chitin of chitin by in situ experiment ................. cece
List of Tables XIV

List of Figures
FIQUIE 0... ccc ee eeccceeeeeeneeee eee ceee eee eentnneeeeeeeee eee tente ned se teeceeeeeteeteteeeneeeneneneeeeg page
Review of Literature
1. Structures of chitin and chitosan .............ccccccceecceeccssceecesccesecceees 35
Chapter ITI (Journal Paper 1)
2. Average cumulative daily gain of steers fed different

protein supplement .................cccecsceceseseeeceeeenneeeeesseteeeeeereseeanenes 51
3. Average cumulative dry matter intake of steers fed different

protein supplements .............eececsssecceeesssnneeeceeeeeeesnnnaneceeeeeseseees 53
4. Average cumulative feed efficiency (gain:feed) of steers

fed different protein supplements ..................cccccssscceceeeseeeeeeeeens 54
Chapter IV (Journal Paper 2)
5. Ruminal ammonia nitrogen and blood urea nitrogen

concentrations of sheep fed diets supplemented
with different protein SOUTCES ©... ccccceeseecesseeeeeseeeeeeeeetneeeeeeens 107
6. Correlation between blood urea nitrogen and ruminal ammonia

nitrogen of sheep fed diets supplemented
with different protein SOUFCES .0............
cece ceeseeesneseceeeeeeeeeeeeneeaeees 109
7. Correlation between blood urea nitrogen and urinary

of sheep fed diets supplemented with different
PTOLEIN SOULCES 0.0... cceccccceescscceceseesenteececcesessseeeeeeescesessasaeeeeeeeeees 111
List of Figures
Chapter V (Journal Paper 3)
8. In situ dry matter degradability of protein supplements ................. 126
9. In situ crude protein degradability of protein supplements ........... 129
10. _ In vitro dry matter digestibility of protein supplements ................. 132
Chapter VI (Journal Paper 4)
11. Effect of steam explosion on in vitro dry matter digestibility

of crab meal and Chitin 2000.0... eee cee eeneceeseneeeecessnaceeeseeseeaneeeeeees 159
12. Effect of steam explosion on degradability of dry

matter of crab meal and chitin i771 Sitte...... ccc cccceeeeccecccee ceeeesecceeeeees 161
13. Effect of steam explosion on degradability crude

protein of crab meal and Chitin 171 SUt1....... cece ccc ccccccccccccceeeenenseneees 166
14. Effect of steam explosion on degradability of

chitin of crab meal and chitin inf Situ... ccceeccccccccccceeeeeeeeettees 171
List of Figures
List of Appendix Tables
Appendix Table ........cccccccccccccceeessesesssessseesseeeeeenenes se deseccaeeeceeaaeeeeeseeanees
1, Dry matter disappearance of protein supplements by

in Situ CXPeTIMENt ...........:ccceeeeeeeees esusacaasacnccecaeeeceeceecauceeeeeeeeceesesees
2. Crude protein disappearance of protein supplements

by 777 Site EXPCTIMENt 00.0.0... cceeccecceeessseeeeeseesensnsaaaeneceeeeeeeeseseeaaeaes
3. Effect of steam explosion on dry matter disappearance

Of crab meal and Chitin 0... cccesseceeeeseseceeeeeseeeeeseeeseeerenanees
4, Effect of steam explosion on crude protein disappearance

of crab meal and Chitin .00.....0 cece ccccccccesseccecesseeeecesneeeeesetseeesaes
5. Effect of steam explosion on chitin disappearance of crab

meal and CHItiN ..........c ce cceecccceessececeeeseesssseeeceeceeeeeeseeseessesseeesessess
List of Appendix Tables xvi

Chapter I
Introduction
Most of the protein and non-protein N (NPN) ingested by ruminants is

converted to microbial protein in the rumen, which is utilized by the animal. Some
protein sources which escape ruminal degradation are utilized more efficiently
than microbial protein. Thus, if these high-quality proteins escape the rumen, the
protein will be utilized more efficiently. Only a portion of the N ingested by
growing ruminants and lactating dairy cows is used for microbial action in the
rumen. In order to maximize the performance of high producing animals, much of
the dietary protein from protein supplements should escape rumen degradation and
be available for absorption in the small intestine.
Studies have been conducted in cattle to assess the escape protein potential
of various protein supplements of waste materials such as feather meal, blood
meal, and distillers dried grains. Little information is available on the escape
protein potential of some commercial protein supplements based on plant and
animal protein, and crab processing waste, a co-product obtained from the sea
food industry. Studies have shown that crab processing waste is a good source of
protein for ruminants which could be fed as dehydrated form (crab meal) or by
ensiling with roughage.
Introduction L
Although crab meal is a good source of protein, it has been shown that the
digestibility of protein in the crab waste-straw silage was lower than that of fish-
waste straw silage (Samuels et al., 1991). The lower digestibility of crab
processing waste diets may be due to chitin, which is a component of exoskeleton
of crustaceans. Chitin, a poly-N-acetylglucosamine, an analogue of cellulose, is
less digestible in ruminants than cellulose. Therefore, a method which would
cleave the chitin -protein matrix would be beneficial for the better utilization of the
waste.
Steam explosion is a technique employed in industries for fractionation of
lignin, hemicellulose, and cellulose. As chitin is structurally similar to cellulose,
perhaps when this material is subjected to steam explosion, there would be a
structural change in the chitin-protein linkage. This technique has been employed
in the field of ruminant nutrition. Steam exploded wheat straw was found to be
better utilized by steers, compared to non exploded straw (Sciaraffia and Marzetti,
1988). However, no information is available on the effect of steam explosion on
the degradability of chitin-protein complex of shell fish wastes. Therefore, the

overall objective of this program was to evaluate the ruminal escape potential of
crab processing waste and other protein supplements and also to investigate the
effect of steam explosion on the nutritive value of crab waste in ruminants.
Introduction 2
Chapter II
Evaluation of Protein Supplements
Protein Degradation in the Rumen. Dietary protein entering the rumen is

extensively degraded by both bacteria and protozoa (Russell et al., 1991). This
degradation involves two steps. Initially, the protein chain is broken by hydrolysis
of peptide bonds (proteolysis), resulting in peptides and amino acids (AA). The
terms degradation and fermentation are often used interchangeably, but there are
differences. Protein degradation is a multistep process involving solubilization,
extracellular hydrolysis, transport, and the formation of endproducts (e.g., NHs,
VFA, CO,, CH,). The term fermentation refers only to the last few steps, which is
a more absolute criterion for complete degradation.
The quality and quantity of ruminal fermentation products are dependent on
the types and activities of microorganisms in the rumen. The ruminal microbial
ecosystem is very diverse (Hungate, 1966). Russell and Hespell (1981) indicated
that the ruminal microbial ecology is complicated by the fact that there are
numerous interrelationships among the various ruminal microbes. The complexity
Review of Literature 3
of the ruminal ecosystem has led many nutritionists to conclude that the ruminal
ecosystem is so complex that it cannot be understood or described in quantitative
terms. |
The proteolytic enzymes appear to be associated primarily with the
bacterial cell wall with a small amount of cell-free activity probably resulting from
cell lysis (Allison, 1970). The protease is present on the outer cell surface and
hydrolyzes protein extracellularly (Blackburn and Hullah, 1974). Proteolytic
enzymes are associated with many ruminal bacteria, and proteolytic activity of
rumen microorganisms is not greatly altered by the diet. Diet can have an effect on
protein degradation in the rumen, perhaps indirectly through altermg pH and
bacterial numbers or types (Allison, 1970).
Protease activity appears to be “trypsin-like” in nature (NRC, 1985). The
liberated amino acids after proteolysis may leave the reticulo-rumen, may be
utilized for microbial growth, or will be degraded to NH; and fatty acids. Amino
acids are rapidly degraded in the rumen, and therefore only limited amounts of AA
will be available for absorption. Chalupa (1976) reported that the half life of eight
essential AA incubated with strained rumen fluid was 2 h or less. Some strains of
rumen bacteria require NH3, even if AA are present in their growth medium.
Bacteroides ruminicola can however, utilize peptides from the growth medium,
suggesting a lack of transport system for the individual AA across the cell wall
(Pittman et al., 1967).
Volatile fatty acids produced by the ruminal fermentation form an
important energy source for the ruminant. Acetate and butyrate are well utilized
for the growth of the animal, while, propionate is used for gluconeogenesis. Slyter
(1976) reported that lactate accumulation leads to ruminal acidosis, lowered fiber
digestion, lowered feed intake and in extreme cases death occurs.
Ammonia Production and Microbial Growth. Most ruminal bacteria are
able to use NH; as a N source for microbial protein synthesis. Normally ruminal
protein fermentation produces more NH; than the microorganisms can utilize. A
portion of the NH; will be utilized by microbes, while the other portion may be
absorbed through the ruminal wall and carried to the liver where it is converted
into urea. A part of the urea thus produced will be recycled back into the rumen
through saliva or directly into the rumen through the rumen wall. The remaining
portion will be excreted through the urine. Since protein is the most expensive
ingredient in most ruminant diets there has been considerable interest in reducing
ruminal protein fermentation. Nolan (1975) reported that in most cases 2 25% of
the protein can be lost as NH3.
Tamminga (1979) reported that if the protein goes into solution quickly, the
rate of enzymatic degradation is often increased. Many of the proteins found in
forages and soybeans are very soluble and are degraded rapidly by ruminal
bacteria. Heat treatment that can denature the protein can decrease solubility and
the rate of protein degradation. Byproduct feeds such as brewers dried grains
(BDG), corn gluten meal (CGM), blood meal (BM), and fish meal (FM) contain
protein of low solubility.
Carbohydrate has little effect on the rate of protein degradation by
extracellular proteinases, but it can greatly affect the end product of AA
metabolism (Russell et al., 1983). Russell and Martin (1984) reported that
microorganisms that utilize non structural carbohydrate take up peptides at the rate
of .07 g of peptide per gram of microorganism per hour, and this N is used for
microbial protein synthesis or NH; production. The diversion of peptides to
microbial protein synthesis or NH; production is regulated by the availability of
carbohydrate. When there is availability of carbohydrate, 66% of microbial protein
comes from peptides and 34% comes from NH3 (Russell et al., 1983). All the
peptide N will be converted to NH; in the absence of carbohydrate.
The capacity of various ruminal bacteria to ferment protein hydrolyzate and
produce ammonia was studied by Bladen et al. (1961). They concluded that
Bacteroides ruminicola was the most important amino acid fermenting bacter1um
in the rumen of cattle. Russell et al. (1983) reported that when mixed ruminal
bacteria were incubated with an excess of casein and with amounts of mixed
carbohydrates that were inadequate to support maximum growth rate, there was a
linear decline in NH; production as the growth rate increased. Studies with ‘“N
indicated that 34% of the NH3 was not influenced by carbohydrate availability.
Excessive NH; production and absorption increases N excretion and the energy
cost of urea synthesis.
Microbial colonization of the digestive system of newborn ruminants
follows a typical ecological succession in that bacteria proliferate in the fluid
phase immediately after birth and colonize the digestive tract (Fonty et al., 1987).
These fluid-phase bacteria facilitate the subsequent sequential colonization of the
fluid by fungi and protozoa (at 8 to 10 and 12 to 20 d, respectively) which results
in the formation of complex multispecies that develop in the fluid, on feed
particles, and on tissue surfaces, and result in the complex digestive activities of
mature ruminants (Fonty et al., 1988; Stewart et al., 1988).
Cheng and Costerton (1980) reported that the types of microbial
populations that develop at various locations of the ruminant digestive tract are
complex, and they depend on the chemical microenvironments provided by
nutrients. Also, once established, they are very stable and will change only when
the nutrients are changed.
Ruminal microorganism derive most of their energy from the fermentation
of carbohydrates, and the ruminal bacteria may be categorized in a general way
according to the type of carbohydrate that they ferment (Russell et al., 1992).
Microorganisms that ferment cellulose and hemicellulose (SC) grow slowly and
utilize NH; as N for microbial protein synthesis. Those that ferment starch, pectin,
and sugars (NSC) grow more rapidly than those that ferment SC and utilize either
NH; or AA as a N source. Nocek and Russell (1988) reported that ruminal
microbial growth yields are a constant function of DMI or OM digestion. The
yield of microbial N per kilogram of TDN was calculated to be 26.12 g (NRC,
1989).
Ammonia N can serve as principal N source for microbial protein synthesis.
The concentration of NH; N ranged from 5 (Roffler and Satter, 1975) to >20
mg/dL (Mehrez et al., 1977) of ruminal fluid. In the case of rapidly degraded
feedstuffs a higher level of NH3 N concentration may be necessary for maximum
digestion and fermentation rates (Hespell and Bryant, 1979). Some microbes
require N in the form of NH3, and the feeding of slowly degraded proteins may
create an NH; deficiency in the rumen (Klopfenstein et al., 1976), but this can be
avoided by urea supplementation to meet microbial NH; needs (Klopfenstein,
1976). Higher ruminal NH; levels (37 mg/dL) were observed at 12 h in lambs fed a
urea control diet.
Lambs fed BM with urea had lower NH3 N (18.2 mg/dL), compared to
natural protein, indicating a lower degradability in the rumen (Stock et al., 1981).
Thomas and Beeson (1976) reported that ruminal NH; N of steers fed soybean
meal (SBM), feather meal (FTM) and hair meal were 14.1, 5.9 and 6.9 mg/dL.
Santos et al. (1984) conducted a trial in lactating dairy cows to estimate the protein
degradability of SBM, CGM, wet brewers grain and distillers dried grains. It was
observed that ruminal NH; N concentrations were 10.1, 5.6, 6.9 and 6.4 mg/dL,
respectively.
Clark et al. (1992) reported that with ruminal NH; N > 5 mg/dL, passage of
microbial N to small intestine is correlated more highly with organic matter truly
digested in the rumen than with the concentration of NH; in ruminal fluid. Further,
the increased proportions of AA passage to small intestine arise from increased
intact protein escaping the rumen. They also reported that with typical dairy diets,
bacterial CP synthesis peaks at 12 to 13 % dietary CP. Kang-Merznarich and
Broderick (1981) suggested that ruminal NH; N concentrations between 3.3 and
8.5 mg/dL were required for maximal microbial growth when diets contained 74%
corn grain. Feeding CGM and BM (combination of 50:50 on N basis) resulted in
NH; N concentrations below 3.3 mg/dL, numerically lower microbial N flows to
the duodenum, and lower microbial efficiency compared with urea, SBM and
SoyPLUS® a high ruminal escape SBM (Ludden and Cecava, 1995).
Schloesser et al. (1993) reported that ruminal NH3 N_ concentration
decreased linearly as BM replaced SBM; however, feeding BM did not affect the
total N, microbial N, or non NH3 N (NAN) flows to the small intestine.
Titgemeyer et al. (1989) reported that replacement of corn starch in diets with
protein sources resulted in decreases in efficiency of microbial protein synthesis.
Ruminal NH; N had the greatest increase when SBM was fed. Blood meal
supplementation resulted in only nonsignificant increases in ruminal NH; N. Urea
supplementation resulted in the highest ruminal NH; concentration, whereas BM
supplementation resulted in the lowest ruminal NH; concentration (Lardy et al.,
1993).
In a study conducted in four steers with ruminal, duodenal, and ileal
cannulas, Cecava et al. (1991), found out that total N flows to the small intestine
were 13.1% greater for a combination of CGM and BM than for SBM because of
increased passage of nonmicrobial N. Feeding SBM vs a combination of CGM and
BM increased efficiency of microbial CP synthesis (27.3 vs 23.3 g of N/kg of OM
truly fermented) and microbial N flow to the small intestine (127.5 vs 112.5 g/d).
Microbial growth yields have often been expressed as microbial N or CP per
kilogram or 100 g of digested organic matter (DOM).
Hsu et al. (1991a) conducted a study in five ruminally, duodenally, and
ileally cannulated sheep to investigate the applicability of using a combination of
defaunation with various combinations of N supplements (SBM, CGM, BM, urea,
and casein). They concluded that defaunation did not limit the supply of free AA
and peptides for ruminal bacterial growth or activity. Defaunation did not have a
consistent effect on ruminal microbial deaminase activity (Hsu et al., 1991b), and
did not decrease the total free AA concentrations in ruminal fluid, but it altered the
AA profile. They also reported that there were no increases 1n total microbial CP
or OM concentrations in ruminal contents even though the defaunation increased
the ruminal bacterial numbers.
Rooke et al. (1986) reported that maximum ruminal bacterial protein
synthesis was with SBM in place of barley in the basal diet. They also reported
that ruminal microbial protein synthesis would be maximally efficient when N
concentration was 27 g N/kg DM intake (~16.8% CP) and thereafter the synthesis
would drop. Microbial protein synthesis was at the peak when the feed contained
50% ruminal degradable N (Cummins et al., 1983). The microbial protein
synthesis exceeded the protein degradation which might be due to the utilization of
recycled N. /n Vitro studies conducted by Strobel and Russell (1986) showed that
there was a significant decline in efficiency of microbial protein synthesis at pH
values < 6.0.
Factors Affecting Microbial Growth. Efficiency of microbial growth is
dependent on the availability of specific substrate for fermentation in the rumen;
pattern, composition and rate of substrate availability; and environmental factors
(NRC, 1985). Also, microbial flow is dependent on rumen volume/passage and
particle size relationships. The factors affecting microbial efficiency are numerous
and complex. Forage intake and rumen dilution rate have been shown to have a
significant impact on microbial flow (Van Soest, 1982).
Interaction of carbohydrate and protein in the diet has a profound effect on
microbial protein synthesis. Harrera-Saldana et al. (1990) determined the influence
of synchronization of protein and starch degradation in the rumen of lactating
cows using barley or milo as the carbohydrate source and BDG or cottonseed meal
(CSM) as the source of protein. Crude protein and starch digestion in the rumen
were greater for diets containing barley than milo but were not affected by protein
source. Efficiency of microbial protein synthesis in the rumen and microbial CP
flow to the small intestine were greatest when a combination of barley and CSM
were fed to cows. This indicates that synchronization of rapid fermentation
stimulated ruminal microbial protein production.
Stokes et al. (1991) studied the effects of different levels of nonstructural
carbohydrate (NSC) and degradable intake protein (DIP) on ruminal microbial
protein production in lactating cows. Diets formulated to contain 31 or 39 % NSC
and 11.8 or 13.7% DIP, as percentages of DM, produced greater microbial protein
synthesis than a diet containing 25% NSC and 9% DIP. They concluded that NSC
greater than 24% and DIP greater than 9% will increase the microbial protein flow
from the rumen. Maeng et al. (1993) and Taniguchi et al. (1993) also reported a
trend that increased levels of rumen available energy and N in diets improved the
efficiency of microbial synthesis in the rumen
Aldrich et al. (1993) formulated diets for cows in early lactation for high
and low ruminal availabilities of NSC and CP. A diet containing 36% NSC, of
which 80% was digested in the rumen resulted in greatest passage of bacterial N to
the small intestine. Decreasing the proportion of NSC to protein digested in the
rumen reduced passage of bacterial N. Henning et al. (1993) provided the same
daily allowance of energy or protein intraruminally to sheep at 12-hourly pulse-
doses or continuous infusion. Better microbial growth was recorded for the
continuous infusion, compared to the pulse-infusion. This study indicated that
continuous availability of energy is more important to microbial growth than the
pattern of energy and N release.
A positive correlation occurs between increased dilution rate and increased
microbial growth (Cole et al., 1976). Rumen dilution rate is defined as proportion
of total rumen volume leaving the rumen per hour. Cole et al. (1976) observed that
there was an increase in dilution rate (3 to 5%/h) along with an increase in
microbial protein synthesis (7.5 to 11.8 g/100 g OMD) when steers were switched
from an all concentrate diet to one containing 14% roughage.
Dietary S affects microbial protein synthesis. Sulfur is required by the
rumen microorganism for the synthesis of methionine and cysteine. Hume and
Bird (1970) reported that when sheep were fed a diet supplying .6 g of S per day
(N:S = 34.3), 82 g of microbial protein were produced daily in the rumen. Raising
S intake to 2 g/d (N:S = 10.9) increased protein synthesis to 94 g/d, but no
improvement was noticed when the ratio was narrowed further to 6.4 (3.4 g/d).
They reported that the optimum N:S ratio is 10:1 for the microbial growth.
Protein Fractions and Degradation. Dietary protein entering the
forestomach is usually extensively degraded by both bacteria and protozoa
(Tamminga, 1979). This degradation involves two steps. Firstly, the protein chain
is broken by hydrolysis of peptide bonds (proteolysis) which results in peptides
and AA and secondly the deamination of AA. Proteolysis has been reported to be
rate limiting (Nugent and Mangan, 1978; Russell et al., 1983). Proteolysis and
deamination were both found to be affected by pH. Conflicting results have been
reported. Lewis and Emery (1962) reported that the optimum pH for both
proteolysis and deamination was between 6 and 7. Hussein and Jordan (1991)
reported that at low pH the degradability of N of SBM will be much lower.
Feed protein is partitioned into three fractions: Fraction A (NPN), Fraction
B ( true protein), and Fraction C (unavailable N) (NRC,1985). Methods have been
developed for assessing the rumen degradability of protein using rate of
degradation and rate of passage (Orskov and McDonald, 1979; Mathers and
Miller, 1981). Passage rate may be influenced by animal (Poppi et al., 1980), and
intake level (Colucci et al., 1982). With high feed intake ruminal degradability
decreases (NRC, 1985). Escape protein values of forages differed between years
and among months within year (Karges et al., 1992). The extent of degradation can
be described as A + kg, x B/(Kg, + kpp), where A is soluble protein, B is degradable
protein, and ky, and kp, are rate constants for degradation rate and passage rate of
degradable protein, respectively. The fraction of total protein that is passed, P =
kp» X B/(kay + kp») + C, where B, kg, and kp, are the same as above; C is
unavailable N.
Methods of Estimation of Ruminal Degradability and Escape Protein.
There are several in vitro and in vivo techniques that have been used to estimate
the degradability of dietary N in the rumen. Degradability experiments with
animals are expensive, time consuming and laborious. Therefore many attempts
have been made to predict the protein degradability of feeds from relatively simple
methods.
In vitro methods based on N solubility (Verite et al. 1979) have been used
for the estimation of ruminal protein degradability. Krishnamoorthy et al. (1983)
proposed a model using commercial proteases. Mahadevan et al. (1987) and Kohn
and Allen (1995) used proteases isolated from mixed ruminal microbes to estimate
protein degradability. Mahadevan et al. (1987) reported that accumulation of NH;
and AA in ruminal in vitro incubations may be misleading indicators of protein
degradation because protein degradation and microbial protein synthesis occur
simultaneously. Broderick (1987) could overcome this handicap by using
inhibitors of NH; and AA anabolism to obtain quantitative recovery of the
products of protein degradation. Aufrere et al. (1991) compared the enzymatic
technique and a method using solubility in a buffer. The prediction was that the
enzymatic was very precise and much better than the solubility method.
An in vitro procedure was developed by Hristov and Broderick (1995) to
estimate rate and extent of ruminal protein degradation using '"NH3 to quantify
uptake of protein degradation products for microbial protein synthesis. They
reported that microbial protein synthesis was a linear function of extent of
degradation.
Chalupa et al. (1991) presented a model-generated protein degradation
system which analyzes protein fractions using borate-phosphate buffer, neutral
detergent, and acid detergent solutions which separate fractions of protein into A,
B,, Bo, Bs, and C. Ruminal degradabilities of fractions (%/h) were A,
instantaneous; B1- 200 to 300; Bz - 5 to 15; B3- .1 to 1.5; and C - 0%. Ruminal
degradation (Ky) of protein is based on literature values where feeds were
incubated either in situ or in vitro with proteolytic enzymes. They generated the
dietary degradability estimates of protein from the above model. They found that
58% of N of alfalfa haylage was in fraction A, large percentages of protein of high
moisture com, SBM and BM were in fraction B2, and 38% of BM protein was
fraction B3. The ruminal degradation of the B, fraction is variable depending on

rates of passage or turnover rates.
Three enzymes, Pronase E, papain and bromelain were compared for the
efficiency of in vitro rumen protein degradation (Tomankova and Kopecny, 1995).
The highest correlation coefficient for protein degradation estimated by the in
sacco method compared with the enzymatic method was obtained for the
bromelain. Correlation coefficients of linear regression equations for rumen
protein degradability estimated by the in sacco method and bromelain were 0.839
for concentrates, .730 for fresh forage, .741 for hays and .876 for silages. They
suggested that feeds with high starch content had to be treated with a-amylase to
obtain the correlation coefficient of .839.
An in vitro method applying Michaelis-Menton saturation kinetics was
developed as an alternative approach for estimating protein degradation rates in the
rumen (Broderick and Clayton, 1992). Non-linear regression (NLR) analysis of the
integrated Michaelis-Menten equation yielded fractional degradation rates, kg,
from direct estimates of the maximum velocity:Michelis constant ratio (kg =
Vinax:Km). They suggested that degradation rates estimated using the NLR method
were more rapid than those obtained with a limited substrate approach. The NLR
rates were found to be more consistent with the in vivo estimates of rumen protein
escape.
The in situ or in sacco method measures the disappearance of feed
components from bag containing the test diet after incubation, for variable periods
in the rumen of an animal fitted with a rumen cannula. This technique has been
used for several years and is the basis of prediction of digestion in several feeding
systems (Waldo and Glenn, 1984). Poos Floyd et al. (1985) reported that among
different methods tested for measuring protein quality, the in situ method has the
highest correlation with in vivo degradability at 12 and 24 h. The major criticism
of this technique is low repeatability as suggested by the diversity of values
obtained by different researchers for similar feed samples (Nocek, 1988). Further,
various factors that may affect the results are, bag porosity, particle size, sample
size, contamination by rumen bacteria and location of the bag in the rumen.
Lindberg and Knutsson (1981) observed increased loss of DM as bag
porosity increased from 10 to 36 um. A similar trend was reported by Weakly et
al. (1983) and Uden and Van Soest (1984). Too small a bag porosity (< 35m)
may cause clogging of pores, bring about a reduction in the solubilization and
degradation of the sample and even accumulation of gas in the bag (Nocek and
Hall, 1984).
There is controversy as to the degree of particle breakdown associated with
microbial digestion (Nocek and Kohn, 1988). Weakley et al. (1983) showed that
DM and N degradation were less with coarse (2000 um) than with finer particle
size (520). Ehle et al. (1982) reported that rate of N digestion of many feed
ingredients are not affected by particle size. Grinding alfalfa and timothy hay
increased digestibility compared to field chopped material (Nocek and Kohn,
1988).
Sample size to bag surface ratio also provides a measure of the appropriate
sample size. Uden et al. (1974) showed that cell wall digestibility decreased from
54 to 34% when sample size was increased from 6.5 to 50 mg/cm’. Mehrez and
Orskov (1977) noticed an increase in DM disappearance from 37.5 to 85% when
sample size decreased from 55 to 15 mg/cm’. Uden and Van Soest (1984)
recommended 6 to 7 mg/cm’. Playne et al. (1978) has reported DM degradation of
forages varies little if the sample size to surface area ratio remains constant.
Potential contamination of test feed with ruminal microflora has been
reported (Kennedy et al., 1984). Concentrate ingredients generally contain little
microbial contamination (Mathers and Aitchison, 1981). Extensive DM
contamination was found on dacron bags that were incubated for prolonged
periods of time in the rumen of steers fed alfalfa hay (Van Milgen et al., 1992).
Use of X-ray analysis showed the presence of hydroxylapatite and synthetic Ca
-Mg phosphate (whitelokite). Van Milgen et al. (1992) reported that DM
contaminant appears as a smooth coating on the dacron fiber, suggesting that
contamination was a gradual process. It was found that DM contamination was
less than .04 g per bag (average bag weight was 1.2 g) during 10 d of incubation.
The effect of bag location on DM digestibility in cattle involved a reduction
of 6 to 10% units from the free to cannula end (Hawley, 1981). The decrease in
DMD in cattle as the bags were located closer to the cannula can be attributed to
the stratification of digesta resulting in a greater digestive capacity in the ventral
than in the dorsal part of the rumen.
The ideal method of estimation of ruminal degradation of feeds is by using
a duodenally-cannulated animal by which bacterial and protozoal CP and
undegraded intake protein can be separated (NRC, 1985). In this method direct
measurement of dietary protein intake and total protein flow at duodenum with
indirect measurement of bacterial crude protein (BCP) are measured. Digesta flow
is determined by use of an indigestible digesta marker (Zinn et al., 1980). The
measurement of digesta flow to the duodenum may be subject to error (NRC,
1985). The amount of undegradable intake protein (UIP) is calculated by the
difference. UIP = duodenal protein - BCP. This calculation does not take into
account endogenous protein.
Procedures for estimating BCP are by utilizing microbial markers such as
nucleic acids, diaminopimelic acid (DAPA), or by aminoethylenephosphonic acid
(AEP). Clark (1977) developed a method using one of the radioisotopes, 5, Pp,
or '°N. This BCP estimate using microbial marker is also subjected to error (NRC,
1985). Since endogenous protein is difficult to estimate, usually this is ignored,

which may lead to an overestimate of UIP.
Stern and Satter (1982) developed another procedure for the estimation of
UIP, which will take care of the endogenous protein. In this method flow of
protein to the duodenum is increased by increasing the intake protein in small
increments. The disadvantage of this method is that it can be used only when the
feed contains high protein.
Factors Affecting Ruminal Degradation of Protein. Several factors affect
the amount of protein in feed that escapes ruminal degradation (NRC, 1985). Two
important factors that determine the ruminal degradation, 1) physical and chemical
characteristics of the protein in feed and 2) methods by which they are processed.
In general, the soluble N fraction of a feed is degraded more rapidly in the rumen
than the insoluble fraction. In the case of SBM a higher proportion of the total N is
in the soluble fraction than for BM, CGM, dried distillers grains (DDG), BDG,
MBM, and dried distillers grains with solubles.

Sniffen (1974) reported that cereal grains and protein supplements contain
four types of protein, albumins, globulins, prolamines, and glutelins. Albumins and
globulins are low molecular weight proteins and because of this property they are
soluble in ruminal fluid. Prolamines and glutelins are higher molecular weight
proteins which render them insoluble in rumen liquor. Nugent and Mangan (1978)
reported that proteins that have low solubility in ruminal fluid and that possess
extensive cross linkages like disulfide bonds are less accessible by proteolytic
enzymes produced by the microorganisms.
Corn contains no albumin and only 5 to 6% globulins (Sniffen, 1974). The
remainder of the protein consists of prolamines and glutelins. Similarly, albumin
content in barley is only 3 to 4% and globulins, 10 to 20 %, the major portion of
the protein being prolamins and glutelins. Hence, corn and barley contain
predominantly prolamins and glutelins which are insoluble and undegradable in
the rumen. Distillers grains, BDG, and CGM are mainly produced from corn and
barley. Since the soluble proteins are removed in the process of fermentation,
mainly undegradable protein fractions will be left in these products.
Losses of lysine and arginine are found to be greater than losses of other
AA in the rumen (Craig and Broderick, 1984; Crooker et al. 1986). Craig and
Broderick (1984) suggested that microbial enzymes that cleave peptide bonds at
lysine residues may be important for regulating protein degradation in the rumen.
Cereal grain byproducts such as distillers grains with solubles, DDG, CGM, and
BDG contain lower concentrations of lysine and arginine than SBM or byproducts
of animal origin. Protein of animal byproducts such as MM, MBM, and FM are
generally resistant to ruminal degradation due to the collagen (connective tissue)
content in the products which renders them inaccessible to microorganism because
of the cross linking (Nugent and Mangan, 1978).
During processing feeds undergo physical changes because of cooking or
drying. In the processing of MM and MBM, considerable loss of rapidly
degradable protein fractions occurs or they are converted into a insoluble form
during cooking grinding and pressing (Bohme, 1982). Bohme (1982) also reported
that decreased surface area for microbial attack and random relinkage of bonds on
cooling renders these animal byproducts more resistant to ruminal degradation.
Heat applied during drying cereal byproduct feeds also coagulates and denatures
the protein which render the protein less soluble and less degradable in the rumen.
Heat processing renders the protein undegradable in the rumen. Treatment with
chemicals such as formaldehyde to lower the rumen protein degradability of
protein supplements have been done (Crooker et al., 1986).
Other factors that affect the rumen degradability of protein are DM intake
and associative effect of other feeds. Zinn and Owens (1983) increased the DM
intake of steers fed a high-concentrate diet containing corn, dehydrated alfalfa
pellets, cottonseed hulls, SBM, and molasses from 1.2 to 2.1% of BW. The protein
that escaped ruminal degradation was increased from 44 to 71%. Tammuinga et al.
(1979) fed lactating dairy cows a mixed diet consisting of long meadow hay and
ground, pelleted concentrates that contained a mixture of proteins. They reported
that as the DM intake of the feeds was increased from 8.2 to 12.9 kg’cow ‘day’,
the fraction of ruminal nondegradable protein in the diet increased from 29 to

45%.
Owens (1986) observed that as feed intake was increased from 1 to 2% of
BW, the quantity of protein in the feed supplemented with SBM that escape
ruminal degradation increased from 16.9 to 27.2%. The explanation for this
increase in ruminal escape was that the decreased retention time of feed in the
rumen resulted in lowered microbial growth which resulted in a lowered
degradation of protein. The effect of DM intake on ruminal protein escape was
lower when the DM intake was increased from medium to high, compared to
lower to medium intake of DM.
Ratio of forage to concentrate in the diet alters the ruminal degradation due
to an altered ruminal fermentation (Owens, 1986). He reported that increasing the
concentrates from 30 to 60% of the diet increased the ruminal escape of protein
from 22 to 29%; which is attributed to lower ruminal pH, causing reduction in
protein solubility. Another reason for increased ruminal escape might be due to a
lowered fiber content in the feed which lowers the microbial mass and hence a
lowered microbial degradation of protein.
Dry matter intake is much greater for high-producing cows than for low
producing cows, steers, and sheep. The concentrate portion of the diet will be high
for dairy cows in early lactation compared to low-producing cows, steers, and
sheep. Protein degradability values determined by using low-producing cows will
be overestimating the degradability of protein in high producing dairy cows.
Nocek and Russell (1988) indicated that the rate of feedstuff degradation in
the rumen will have a profound effect on fermentation end product and animal
performance, if, 1) the rate of protein degradation exceeds the rate of carbohydrate
fermentation, large quantities of N can be lost as NH3; 2) the rate of carbohydrate
fermentation exceeds the protein degradation rate, microbial protein production
can decrease; 3) feedstuffs are degraded slowly, rumen fill will decrease intake
and 4) the degradation rate is low, some of the feed may escape ruminal
fermentation and pass directly to lower gut.
The fate of the feed is ultimately determined by the relative rates of
fermentation and passage (Waldo et al., 1972). Russell et al. (1992) indicated that
fermentation rate is an inherent property of the feed and is described by first order
kinetics (substrate-limited, enzyme excess) and specific rate constants (hours’’).
Passage rates (hours’) are regulated by feed intake, processing (chopping,

grinding, and other means of particle size reduction), and the type of feed that is
consumed (e.g., forage vs cereal grain).
Protein Supplements. High resistance to ruminal degradation is seen in by-
product protein sources such as BDG, DDG, CGM, FM, BM, and MBM (NRC,
1985). The feeding of optimum undegradable protein in diets seems to be
important. Too high a protein supply in the form of highly degradable protein such
as SBM may result in poor utilization of the extra protein.
The demand for undegradable protein is less in low-producing animals and

more in high-producing animals. According to Waldo and Glenn (1984)
undegradability of the feed must be specified in addition to a total protein
requirement. As high milk production requires more total protein and available
degradable intake protein (DIP), more undegradable protein sources increase the
efficiency of protein use.
Many animal byproduct feeds have a high level of undegradable intake
protein (UIP), which could be utilized to increase the total UIP concentration in
dairy heifer diets as well as high proportion of essential AA that may be required
by ruminants (Amos, 1986). Amos (1986) also reported that ADG has been
enhanced when UIP percentage in the ration was increased from 36 to 56% while
total DM intake did not change. A linear relationship between UIP level and
weight gain has been reported in beef cattle (Newbold et al., 1987).
Undegradable proteins, when fed as the major N source may sometimes reduce the
microbial growth (Polan, 1988).
Corn gluten meal is the residue remaining after removal of the larger part of
the starch and germ and separation of the bran (Church, 1991). The nature of
protein resembles that of the parent grain in that lysine and tryptophan are usually
the most limiting amino acids, but S containing AA are found in higher
concentrations than in the grains. Much work has been conducted in feeding of
CGM with finishing steers and with lactating cows, considering its higher ruminal
escape nature of protein compared to SBM.
Seymour et al. (1992) observed a higher duodenal flow of total N, total
protein N, microbial N, and duodenal recovery of ingested N for the SBM and
CGM diet, compared to a SBM diet. Higher levels of ruminal NH; N, plasma urea,
ruminal butyrate and valerate were recorded for the SBM diet. Stock et al. (1981)
reported that SBM is degraded to a greater extent than more slowly degraded
proteins such as CGM.

Romagnolo et al. (1994) conducted electromagnetic and densitometric
analysis on the CGM residues after ruminal incubation for 72 h. The prolamin
fraction, zein for corn and CGM were more resistant to ruminal degradation than
albumins, globulins, and glutelins. The total degradability of protein fractions of
CGM was 18.6%. Zorrilla-Rios et al. (1991) conducted a trial in 80 steers to

determine protein-sparing value of the N added to wheat straw during the
ammoniation process and to determine the effects of supplementing ammoniated

straw diets with energy and ruminal escape protein. Addition of CGM did not alter
the straw consumption but ADG was increased by .35 kg. Ruminal and blood N
concentration indicated that the N from ammoniated straw was contributing to the
ruminal N pool and that CGM was compensating for microbial protein deficiencies
postruminally.
Over 1% of the corn crop in the United States is used for manufacturing
beer and distilled liquors, and most of the residue, minus part of the starch, is
returned as animal feed (Church, 1991). In the manufacture of liquors various

cereal grains such as, corn, rye, and malted barley in different proportions are
cooked, cooled and fermented. Following fermentation and distillation, solids are
recovered and dried. Various feeds produced are, dried distillers solubles, distillers
dried grains, and distillers dried grains with solubles. Distillers dried grain account
for the largest volume of distillery byproducts. Distillers dried grains have a
protein content of 27 to 29%, which is relatively deficient in lysine and
phenylalanine. Phosphorus, S, and Se levels are relatively high.
Protein sources from the beer industry include BDG dried grains, dried
spent hops, malt sprouts, and brewers yeast (Church, 1991). Brewers dried grains
are the dried residue of the barley malt and other grains that have been used to
provide maltose and dextrins for fermenting. Protein content varies from 26 to
29%. Lysine is most limiting, while tryptophan is relatively high in all brewery
byproducts. Dried grains have proteins that are resistant to degradation by rumen
microorganisms.
Ruminal digestion (predicted from in situ degradation) of BDG, brewers
wet grains and SBM was 42, 73 and 83%, respectively (Armentano et al., 1986).
This indicates that BDG deliver more protein and DM to the duodenum than
isonitrogenous amounts of SBM. Release of N from BDG is slower and more
sustained than that from SBM. The sustained release may have greater impact
when protein supplement 1s fed less frequently.
Santos et al. (1984) conducted a trial in lactating dairy cows to estimate the
protein degradability of SBM, CGM, wet brewers grain and DDG. It was observed
that ruminal NH; N concentrations (mg/dL) were 10.1, 5.6, 6.9 and 6.4
respectively. Protein degradation in the rumen was higher for the SBM diet (70%)
than for CGM (45%), wet brewers grain (WBG) (52%) and DDG (46%) diets.
Apparent absorption of AA from the small intestine was 70, 77, 71 and 66% of the
AA entering the duodenum for the SBM, CGM, WBG and DDG diets,
respectively. Actual amounts absorbed (g/d) were lowest for the SBM diet. It was
concluded that diets containing CGM, WBG and DDG will supply more AA to the
intestine than a diet containing SBM.
Firkins et al. (1983) reported that non-NH; non-bacterial N reaching the
duodenum was lowest for steers fed a urea-supplemented diet (20.9 g/d) whereas
the values were higher for wet distillers grain (64.1 g/d) and dried distillers grain
(74.7 g/d) when compared to wet corn gluten feed (41.3 g/d) and dry corn gluten
feed (32.7 g/d). They reported that percentage protein apparently escaping rumen
fermentation was greater for steers fed wet distillers grain (47%) or dried distillers
grain (54%) than for those fed wet corn gluten feed (26%) or dry corn gluten feed
(14%).
In a study conducted in Holstein cows, Cozzi and Polan (1994)

supplemented the diet with SBM alone or with CGM or BDG. Ruminal isovalerate
was found to be highest in the diet supplemented with CGM because of its high
leucine content. Dried brewers grain gave good production response due to a more
favorably balanced AA profile in the ruminally undegraded protein.
Hydrolyzed poultry feathers is the product resulting from the treatment

under pressure of clean, undecomposed feathers from slaughtered poultry free of
additives, and/or accelerators (Boehme, 1982). Not less than 75% of its CP content
must be digestible by the pepsin digestible method The American rendering
industry in 1978 produced 318,000 t of FTM (Boehme, 1982). In the process of

preparation of hydrolyzed FTM, the feathers are cooked under steam pressure for
30 to 45 min and the resulting slurry is cooked for an additional hour to drive off
excess moisture. The material is then transferred to a steam tube or hot air dryer
where the material is dried to 6 to 8 % moisture (Ellingson, 1993).
Feather meal is limiting in methionine, lysine, histidine, and tryptophan, but
it has a greater amount of several other AA, particularly cystine (Baker et al.,
1981). Cystine disulfide bonds within the keratin contribute to the insolubility and
indigestibility of the protein. Therefore, these bonds have to be cleaved before
feather protein can be digested by animals (Moran et al., 1966). Extensive studies
have been conducted with FTM as a source of protein in poultry diets. Feather
meal has been used as a protein supplement for ruminants too. Jordan and Croom
(1957) reported that FTM could replace SBM as a supplement to a corn and hay
diet without reducing lamb performance.
Karges et al. (1992) fed steers on summer pasture with increasing levels of
ruminally degradable (.15, .27 and .37 kg/d) and escape protein (.07, .14 and .21
kg/d) replacing a cornstarch and molasses (energy control) supplement. Treated

SBM and FTM were the sources of escape protein. Corn steep liquor supplied the
degradable protein. They observed that low level of degradable protein produced
numerically the greatest ADG (1.06 kg), compared to the high level (.96 kg). A
linear increase in ADG was also observed in steers fed escape protein in addition
to ruminally degradable protein (1.02 vs 1.12 kg). An in vitro study indicated that
more microbial protein was synthesized from the energy supplement than from the
degradable protein. As level of corn starch mixture decreased in the supplement

and level of steep liquor increased, gains by steers fed degradable protein
treatments tended to decline.

Thomas and Beeson (1977) reported that N digestibility was reduced from
68% to 59% when FTM replaced SBM in the diet. Digestibility of protein from
SBM, FTM and hair meal were 90, 62.8 and 49.7%, respectively. Dry matter and
gross energy digestibility tended to be higher on the SBM supplemented diet.
However, steers fed the FTM protein supplement retained a higher percentage of
absorbed N than those fed SBM. Church et al. (1982) reported that protein
digestibility was reduced by 5.7 and 8.5 percentage units in two digestion studies
when FTM replaced 70% of the SBM protein in the diet. The replacement of SBM
by FTM may have reduced the amount of protein degraded in the rumen and
ruminal NH; concentration may have been reduced below the optimal level for
maximal ruminal digestion.
Ruminal NH; concentration of animals fed SBM, FTM and hair meal were
14.1, 5.9 and 6.9 mg/dL, respectively (Thomas and Beeson, 1977). The ruminal
NH; concentration of animals fed with the SBM supplement diet were higher at 1,
2, 4 and 6 h. If the ruminal NH; values appear to be too low DM and energy
digestibility may have been limited by the lack of N (Klopfenstein et al., 1976).
Satter and Roffler (1975) reported that ruminal NH; N levels greater than 6 mg/dL
ruminal fluid may have had no beneficial effect on N balance.

When growing calves were fed a basal diet of corn cob:sorghum silage
(61:39) and were individually supplemented with DDG, heat damaged DDG,
feather meal (FTM), or urea, the plasma concentration of methionine responded
linearly and quadratically to increased escape protein (Gibb et al., 1992). There
was a linear and quadratic decrease in plasma concentration of lysine with an
increase in supply of DDG. They reported that plasma AA responses may identify
AA that become limiting with increasing escape protein. Of the essential AA,
threonine, valine and isoleucine were more resistant to ruminal degradation;
methionine, cysteine, histidine and arginine were more extensively degraded than
the total AA supply (Titgemeyer et al., 1989).
Blood meal, conventional cooker dried is produced from clean fresh animal
blood, exclusive of all extraneous material such as hair, stomach belchings, and
urine except in such traces as might occur unavoidable in good manufacturing

processes (Boehme, 1982). The protein content of BM is about 85 % and is a good
source of AA except isoleucine (Church, 1991). Blood meal is low in minerals
except iron. By virtue of its low rumen degradability of protein, the material is a
potential protein supplement for high producing dairy cows.
Titgemeyer et al. (1989) reported that replacement of corn starch in diets
with protein sources resulted in decreases in efficiency of microbial protein
synthesis. Ruminal NH3 N had the greatest increase when SBM was fed. Blood
meal supplementation resulted in only a nonsignificant increase in ruminal NH3N.
Soybean meal had the lowest proportion of N escaping ruminal degradation (21%).
Corn gluten meal-N (86%) and BM-N (92%) escaped ruminal degradation to the
greatest extent, and FM-N was intermediate (68%). They also reported that
absorbable non bacterial N from the small intestine followed the same trend, 13,
69, 68 and 50%, respectively for SBM-N, corn gluten-N, BM-N, and FM-N,
respectively.
Lambs fed BM with urea had lowest ruminal NH3 N (18.2 mg/dL),
indicating a lower digestibility in the rumen (Stock et al., 1981). Lardy et al.
(1993) reported that urea supplementation resulted in the highest ruminal NH;
concentration, whereas BM supplementation resulted in the lowest ruminal NH;
concentration. Blood meal resulted in greater dietary protein flow to the duodenum
than the other treatments. They also reported that the ruminal escape value of
rapeseed meal was greater than that of SBM but less than BM.
Schloesser et al. (1993) did not find any advantage of feeding BM over
SBM in sheep fed grass hay diets. They reported that ruminal NH3 N
concentration decreased linearly as BM replaced SBM; however, feeding BM did
not affect the total N, microbial N, or non NH; N flows to the small intestine.
They also reported that the different dietary treatments (SBM, 2/3 SBM:1/3 BM,
1/3 SBM:2/3BM; or BM) had no influence on ewe BW or body condition score

changes. Protein source did not influence serum protein, urea N, creatinine,
glucose, or nonesterified fatty acid concentrations.

Rusche et al. (1993) reported that when cows were fed a diet that contained
high CP (150% NRC recommendations) which contained gluten meal/BM (high
escape) as the principal supplemental protein source, there was an increase in
ADG as well as a decrease in plasma glucose and urea N. They also reported that
calves had an increased ADG from 0 to 97 d. Calves did not have access to cows’
diet and they assume that the increased milk production might be one of the reason
for having an increased ADG. In contrast, Wiley et al. (1991) reported no effect on
milk production or gain in calves from supplying additional CP from a high-escape
source.
In a study conducted in Holstein calves, Swartz et al. (1991) observed that
calves fed the highest undegradable protein (BM) diet had the least amount of CP
intake, 553 g/d compared to 589 g/d for the lowest undegradable protein. Feed
efficiency (kg feed/kg gain) was increased with increasing rumen undegradable
protein, 3.99, 3.77 and 3.39, respectively, for low, medium and high undegradable
protein intake.
Petersen et al. (1982) reported that escape protein from BM did not improve
winter weight gains in cows or steers. Cattle performed similarly when they were
fed 40% CP supplements formulated with BM-urea and SBM-urea although BM-
urea contained three times the quantity of escape protein as the SBM-urea
supplement. The escape protein values were calculated to be 16.5 and 54.2%,
respectively for SBM-urea diet and BM.
Meat meal is defined as the dry rendered product from mammal tissues,
exclusive of hair, hoof, horn, hide trimmings, manure and stomach contents,
except in such amounts as may occur unavoidably in good factory practice
(Boehme, 1982). It should not contain added BM, and not more than 14% pepsin
indigestible residue, and not more than 11% of the CP in the product shall be
pepsin indigestible. In the case of MBM the only difference from MM is that it
may contain more than 4.4% P. Usually the products are marketed to contain 45,
50, 53 to 55 % CP (as fed). The protein quality is generally considered to be lower
than that of FM because of lower isoleucine (Church, 1991).
Anderson et al. (1988), in a study conducted in eight ruminally-cannulated
heifers on wheat pasture, recorded an increase of 39% DM intake (expressed as
percentage of BW) by the supplementation of MM. There was no effect on N

balance by supplementation of MM. The ruminal escape of MM was estimated to
be 56 % (Vogel et al., 1988). Loerch et al. (1983) reported that percentage of N
escape of MBM was 49.3 compared to 28.7 that for SBM.
Fishmeal is defined as the clean, dried, ground tissue of undecomposed
whole fish or fish cuttings, with or without extraction of part of oil (Church,
1991). It must not contain more than 10% moisture and, if it contains more than 3
% salt, the amount of salt must be specified and in no case should salt content
exceed 7%. Fish meals are excellent sources of proteins and essential AA
especially lysine which is deficient in cereal grains. They are a good source of B-
vitamins and most of the minerals. Therefore, FM is an excellent protein
supplement for swine and poultry feeds. Fish meal is being used in ruminant
feeding also by virtue of its slowly degraded protein fraction (NRC, 1985).
Fish meal is processed by cooking, pressing, drying and grinding of fish
(Barlow and Windsor, 1983). Fish 1s cooked to coagulate proteins, free water and
fat from the tissues, and to sterilize the material. Cooked fish is then pressed to
remove the water and fat. The pressed material is then dried to about 90% DM.
Barlow and Windsor (1983) reported that CP content of FM varied from 60.4 to
72% and is higher in long chain (20 carbon atoms or more) polyunsaturated fatty
acids than vegetable fat. They also reported that FM is rich in essential AA
specially S containing amino acids. Although there are differences in CP content
among FM of different origin, AA profiles are similar.
Ruminal CP degradation of FM ranges from 30 to 70% (Orskov et al.,
1971). The ruminal CP degradation of FM depends on several factors such as,
storage period of the raw fish before processing, dryer types, formaldehyde
addition, heating duration and proportion of fish solubles added to the meal
(Kaufmann and Lupping, 1982)
Growing lambs fed FM diets had higher ADG than those fed SBM diets
(Orskov et al.,1970; Adam et al., 1982). Beerman et al. (1986) reported a 16%
increase in ADG when FM replaced approximately one-third of SBM protein in

com-based diets. In contrast, Hussein and Jordan (1991) did not observe any
difference in ADG when growing wether lambs were fed FM or SBM. Oldham
and Smith (1982) also reported greater ADG for young growing heifers (weighing
less than 200 kg) fed diets supplemented with FM than those fed diets
supplemented with urea or SBM. In contrast, Steen (1989) did not find any
difference in ADG or feed efficiency in finishing steers (weighing 450 kg) when
FM was replaced with SBM or sunflower meal.
Donaldson et al. (1991) reported that when steers on pasture were fed high
ruminal escape protein (FM and dried brewers yeast), low ruminal escape protein,
or corn, the escape protein flows were 1137, 1027 and 844 g/d, respectively.
Similarly, abomasal non NH; N (NAN) tended to be greater for steers receiving
high ruminal escape protein. Non NH; N flows were 1044, 955 and 777 g/d for
steers receiving high ruminal escape protein, low ruminal escape protein and corn,
respectively. The results show that escape protein can increase post ruminal
protein flow and also showed that there were no adverse effect on fiber digestion
in steers grazing annual rye pasture.
Feeding diets supplemented with either SBM or FM to cows in early
lactation resulted in similar milk yields (Zerbini et al., 1988; McCarthy et al.,
1989). Their studies using ruminally and intestinally cannulated dairy cows
indicated that replacing SBM with FM in the diet did not affect NAN flow to the
small intestine. This showed that the increased duodenal flow of dietary N as a
result of FM supplementation was balanced by a depression in ruminal microbial
protein synthesis. Orskov et al. (1981) fed high-producing dairy cows in early
lactation with high- or low-concentrate diets supplemented with groundnut meal or
FM. Milk yield was not affected when FM supplemented high-concentrate diets,
but was increased by 1.5 kg/d with the low-concentrate diets. The explanation
given for this effect was due to the increased dietary AA flow to the duodenum
and (or) improved ruminal fiber fermentation through a continuous release of
nitrogenous substrates from FM to the ruminal microbes.
Milk protein yield increased, 61, 95, and 130 g/d with SBM, high soluble
FM, and low soluble FM versus no supplement in dairy cows (Broderick, 1991).
They also reported that ruminal escape protein (estimated in vitro) was 27%
(SBM), 43% (high soluble FM), and 63% (low soluble FM). The results indicated
that greater rumen escape of FM protein, relative to SBM, increased efficiency of
protein utilization in lactating cows fed alfalfa silage. Fish meal supplementation
increased dietary N intake and significantly increased the flow of total N, NAN
and AA at the duodenum (Dawson et al., 1988). Shqueir et al.(1984) reported that
the feeding of liquified fish (LF) resulted in increased rumen fluid-N (NH; N)
(16.2 mg/dL) compared with cottonseed meal (CSM) (9.6 mg/dL) and feather meal
(FM) (10.6 mg/dL).
Combinations of Different Protein Supplements. In a study conducted in
four lactating Holstein cows fitted with duodenal cannula, Erasmus (1994) fed
different protein supplements, BM, CGM, a combination of BM and CGM and
sunflower meal. They observed that there were no differences in DM intake or in

milk yield. Ruminal NH; N concentration was higher for the sun flower diet, while
molar percentage of propionate decreased with the BM diet. Bacterial N flows

were 46.0, 45.3, 46.4, and 65.8% of NAN for the respective diets. The essential
AA profiles of the duodenal digesta and duodenal flow of individual AA closely

reflected the AA composition of the respective protein supplements, indicating that
the composition of rumen undegradable protein profoundly affected the
composition of protein entering the intestine when supplemental protein provided
35% of total CP intake.
Grummer et al. (1994) reported that Holstein cows fed a combination of
MBM plus BM had a higher milk yield (45.5 kg/d) over those received raw
soybeans (43.2 kg/d). Increasing the ratio of undegradable to degradable dietary
protein also increased yields of milk protein and fat, but, DM intake was not
influenced by treatment. A growth trial conducted by Blasi et al. (1991) with

grazing calves using combinations of BM and FTM showed a complementary
effect in calves fed 87.5:12.5 FTM:BM on a CP percentage basis. Calves fed the
control diet that contained urea gained .38 kg/d, whereas, maximal gain due to
protein supplementation was .7! kg/d. They attributed the increase in gain above
the control to the ruminal escape protein supplied by FTM and BM. Goedeken et
al. (1990) reported that the escape protein value for FTM (69.1%) was less than
the values for BM (82.8%) and CGM (80.4%) but greater than that for SBM
(26.6%). They also reported that protein efficiency was similar for BM:CGM,
BM:FM and BM:FM:CGM combinations.
Sindt et al. (1992) reported that calves supplemented with a combination of
BM/FTM/urea were 6% more efficient during the first 41 d than calves
supplemented with urea alone. At 76 d and by the end of the trial (164 d) calves
fed diets supplemented with natural protemn and urea were 7 and 6% more
efficient, respectively, than calves fed diets supplemented with urea alone (Sindt et
al., 1993). Calves fed dry-rolled corn or high-moisture corn basal diets
supplemented with BM25/FTH25/ureaSO0 were 10 and 8% more efficient,
respectively, than calves supplemented with urea alone.
Crab Processing Waste
Crab processing waste (CPW) is a good source of protein for ruminants. It

can be utilized for feeding of animals either as dehydrated meal (Patton et al.,
1975; Velez et al., 1991) or by ensiling with roughage (Samuels et al., 1991;
1992). Low digestibility for the crab waste straw silage (55%) compared to fish
waste straw silage (83.6%) was observed by Samuels et al. (1991). There was a
lower rate of ruminal degradation and/or deamination of the acetic acid treated
CPW silage. It is expected that CPW could be a potential source of escape protein
supplying AA to the lower intestinal tract. Apparent digestibility of DM, OM, CP,
energy, NDF, cellulose, and hemicellulose of silage containing CPW decreased
linearly with increased levels of CPW (Ayangbile, 1989). The decreased
digestibility of CPW was due to the very low digestibility of chitin ( 15 to 29%).
Samuels et al. (1991) observed that urinary N excretion was greater in
sheep fed fish waste silage diets than by those fed CPW silage diets. As a
percentage of absorbed, N retention was greater (27.9%) by sheep fed crab waste -
straw silage (60:40) when compared to 9.7% of retention for those fed diets
containing fish waste-straw silage (70:30). Ayangbile (1989) reported that N
retention expressed as percentage of intake or absorbed, was similar among the
sheep fed diets with different levels of crab waste straw silage.
Ayangbile (1989) reported that N intake increased linearly (P < .05) with
level of CPW. Total N excretion increased linearly with level of crab waste straw
silage. Nitrogen retention, expressed as g/d, increased linearly with level of crab
waste-straw silage. This is in agreement with the study of Abazinge et al. (1994).
Lubitz et al. (1943) stated that the quality of N from shell fish waste is superior to
that from fish waste.
Ruminal NH3 N was lower for sheep that were fed the acetic acid treated
crab waste silage, compared to those on molasses treated crab waste silages
(Abazinge et al., 1994). Patton and Chandler (1975) found that solubility of DM
and CP of crab meal in the rumen ranged from 25 to 42% and 37.5 to 50.2%,
respectively.
The chitin-N forms about 20 % of total N content of CPW (Lovell et al,
1968). Chitin, which is a component of exoskeleton of crustaceans is a (1-4)-B-

linked homopolymer of N-acetyl-D-glucosamine, an analogue of cellulose (Figure
1), which is less digestible than cellulose in ruminants (Velez et al, 1991). The
deacetylated chitin is chitosan. The lower digestibility of crab processing waste
diets may be due to chitin. Chitin has been shown to be resistant to degradation by
CH3
co
\
NH CH20H
oO HO 0
HO NH 9 CH20H ya
bo co
1 \
CH3 CH3
H NH
Wo 0 INT 8 O
HO NH2 CH20H © HO NH
Figure 1. Structures of chitin (a) and chitosan (b)
(Gooday, 1990)
microorganisms in the gastrointestinal tract of ruminants (Morgavi et al., 1994).

The CP digestion coefficients for the crustacean waste meal diets were 8 to 18%
lower than that for an isonitrogenous diet with SBM as the protein source.
Steam Explosion
The steam explosion process has been studied widely as it is an effective

method to increase the accessibility of cell wall components (Chornet and
Overend, 1985; Kokta, 1988). The process as it is now practiced is best
characterized as a “flash hydrolysis". The steam explosion method is a thermo-

mechano-chemical process in which the combined presence of heat (as steam),
mechanical forces (shear effects) and chemistry (as hydrolysis) results in massive
disruption of the lignocellulosic structure. The process combining thermal,
35
mechanical and chemical effects can be traced back to the early patents of Mason
(1929).
In the steam explosion technique the material is introduced into the reactor
and heated under steam pressure of 20 to 50 bars (unit of pressure, bar = one
million dynes / cm’) and 200 to 260°C for a few minutes (Toussaint et al., 1991).
During this process the hemicelluloses are hydrolyzed and become water soluble,
the cellulose is slightly depolymerized, the lignin melts and is depolymerized. The
reaction is immediately stopped by opening the reactor to depressurize the material
which can be expelled through a bar nozzle. Materials such as poplar wood
(Toussaint et al., 1991), wheat straw (Sciaraffia and Marzetti, 1988; Beltrame et
al., 1992) and other lignocellulosic materials (Grethlein and Converse, 1991) have
been studied.
Augustin et al. (1991) reported that steam explosion technique could alter
profoundly the microstructure of cellulose. A gel type compound was formed. This
shows that physical action of this method induces a structural change to the
cellulose. Kamide and Okajima (1987) have shown that when an aqueous
suspension of cellulose was treated for a very short time at high temperature
(~250°C) and the pressure suddenly released by opening a discharge valve, the
cellulose undergoes structural changes, which were not identified as H-bonding
modifications.
The changes were probably induced by structural H-bond or shifts due to

enhanced water penetration between adjacent cellulose molecules. This effect was
due to the sudden decompression. They also noticed that direct exposure to 250°C
without decompression did not lead to structural changes.
The explosion treatment process results in a profound change in the enzyme
accessibility, compared to the untreated wood (Maschessault and St-Pierre, 1980).
They also claimed that the morphological modification involving disruption into
microfibrillar elements of the cell wall as well as the fiber fracture would be
responsible for this phenomenon. Steam explosion process leads to a major
increase in the enzyme accessible surface. This factor is responsible for enhanced
enzymatic degradation of steam exploded material.

Sciaraffia and Mazetti (1988) conducted an in vivo digestion trial with
steam exploded wheat straw with sheep for 4 wk. The straw was steam exploded at
210°C for 2 min. Due to high moisture content, the steam exploded material was
conserved as alkaline silage (adding urea). The digestibility of steam exploded
straw was up to 58% compared to 45% to that for untreated straw. It was also
showed that the steam explosion did not negatively affect the blood parameters
considered. There was an increase in the nutritional value and palatability of straw.
It was found from the in vivo studies, that steam exploded wheat straw could be a
promising feed for ruminants because of its better palatability.

Chitin-protein matrix (Complex) of crab cuticle shows a supra molecular
organization geometrically comparable to a "Twisted Plywood" appearance
(Bouligard, 1972). Fully grown crab cuticle is composed of four distinct

superimposed layers, the outer epicuticle with a layer of lipoprotein coating and
the three layers of procuticle whose general structure is stratified. The organic
fibril forming the procuticle are composed of chitin crystallites and protein
(Giraud-Guille and Bouligard, 1986). They also reported that a chitin-protein

microfibril structure consists of a crystalline chitin core surrounded by a sheath of
ordered proteins. Chitin is considered as forming clear rods embedded in a dark
protein matrix, but there is high variation, depending upon the chitin content
(Hackman, 1984)
Covalent links between chitin and protein occur via aspartic acid
(Hackman, 1984). Brine and Austin (1981) showed that predominant AA in the
residual chitin after vigorous alkaline hydrolysis were aspartic acid, serine and
glycine which may be involved in the chitin-protein linkage.
A part of N in the shell fish waste is present in the chitin which may not be
available to the animal. Lovell (1968) reported that crayfish contained 40% CP.
The extracted chitin contained 19.8% of the total N of the waste. Only 32.2%
formed the corrected CP. This value is similar to the value of 32.7% CP obtained
for crab meal (Lubitz et al., 1943). The mean digestion coefficients of CP for cray
fish was lower (70.2), compared to soy protein (85.7) when the diet containing 8%
of either of the above ingredient was fed to rats. Ayangbile (1985) reported that
apparent digestibility of DM, OM, CP, Energy, NDF, ADF, cellulose and
hemicellulose in silage containing crab waste decreased linearly (P < .01) with
increased level of crab waste straw silage.
From the foregoing literature it is seen that as the chitin content in the feed
increases there is a linear decrease in the overall digestibility. If chitin complex
could be cleaved the nutrients would be available to the animal. Therefore, the
proposed research was to explore the possibilities of any structural changes in

chitin by steam explosion techniques so that the material would be better utilized
by ruminants.
Objectives
The overall objective of the research was to evaluate the ruminal escape
potential of crab meal and other protein supplements and to study the effect of
steam explosion on physico-chemical characteristics of crab processing waste
and rumen degradability.
Specific objectives:
I To determine the relative value of different protein supplements included in
high-roughage diets on :
e Performance of cattle
° N utilization by lambs
e Blood urea and ruminal ammonia
e Ruminal degradability of DM and protein
Il To investigate the effect of steam explosion on:
e Ruminal degradability of DM and chitin

e Ruminal escape potential of crab meal
Objectives 39
Chapter III. Journal Paper 1. Effects of Feeding Crab
Processing Waste and Other Protein Supplements on Growth
and Ruminal Characteristics of Steers Fed High-Roughage
Diets |
Abstract: A growth trial was conducted with 48 Angus x Hereford and Angus x
Simmental steers (avg. BW, 223 kg) for 126-d to study the relative value of crab
meal and other protein supplements included in high roughage diets on growth and
feed efficiency. Steers were blocked by weight and breeding into eight blocks, and
randomly allotted within blocks to six supplements (eight steers per treatment).
Diets were formulated to contain 10.5% CP and 63% TDN, DM basis. Ingredients
were orchard grass hay, cottonseed hulls, corn grain, and protein supplements. The
six supplements were, 1) soybean meal (SBM), control; 2) supplement based on
industrial byproducts of both plant and animal origin (IPA); 3) experimental
supplement based on byproducts of animal origin (ESA); 4) hydrolyzed
supplement No 3. (HESA); 5) commercial supplement based on animal protein
(CS), Pro-Lak®; and 6) crab meal (CM). In each diet, one third of the N was
supplied by the protein supplement. Steers were individually full fed during a 16 h
period daily. Average daily gains were 1.16, 1.13, 1.23, .98, 1.26 and 1.19 kg,
Journal Paper 1. Growth studies with steers 40

respectively, for the cattle fed the six supplements. Gain/feed values were .138,
.148, .141, .118, .147, and .146, respectively. There were no significant positive
responses (P > .05) for any of the protein supplemented groups over those fed
SBM. However, there was a trend (P < .1) for a positive response in gain:feed for
steers fed CS and IPA, compared to SBM fed steers. Lower (P < .05) weight gain
and feed efficiency were recorded for the cattle fed the HESA supplement. Steers
fed ESA and CM had intermediate gain:feed values. Steers fed the CM diet had a
numerically higher growth and feed efficiency than those fed the SBM diet. The
blood urea N and ruminal pH at 56 d and 126 d of the trial showed no significant
differences (P > .05) among the treatments. At 126 d ruminal NH; N was higher (P
<.05) for the steers fed SBM than for those fed the IPA and HESA supplements.
Blood urea was numerically highest for the steers fed the ESA and lowest for those
fed the HESA. There were no differences (P > .05) in the total VFA, propionate,
isobutyrate, butyrate, or valerate concentrations. Numerically, highest ruminal
acetate concentration (72.4 mol/100 mol) was for the steers fed CM. Substituting
other protein supplements used in this study for SBM did not elicit a significant
positive response in weight gain or gain:feed in growing steers on a high-roughage
diet. Crab meal was comparable to SBM or other products as a protein supplement
for steers.
Key Words: Protein Supplements, Steers, Feed efficiency, VFA
Introduction
It has been shown that quantity of microbial protein synthesized in the

rumen is inadequate for rapidly growing steers (Chalupa, 1975) or high producing
dairy cows (Santos et al., 1984; Keery et al., 1992). There may not be any
Journal Paper 1. Growth studies with steers 4)

any advantage in increasing the protein content which is highly degradable in the
diet for high producing animals and fast growing steers, unless the diet contains
sufficient energy for maximum microbial growth. Diets supplemented with protein
sources that will degrade slowly in the rumen has improved N and amino acid
(AA) flows to the small intestine. (Santos et al., 1984; Stern et al., 1985;
Titgemeyer et al., 1989; Coomer et al., 1993; Ludden and Cecava, 1995).
Several byproduct feeds of plant, animal and marine origin have been
shown to be good protein supplements for high-producing animals due to
resistance to ruminal degradation. Most of these byproducts are deficient in one or
more essential AA. Corn gluten meal (CGM) and brewers dried grains (BDG) are
deficient in lysine, while fish meal (FM) is rich in lysine. Microbial protein has
been found to be most limiting in lysine, methionine, and threonine for growth in
steers (Richardson and Hatfield, 1978). Therefore, these deficiencies have to be
met by supplementing the diet with protein sources which can provide these AA.
Studies have been conducted to elicit the complimentary responses by feeding
combinations of the rumen undegradable protein sources such as blood meal (BM)
and CGM (Stock et al., 1981), or BM and feather meal (FTM) (Goedeken et al.,
1990; Blasi et al., 1991).
Another protein source, “crab processing waste (CPW), a_ byproduct
obtained from seafood industry, has been shown to be a good protein supplement
for ruminants. Crab processing waste can be fed either as a dehydrated meal
(Patton et al., 1975; Velez et al., 1991) or by ensiling with roughage (Samuels et
al., 1991; 1992). Abazinge et al. (1994) suggested a lower rate of ruminal
degradation and/or deamination for the acetic acid treated crab waste silage. This
property reflects its potential as a source of escape protein.

The objective of this study was to evaluate crab processing waste and other
protein supplements on growth, feed efficiency, and ruminal and blood parameters
in steers.
Experimental Procedures
Forty eight Angus x Hereford and Angus x Simmental steers (avg. BW 233
kg) were used in a growth study. Steers were blocked by weight and breeding into
eight blocks and were randomly allotted within blocks to six diets containing the
following protein supplements: 1) soybean meal (SBM), control; 2) supplement
based on industrial byproducts of both plant and animal origin (IPA); 3)
experimental supplement based on byproducts of animal origin (ESA); 4)
hydrolyzed supplement No 3. (HESA); 5) commercial supplement based on animal
protein (CS), Pro-Lak®, and 6) crab meal (CM).

The hydrolyzed supplement No 3. (HESA) was processed in a high
intensity mixer and hydrolyzed under basic conditions at atmospheric pressure for
approximately 10 min. Temperature of the product reached 110°C (Meacham,
1995). Protein supplements, IPA, ESA, and HESA were obtained from Harmony
Products Inc. Chespeake, Virginia. The commercial supplement (CS) is
manufactured and marketed as Pro-Lak® by H. J. Baker and Bro. Inc. N.Y, NY.
Crab meal was obtained from Graham and Rollins, Hampton, Virginia. The other
ingredients were orchard grass hay, cottonseed hulls, corn grain and mineral and
vitamin supplements. The mixed protein supplements, IPA, ESA, and HESA were
formulated as shown in Table 1. The chemical composition of protein supplements
and other ingredients are presented in Table 2. The CP content of protein
supplements used in this study was 24.0, 68.1, 64.3, 76.6, 38.4, and 49.3%,

Table 1. Composition of mixed protein supplements”
Protein supplement*
Ingredient IPA ESA HESA
%
Ground corn grain 17.50 - -
Wheat middlings 31.50 - -
Corn gluten meal 8.75 - -
Distillers dried grains 5.00 - -
Feather meal - 73.20 73.20
Blood meal 8.75 9.15 9.15
Megalac®* 20.00 - -
Fat® - 9.15 9.15
Sodium bicarbonate 5.00 5.00 5.00
Monoammonium phosphate 2.50 2.50 2.50
Yea-sacc' 30 50 0
Niacin® 30 30 .30
Dairy flavor" 15 15 15
“As fed basis
"Harmony Products Inc. Chespeake, VA.
“IPA = supplement based on industrial byproducts of both plant and animal origin; ESA =
experimental supplement based on byproducts of animal origin; HESA = hydrolyzed supplement
No 3.
4Ca salts of palm oul fatty acids, Church and Dwight Co., Inc., Princeton, NJ.
“Mixture of animal and plant fats
‘Alltech Inc.,
£2767 mg/kg
"Feed flavor, Inc.

45
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respectively for IPA, ESA, HESA, CS, CM, and SBM. Crab meal contained an
average of 15.7% chitin, DM basis. Since ADF is a measure of chitin in CM

(Ayangbile, 1989), chitin content in CM is the value obtained for ADF content. It
has been reported that chitin contains 6.9% N (Black and Schwartz, 1950). Thus
nonchitin CP of CM was calculated to be 31.7%.
Diets were formulated to contain 10.5% crude protein (CP) and 63% TDN,
DM basis. In each diet one third of the N was supplied by the protein supplement.
Diets were formulated to meet or exceed NRC (1989) requirements. Diet
formulation was adjusted periodically based on the chemical composition of
ingredients and the mixed diets. At every mixing of diets, samples of hay, protein
supplements, feed ingredients, and diets were collected. These samples were
ground through a | mm mesh in a mill (Thomas-Wiley, Laboratory Mill, Model 4,
Arthur H. Thomas Co. Philadelphia, PA.). Samples were analyzed for DM and CP
by AOAC (1990) procedures. Calcium was estimated by atomic absorption
spectrophotometer (Perkin Elmer 5100, Norwalk, CT) and P by the colorimetric
method of Fiske and Subbarow (1925) after wet ashing (Sandel, 1959). Diet
samples were also analyzed for NDF (Van Soest and Wine, 1967), ADF (Van
Soest, 1963), lignin, cellulose, and hemicellulose (Van Soest and Wine, 1968) and
ash (AOAC, 1990). The ingredient and chemical composition of diets are
presented in Table 3
All steers were dewormed with Ivomec®(1 ml/50 kg body weight, s.c.,
MSD, Division of Merck & Co., Inc., Rahway, New Jersey) before the trial began.
Initial and final weights were averages of two consecutive daily weights taken 16 h
after removed from water. Steers were individually fullfed during a 16 h period
daily. The amount of feed was adjusted as needed to minimize refusals. Animals
were weighed every 14 d. Ruminal fluid and blood samples were collected at 56 d

Table 3. Ingredient and chemical composition of diets fed to steers
Supplement*
Item SBM IPA ESA HESA CS CM
%
Ingredient composition”
Hay 31.86 48.96 22.26 25.41 25.48 29.39
Cottonseed hulls 28.76 14.19 32.67 31.82 33.39 26.33
SBM 7.11 - - - - -
IPA - 14.24 - - - -
ESA - - 5.04 - - -
HESA - - - 5.43 - -
CS - - - - 4.57 -
CM - - - - - 9.31
Corn grain 30.53 21.63 37.50 34.92 35.09 34.23
Trace mineralized salt 50 50 50 50 50 50
Dicalcium phosphate 81 .30 75 .46 43 23
Lime stone 45 18 52 .69 54 -
Vitamin mineral premix - - .80 .80 - -
Chemical composition?
Dry matter® 89.06 89.51 89.20 89.11 89.08 89.47
Organic matter 94.19 92.82 93.73 93.60 94.53 91.86
Crude protein* 10.45 10.47 10.78 10.69 10.79 10.63
RUP, %CP* 38.90 53.00 55.20 52.00 52.70 47.20
NDF 70.53 67.03 67.50 62.00 68.80 68.10
ADF 35.62 33.01 27.57 33.86 35.09 32.22
Lignin 6.89 550 438 627 2.37 £4.16
Cellulose 28.83 25.82 23.28 28.45 31.21 27.36
Ash 5.81 7.18 627 640 5.47 ~~ 8.14
Calcium 0.52 055 053 O55 054 = 1.13
Phosphorus 035 032 038 036 0.35 0.36
“SBM = soybean meal, control; IPA = supplement based on industrial byproducts of both plant
and animal origin; ESA = experimental supplement based on byproducts of animal origin; HESA
hydrolyzed supplement No 3.; CS = commercial supplement based on animal protein; and CM
crab meal. All diets were supplemented with 2200 IU Vitamin A / kg
*DM basis
"Average of 18 samples
“Contained 98% NaCl, .35% Zn, .34% Fe, .2% Mn, .033% Cu, .007% I, .005% CO
“RUP = rumen undegraded CP; based on measured ruminal escape N of protein supplements
and reported values for other ingredients.

and at the end of the trial (126 d). Ruminal liquor was collected from each steer
using a stomach tube with the help of a vacuum pump, filtered through eight layers
of cheese cloth and pH of the fluid was measured immediately after the collection
(Accumet®, Mini pH Meter, Model 640 A, Fisher Scientific Company). Samples
for the estimation of VFA and NH3N were taken in 15 mL tubes containing ImL
of 25% metaphosphoric acid and 1 drop of concentrated H2SO,, respectively.
Samples were centrifuged at 1,800 x g for 15 min. Volatile fatty acids were
determined by gas chromatography (Varian Vista 6000 gas chromatograph). A
glass columm packed with 10% SP-1200/10% H3PO, liquid phase on 80/100
chromosorb WAW packing (Supelco Inc. ) was used to separate acetic, propionic,
butyric, valeric, isobutyric, and isovaleric acids. The detector temperature was
175°C, the column temperature as 125°C, and inlet temperature was 180°C. The
VFA concentrations were determined by integration, using a VFA standard

containing acetic (51.66 pmol/mL), propionic (30.63 Lmol/mL), butyric (10.4
umol/mL), valeric (5.18 ~mol/mL), isobutyric (4.96 pumol/mL), and isovaleric
(4.95 pumol/mL) acids. Ruminal NH3 N was determined by the method described
by Beecher and Whitten (1970).
Blood was drawn by jugular venipuncture from all steers in vacutainer
tubes and was placed on ice until centrifuged at 1,800 x g for 15 min. Blood urea
nitrogen (BUN) in serum was estimated in an Autoanalyzer, Centrifichem® System

500, using BUN (Rate) reagent. Sigma Diagnostic, St. Louis, MO.
Statistical Analysis
All data are presented as least squares means. Data were analyzed using the
GLM procedure of SAS (1989) as a completely random arrangement of treatments.
All parameters were subjected to the design shown in model. The model included
block and diet.

Model. Randomized block design
Yj = utb+tt e
Where,
Y;, = observation of steer in the i" block given j" diet

ut = unknown constant
b; = effect of i" block, where i = 1, 2, ....8.
t, = effect of j' diet, where j = 1, 2,....6
e; = Yj - (+b; + t) is the experimental error of observation
of steer randomly assigned to diet j 1n block 1

Tukey’s Studentized Range (HSD) Test was used for comparing the treatments for
different variables. Significance of (P < .05) was used throughout unless otherwise
mentioned.
Results
Data on ADG, DMI, and feed efficiency are presented in Table 4. The DMI
averaged 2.8% BW. Daily gains averaged 1.16 kg/d. Numerically, highest weight
gain was recorded for steers fed CS (1.26 kg/d) and the lowest were for the steers
fed HESA supplement (.98 kg/d) which was lower (P < .05) than those fed other
protein supplements except IPA (Figure 2). Differences in rate of gain were
similar throughout the trial. There was a trend for a higher (P= .09) rate of gain for
steers fed CS compared to those fed IPA supplement (1.13 kg/d). Lowest DMI
were for the steers fed IPA supplement (2.61% BW) which was lower (P < .05)
than for those steers fed SBM, ESA, and CS, expressed as kilogram per day and
lower (P < .05) than for ESA and HESA, expressed as percent of BW. Expressed
as percent of BW, there was a trend for intake to be higher (P < .10) for steers fed

30
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SBM (2.82% BW), CS (2.81% BW) and CM (2.69%) than those fed the IPA
supplement. The steers fed CM supplement tended to have a higher (P < .10) DMI
(8.09 kg/d) compared to those fed the IPA supplement (7.59 kg/d). Differences in
DMI were present after 56 d and followed a similar pattern until the end of the
experiment, but differences were larger at 126 d (Figure 3).
Gain:feed was lower (P < .05) for steers fed HESA than for those fed the
other supplement. There was a trend for a higher (P < .10) gain:feed for steers fed
supplements IPA, CM and CS than those fed the SBM supplemented diet. Steers
fed CM supplement had numerically higher gain:feed (.146) than those fed SBM,
ESA, and HESA supplements, also. Throughout the trial period of 126 d, the steers
fed HESA supplement recorded the lowest (P < .05) gain:feed and the other trends
remained constant (Figure 4). In general, gain:feed of the steers tended to decline
as experiment advanced because of linear increase in DMI with almost a constant
ADG.
Data on average ruminal pH, ruminal NH; N, and blood urea N (BUN) at
56 d and 126 d of the experiment are presented in Table 5. Ruminal pH averaged
7.0 and 7.3 at 56 d and 126 d, respectively. No significant differences (P > .05)
were observed in pH, at either 56 or 126 d among the steers fed different protein
supplements. Steers fed SBM had numerically highest ruminal NH; N (10.05
mg/dL) and lowest values were for the steers fed ESA and HESA supplements
(4.00 mg/dL, for each) at 56 d. However, values were not significantly different (P
> .05), perhaps due to individual variation within the treatment (SEM=1.54). At
126 d, highest ruminal NH3 N were also for the steers fed the SBM (21.03 mg/dL)
which was higher (P < .05) than those fed the IPA and HESA supplements (8.86
and 9.39 mg/dL, respectively). Steers fed ESA, CS, and CM had intermediate NH;
N concentrations (12.29, 19.28, and 19.16 mg/dL, respectively).

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Blood urea N averaged 3.84 and 4.22 mg/dL at 56 and 126 d, respectively.
The steers fed the HESA supplement had numerically lowest BUN, 2.94 and 3.52
mg/dL, respectively at 56 d and 126 d, among the steers fed various supplements.
However, there were no significant differences (P > .05) in BUN
Data on the ruminal VFA of steers fed various supplements at 56 and 126 d
are presented in Table 6 and 7, respectively. Total ruminal VFA averaged 46.23
and 43.06 umol/mL, respectively at 56 and 126 d, which were not significantly
different. At 56 d, lowest (P < .05) acetate concentrations (61.72 mol/100 mol)
were for the steers fed CS supplement, compared to those fed other supplements.
Highest propionate concentrations (22.48 mol/100 mol) were observed for the
steers fed CS supplement which were higher (P < .05) than those for the steers fed
other supplements except that for HESA supplement. Isobutyrate concentration
was lowest for the steers fed IPA and CM supplements. Values were lower (P <
.O5) than for steers fed the ESA supplement. Butyrate concentrations were lower
(P < .05) for the steers fed IPA supplement (10.10 mol/100 mol) compared to
those fed ESA and CM supplements (13.64 and 13.37 mol/100 mol, respectively).
Isovalerate was highest (P < .05) in steers fed ESA supplement compared to those
fed other supplements except for CS. Molar concentration of valeric acid was
shown to be numerically highest in steers fed CS supplement and lowest in those
fed IPA and HESA supplements. However, these values were not significantly
different (P > .05). Higher (P < .05) acetate to propionate ratio was for the steers
fed ESA, IPA, and CS supplements (4.36, 4.1, 4.06, respectively) compared to
those fed CS (2.87).
At 126 d, highest acetate levels were for the steers fed CM supplement
(72.39 mol/100 mol) which were higher (P < .05) than those fed ESA and CS
supplements (67.91 and 68.26 mol/100 mol, respectively). Propionic acid,

57
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isobutyric acid, butyric acid, and valeric acid and the acetate to propionate ratio
were not significantly different (P > .05) among the steers fed the different
supplements. Steers fed SBM and IPA supplements recorded lower (P < .05)
isovaleric acid (1.24 and 1.13 mol/100 mol, respectively) compared to those fed
ESA, HESA and CS supplements (1.73, 1.77 and 1.86 mol/100 mol, respectively).
Discussion
The lower DMI of IPA fed steers might be due to the low density of the diet
as a result of low level of cottonseed hulls and high levels of hay compared to that
of other diets. It was observed that as the level of cottonseed hulls increased there
was an increase (numerical) in DMI expressed as both kilograms per day as well
as percentage of BW. Nonsignificant lower DMI were observed for the steers fed
CM supplement. These results tend to agree with the previous findings of Velez et
al. (1991) who reported that DMI of Holstein heifers fed crab waste meal were
significantly lower from those fed SBM supplemented diet. In the present study,
lack of large and consistent differences in DMI of steers fed different protein
supplements is in agreement with the previous reports of Harvey and Spears
(1989), Sun et al. (1990), Johnson et al. (1994), and Mansfield et al. (1994), while
Donaldson et al. (1991) found that escape protein supplementation (combination
of fish meal and distillers dried grains) increased DMI in steers.
Substituting rumen undegradable protein sources for SBM did not elicit a
profound effect in growth or gain:feed in growing steers. These results agree with
the results of Coomer et al. (1993) who did not record any differences in ADG,
feed efficiency or CP efficiency (defined as the daily gain observed above the urea
control per unit of natural protein supplemented) when calves were fed different
diets supplemented with SBM, heated SBM (HSBM), CGM, or combination of

HSBM and CGM. Adam et al. (1988) and Derouen et al. (1988) also reported that
feeds supplemented with undegradable intake protein (UIP) did not affect the
growth of beef heifers and Holstein heifers. However, an increased ADG and feed
efficiency has been reported in Holstein heifers fed increased UIP supplied by
CGM and dehydrated alfalfa meal (Amos, 1985). Ewes gained more weight when
they were fed a combination of BM and SBM, compared to those fed either SBM
or urea (Hoaglund et al., 1992).
Results of the present study agree in part to Ludden et al. (1995) who fed
three groups of steers, in a 175 d trial, with SBM; SoyPLUS®, a high rumen escape
soybean product; and a 50:50 CGM and BM at different levels of supplemented
true protein in the diet (20, 30, or 40% of total dietary protem by altering the
proportions of true protein and urea in the supplement). They observed no
differences in ADG, DMI or efficiency.
The inability to detect responses to feeding ruminal escape protein sources
such as IPA, ESA, HESA, and CS compared to that of SBM in the present study
may be related to a relatively high escape N content for SBM. Ludden et al (1995)
reported that the estimate of the ruminal escape N content of SBM was more than
twice as great as the average estimate of 28% reported by NRC (1989). Perhaps,
the relatively high escape of N of SBM may be related to the ruminal environment
of the diet (70% corn grain) in that study. In an another study (Chapter V), the
escape N of SBM estimated by in situ technique was only 23% when bags were
incubated in the rumen of a cannulated steer fed 50:50 alfalfa grass hay diet. The
diet formulated for the steers in the present study contained on an average 30%
ground corn which would have affected the degradability of SBM.
Loerch et al. (1983) observed that the decrease in ruminal pH associated
with feeding diets based on cereal grains may reduce the protein degradability of

SBM. The escape N content of SBM was increased to 54.2% when bags were
incubated in the rumen of steers fed 80% high moisture corn-based diets. When
Ludden et al. (1995) incubated SBM in the rumen of steers fed high corn diet with
a ruminal pH of 6.2, escape protein was found to be 66.0%. In the metabolism
study conducted, the average ruminal pH of the SBM fed sheep was 6.39 (Chapter
IV). Hussein and Jordan (1991) reported that at low pH the escape of N in SBM
will be much higher and it is close to the escape value of fish meal. However, in
the present study no differences of ruminal pH were observed for SBM fed steers
compared to those fed other protein supplements. Further, average ruminal pH of
steers fed different protein supplements was higher (7.0 and 7.3, respectively at 56
and 126 d) than expected. Veen (1986) suggested that in general the pH will be
higher immediately before morning feed than afterwards. One possible explanation
given by Veen (1986) is that before feeding there is no easily fermentable
substrate remaining in the rumen. Another explanation may be the fact that the
concentration of NH; rises before the morning feed through the endogenous
metabolism of bacteria and/or through lysis of bacteria (Wohlt et al. 1976). In
addition, the concentration of NH; may increase again many hours after feeding,
as a result of increased salivary secretion (Wohlt et al., 1976). In the present study
for most of the steers there was a lapse of at least 12 h between the time the steers
were fed and sampling of ruminal fluid which may have caused the ruminal pH to
be higher.
At a low ruminal pH, ruminal degradability of protein decreases due to
reduction of electrostatic repulsion between neighboring protein molecules due to
the lack of net charge (Clark, 1975). As a result, the reduction in ruminal pH
observed when concentrate-based diets are fed, could increase the escape N
content of certain proteins by reducing solubility of these proteins in the rumen.

Thus, low ruminal pH would enhance the escape nature of protein in the feed-
stuffs. The incorporation of corn in the present study probably lowered the
degradability of SBM.
Loerch and Berger (1981) recorded, numerically, higher ADG and DMI
when SBM was supplemented to a 75% corn based diet, compared to BM and
MBM supplements. Sindt et al. (1993a) did not observe any differences in ADG or
feed efficiency when urea was replaced with a combination of BM and FTM in
finishing calves although there was a small response in the beginning. Similar
results were obtained by Swartz et al. (1991) when Holstein calves were fed
different levels of undegradable intake protein (UIP). They did not observe any
difference in ADG in calves fed different levels of UIP (low, 30%; medium, 34%;
high, 38% of the feed) but, significant differences in feed efficiency for the calves
fed different levels of UIP were observed due to differences in DMI.
Ludden and Cecava (1995) could not explain the reason for not having a
significant response to escape protein supplementation. They suggested that the
reason might be due to increased availability for microbial protein synthesis by
feeding concentrates. Another reason for not getting a response may be due to the
escape nature of com grain protein, zein, which is 58 to 73% undegraded when
feed contain proportionately high levels of corn in the diet (Zinn and Owens,
1983). In the present study, the levels of corn in the diet (~30%) would have
contributed considerable escape protein. Further, ruminal NH; concentrations were
much higher at 126 d than the minimum concentration of ruminal NH; N of 5
mg/dL required for the optimum microbial protein synthesis, as suggested by
Satter and Slyter (1974). This suggests that optimum microbial protein synthesis
occurred in the steers fed the different supplements.

In the current study, steers fed protein supplement based on plant protein
and BM had a lower DMI compared to those fed SBM, but the daily gain of the
steers were similar. Feed efficiency of the steers fed IPA was numerically higher
than for those fed SBM, however, the differences were not significant. Similar
trends were observed in ESA, CS, and CM supplemented steers. This trend for a
response, perhaps due to the complementary effect of protein sources.
Goedeken et al (1990a) reported that when steers were fed a diet containing
72% ensiled corn cobs and 20% alfalfa (DM basis), supplemented with BM, FTM,
BM with CGM, BM with FTH and a combination of BM, FTH and CGM, they
gained faster than steers supplemented with urea. They suggested that BM may
have supplied needed lysine while CGM may have provided needed sulfur amino
acids. They also reported that protein efficiency for BM and CGM was improved,
compared to BM or FTM alone, but it was similar to BM and FIM and
combination of BM, FTM, and CGM. Stock et al. (1981) reported a 22%
improvement in protein efficiency when BM and CGM were fed, compared to BM
alone.
In an another study, Goedeken et al (1990b) fed steers diets containing 50%
ground corn cobs and 40% corn silage supplemented with protein supplements
such as SBM, FTM, BM and a combination of FTH and BM, compared to urea
control. They suggested that the most efficiently used protein sources were BM
and FTM and BM combinations compared to SBM. Also, a complementary effect
has been observed when BM and FTM were fed together as the protein efficiency
for the mixture was improved compared to the calculated average of BM and FTM
treatments. In the current study the steers fed ESA, which is a combination of BM
and FTM recorded numerically higher weight gain and gain:feed compared to
Journal] Paper 1. Growth studies with steers 63

those fed SBM. This trend for a response perhaps was due to the complementary
effect of FTM and BM to provide a better balance of AA.
Microbial protein which is limiting in methionine and lysine (Nimrick et al.,
1970; Fendenon and Bergen, 1975; Richardson and Hatfield, 1978) would have
been complemented with high levels of lysine (BM) and sulfur AA (CGM and
FTM) from the protein sources. Blasi et al. (1991) reported that steers fed 100%
BM had maximum protein efficiency compared with those fed urea, FTH, and
combination of BM and FTM. They also reported that no differences were
observed in protein efficiency among the supplements containing the various
proportions of FTH:BM. Protein value of FTH for growing ruminants can be
dramatically improved with small additions of blood to FTH during the processing,
so that deficiency of AA profile of microbial protein can be complemented (Blasi
et al., 1991).
A linear increase in body weight was observed when wether lambs were fed
FTM, compared to SBM in a 56 d experiment (Thomas et al., 1994). The authors
suggested that, the growth response obtained from a linear increase in dietary
protein supplied by FTM was due to an increased availability of protein and not
due to sulfur containing AA though there was a linear increase in the S
concentration in the diet as the FTM level increased in the diet.
Gibb et al. (1992) reported that as the level of FTM increased in the diet,
level of histidine also decreased, because of its low histidine content. In our study,
although AA acids were not estimated, ESA which is a combination of FTM and
BM is a rich source of S AA produced similar weight gain and feed efficiency as
other protein supplements except for HESA.
Complementary effects of feeding combinations of FTM and BM with urea
supplementation have been reported by Sindt et al. (1993a). They reported that,

although not significant, calves finished after grazing corn stalks and supplemented
with SBM were 7% more efficient than calves supplemented with urea alone.
They also suggested that supplementing SBM/FTM/urea or BM/FTM/urea
improved feed efficiency compared with supplementing FTM/urea alone. These
data show that supplementing the diet with combinations of protein sources are
more beneficial than feeding single source. In an another study Sindt et al (1993b)
reported that calves supplemented with urea-BM/FTM were more efficient during
the first 41 d of the trial than were calves supplemented with urea alone (basal diet
contained either dry rolled corn or dry rolled grain sorghum diets). However, ADG
and feed efficiency were not affected from 42 d to slaughter (188 d) by protein
sources such as urea, urea + 60:40 combination of BM and FTM. The data
strongly suggest that calves in the early stage, 1.e., from 0 to 41 d were deficient in
metabolizable protein (MP) in the urea fed calves, but were adequate in MP from d
42 to slaughter (188 d). The data also show that supplementing urea-BM/FTM
improved feed efficiency when the diet was limiting in MP and not when the MP
was adequate
Although the above study cannot be compared in foto with the present
study due to urea incorporation in their diets, the results of the present study agree
with this trend. The SBM supplemented diet perhaps provided adequate MP for
the optimum feed efficiency for the calves, so the other protein supplements did
not elicit a pronounced response when compared to SBM. The findings agree with
the suggestion of Chalupa (1975) who reported that the potential of rumen escape
protein is shown highest in young, growing ruminants, where microbial protein
synthesis may not be sufficient for the optimum requirement for rapid growth.
Earlier, Orskov et al. (1971) observed increased ADG and gain:feed when post
weaning lambs were fed FM substituted for SBM. Oldham and Smith (1982)

suggested that growing cattle weighing more than 200 kg fed a diet with adequate
energy intake can meet their protein requirements from the MP synthesized in the
rumen. They postulated that the protein sources that are rapidly degraded in the
rumen will be sufficient to meet the N requirement through MP synthesis.
The reason for the lower (P < .05) ADG and gain:feed for steers fed HESA
compared to other protein supplements is not known. One reason might be the low
degradability of the protein which would have created a deficiency of NH3 N and
would have resulted in low microbial growth. In the present study ruminal NH; N
concentration was only 4.0 mg/dL which is lower than the minimum concentration
of 5 mg/dL for the optimum microbial growth as suggested by Satter and Slyter
(1974). However, concentration of NH; N was higher than this at 126 d. Further,
the digestibility of the material in the lower tract would be low compared to the
other supplements. Metabolism studies conducted in sheep revealed that sheep fed
HESA supplement had the lowest DM, OM, and cellulose digestibility among the
sheep fed different protein supplements (Chapter [V). Disulfide bonds in cystine
of FTM cause poor digestibility (Moran et al., 1966). Correct balance of time and
temperature during hydrolysis of feathers is necessary for optimum protein
digestibility (Morris and Balloun, 1973; Latshaw, 1990). Cystine is converted to
lanthionine during processing (Robbins et al., 1980). As processing increases,
more lanthionine is produced which limit the bioavailability of S AA (Robbins et
al., 1980) and possibly other essential AA as well (Papadopoulos et al., 1986).
Goedken et al. (1990b) indicated that protein quality of FTM and BM combination
will be reduced if the BM is added before hydrolysis of FTM due to reduced
ruminal protein escape and total tract protein digestibility. In the present study,
perhaps HESA supplement was processed for too long a period of time.

Numerically, higher feed efficiency was obtained for steers fed the CM
supplemented diet compared to those fed SBM supplemented diet. Higher feed
efficiency has been reported by Ayangbile (1989) when cattle were fed 30% crab
waste straw silage compared to a control diet. Similar results were obtained by
Patton et al. (1975) who observed no reduction in weight gain, but improved feed
efficiency for steers fed CM. In contrast, Brundage (1986) reported a reduction in
daily gain in steers fed 7.5 to 22.5% tanner crab meal. Lower weight gains and
poor feed efficiency has been reported in minks fed 10 and 20% crustacean meal
(Watkins et al., 1982). Results of the present study contradicts the findings of
Velez et al. (1991) who reported that DMI and feed conversion efficiency of crab
waste meal fed Holstein heifers were significantly lower than those fed SBM
supplemented diet. They reported that overall performance of Holstein heifers on a
crab waste meal diet was inferior to those that received a SBM supplemented diet
(Velez et al., 1991). They suggested that crab waste meal was inferior to SBM
when supplemented in corn silage based diets. The inferiority of the crab waste
meal, according to the authors, might be due to less ruminal microbial degradation
leading to low microbial protein synthesis. Another possibility might be due to
possible mineral imbalance. In the present study we did not observe any negative
response compared to those fed SBM.
Preston and Bartle (1990) reported that maximum gain and efficiency were
obtained when steer calves were fed a basal diet comprised of cottonseed hulls
(25%), corn silage (40%) and steam flaked sorghum grain (23.4%) supplemented
with protein having 60% ruminal escape protein (41% of total CP). This
conclusion was further strengthened by the results of Clark et al. (1992) who
suggested that diets should contain at least 35% of the dietary protein from the
protein supplement to elicit a positive response. In the present study proportion of

the diet was only 33.3% which was marginal to elicit any response as suggested by
Clark et al. (1992).
Ruminal pH was higher than expected in all the treatments. Similar results
were obtained when different UIP sources, SBM, heated SBM, CGM and
combination of heated SBM and CGM were fed to steers (Coomer et al., 1993),
and dairy cows ( Seymour et al., 1992; Robinson and McQueen, 1994). Increased
rumen pH when cows were fed rumen undegradable feeds have been reported with
different protein sources (Zerbini et al., 1988; Broderick et al., 1993; Christenson
et al., 1993). Rodriguez (1994) reported a higher pH when Holstein cows were fed
41% RDP diets compared to those fed 29% RDP diets.
The high ruminal NH; N in steers fed the SBM diet shows that SBM is
more degradable than other protein supplements. Differences in protein solubility
in rumen resulted in a significant increase (P < .01) in ruminal NH; N from 5.57 to
23.53 mg/dL (Sniffen, 1974). He reported that an increase of 2.7 times in
solubility of intake protein would result in 4.2 times the production of ruminal
NH3.
Several workers have reported that ruminal NH3 N decreased linearly as
FTM replaced SBM (Thomas and Beeson, 1977; Daugherty and Church, 1982;
Ellingson, 1993; Thomas et al., 1994). The authors suggested that ruminal
NHN concentrations in wethers fed FTM supplements were probably related to a
lower ruminal degradation of FTM. Feeding low ruminal degradable protein
sources in dairy cows resulted in low levels of ruminal NH3 N (McCarthy et al.,
1989; Klusmeyer, 1990; Stokes et al., 1991)
Titgemeyer et al. (1989) reported decreased ruminal NH; N concentration in

steers fed CGM compared with those fed SBM. Coomer et al. (1993) observed that
ruminal NH; N peaked at 2 h after feeding and were 16.9, 16.2, 11.2, and 13.4

mg/dL for SBM, heated SBM, CGM, and combination of heated SBM and CGM.
They observed a reduction (P < .01) in ruminal NH; N in steers fed CGM,
compared to those fed SBM. Koeln and Paterson (1986) also reported a lower
ruminal NH; N from in vitro fermentation of heated SBM and CGM than from
SBM. This low availability of NH; N would have directed microbes to use other
sources of N such as peptides and AA (Chen et al., 1987).

In our study, BUN did not show any significant differences among the
steers fed different protein supplements. This is in agreement with the findings of
Swartz et al. (1991) who reported that when calves were fed three levels of UIP,
33, 37, and 46% up to 12 wk, and 30, 34, and 38% from 12 to 28 wk, there were
no differences in BUN. They suggested that plasma urea N did not change with
diet and suggested that when total CP in the diet was the same, protein utilization
in the body is not likely changed by varying degradable protein in the diet.
Although the sources of protein were different in the present study, total protein
concentration in the diet was same in all the diets. Steers fed the SBM
supplemented diet showed higher ruminal NH; N concentrations even though there
were no differences in BUN among the steers fed different protein supplements.
These results do not agree with the reports of Preston et al. (1965), who found a
high correlation between ruminal NH; N and blood urea. Jordan et al. (1983) has
reported that increased plasma N concentrations were observed when increased
levels of CP were fed. They suggested that high levels of plasma urea were
expressed because of high amounts of NH3 formed from high levels of CP with
high ruminal degradability. Corbett and Edey (1977) observed lower plasma urea
N in ewes fed formaldehyde-treated casein, compared with ewes fed
isonitrogenous diets supplemented with untreated casein.

Feeding different protein supplements did not alter the total VFA at the
middle and end of the experiment. These results are in agreement with those
obtained by Wandersee et al., (1990) when cows were fed UIP sources such as
FTM. In the present study, individual VFA were not affected except for acetate
and valerate when steers were fed different protein supplements. Higher
concentration of acetate in CM diet fed animals may be due to the relatively higher
amount of acetate formed from the chitin degradation in the rumen. Acetate has
been reported to be the major end product of chitin fermentation (Pel et al., 1990).
Molar concentration of isobutyrate was not affected, while isovalerate was
higher for steers fed CS, HESA, and ESA supplemented diets, compared to those
fed SBM. Hussein et al. (1991b) reported that higher concentration of isobutyrate
and isovalerate were detected for diets containing SBM than for those containing
slowly degraded protein sources such as FM. Hussein et al. (1991a) suggested that
the higher levels of these branched VFA were produced as a result of higher
degradation of SBM. Isobutyrate and isovalerate are produced from the
deamination of the branched-chain AA, valine and leucine (Harwood and Canale-
Paula, 1981). The reason for lower molar concentration of isovalerate for the
steers fed SBM is not known. Ratio of acetate to propionate did not differ for
steers fed different protein supplements in the present study. Rodriguez (1994)
also did not observe any difference in acetate:propionate ratio when cows were fed
41% and 29% rumen undegradable protein (RUP) diets, while, valerate
concentration was found to be lower (P < .05) in cows fed a higher RUP (41%).
Reduced concentration of butyrate and valerate with high RUP diets were also
observed by Seymour et al. (1992). The authors suggested that this low
concentration of butyrate and valerate may indicate a decreased ruminal

fermentation of AA. In the present study no differences were observed for
butyrate and valerate among steers fed various protein supplement diets.
Implications
Substituting SBM with other commercial protein supplements did not elicit
a significant difference in weight gain or gain:feed in growing steers on a high
roughage diet. However, there was a trend for positive response in gain:feed for
the protein supplements, IPA, and CS. Crab meal appears to be similar to SBM
and other protein supplements for growing steers. Feeding HESA (hydrolyzed
feather meal and blood meal) had a negative response. It can be concluded that
protein supplements such as IPA, CS, ESA, and CM may be beneficial in replacing
SBM in the diets for growing steers.
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Chapter IV. Journal Paper 2. Effects of Feeding Crab Meal
and Escape Protein Supplements on Nitrogen Balance,
Digestibility and Calcium and Phosphorus Availability in
Sheep
Abstract: Two metabolism trials were conducted with 24 wether lambs to

investigate the effects of feeding crab meal and other protein supplements on N
utilization, digestibility and Ca and P balance in sheep. The lambs (avg. BW, 25
kg) were randomly allotted to eight diets in each of two trials. The supplements
were: 1) none, negative control (NC); 2) soybean meal (SBM), control; 3)
supplement based on industrial byproducts of both plant and animal origin (IPA);
4) experimental supplement based on byproducts of animal origin (ESA); 5)
protein (CS), Pro-Lak®; 7) crab meal (CM); and 8) urea (U). The supplements
supplied 33% of the total dietary N (CP, 9.8%; DM basis). Lambs fed NC diets
had lower (P < .05) DM and OM digestibility. There were no differences in DM
and OM digestibilities among the different protein supplement groups. Lower (P <
Journal Paper 2. Metabolism studies with sheep 80

.05) apparent absorption of N was recorded for the lambs fed the HESA and NC
diets. Numerically, higher Ca absorption was observed for lambs fed IPA, ESA,
and CS compared to those received SBM. Sheep fed CM had lower Ca absorption
compared to SBM. Highest (P < .05) P absorption was observed for lambs fed CS
and CM and lowest for U and NC diets. Sheep fed CM had higher (P < .05) VFA
concentration (65.7 ymol/mL), compared to those fed ESA, CS, and NC diets
(47.3, 49.8, and 49.5 umol/mL, respectively). There were no differences (P > .05)
in propionate, butyrate and valerate concentrations among animals fed the different
protein supplements. There were no differences in ruminal fluid pH for lambs fed
the different diets. Highest (P < .05) ruminal NH; N (29.6 mg/dL) was observed in
lambs fed the U diet, while those fed the NC diet had the lowest (P < .05) average
value ( 7.66 mg /dL). Lambs fed the U diet had the highest blood urea N (10.67
mg/dL) and those fed the NC diet had the lowest (P < .05) average value (3.06
mg/dL). The other protein supplements, CS, ESA, IPA, and CM may be
advantageous in supplementing the diet for the growing steers, considering the
numerically higher retention of N compared to SBM.
Key Words: Escape Protein, Sheep, Protein Supplement, Digestibility, N

Balance
Introduction
Feeding combinations of different escape protein supplements such as

blood meal (BM) and feather meal (FTM) (Blasi et al., 1991; Sindt et al., 1993)
and BM and corn gluten meal (CGM) to growing calves (Ludden and Cecava,

1995) have been shown to be more efficient than feeding these protein
supplements separately. These protein supplements of high rumen escape potential
are deficient in one or more amino acids (AA). Corn gluten meal and FTM are
high in S AA especially methionine while, BM is high in lysine. These AA have
been found to be limiting in microbial protein (William and Smith, 1974). Various
combinations of different protein supplements are available commercially, but
their rumen escape potential and their effect on digestibility are not known.
Several studies conducted in this laboratory (Ayangbile, 1989; Samuels et
al., 1991; 1992; Abazinge et al., 1993; 1994) and elsewhere (Lubitz et al., 1943;
Patton et al., 1975; Velez et al., 1991) have shown that crab processing waste
could be processed and fed as protein supplement either in the form of a silage in
combination with roughage or as dehydrated meal for ruminants. Lubitz et al.
(1943) reported that quality of crab meal protein is higher than that of fish meal
(FM) protein. Later, Patton et al. (1975) reported that there were no significant
reductions in digestibility of DM, N, and Ca when cattle were fed 10 or 20% CM.
Higher DM and OM digestibilities were observed when sheep were fed silage
containing 60% crab waste than those fed silage containing 40% crab waste
(Samuels et al., 1992). Higher N retention has been reported when sheep were fed
crab waste-straw silage compared to wheat straw silage (Samuels et al., 1992;
Abazinge et al ., 1994). Ayangbile (1989) reported that DM, OM, CP, energy,
NDF, ADF, cellulose and hemicellulose decreased linearly (P < .01) with
increased levels of crab waste-straw silage. Nitrogen retention increased linearly
(P < .05) with increased levels of crab waste-straw silage.
An experiment was conducted to determine digestibility, N balance, and Ca
and P metabolism in lambs fed crab meal and different protein supplements.

Two metabolism trials were conducted each with 24 wether lambs (avg.
BW, 25 kg). In each trial, the lambs were blocked into three blocks of eight
according to BW, and were randomly allotted within blocks to eight experimental
diets containing the following supplements: 1) none, negative control (NC); 2)
soybean meal (SBM), control; 3) supplement based on industrial byproducts of
both plant and animal origin (IPA); 4) experimental supplement based on
byproducts of animal origin (ESA); 5) hydrolyzed supplement No 4. (HESA); 6)
commercial supplement based on animal protein (CS), Pro-Lak®; 7) crab meal
(CM); and 8) urea (U). In randomizing the lambs for the second trial the lambs
were not allowed to receive the same supplement as in the first trial. The
ingredient and chemical composition of different experimental diets are presented
in Tables 8 and 9, respectively. Diets were isonitrogenous (9.8%, CP) and
isocaloric (58%, calculated TDN), DM basis, except for the negative control diet
in which the CP was 6.5%.
Lambs were kept in metabolism stalls similar to those described by Briggs
and Gallup (1949) designed for separate collection of feces and urine. All animals
were treated with Ivomec® (1 ml/50 kg BW, s.c.; MSD, Division of Merck and
Co., Inc. Rahway, New Jersey) for internal parasites and were given 500,000 I.U.
of vitamin A and 75,000 I.U of vitamin D, i.m. before starting the first trial.
Metabolism trials consisted of 7 d adaptation, 2 d transition, 10 d
preliminary, and 10 d collection periods. Each lamb was fed 700 g feed daily in
equal portions at 12 h intervals at 0800 h and 2000 h . Proportionate quantities of
hay, cottonseed hulls, and concentrate (combination of other ingredients of the
respective diets) were weighed separately for each lamb at each feeding. Water

84
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Samples of feed (hay, cottonseed hulls, and concentrate) were collected beginning
2 d prior to start of the trial until 2 d prior to the end of the trial. At the end of each
trial, the feed samples (10 d) were composited, subsampled and ground in 1 mm
mesh in a Wiley mill (Thomas Wiley, Laboratory Mill Model 4, Arthur H. Thomas
Co. Philadelphia, PA.) for chemical analysis.

Feces were collected daily in metal pans and dried in a forced draft oven
maintained at a maximum of 60°C. The daily collections of feces of each lamb

were composited in plastic buckets with loose fitting lids for equilibrating with
atmospheric moisture. At the end of the trial, dried feces were subsampled and
ground in a Wiley mill through a 1 mm mesh screen. Urine was collected in 4 L
plastic jars containing 15 mL of 1:1 (w/w) concentrated H,SO, and HO plus
approximately 500 ml of H,O. Urine was collected once daily, diluted to a fixed
weight (5000 g) with H,O and a 2% (100 mL) aliquot was taken from each lamb
and placed in a bottle and refrigerated. At the end of the trial urine samples were
subsampled and kept frozen for further analysis of N and Ca. At the end of the
trial ruminal fluid samples were collected 2 h post feeding using a stomach tube
and vacuum pump. Ruminal fluid was strained through eight layers of cheese cloth
and pH was immediately measured using a portable pH meter (Accumet® Mini pH
Meter, Model 640A, Fisher Scientific Company). Samples (5 mL each) for VFA
and NH; N determination were collected in 15 ml tubes containing 1 ml of 25%
metaphosphoric acid or one drop of sulfuric acid, respectively. Blood was drawn
by jugular venipuncture from all wethers 6 h after feeding and was centrifuged at
1800 x g for 15 min. and serum was separated. Urea N in serum was determined in
an Autoanalyzer, Centrifichem®System 500, using BUN (Rate) reagent, Sigma

Diagnostic, St. Louis, MO.

Feed components and feces were analyzed for DM and ash (AOAC, 1990),
Ca (atomic absorption Spectrophotometer, Perkin Elmer 5100 PC, Norwalk, CT),
P (colorimetric method of Fiske and Subbarow, 1925), NDF (Van Soest and
Wine, 1967), ADF (Van Soest, 1963), Cellulose (Van Soest and Wine, 1968).
Feed, fecal and urinary N were determined by kjeldahl method (AOAC, 1990).
Ruminal NH3 N was determined by the method described by Beecher and Whitten
(1970). Volatile fatty acid analyses were performed by gas chromatography
(Varian Vista 6000 gas Chromatograph, column packed with 10% SP-1200 /10%
H3PO, on 80/100 chromosorb WAW). The detector, column, and inlet
temperatures were 175, 125, and 180°C, respectively. Sample VFA concentrations
were determined by integration, using a VFA standard containing acetic (51.66
umol/mL), propionic (30.63 umol/mL), butyric (10.4 umol/mL), Valeric (5.18
umol/mL), isobutyric (4.96 umol/mL), and isovaleric (4.95 mol/mL) acids.
Statistical Analysis
All data are presented as least squares means. Data were analyzed using the
GLM procedure of SAS (1989). All parameters were subjected to the design
shown in model. The model included trial, block, and diet.
Model. Randomized block design
Yix = Wt dD + dit tk + ej
Where,
Yi, = observation of steer in the i" block given j" diet

LL = unknown constant
b; = effect of i block, where i = 1, 2, ....6.

d; = effect of j" diet, where j = 1,2,....8
t. = effect of kth trial, where k = 1 and 2
ej = Yi - (4 + b; + dj+ t,) is the experimental error of the
observation of the sheep randomly assigned to diet j in

blocki in trial k.
Tukey’s Studentized Range (HSD) Test was used for comparing the treatments for
different variables..
Results and Discussion
Apparent Digestibility. Data on apparent digestibility of DM, OM, ADF,

cellulose, and CP and Ca and P availability are presented in Table 10. Apparent
digestibility of DM ranged from 57.0 to 62.5% across diets. The lowest numerical
value for DM digestibility was for the lambs fed the diet with no supplemental N
which was lower (P < .05) than values for diets supplemented with SBM, IPA,
ESA and CS. The lower digestibility of DM for steers fed the diet with no
supplemental N might be due to lower microbial activity in the rumen. Christensen
et al. (1993) did not observe any differences in DM digestibility when protein with
low ruminal degradability (55%) was fed as the sole supplemental protein,
compared to a high ruminally degradable protein (70%). Results of the present
study agree with their findings. Blasi et al. (1991) reported that DM digestibility of
the BM diet was higher than that of the SBM and urea diets. In the same study, the
DM digestibility of the diet containing FTM hydrolyzed for 18 min was greater
than that of the diet containing FTM hydrolyzed for 10 min and slightly higher
than that of the diet containing FTM for 12 and 15 min. Stock et al. (1981)
reported that lambs fed a urea control diet had lower (P < .05) DM digestibilities

89
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contrast, Goedeken et al. (1990) detected no differences (P > .20) in DM

digestibilities when lambs were fed urea, SBM, BM, FTM or CGM supplemented
diets.
Apparent digestibility of OM was in the range of 57.8 to 63.0%. The
pattern of OM digestion was similar to that of DM digestion. Digestibility of OM
was lower at (P < .05) for the negative control diet than for the other diets. The
value of the IPA supplemented diet was higher (P < .05) than the diets
supplemented with HESA and U. Values for the diets supplemented with SBM,
ESA, CS, and CM were intermediate. Keery et al. (1993) found that whole tract
digestibilities of OM were not influenced by supplemental protein sources (SBM,
heated SBM, menhaden FM). Ludden and Cecava (1995) did not observe any
difference (P > .10) in total tract OM digestibility for diets supplemented with BM
compared to SBM, urea and SoyPLUS®- a high ruminal escape SBM. The authors
reported that animals had an acceptability problem with BM which caused a
slower rate of consumption of CGM-BM combination. They suggested that slower
rate of intake of CGM-BM diet would have reduced passage rate and as a result
increased OM digestibility.
Trends for ADF and cellulose digestibilities were similar as the OM
digestibility. Apparent digestibility of ADF ranged between 40.5 and 46.0%, and
that of cellulose was from 52.9 to 57.6%. When cows were fed a diet
supplemented with CGM, Klusmeyer et al. (1990) did not find any decrease in
digestibilities of OM, starch, NDF, and ADF compared to those fed a SBM
supplemented diet. Hussein et al. (1991) suggested that replacing high ruminally
degradable protein sources in the diets may improve ruminal fiber digestion.
Contradictory to the above findings, McAllan and Griffith (1987) reported that

ruminal fiber digestion was inversely related to ruminal protein degradation when
diets containing casein, SBM, or FM were fed to steers. Veen (1986) suggested
that feeding protein low in degradability will produce gradual release of NH; N,
peptides, and branched chain VFA. Therefore, cellulolytic bacteria can utilize
steadily these essential nutrients for a longer period of post feeding time.
The CM = supplemented diet was comparable with other protein
supplemented diets with regard to DM and OM digestibilities (59.7% and 61.4%
respectively). These results are not in agreement with the results obtained by Velez
et al. (1991). They reported that DM digestibility of a diet supplemented with CM
was lower (P < .05) than for a diet supplemented with SBM. In the present study
sheep fed the CM diet had a lower, numerically, ADF digestibility (42.6%)
compared to those fed SBM supplemented diets (45.7%). However, there was no
significant difference. This is in agreement with the results obtained by Velez et al
(1991). In the present study, numerically lower ADF digestibility might be due to
the low digestibility of chitin which forms 15.7% of CM, DM basis. The fiber
content of crustacean waste meals is a reliable estimate of the amount of chitin
present (Lovell et al, 1968; Ayangbile, 1989). A lower (P < .01) apparent
digestibility of DM, OM, CP, energy, NDF, ADF, cellulose and hemicellulose
with increase in the level of crab waste-straw silage for sheep has been reported
(Ayangbile, 1989).
Nitrogen intake was lower for the sheep fed no supplemental N and intake
were similar for the lambs fed the supplements (Table 11). The highest (P < .05)
fecal N excretion was for the steers fed HESA supplement (5.82 g/d) which shows
that N that passed to the lower gut was not digested as efficiently that of other
supplements. Lowest fecal N excretion was for the steers fed no supplemental N
(which was a reflection of low N intake) and IPA supplement. Sheep fed ESA, CS,

92
(SO'>d) JFIP si9ye] Jduossodns JUaIOJZIP YAA MOI DUIS OY} UT SIAqUINN,
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jeuowiedxe = ySq ‘UIsuO [euNUe pue jueld Ylog Jo syonpordAq jeLsnput UO paseq JuaWafddns = Vd] *JOI}UOD ‘Teoul URagsos = WAS,
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and CM had higher (P < .05) fecal N excretion compared to those fed IPA and no
supplemental N. This shows that N of IPA, which is a combination of plant protein
and BM is more digestible than that of FTM-BM combination (ESA) and the other
animal protein based supplement. Sheep fed SBM and U supplement had
intermediate values between those fed ESA, CS and CM supplement and IPA and
no supplemental N.
Apparent absorption of N ranged between 28.2 to 52.3%. Expressed as g/d
or as % of intake apparent absorption was lowest (P < .05) for the sheep fed no
supplemental N. There were no differences in apparent absorption of N for the
sheep fed different protein supplement except for those fed HESA supplement,
which were lower (P < .05) than others. Feeding HESA supplements depressed the
N absorption by 19.2% when compared with that of SBM fed animals. Cecava and
Hancock (1994) found that when steers were fed diets supplemented with
combinations of SBM and FM, N digestion was decreased (P < .05), compared to
that for a urea-supplemented diet. Blasi et al. (1991) observed a lower (P < .05)
protein digestion for hydrolyzed FTM diets than for BM, SBM, and urea diets.
Apparent protein digestbilities of FTM hydrolyzed for 10 and 18 min were higher
(P < .05) than those of FTM hydrolyzed for 12 and 15 min. The authors suggested
that hydrolysis time is not a criterion in determining the quantity of CP digested
and the quality of the raw materials are the primary factors. They suggested that
quality factors include deterioration with time before hydrolysis and contamination
with heads and feet.
Numerically, lower absorption was observed for the sheep fed ESA
supplement which is a combination of FTM and BM compared with those fed IPA
and SBM supplements. This tends to agree with the findings of Thomas and
Beeson (1977) who reported that N digestibility was reduced from 68% to 59%

when FTM replaced SBM in the diet. Church et al.(1982) reported that protein
digestibility was reduced by 5.7% and 8.5% units in two digestion studies where
FTM replaced 70% of the SBM protein in the diet. In their studies FTM
substitution for SBM would have produced lower ruminal NH3 N which could
have caused lower microbial protein activity and thus caused a low digestion.
Lardy et al. (1993) reported that supplementation with BM resulted in lower
ruminal and total tract CP digestibilities than SBM and rapeseed meal, while U
supplementation resulted in highest total tract CP digestibility. In contrast to the
above findings, Goedeken et al. (1990) observed no differences in total tract N
digestibility when lambs were fed urea, SBM, BM, FTM or CGM supplemented
diets.
Numerically, a lower N digestibility of CM supplement (48.3%) was
observed, compared to those fed SBM (51.0%), however, the values were not
significantly different. These results tend to agree with those of Velez et al. (1991)
who reported a lower (P < .05) CP digestibility of 51.1% for CM compared with
that of 55.7% for an iso-nitrogenous diet supplemented with SBM. Patton et al.
(1975) determined N digestibility of 67.8% for CM. However, the CP digestibility
coefficient of CM observed in the present study does not agree with the higher
value that was obtained for Patton et al. (1975). Lovell et al. (1968) reported that
N content of chitin is indigestible and has no value for nonruminant animals. The
N content of chitin is 6.9% and accounts for 19 to 20% of the total N in crustacean
meals (Lovell et al., 1968). Velez et al. (1991) reported that the wide variation in
the response may be because of the variability in the commercial CM obtained
from different sources.
Highest (P < .05, 4.90 g/d) urinary N excretion was for the steers fed U and
the lowest (P < .05, 2.05 g/d) was for those fed no supplemental N. Wethers fed

the ESA supplemented diet had a lower (P < .05) urinary N excretion (3.61 g/d)
than those fed SBM supplemented diet (4.28 g/d). Stock et al. (1981) reported that
urinary N excretion did not differ (P > .05) among lambs fed diets supplemented
with various protein sources, however, N excretion was higher in those fed urea
and SBM-urea diets. Cecava and Hancock (1994) observed higher urinary
excretion of N in steers fed a urea diet compared to a SBM-FTM combination. In
the present study, total N excretion was highest in steers fed U supplement, which
was higher (P < .05) than for steers fed IPA, ESA, CS, and CM. This is probably a
reflection of the lower efficiency of N utilization of urea. Sheep fed no
supplemental N recorded the lowest (P < .05) total N excretion, due to low N
intake.
Nitrogen retention (% of intake) ranged from -3.35 to 12.73%. A negative
N balance was observed for those fed no supplemental N (-3.4%) which was lower
(P < .05) compared to all other experimental groups, except U and HESA. This
indicated that considerable amount of N was mobilized from body tissues. Higher
(P < .05) N retentions (% of intake) were observed in ESA and CS (12.7% and
12.2%, respectively) compared to those fed U, NC, and HESA diets. Sheep fed
IPA, CM, and SBM had intermediate values (11.15, 9.64, 7.26%, respectively).
Retention of N was numerically higher for wethers fed CM compared to those fed
SBM supplemented diet. These results agree with those reported by Abazinge et
al. (1994). In the present study, sheep fed urea had lower (P < .05) N retention
(3.8%) compared to ESA and CS fed sheep (12.7 and 12.2%, respectively). This
shows that when urea was supplied as the sole source of supplemental CP with a
moderate source of metabolizable energy, urea was used with low efficiency. In
sheep fed the urea supplemented diet, urea would have been rapidly converted into
NH in the rumen and would have been converted into urea in the liver, causing

increased urinary N loss and decreased efficiency of N utilization compared with
other protein supplements of true protein nature. Sheep fed ESA, CS, IPA, and CM
had retained a numerically higher percentage of absorbed N (25.56, 25.06, 20.02
and 18.66 mg/dL) than steers fed SBM (14.21 mg/dL). Thomas and Beeson (1977)
observed a similar trend in N retention as percentage of absorbed for animals fed
SBM and keratin protein supplements (FTM and hair meal). Stoke et al. (1981)
reported that N retention was greater (P < .05) for lambs fed meat meal(MM)-urea,
CGM-urea, CGM-MM-urea, CGM-SBM-urea, and CGM-BM-urea compared to
those fed urea diet. They suggested that some complementary effects might exist
between the protein in CGM and that in SBM and BM.
Although N digestibility was numerically lower for ESA (which 1s a
combination of FTM and BM) compared to SBM, there was numerically a higher
N retention for ESA (12.7%) over SBM (7.3%). Feather meal N is less digestible
than SBM N, but FTM would have supplied AA of better profile than SBM
(Cecava and Hancock, 1994).
Calcium intake was higher for the lambs fed CM supplement, and intake
were similar for the lambs fed the other supplements (Table 12). Fecal excretion
was higher (P < .05) for lambs fed CM, a reflection of higher intake. Among sheep
fed protein supplemented diets, Ca absorption was similar, but expressed as
percent of intake CM fed sheep had a numerically lower Ca absorption. Urinary
Ca excretion was low for the lambs fed all supplements with no difference among
the different supplements. Absorption and retention of Ca were lower for sheep
fed diets supplemented with U or no supplemental N, but differences were usually
not significant. Absorption of Ca is dependent on its solubility at the point of
contact with the absorbing membranes irrespective of the forms in which Ca is
ingested (Maynard et al., 1979). Furthermore, animals absorb Ca from their gut

97
(SO > d) JQP S199] }dudsIadns JUSIaYIp YIM MOI sues dy} UT SJOqUINN,,.,,
‘JROU Guid = JD pue ‘ulojosd jeumue uo paseq
quawayddns jersyouw0d = Sd ‘'¢ ON JOWs;ddns pozAjompAy = WSAH ‘USO eutue Jo syonpoidAq uo paseq Jusuajddns jequautiedxs
= WSq ‘ulduo jeuue pue juejd oq Jo syonposdAq jeusnpur uo paseq juowlojddns = Vd] ‘Jouos ‘eau ueaqgdos = WS,
dooys XIs JO URDU OY} SjUsoIdoJ onjeA Ye],
0O00r 669 €868 Z2OSL 898 @rI6 8S9L O00S paqiosqe jo %
61T COT — po PO'V —poSTB pol OS — SHOT = TTT EOL oll’ IABIUL JO %
Ol’ 370° pos & pave’ pC pos" 8h" pol 3£0" p/3
UOTUI}0Oy
BVT pp6h'T —po0'S 816g 989 OS IT 6611 1S'8 3651 IABIUT JO %
Ol OT poe po8e po8C 0S” pls phe 390°

p/s
uondiosqe juaieddy
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Z0° 390° 380° v0" 3v0" 360° 560" 390° 3£0° Aleuuiy)
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3/8 ‘UONIIOX7
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,ssuawajddns u1ajo1d yuasajjip pay daays Aq aouvyeq wine) *Z] afqu LL
according to their need and they can alter the efficiency of absorption depending
upon the need (McDowell, 1992). Thus, lower absorption of Ca, expressed as
percent of intake for the CM supplemented lambs is a reflection of intake
exceeding the requirement. Excessive dietary Ca fed to dairy calves affected
concentration of Zn, Fe, Cu, and Mn in some body tissues (Alfaro et al., 1988). In
the present study Ca content of the CM diet was almost double (1.34%) than the
average value for the other experimental diets (.66%).
The lowest P excretion were in sheep fed CS and CM which were lower (P
< .05) than those fed HESA, U, and no supplemental N (Table 13). Highest P
excretion was for sheep fed no supplemental N which was higher (P < .05) than
for those fed SBM, IPA, CS, and CM supplements. The highest absorption of P
were for the sheep fed CS and CM (27.9 and 25.0%, respectively) and the lowest
was for those fed U and no supplemental N (5.5 and 5.3%, respectively). The
values for sheep fed diets supplemented with SBM, IPA, ESA, and HESA were
intermediate. The CM fed sheep had numerically a higher P absorption (25%) over
those fed SBM (16.8%).
Volatile Fatty Acids. Total VFA ranged from 47.3 to 65.7 umol/mL. Sheep
fed CM had a higher (P < .05) total VFA (65.7 umol/mL) compared to those fed
NC, ESA and CS ( 49.5, 47.3 and 49.8 pmol/mL, respectively) (Table 14).
Weathers fed SBM, IPA, and HESA had intermediate values. Acetate proportions
were higher (P < .05) in wethers fed IPA (69.3 mol/100 mol) compared to those
fed U and NC diets (59.9 and 60.7 mol/100 mol, respectively). However, there
were no differences (P > .05) among the sheep fed diets supplemented with
different protein sources. In contrast, Cheery et al. (1993) found that concentration
of acetate was higher (P < .10) in steers fed the SBM diet (96.8 m///L) than those
steers fed the heated SBM, menhaden FM, and combination of protein sources

99
po}oajap oq JOU pfnod uONUD}o1 se posJopIsuod aq Avul UOI}dIOSge Jo onjeA ay} DoUDY ‘oSueI 9[QuIO9}0p & UI JOU S19A\ OULIN UT SMIOYydsoYy,
(SO > d) J9HIP Sion] }duossodns yuosaygIp YPIa MoI sues OY} UT SIdQUINN,.,...,
‘yeowl qeio = JD pue ‘ursjold
Jewurue uo poseq yuowsddns jersowwio0s = SD ‘¢ ON Jowsjddns pozAjompAy = WSAH ‘UIs yeuNUe Jo syonpoidAq uo poseq juoWa;ddns
Jejuoutiadxa = WSq ‘uIsuo Jewrue pue yuejd yI0q Jo syonposdAq JeLsnpul uo paseq Juoursfddns = Wa] ‘JoUOO ‘[eow UBaqdos = GS,
dooys xis JO Uva SY) SyuOSoIdol onyeA yey,

OVE OVS pEOST ~BBLT poSSST pol OST poCELI pe89I 97S SALUT JO %
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[euouodxe = WOq ‘Uso ewe pue jued yIOq Jo sjonpordAq [eLNsnpur UO paseq quowajddns = VJ ‘[oNUOD ‘eau UvaqAos = WS,
dooys xIs JO uvowl oy} syudsordal onyeA yorg
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sheep
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Journal Paper 2. Metabolism stud ies

‘WHA [210.1
as A WO SO _-VSHH vSa Vdl Was ON w3}]
_ guewisjddng
,S)3Ip yuawaddns ula701d yustayIp ay) poy days jo sproe Ajyjej BJIVVjOA [VUIUINY “pT 21GB
supplemented diets (83.5, 80.8, and 78.9 mM/L, respectively). Ayangbile (1989)
reported that total VFA and acetate tended to be higher for sheep fed 50% crab
waste-silage compared to those fed basal diet without crab waste.
Wethers fed the U diet had higher (P < .05) propionate proportions (30.1
mol/100 mol) compared to those fed CM, IPA and SBM supplements. Molar
concentration of different volatile fatty acids for the sheep fed CM supplement
were not significantly different from those fed SBM supplement. Ayangbile (1989)
observed lower propionate, butyrate, and isovalerate concentrations for the sheep
fed 50% crab waste-silage diet compared with those fed a basal diet without crab
waste. Khorasani et al. (1994) reported a lower (P < .06) ruminal concentration of
propionate, isobutyrate and valerate when cows were fed slowly degradable
protein sources (FM, CGM, and MM) substituted for rapidly degradable protein
sources (canola meal and SBM). Keery et al. (1993) found that rumen
concentrations of isobutyric acid and isovaleric acid were lower (P < .10) in steers
received heated SBM and combination of protein sources diets than in steers
received menhaden FM. Similar results have been obtained by Pena et al. (1986).
When cows were fed diets supplemented with heat-treated cottonseed (roasted or
extruded), concentration of isobutyrate and isovalerate were lower than in cows
fed diets supplemented with raw cotton seed. In the present study generally,
numerically lower proportions of branched chain VFA such as_ valerate,
isovalerate, and isobutyrate were observed among the groups fed different protein
supplemented diets compared to those fed SBM. For the optimum growth of
cellulolytic bacteria branched chain fatty acids are needed. The branched chain
fatty acids are formed from the fermentation of branched chain AA (Russell and
Sniffen. 1984). Leng (1973) suggested that the high level of isovaleric acid may
be an indication of ruminal proteolytic activity. The SBM supplemented diet

produced numerically higher isovalerate compared to other protein supplemented
group, which agrees with the above statement. In the present study, there were no
differences (P > .05) in the ratio of acetate to propionate among the sheep fed diets
supplemented with different protein sources. However, lower (P < .05) ratios were
observed in those sheep fed U and NC diets compared with those fed SBM diet.
Contradictory results were obtained by Ludden and Cecava (1995) who
reported that feeding SoyPLUS®, high ruminal escape SBM increased total VFA
and branched chain and isoacids. It has been shown that branched chain VFA
stimulates microbial growth in vitro (Argyle and Baldwin, 1989). Khorasani et al.
(1994) observed higher (P = .06) total branched chain fatty acids when cows were
fed diets supplemented with slowly degradable protein sources, compared to those
fed diets supplemented with highly degradable protein sources.
Other Ruminal and Blood Parameters: The ruminal pH of sheep fed
different experimental diets averaged 6.38 which was lower than observed for
steers in the growth study fed different experimental diets (Chapter III). In this
study ruminal pH ranged from 6.24 (HESA) to 6.52 (U) (Table 15). There were
no differences in ruminal pH for sheep fed different experimental diets. These
results agree with the results of several studies conducted with protein
supplements. Cozzi and Polan (1994) observed no differences in ruminal pH when
cows were fed diets supplemented with SBM, CGM, or brewers dried grain.
Titgemeyer et al. (1989) also reported that ruminal pH was not altered due to
feeding escape protein supplements such as CGM or FM over SBM. Similar
results of not having any change in ruminal pH by supplementing diets with
different protein supplements have been reported (Seymour, et al., 1992;
Schloesser et al.,1993; Sindt et al., 1993; Khorasani et al., 1994; Ludden and

103
(SO’ > d) JagIp siduosiodns yusJayIP YIM ALOI OUTS 94} UI SIOQUINN,,...,
‘yeoul geld = WD pure ‘ulajosd yewnue uo poseq
quowijddns yetssouMuod = Sd “¢ ON JuoWajddns pozAjompAy = YSqH ‘usu yeumue jo syonpoidAq uo poseq juoWwosddns jequsWadxo
= VSd ‘uiguo jeulue pue jueld yjoq Jo syonposddq jeLsnput uo poseq yuowsjddns = Wd ‘JOsjUOD ‘Jeou UvaqAos = WAS,
daoys xIs Jo UvdWI oY SJUasoIdol anyeA yorq
CL aL OL IVD = »pSTO = »pBTS = pIDS =, 0S 9 pI L 990 £ Tp/sui “‘N-ea1n poojlg

Col 696C pCL OT ,SEST »pSHVE »pSLCI apLl vl = p8S°LI 99 L Tp/sui ‘N -€HN peuluny
LO ocS 9 oot 9 s0£ 9 sbe 9 o£ 9 Ib 9 s0£ 9 ocb 9 Hd jeuwny
ds fh WO SO VSHH VWSH Vdl WdS ouON Wd}
qquoulajddng
_S)0Ip [eyusUuIadxa pay daays jo uado.1}IU BlUOWWeE pus Fd UaWNA pue ‘uad01}IU BIN PooTg “S] 21qGRL
Cecava, 1995). Ellingson (1993) observed low ruminal pH in heifers fed diets
supplemented with SBM, CGM or FM. Optimum pH for high cellulolytic activity
has been proposed to be 6.8 (Terry et al., 1969). Increased ruminal pH values have
been reported when cows were fed rumen undegradable feeds with different
protein sources (Zerbini et al., 1988). A higher pH has been reported for sheep fed
crab waste-straw, compared to a diet that fed without crab waste (Ayangbile,
1989).
Ruminal NH; N of sheep fed different diets ranged from 7.66 to 29.59
mg/dL. Highest (P < .05) NH; N values were observed for sheep fed the U
supplement (29.59 mg/dL) and sheep fed no supplemental N, numerically, had the
lowest NH; N (7.66 mg/dL). Ruminal NH; N for sheep fed SBM, CS, and CM
were higher (P < .05) than those not fed a N supplement. This is in agreement
with the results reported by Khorasani et al. (1994). Ruminal NH; N observed in
all the diet treatments were higher than the minimum concentration (5 mg/dL)
reported to be essential for optimum ruminal microbial growth (Satter and Slyter,
1974). It has been shown that to achieve maximum microbial activity much higher
NH3 concentrations are required than for the maximum microbial protein
production may be required (Mehrez et al 1977). Satter and Roffler (1975)
reported that under normal feeding conditions this minimum level of 5 mg/dL
rumen fluid will be achieved with a dietary CP between 11 and 14% in the diet
DM. Kang-Merznarich and Broderick (1981) suggested that ruminal NH; N
concentrations between 3.3 and 8.5 mg/dL were required for maximal microbial
growth. From these studies it can be implied that ruminal NH; N produced by the
various diets supplemented with different protein sources in the present study
appears to be sufficient for maximum microbial activity.

Ludden and Cecava (1995) reported that free a-AA N decreased (P < .10)
as the ruminal degradability of the preformed protein source decreased. They also
observed that concentration of peptide-associated « amino N and the relative size
of the peptides decreased (P < .10) when steers were fed SBM supplemented diet.
The authors suggested that the peptides formed as a result of SBM degradation are
immediately utilized by ruminal microbial colony which boost microbial growth.
Stock et al. (1981) reported that ruminal NH; levels were equal at 2 h post
feeding for sheep fed urea, SBM-urea, MM-urea, BM-urea, CGM-urea, CGM-
SBM-urea, CGM-BM-urea, and CGM-MM-urea. The reason for not having a

difference in ruminal NH; N concentration would be because of urea incorporation
in all the diets (40% of N). Later, at 4 and 6 h post feeding, lambs fed urea control
diet had higher (P < .05) concentration of ramen NH; N than those fed the other
seven diets. In that experiment urea masked any difference in in vivo rate of
degradation of protein supplements. They also reported that ruminal NH; N levels
in lambs on the urea treatment exceeded (P < .05) the average of the means of the
lambs fed, SBM-urea, BM-urea and MM-urea. Lambs fed BM had the lowest (P <
.05) ruminal NH; N compared to those fed urea, SBM-urea, and MM-urea. Lambs
fed SBM had intermediate values at 3 h of post feeding.

Feeding more resistant protein will result in lower NH3 N levels (McCarthy
et al. 1989) in ruminal fluid and even NH3 N may fall below the minimum level
required for maximum microbial protein synthesis (Wohlt et al., 1976).
Titgemeyer et al. (1989) reported that ruminal NH3 N was 12.5 mg/dL when steers
were fed a basal diet without any protein supplements. Addition of SBM caused
the greatest (P < .05) increase in ruminal NH; N concentration and BM addition
resulted in only a nonsignificant (P > .05) increase. They also reported that an
increase in ruminal NH; N concentration was similar between lambs fed CGM and

FM. Soybean supplementation resulted in greater NH3 production in the rumen
than did a diet containing CGM (Klusmeyer et al., 1990), CGM and BM ( Cecava
et al., 1991; Ludden and Cecava, 1995), CGM or brewers dried grain (Cozzi and
Polan, 1994), CGM, wet brewers grain, or distillers dried grain (Santos et al.,
1984), heated SBM or combination of protein sources (Keery et al., 1993) and
FTM or hair meal (Thomas and Beeson, 1977). A higher ruminal NH; N has been
observed in sheep fed crab waste-straw silage over those fed a diet without crab
waste (Abazinge et al.,1994). Ludden et al. 1995 reported that feeding a CGM-BM
combination resulted in lower microbial N flows to the duodenum and lower
microbial efficiency, compared to urea, SBM and SoyPLUS®. Schloesser et al.

(1993) observed a linear decrease in ruminal NH; N as they increased the level of
BM replacing SBM in the diet for ewes. They also reported that ruminal NH; N
concentration and other growth factors were in sufficient concentrations to meet
the requirements for microbial protein synthesis when wethers were fed a diet
supplemented with BM replacing SBM.
Lardy et al. (1993) observed highest (P < .01) mean ruminal NH3 N
concentration when they supplemented the diet with urea and lowest for those
supplemented with BM. These results support the data obtained in the present
study for the ruminal NH; N release in the animals fed diets supplemented with
urea and other protein sources.
Blood urea N of sheep fed different experimental diets ranged from 3.06 to
10.67 mg/dL . Lowest BUN were for the sheep fed no supplemental N, which was
lower (P < .05) than the value for those fed U supplement (10.7 mg/dL). The
values for the other supplements were intermediate. The same trend as that was
observed in ruminal NH; N was observed in BUN (Figure 5). As the level of
ruminal NH; N increased there was a proportional increase in BUN. These results

Jaygip 19y}9] UOWIOD B SUIyde] SIeQ “IDJOUreIed UI AA [RoW
qeid = WO pur ‘ulajoid eutue uo paseq juswiajddns jeissaumi0od = Sd “pf ON Juowsddns pozAjospAy = WSqH ‘ul3u0 jeutue jo syonposdAgq
107
uo paseq juousjddns jejuourtiadxa = ySq ‘ulsio jeunue pue yuejd yjoq jo syonposdAq jejsnpul uO poseq yuowajddns = Wq] ‘;eow uesq.os
= WGS ‘eon = 9 (ON) N Jequowsjddns ou yy sjoIp pay doays Jo suoneijUs.U0. vIn poo;g puke UosOIIU eUOWWe jeuTUNY *¢ o1ndI4
sjuewejddns ulejoid
W oO oO VSSH VSd Vdl Was @UON
~
L
OO
Ly
9
1p/5w ‘N €HN feulmnY

N
©
j

71 0&
OV
NNGd YZ N HN feulwing
are in agreement with the results of Thomas et al. (1984). Thomas and Beeson
(1977) reported that plasma urea N concentration was higher (P < .05) for animals
fed SBM compared to those fed FTM and hair meal. Christensen et al. (1993)
found that concentration of urea in plasma was reduced by decreasing the ruminal
degradability of the dietary CP. Effect of protein source contribute mainly to the
level of BUN. Greater concentration of BUN would be expected when protein of
high ruminal degradability was fed. In the present study BUN followed the same
trend. Corbett and Edey (1977) reported a low BUN when ewes were fed
formaldehyde-treated casein compound compared to those fed a isonitrogenous
untreated casein.
Higher BUN obtained in this study when U was fed is in agreement with
Cecava and Hancock (1994) who observed higher (P < .05) plasma urea-N when
they fed urea or SBM-FTM combination in the diet for steers compared to those
fed a diet based on corn silage and corn supplemented with urea. Higgingbothams
et al (1989) found an increase of 2.6 mg/dL in BUN in cows fed isonitrogenous
and isocaloric diets by increasing DIP from 58 to 65% of dietary CP. They
suggested that ruminal and post ruminal excess of N are eliminated from the body
in the same process of hepatic urea synthesis; therefore, excess protein, whether
DIP or UIP, will elevate BUN.
Rusche et al. (1993) observed increased plasma urea N concentration with

increased dietary protein which they explained that it may be largely by increased
absorption of ruminal NH; N, resulting in greater quantities of NH3 being
converted into urea in the liver. In the present study a correlation of r = .79 has
been recorded for ruminal NH; N and BUN (Figure 6). The level of BUN is
relatively proportional to the concentration of ruminal NH; (Preston et al., 1965).
Contrary to these reports, Kwak (1990) reported that concentrations of BUN did

‘Jeo qesd = WD pure ‘ursjoid jewue
UO paseq juswio|ddns [etosauNUOD = Sd ‘p ON JuoUIa{ddns pozAjompsy = WSAH “UISLIO jeUMIUE Jo syonpo1dAq uo paseq
yuows;ddns jeUsUILIadxa
109
= VSq ‘uidu0 jeuue pue queyd yjoq Jo syonpoidAq jeusnput uo poseq juowsjddns = vq] ‘vou! ueagAos = wg ‘kein
= (¢) (DN) N UU ‘s2ounos
ulajo1d JuoayyIp FIM poyuowayddns syorp pay daays yo usZorTu eruounue yeu pure uadouIU Bain poojq usamjaq UONRIaI07) “9 21Nn31y
(7p/6w) NNg
ol Ob 8 9 v G 0
I | I !
(7p/6w) N EHN /euwny

6/'=4
Ov
not vary consistently with ruminal NH; N levels. Thornton and Wilson (1972)
suggested that urinary N was linearly related to plasma urea concentration (r =
.97). The present study also agrees to these results (r = .62) (Figure 7), but BUN
was more correlated to ruminal NH; N than urinary N. Kwak (1990) suggested
that BUN concentrations reflected urinary N excretions instead of ruminal NH; N
levels.
Implications
Protein digestibilities of diets supplemented with experimental supplement

based on feather meal blood meal; supplement based on animal protein, Prolak®;
supplement based on plant protein and blood meal; and crab meal are comparable
with that of soybean meal. The hydrolyzed experimental supplement was found to
be lower to all other protein supplements tested. There seems to be no advantage
in hydrolyzing feather meal-blood meal combination in improving the nutritive
value of the material for ruminants might be because of varying processing
conditions. The retention of N of the above protein supplements and crab meal
have numerically higher retention of N compared to soybean meal and may be
advantageous in feeding of growing steers.

‘Jeoul quid = WD pue ‘ulojold jewue uo poseq Juowaddns jedsou0d = SD ‘p ON JUoWD]ddns pozAjoIpAy = WSqH ‘ulsu0
jeune jo sjonpoidAq uo poseq yuawojddns yeyuowuedxa ~ ySq ‘usu [euue pur ued YyIOg Jo sjonpoidAq [eLsnpul uO paseq juouiajddns =
11]
Wd] :[eow ueagqdos = Ws ‘eon = 9) (ON) N [equowsddns ou wim sjorp pay dasys Jo suonenuasuod vain poo|q pue udso0jIU BuO jeuTUNY
"S9OINOS UIa}OId JUDIOIIP YIM poyuowsjddns sjorp poy dooys Jo uodosu AreuN pue ussoIIU vaIN Poo;q UddA\Joq UONRIAIOD “{ 91NFI 1
(qp/6w) nnd
ol Ot 8 9 V o 0
TT T T T 1 0
(p/6) N Aseuun
Journa] Paper 2. Metabolism studies with sheep

c9 =4
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Chapter V. Journal Paper 3. Jn Situ Dry Matter and Crude
Protein Degradability and Jn Vitro Dry Matter Digestibility of
Crab Processing Waste and Other Protein Supplements.
Abstract: An in situ dacron bag experiment, consisting of two trials, was

conducted with a ruminally cannulated steer to determine the ruminal
degradability of DM and CP of crab meal and other protein supplements used in
the feeding trial. Protein supplements were, 1) soybean meal (SBM); 2)
supplement based on industrial byproducts of both plant and animal origin (IPA);
3) experimental supplement based on byproducts of animal origin (ESA); 4)
protein (CS), Pro-Lak®; and 6) crab meal (CM). In vitro DM digestibility
(IVDMD) of the above protein supplements was estimated also with ruminal fluid
from the ruminally cannulated steer. The highest in situ ruminal DM degradability
was for SBM (70.9%) which was higher (P < .001) than for other supplements,
viz., IPA (38.8%), ESA (17.0%), HESA (26.8%), CS (24.5%), and CM (29.1%).
Dry matter degradability of ESA was lower (P < .01) than IPA, while values for
HESA, CS, and CM were intermediate. The in situ ruminal escape of protein was
lowest for SBM (23.2%) which was lower (P < .01) than CM (48.4%). The highest
value was for the ESA supplement (79.8%) which was higher (P < .05) than IPA.
Journal Paper 3. In situ and in vitro studies 118

Values for the HESA and CS (67.3 and 69.8%, respectively) were intermediate.
The rate of degradation of degradable B (kg) of N was highest (P < .0001, 12%/h)
for SBM compared to IPA (4%/h), ESA (2.3%/h), HESA (2.4%/h), CS (3.4%/h),
and CM (5.2%/h), however, the values for the latter supplements were similar (P >
.05). The IVDMD were 89.0, 78.8, 40.5, 50.9, 63.4, 67.0, respectively, for SBM,
IPA, ESA, HESA, CS, and CM. Values were different (P < .0001) among different
supplements except between CS and CM (P > .05). A protein supplement based on
feather meal and blood meal was found to have the highest ruminal escape protein.
These data shows that CM is a digestible moderate escape protein source for
ruminants.
Introduction
Advantages of feeding escape protem for enhancing performance of

ruminants have been reported (Stock et al., 1981; Harvey and Spears, 1989;
Titgemeyer et al., 1989; Blasi et al., 1991; Sindt et al., 1993; Ludden et al., 1995).
It has been shown that amino acid (AA) requirements at the tissue level of the
ruminant animal increases as production level increases (Hussein et al., 1991).
Microbial protein has been found to be most limiting in lysine, methionine, and
threonine for growth in steers (Richardson and Hatfield, 1978). Several byproduct
feeds of plant, corn gluten meal (CGM) and brewers dried grain (BDG); and of
animals, blood meal (BM), feather meal (FTM), meat meal (MM), meat and bone
meal (MBM); and marine, fish meal (FM) have been used as protein supplements
for high producing cows due to their high ruminal escape potential and AA profile
(Waltz et al., 1989; Hussein et al., 1991; Christiensen et al., 1993; Cozzi and
Polan, 1994). Complimentary responses by feeding combinations of these
Journal Paper 3. Jn situ and in vitro studies 119

byproducts such as BM and CGM (Stock et al., 1981), or BM and FTM
(Goedeken et al., 1990b; Blasi et al., 1991) have been studied. Several commercial
escape protein supplements of different combinations of these byproducts of
different proportions are now available, but their ruminal degradability and escape
potential are not known.
Another product of interest as a protein supplement for ruminants is crab
processing waste which is a byproduct of sea food industry. Several studies have
shown that crab waste meal can be fed to ruminants as a protein source. Patton and
Chandler (1975) observed rumen degradation of crab waste meal to be 35.9% for
DM and 50.2% for CP. Patton et al. (1975) observed no differences in average
daily gain, feed intake, feed conversion, N retention, or digestibility of DM, crude
fiber or N when Holstein heifers were fed 10 or 20% crab waste meal compared to
those fed a diet with no crab waste meal. The apparent digestibility of DM and CP
of silage containing crab processing waste has been shown to decrease linearly
with increased levels of CPW silage in the diet (Ayangbile, 1989). Velez et al.
(1991) observed lower rumen degradability of DM and CP of crab processing
waste compared with those values obtained by Patton and Chandler (1975).
To measure the rumen DM and CP degradability, in situ (nylon bag, in
sacco) technique has been the common method employed since it was first
developed by Quin et al. (1938). Previously, this nylon bag technique was used
primarily for forage evaluation. This method has been employed to evaluate DM
degradation (Mehrez and Orskov, 1977; Nocek et al., 1979; Lindberg, 1989) and
for the rate of protein degradation (Mehrez et al., 1977; Nocek et al., 1979;
Weakly et al., 1983).
The objectives of this study were to determine the ruminal disappearance
of DM and CP of crab meal and other protein supplements by measuring in situ

degradability and to determine the in vitro dry matter digestibility of the
supplements.
In Situ Experiment. A ruminally fistulated steer ( 550 kg, BW) was used to
determine in situ DM, CP degradabilities of the protein supplements and crab
meal. The steer was fed equal portions of alfalfa and orchard grass hay beginning
15 d before the start of the first in situ trial. Protein supplements tested were, 1)
soybean meal (SBM); 2) supplement based on industrial byproducts of both plant
and animal origin (IPA); 3) experimental supplement based on byproducts of
animal origin (ESA); 4) hydrolyzed supplement No 3. (HESA); 5) commercial
supplement based on animal protein (CS), Pro-Lak®; and 6) crab meal (CM). The
supplements were ground through I mm mesh in a Wiley mill (Arthur Thomas
Company, Philadelphia, PA).
Polyester bags of 10 x 23 cm size with 59 + 2 «um porosity were used.

Calculated sample size to bag surface area was 15 mg/cm? (Nocek, 1985). Six
grams of samples were placed in each polyester bag. Bags were closed with 10 cm
Bar-Lok® Cable ties (Dennison Manufacturing Co., Framingham, MA). Duplicate
bags were tied with another Bar-Lok® Cable tie of 10 cm length. Duplicate bags of
the supplements were tied to the metal chain attached to the polyester thread with
bigger, 20 cm Bar-Lok®Cable ties. Bags for the 72 h incubation were tied to a
thick polyester string (to which subsequent groups of bags were attached) of
approximately 75 cm in length which was attached to a metal chain 20 cm in
length. The incubation periods were 0, 2, 6,12, 24, 48, and 72 h. The bags were
introduced in the rumen in the reverse order. Prior to incubation, bags were soaked
Journal Paper 3. In situ and in vitro studies 12]

in lukewarm water (~ 38° C ) for 15 min (Rodriguez, 1994) and then introduced
into the rumen through the rumen cannula. Eighty four bags were suspended in the
rumen. The bags for the 0 h incubation were not introduced in the rumen, instead,
bags were soaked in lukewarm water for 15 min. After 72 h all bags were
retrieved, washed in running tap water and duplicates were seperated. Bags were
washed thoroughly until rinse water was clear. Bags were dried at 60°C in a forced
draft oven for 48h. Bags with dried residues left in the bags were weighed and DM
disappearance was estimated. Residues left in the bags were used for the analysis
of N by Kjeldahl method (AOAC, 1990).
Ruminal degradation of DM and CP were classified into three main
fractions as proposed by Armentano et al. (1986). For CP, the fractions are A, B,
and C which are as follows,
A = NPN or true protein that is solubilized rapidly in warm water

and is calculated by subtracting the percent remaining at 0
time from 100.
B = Protein that is degraded at a rate similar to the rate of
passage and is calculated by subtracting sum of A and C
from 100.
C = Protein that is undegraded in the rumen, which is the percent

remaining at 48 h.
For DM, the definition was the same as above. Degradability of DM or CP
depends on rate of passage of digesta (k,,) which is expressed as percentage per
hour (%/h) and rate of degradation (ky,). The rate of passage in this study was
assumed to be 5%/h. The residue at 48 h incubation was considered as C fraction
and there was no change for this fraction. The B fraction was calculated as the
difference between 0 h and 48 h sample weight. Then, the residues (expressed as
Journal Paper 3. J7 situ and in vitro studies 122

percentage of the material inserted) at each incubation time minus C fraction were
converted into natural log, and subjected to linear regression. The degradation
constant (kg,) is represented by the slope of this line. The /n situ ruminal
degradability of DM and escape of CP of protein supplements were estimated
applying the model as prescribed by Orskov and Mc Donald (1979) considering
the ruminal passage rate (k,,).
D = A+(Bx kap)/(kap + kpp)
where,
D = Protein degradability, (“%)

A = Fraction A, readily degradable, (“%)
B = Fraction B, slowly degradable, (%)
ka, = Degradation rate constant of degradable B
kp = Rate of passage (assumed to be 5%/h)

The escape of protein was also estimated by applying the model proposed by
Orskov and McDonald (1979).
Escape = kp» x B/ (ky, + kpp) + C
Where,
B, kay, and k,, are stated as in above model
C = Fraction that 1s undegraded
In Vitro Experiment. The in vitro procedure according to Tilley and Terry

(1963) was followed for the estimation of DM digestibility of the protein
supplements. The experiment was replicated. The protein supplements were
prepared as for the in situ method. The ruminally cannulated steer was fed a diet
containing equal portions of alfalfa and grass hay. The steer was fed this diet for a
minimum of 15 d prior to the collection of rumen liquor. Ruminal fluid was

collected through the cannula and filtered through eight layers of cheese cloth into
a prewarmed thermos flask. Fluid was then transported to laboratory where it was
added to the buffer solution, which has been prepared prior to the collection of
rumen liquor.
The buffer solution (McDougall, 1948) was prepared as follows.
NaHCO; 9.80 g
Na ,HPO,4.12H20 =: 9.38 g
NaCl A7 g
Kel 72
MgCl, .06 g
The above salts were thoroughly dissolved in about 500 mL of distilled
water and adjusted to a volume of 1 L. pH of buffer at this time was around 8.2 to
8.4. Just prior to use, .04 g of CaCl, and enough (NH,4)2SO, to make the solution
.02 M (2.64 g/L) were added to the buffer and thoroughly dissolved. Temperature
of the buffer was adjusted at 38 to 39° C and CO, was bubbled through the buffer
to bring down the pH to 6.8 to 7.0. Rumen liquor was added to the buffer in the
ratio 1:4. Thirty five mL of rumen liquor:buffer mixture were placed in the tubes
containing protein supplements and flushed with CO, to create anaerobic
condition. Tubes were closed with Bunsen valves, then incubated in a water bath
for 48 h at 39°C, centrifuged at 1800 x g for 15 min and decanted. The tubes were
again incubated in water bath for 48 h at 39°C after adding acid pepsin (2 g pepsin
plus 83.3 mL of HCI per L of solution) loosely fitted with Bunsen stoppers. At
the end of incubation, tubes were centrifuged at 1800 x g for 15 min, decanted,
and dried at 60°C for 24 h. Percentage in vitro DM digestibility (VWDMD) was

calculated using the formula:
% IVDMD = wt of sample - (wt of residual - blank) / wt of sample x 100

Statistical Analysis: A\l data are presented as least squares means. Data
were analyzed by analysis of variance using the GLM procedure of SAS (1989).
Tukey’s Studentized Range (HSD) Test was used for comparing the treatment for
different variables. Significance of (P < .05) was used throughout unless otherwise
stated.
Results
In Situ Experiment. Dry matter degradability of protein supplements at

different incubation times is presented in Figure 8. Over 90% DM of SBM was
degraded in first 12 h, whereas the values were only 40 to 45 % for ESA, HESA,
and CM. High degradability (60%) was observed for HESA at 0, and 2 h, then the
degradability plateued. More than 90% of DM of SBM was degraded in the first
12 h, whereas only about 40% of DM was degraded in the case of ESA, CS, and
CM. About 60% of DM of HESA was degraded in the first 2 h and thereafter
degradability was plateued and only 10% was further degraded in another 46 h.
Similar but slightly higher degradability was seen for IPA where almost 65% DM
was degraded in first 6 h and in the next 42 h only 20% was degraded.
The data on fractions, degradation rate constant of B fraction, and
degradability of DM of protein supplements are presented in Table 16. The soluble
A fraction ranged from 8.8 to 33.9%. Highest soluble A fraction was for the HESA
supplement (33.9%) which was higher (P < .05) than that for IPA (19.7%), CM
(12.2%), CS (10.7%), and ESA (8.8%), while SBM had an intermediate value
(25.0%). Degradable B fraction was highest in SBM (74.3%) which was higher (P
< .05) than ESA (43.9), HESA (33.3%), and CS (47.7%) . Lowest (P < .0001)
undegradable C fraction of DM was observed in SBM (.7%). The supplements
Journal Paper 3. /n situ and in vitro studies 125

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(30.2%) which was higher (P .001) than IPA (15.0%).
Degradation rate constants (ky) of the B fraction of DM of feedstuffs
ranged from 2.4 to 12.7 %/h. The ky, of SBM was the highest (P < .01; 12.7%/h)
compared to other supplements where kg, ranged between 2.4 and 5%/ h. Among
these protein supplements, numerically, higher ky, was for CM (5%/h) and the
lowest was for ESA (2.4%/h), however, differences were not significant (P > .05).
Dry matter degradability was highest (P < .001, 70.9%) for SBM among the
different protein supplements tested. The lowest value was for the ESA (17.0%)
which was lower (P > .01) than IPA (38.8%). Values for HESA, CS and CM
(26.8, 24.5, and 29.1%, respectively) were intermediate.
Similar trends were observed in the CP fractions of SBM, but the trend
varied among other protein supplements (Figure 9). Crab meal, which had a lower
DM _ degradability, showed a _ higher degradability of protein. Highest
disappearance was for SBM.
Lowest soluble A fraction was for the ESA (6.2%) which was lower (P <
.01) than that of IPA (21.3%), HESA (23.2%) and CM (20.7%) (Table 17). Values
for SBM and CS were intermediate. Degradable B fraction was highest (P < .0001)
in SBM (87.1%). Degradable B fraction of CM (59.8%) was higher (P < .0001)
than for ESA and HESA. The value for CS was intermediate.
Undegradable C fraction was negligible (.2%) for SBM while ESA had the
highest (53.2%), which was higher (P < .01) than for the other supplements.
Undegradable C fraction of CM (19.5%) and IPA (21.7%) were lower (P < .01)
than for HESA and CS. Degradation rate constant (kg,) of the degradable B
fraction of the CP of protein supplements showed a similar trend as that of rate
degradation of DM. Rate of degradation was highest (P < .01) for SBM (14%/h)

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compared to other protein supplement, where the rate of degradation ranged from
2.3 to 5.2%/h Rate of degradation of CM (5.2%/h) was highest (P < .01) than for
ESA and HESA (2.3 and 2.4%/h). Values for IPA and CS were intermediate (4
and 3.4%/h)
The ruminal escape of CP was highest (79.8%) for ESA which was _ higher
(P < .05) than IPA (60.6%), SBM (23.2%) and CM (48.4%). The escape of CP of
CM was lower (P < .05) than IPA, whereas the values for HESA, and CS (67.3and
69.8%, respectively) were intermediate. Lowest (P < .001) escape value was
observed for SBM (23.2%).
In Vitro Dry Matter Digestibility (IVDMD). In vitro DM digestibility of
different protein supplements ranged from 40.5 to 89% (Figure 10). Highest (P <
.0001) DM digestibility was for SBM (89%) and the lowest (P < .0001) value was
for ESA (40.5%). Respective values for IPA, CM, CS and HESA were 78.8, 67,
63.4, and 50.9%, which were different (P < .0001) except between CS and CM (P
> .05).
Discussion
Degradation pattern of CP of SBM agrees with the findings of Kwak (1990)

who reported that the rate of degradation of degradable B fraction of SBM was
10.3%/h, while Zerbini and Polan (1985) reported a still closer value of 12.2%/h
compared to the value obtained in this study (14%/h). Blake and Stern (1988)
reported a low kg, of 6%/h. The present study reveals that the degradable fraction
B of CP of all the protein supplements was rapidly degraded during the first 2 h,
then slower up to 12 h and from 12 to 48 h degradation was still lower. The 2 h N
disappearance was assumed to contain soluble NPN plus a rapidly degradable

132
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protein fraction (Nocek et al., 1979). These rates will be referred to as 2h N kgp. In
the present study N disappearance up to 2 h was rapid, especially for protein
supplements of byproduct nature compared to that of SBM, The 2 to 12 or 2 to 24
h N kg, represents a slowly degradable protein fraction which is assumed to be
degraded linearly. A two sloped degradation charecteristic has been demonstrated
in wide variety of feeds (Mathers et al., 1977; Crawford et al., 1978). Nocek et al.
(1979) reported that 2 h ky, for N show a wide range of degradation rates in feed
ingredients ranging from 3.5%/h to 27.9%/h. Therefore, to eliminate this
discripency, Nocek et al. (1979) excluded 2 h kg from the calculation of
correlations. They also reported that N of cottonseed meal and dehydrated alfalfa
disappeared rapidly within the first 2 h, while other processed protein supplements
such as SBM, distillers grain, and fish meal had lower 2 h kgp.
Dry matter disappearence was higher starting from 6 to 12 h period, with

significant difference (P < .01) at 12, 24, 48 and 72 h ruminal incubation between
the SBM and all other supplements. The same trend was observed for CP also, but
the disappearance was rapid i.e., higher disappearance of SBM N compared to
other supplements was shown starting 2 to 6 h period with significant difference
(P < .01) at 6 h and all other incubation periods thereafter. This indicates that SBM
was more susceptible to microbial degradation in the rumen. At 0 h, numerically
higher disappearance of CP was observed for IPA, HESA, and CM (23.8, 23.2,
and 20.7%, respectively) than SBM (12.7%). This indicates the presence of highly
soluble N fraction in these supplements. Commercial supplement (CS) had similar
CP disappearance at 0 h compared to that of SBM. The ESA had the lowest (6.7%)
disappearance of CP at 0 h, showing a relative lower content of soluble N fraction
than all other protein supplements.

The soluble N fraction of a feed is degraded more rapidly in the rumen than
the insoluble fraction (Clark et al., 1987). Clark et al. (1987) reported that more
total N in the SBM is in the soluble fraction compared to those in BM, CGM or
meat and bone meal. Sniffen (1974) reported that cereal grains and protein
supplements contain four types of proteins, albumin, globulin, prolamin, and
glutelin. They indicated that albumins and globulins are low molecular weight
proteins which are soluble in ruminal fluid, whereas prolamins and glutelins are
high molecular proteins that contain disulfide bonds which render them less
soluble in ruminal fluid. Nugent and Mangan (1978) reported that protein that
have a low solubility in ruminal fluid and that contain extensive cross linking such
as disulfide bonds are less accessible to proteolytic enzymes and are relatively
resistant to degradation. They also reported that albumin and globulin have better
AA profile than glutelin and prolamin but they are degraded in the rumen. Sniffen
(1974) reported that the largest portion of protein of corn and barley are prolamin
and glutelin. Soybean meal contains largely globulin, hence the meal is highly
degraded in the rumen.
Nitrogen disappearance of SBM in first 2 h in this study agree with results
of Nocek et al. (1979). Nitrogen solubility has been considered as an indication of
N degradability on the assumption that if N compounds are highly soluble, then
these also will be highly degradable (Sniffen, 1974; Wohlt et al., 1976). Mertens
(1977) proposed that in vivo all of the soluble N is degraded, and 40 to 50% of the
insoluble N is degraded in the rumen. Some workers have disagreed to the above
statements and reported that protein solubility is a poor indication of protein’s
resistance or suceptibility to degradation in the ramen (Mahadevan et al., 1980;
Stern and Satter, 1984; Nicholson and Johnson, 1991). However, Nocek et al.
(1979) reported that neither N solubility nor 2 h N disappearance are reliable

indicators of total N degradation of a feedstuff during ruminal residence. Solubility
of protein is not a guarantee nor a prerequisite for degradability (Chalupa, 1982).
Leng and Nolan (1984) suggested that soluble proteins such as serum albumin,
ovalbumin, chloroplast protein extract, and soluble protein from SBM and
rapeseed meal are degraded to varying degrees. It has been shown that the
structure of protein is an important determinant of degradability (Chalupa, 1982;
Leng and Nolan, 1984). Proteins which have no terminal amino or carboxyl groups
(i.e., ovalbumin ) and those with excessive cross linking (i.e., Ribonuclease A)
appear to be less accessible to proteolytic enzymes.
The same trend as seen in CP degradation was shown in DM also. Nocek et
al. (1979) reported that there was a positive correlation (1° = .81) between the 2 h
and 2 to 48 h ky, of DM of various feed ingredients. Therefore, they suggested that
more reliable prediction of degradation of DM from DM solubility than from N
solubility.
Ruminal degradability of CP of SBM obtained in this study agrees well
with previous reports for the ruminal N degradability of SBM. Literature values
for SBM N escaping ruminal degradation have ranged between 15 and 43% (Zinn
et al., 1981; Zinn and Owens, 1983; Loerch et al., 1983a; Kirkpatrick and
Kennelly, 1986; Kwak, 1990; Zerbini and Polan, 1990; Rodriguez, 1994). The
escape value determined (23.2%) for the SBM N in the present study agrees with
previously reported values. Sindt et al (1993) reported ruminal N escape of 52.3%
for SBM where k,, was estimated to be 6.3%/h while, for BM and FTM values
were 4.7%/h and 5.2%/h, respectively. The high escape of N of SBM in their study
might be due to increased rate of passage which reflected on a lower ka, of 5.7%/h
compared to 14%/h in the present study. Orskov et al. (1983) reported that the rate
of outflow of protein can range from approximately 1%/h with a low level of

feeding of ground diets to approximately 10%/h with very high levels of feeding of
mixed diets. Normally, k,, of 5%/h was considered optimum at maintenance level
of feeding (Miller, 1982). They also reported that degradability cannot be

expressed as a single value and will vary considerably with outflow rate. Their
estimate of escape N in SBM (52 or 43%, depending on passage rate estimate) was
higher than the value obtained in the current study (23.2%) where the steer was
fed 50:50 alfalfa grass hay diet. Their low ruminal degradation values for the SBM
N might be due to a low ruminal pH. Loerch et al. (1983a) reported that 12 h in
situ escape N of SBM increased from 33 to 55% as level of high-moisture corn in
the diet increased from 20 to 80%. Ruminal pH decreased with increased high-
moisture corn level in the diet. Loerch et al. (1983a) suggested that this low
ruminal degradation might be due to a low pH when cattle were fed high grain
diets. They also suggested that this low pH might have reduced the number of
proteolytic bacterial species. However, it 1s not likely that the ruminal pH of the
steers used in the present study was low since the diet did not include
concentrates.
A high level of escape protein for SBM (66.0%) has been reported by
Ludden et al. (1995). Escape of protein of a combination of CGM and BM was
90.8%. Rate of degradation of SBM and combination of plant protein and BM was
also lower than observed in the present study. One possibility for this high escape
of SBM in their experiment would be due to the diet which contained 70% corn
grain whereas, in the present study the host animal was fed a complete roughage
diet (50:50, alfalfa - grass hay). A similar result as observed by Ludden et al
(1995) has been reported earlier by Barrio et al. (1986) who found that the rumen
escape value for DM and N in SBM were lower when a diet with 80% as opposed
to 40% concentrate was fed to cattle. Loerch et al. (1983b) had reported earlier
Journal Paper 3. J situ and in vitro studies 136

that the decrease in ruminal pH by feeding diets based on cereal grains may reduce
the protein degradability of SBM. Ward et al (1986) reported that the amount of
dietary protein that escaped ruminal degradation in lactating dairy cows fed
concentrate, alfalfa hay and sorghum silage was about 2.5 times greater (46 vs
18%) when concentrates supplied 65% rather than 35% of the dietary DM. Owens
(1986) indicated that increasing concentrate from 30 to 60% of the diet increased
protein that escaped ruminal degradation from 22 to 29%, which may be attributed
to lower ruminal pH, which reduces solubility, thus reducing the accessibility of
protein for degradation (Loerch et al. 1983a) or due to a reduction in the fiber
content of the diet, which inhibits microbial degradation of protein (Weakely et al.,
1983).
Sindt et al. (1993) reported that estimated escape N determined by
combining passage and digestion rates were highest for BM and FTM and lowest
for corn silage while, SBM had an intermediate value. Reported values for N
escape for BM were 85, 82, 81.4, 92, and 93.2 %, respectively, by Stock et al.
(1981), Loerch et al. (1983b), Palmquist et al. (1983), Titgemeyer et al (1989), and
Rodriguez (1994), while those for FTM were 65, 85.1 and 91.7%, respectively, by
Titgemeyer et al. (1989), Palmquist et al. (1993) and Sindt et al. (1993).
Titgemeyer et al. (1989) reported that BM contained the largest amount of N
(22%) that was not available for either ruminal degradation or small intestinal
absorption. whereas SBM contained the least nonavailable N (6.5%) followed by
FTM (13%). The escape N for urea-BM/FTM combination was 59.2% (Sindt et al.
1993) which was lower than the value obtained for combination of FTM and BM
in the present study, the reason for their low value might be due to the
incorporation of urea.
Journal Paper 3. /n situ and in vitro studies 137

The present study indicated that the CP of ESA which is a combination of
BM and FTM was only about 25% as degradable as the protein in SBM, whereas
for the HESA and CS, degradability was 43 and 39%, respectively, as degradable
as the SBM protein. In the present study the rumen escape of CP of ESA and
HESA supplement were 79.8 and 67.3%, respectively. Although both products are
combinations of BM and FTM, the later was hydrolyzed with little differences in
composition between the two. The escape of N of HESA was similar to that of CS
(69.8%), but the nutritive value of HESA was significantly lower than CS
(Chapter III and IV). This suggests that the hydrolyzed product of FTM and BM
combination might be poorly digested postruminally nor had a poor AA profile. In
fact CP digestibility was very low (41%) for the HESA supplemented diet
(Chapter IV). These results agree with findings of King et al. (1990) who reported
that BM N was poorly digested post ruminally, especially when BM _ was batch
dried at high temp which may decrease available protein more than would spray or
roller drying.
Garret et al. (1987) calculated undegradable intake protein (UIP) of BM and
SBM as 58 and 30%, respectively, which indicated that various sources of BM
have a lower UIP value than that reported (82%) by NRC (1989). Titgemeyer et al.
(1989) reported UIP of 92% for BM. Howie et al. (1994) suggested that these
inconsistencies may be due to variation among samples of BM processed by
different methods or estimation of ruminal CP degradation.
Goedeken (1990b) reported that escape protein of FTM (73.2%) was
greater than that of SBM (24.6%) but less than that of BM (90.0%). A
combination of BM and FTM had an escape of 82% which was comparable with
the values (80%) obtained in the present study. They also reported that weighted
average of ruminal escape of FTM and BM is similar to the protein escape value

for the FTM + BM (79 vs 82%). Hydrolyzing blood possibly increased ruminal
fluid solubility and reduced ruminal escape (Goedeken et al., 1990b). They
indicated that adding raw blood to feathers before hydrolyzing did not increase the
escape above that of FTM without blood, whereas adding blood after hydrolyzing
feather increased ruminal escape compared with FTH. The same workers
(Goedeken et al., 1990a), in another experiment, estimated the in situ N
undegradability of FTM and BM to be 69 and 83%, respectively. Waltz et al.

(1989) reported that grain mixes containing FTM and BM + FTM had lower rates
of N disappearances and protein degradation compared with the grain mix
containing SBM. They also reported that rate of N disappearance and extent of
protein degradation were lower in the grain mix containing the BM than in the
grain mix containing SBM but was higher than in the grain mix containing FTM.
Method of processing of BM had an effect on degradability of BM (Harvey and
Spears, 1989). Waibel et al. (1977) reported that the conventional method of
processing BM by vat drying is less expensive than the ring dried but lysine has
been shown to be lower in vat dried BM.
Hvelplund et al (1993) reported that rumen undegraded protein (RUP)
should be partitioned into intestinally available and unavailable fractions. The
NRC assumes all the escape proteins are equally digested in the small intestine.
The NRC models consider only RDP and RUP, ignoring the nature of protein, 1.e.,
bound or unavailable that is present in RUP. Since bound protein may not be
digested in the small intestine, the nutritional value and digestibility of RUP may
be variable. This shortfall has been recognized by the Cornell Net Carbohydrate
and Protein System for evaluating cattle diets. This system contains a sub model
whereby protein is partitioned into six fractions. Fraction C represents proteins
that are insoluble in acid detergent and considered to be indigestible in the

intestine (Fox et al., 1990). Although intestinal measurements have not been
studied in this experiment, it is assumed that intestinal digestion of CP of HESA
may be poor when the results of this experiment is related to the balance
experiments and growth trials conducted (Chapter III and IV).
Wallace (1988) noted that proteins of animal origin are generally broken
down rapidly but incompletely, whereas plant proteins are degraded more slowly,
but potentially degraded almost completely. Results of the present study do not
agree entirely with the above statement because CP disappearance of ESA and CS
was lower than SBM, which might be due to collagenous nature of protein of FTM
and other proteins of anima] origin which are resistant to ruminal degradation.
However, in the present study N disappearance from CM, CS, ESA, and HESA
tended to be higher compared to plant protein supplement such as SBM and a
combination of plant protein and BM for the Ist 2 h of incubation.
Clark et al. (1987) reported that protein of animal byproducts are less
degraded compared to cereal by products. The low degradability of meat or meat
and bone meal which contains relatively large amount of collagen, and is low in
lysine (Happich, 1975). Therefore, these products will have a shortage of trypsin-
sensitive peptide bonds. Accessibility of certain AA residues within the protein
molecule may influence both rate and extent of protein degradation in the rumen
(Craig and Broderick, 1984). Losses of lysine and arginine have been shown to be
greater than average loss of other AA when proteins were degraded by ruminal
microbes. (Craig and Broderick, 1984; Crooker et al, 1986). It has been shown
that microbial enzymes cleave the peptide bonds at lysine residues, similar to the
mammalian trypsin enzyme (Craig and Broderick, 1984). Obviously, a feedstuff
with low lysine content (cereal grain by product) will have a slow degradation in
the rumen. By product feedstuffs of animal origin such as FTM and hair meal are

extensively cross linked with disulfide bonds, which are not very susceptible to
proteolytic enzymes (Clark et al, 1987). These cross linkages render these products
resistance to ruminal degradation (Nugent and Mangan, 1978). The low ruminal
degradation of the ESA and HESA supplements are explained by the above reason.
Processing of byproduct feeds undergo cooking or drying which denatures
and coagulates the protein, making it insoluble (Boehme, 1982). Similarly, cooling
causes a random relinkage of the protein molecule (Bendall, 1964). He also
reported that decreased surface area for microbial attack and the formation of
chemical bonds upon cooling render the protein in these byproduct feeds more
resistant to ruminal degradation. Heat applied during drying of cereal byproduct
feeds also denatures and coagulates protein, making it less degradable in the
rumen.
In the present study ruminal DM degradability was lower (29.1%) for CM
compared to that for CP (51.6%). Relatively higher degradability of CP of CM
was observed compared to other protein supplements. However, the escape protein
was found to be over twice as that of SBM. A similar trend was observed by Velez
et al. (1991) but a lower DM and CP degradability of 18.3 and 34.9%,
respectively, were reported for crab waste meal. They also reported a relatively
lower degradability of DM and CP of SBM (55.9 and 63.6%, respectively). A
lower rate of degradation of DM and CP of crab waste meal (.57%/h and .79%/h,
respectively) have been reported (Velez et al., 1991), compared to higher values
of 5%/h and 5.2%/h, respectively, for the present study. The differences observed
in these two studies might be explained by the differences in the ruminal
environment due to the diet variation or due to differences in the procedures.
Degradability observed in this study for CM agrees with the findings of Patton and
Chandler (1975) who reported the DM disappearance at 12, 24, 36, 48, and 60 h
Journal Paper 3. Jn situ and in vitro studies 14]

for crab waste meal as 26.7, 33.2, 36.2, 41.2, and 38.3%, respectively. The low
degradability of DM relative to CP might be due to high levels of ash (45%) which

may be poorly soluble in rumen.
As expected, almost 90% of the DM of SBM was digested by in vitro
estimation of DM digestibility ((VDMD). Feather meal and BM combination
(ESA) had the lowest DM digestibility whereas the hydrolyzed product had a
higher DM digestibility but this difference was not revealed in in vivo estimation
(Chapter IV), perhaps because of the nutrient dilution. These results agree with the
findings of King et al. (1990) who reported that BM N was poorly digested post
ruminally, especially when BM was batch dried at high temperature. Relatively
higher level of DM digestibility and ruminal escape of N for the CM, shows that it
will be advantageous to use CM as a protein supplement for ruminants.
Implications
Crab meal contains over twice the ruminal escape protein of SBM. This
seafood waste is a digestible moderate escape protein source that may be useful in
diets for ruminants. Combination of feather meal and blood meal (ESA) had the
highest escape protein among the supplements tested which shows its potential use
as an escape protein supplement. Although hydrolyzed product of feather meal and
blood meal combination had almost thrice the ruminal escape protein of SBM, its
nutritive value is significantly lower than all the other protein supplements tested.
This implies that caution should be taken to evaluate the feedstuffs based on
escape value alone and it is suggested that other criteria such as amino acid profile
and whole tract digestibility may be considered in evaluating the feedstuffs as a
protein supplement for ruminants.

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Chapter VI. Journal Paper 4. Effect of Steam Explosion at
Two Levels of Severity on Dry Matter and Chitin
Digestibilities and Escape of Protein as Measured by In Situ

and In Vitro Methods
Abstract: The potential was explored of the steam explosion technique to

enhance the nutritive value of crab meal for ruminants. Crab meal and laboratory
grade chitin (isolated chitin) were steam exploded in a 25 L, stainless steel, batch
steam explosion reactor at reactor temperature 193° C, pressure 12.7 kg/cm2, (low
severity); and reactor temperature 226° C, pressure 25.5 kg/cm’, (high severity);
for 3 min and 30 sec. Steam explosions for each ingredient for each level of
severity were replicated. Steam explosion decreased N content of crab meal by
20% in both severities. A ruminally-cannulated steer (550 kg, BW) was fed a diet
containing 40 parts alfalfa hay, 40 parts grass hay, 10% ground com, and 10%
crab meal starting 15 d prior to the experiment. The materials tested for in vitro
and in vivo experiments were, |) crab meal, 2) steam exploded crab meal at low
severity, 3) steam exploded crab meal at high severity, 4) isolated chitin, 5) steam
exploded isolated chitin at low severity, and 6) steam exploded isolated chitin at
high severity. An IVDMD experiment was replicated. Steam explosion at low and
Journal Paper 4. Studies on steam exploded crab meal 149

high levels of severities improved (P < .01) IVDMD (71.0 and 75.2%,
respectively) compared to unexploded (65.9%), while IVDMD of isolated chitin
was reduced by 40% by steam explosion. Steam explosion did not show any effect
on ruminal DM degradability. The escape protein of crab meal was estimated to be
37.4%. Steam explosion did not improve the escape value of crab meal protein
(41.2% and 34.7%, respectively for crab meal steam exploded at low and high
severities). Chitin content in the crab meal was 14.7%. A 60% increase in chitin
degradability was observed for crab meal exploded at high severity. Chitin
degradability in isolated chitin exploded at high severity was more than twice that
of crab meal, but steam explosion lowered chitin degradability (P < .05). Steam
explosion does not seem to be a promising processing method for better utilization
of crab waste for ruminants.
Introduction
Substantial quantities of wastes are produced as byproduct from seafood

processing plants around the world. It has been estimated that global annual
production of dry shell wastes from crab, lobster, shrimp, krill, and clam/oyster, is
1.46 million t, DM basis (Knorr, 1991). The waste produced by seafood producing
plants is highly perishable and has become a menace to the processor both in
developed and developing countries due to the environmental problems. Shell fish
wastes have been found to be suitable for feeding ruminants as dry meal (Patton et
al., 1975; Velez et al, 1991) and as silage with wheat straw (Samuel et al., 1991).
Although crab meal (CM) is a good source of protein, it has been shown that the
digestibility of protein by crab waste-straw silage was lower than that of fish
waste straw silage (Samuels et al., 1991). Further, Velez et al. (1991) reported that

CP digestibilities for the crustacean waste meal diets were 8 to 18% lower than for
isonitrogenous diets with SBM. Digestion trials conducted with sheep showed that
animals fed 50% crab waste-straw silage and 50% basal diet had a lower
digestibility (37%) of chitin than for sheep receiving 100% crab-waste straw silage
(58%) (Ayangbile, 1989). Apparent digestibility of DM, OM, CP, energy, NDF,
ADF, cellulose and hemicellulose in silage containing crab waste decreased
linearly with increased level of crab waste straw silage.
The lower digestibility of crab processing waste diets may be due to chitin
which is (1-4)-B-linked homopolymer of N-acetyl-D-glucosamine, an analogue of
cellulose. Chitin has been shown to be resistant to degradation by microorganisms
in the gastrointestinal tract of ruminants (Morgavi et al., 1994). This lower
digestibility of chitin and protein is perhaps due to lower accessibility of microbial
enzymes. Chitin and protein are covalently linked via aspartic acid (Brine and
Austine, 1981; Hackman, 1984). If chitin-protein complex could be cleaved,
perhaps more nutrients would be available due to more accessibility to enzymes.
Therefore, the proposed research was to explore the possibilities of any structural
changes in the components of crab waste by steam explosion technique.
Steam explosion is a technique employed in industries for fractionation of
lignin, hemicellulose, and cellulose. In this method biomass is treated with high
pressure steam followed by explosive release of pressure (Foody, 1982). Steam
explosion of biomass normally produces a fibrous material that can be fractionated
into water-soluble hemicelluloses, alkali-soluble lignin, and an insoluble cellulose-
rich residue in proportions of 25, 25, and 50%, respectively (Jain and Glasser,
1992). Steam explosion technique could alter profoundly the microstructure of
cellulose and it has been shown that physical action of this method induced a
structural change to the cellulose. Kamide and Okajima (1987) have shown that

these changes were not due to H-bonding modification, but probably induced by
enhanced water penetration between adjacent cellulose molecules which was due
to sudden decompression. The changes are due to the explosive depressurization
process which combines the chemical action of autohydrolysis with mechanical
action (Grethlein and Converse, 1991). Steam explosion process leads to a major
increase in the enzyme accessibility because of the morphological modification
involving disruption into microfibrillar elements of the cell wall as well as the
fiber fractions (Machessault and St-Pierre, 1980).
Steam explosion technique has been employed in the field of ruminant

nutrition. Steam exploded wheat straw was found to be better utilized by steers
compared to unexploded straw (Sciaraffia and Marzetti, 1988). Although no report
is available on steam explosion of crustacean meal, it is assumed that there would
be some structural changes for the chitin-protein complex and may improve
digestion by ruminants. Therefore, the objectives of this experiment were 1) to
study the effects of steam explosion at two levels of severity on ruminal
degradation of DM, CP, and chitin of CM by in situ experiment and DM
digestibility by in vitro method, and 2) to estimate the ruminal escape potential of
CP of CM.
Crab meal was obtained from Graham and Rollins, Hampton, Virginia and
isolated chitin (practical grade) from crab shells was obtained from Sigma
Chemicals Co, St Louis, MO. Steam explosion of CM and isolated chitin was
performed in a 25 L, stainless steel, batch steam explosion reactor, located in the

Pilot/Demonstration Laboratory of Thomas M. Brooks Forest Products Center

located at Virginia Polytechnic Institute and State University, Blacksburg. Steam
explosion of two severities were tested. 1) Low severity, reactor temp 193°C,
pressure 12.7 kg/cm’ 2) High severity, reactor temp 226°C, pressure 25.5kg/cm”.
Both treatments were for 3 min and 30 sec. For each level of severity (low and
high) duplicate explosions were carried out with 1 kg of each material. Steam
exploded material was collected in plastic buckets of 18 L capacity and
transported to the laboratory. Steam exploded material contained moderately high
water content due to the condensation of steam and washings. Samples of
approximately 1 kg (to get approximately 200 g of DM for in situ and in vitro
experiments) were collected from each exploded material and were frozen in ice
trays. The frozen materials were freeze dried and kept for analysis for Kjeldahl N
by AOAC (1990) and chitin (procedure for ADF estimation; Ayangbile, 1989) .
In Vitro Experiment. The IVDMD were performed according to Tilley and
Terry (1963) on the following materials: 1) crab meal, 2) steam exploded CM at
low severity, 3) steam exploded CM at high severity, 4) isolated chitin, 5) steam
exploded isolated chitin at low severity, and 6) steam exploded isolated chitin at
high severity. The experiment was replicated. A ruminally-cannulated steer (550
kg, BW) was fed a diet containing 40 parts alfalfa hay, 40 parts grass hay, 10 parts
ground corn and 10 parts crab meal, starting 15 d prior to the collection of rumen
liquor. Rumen fluid was collected through rumen fistula and filtered through eight
layers of cheese cloth into a pre-warmed thermos which was then taken to
laboratory. Ruminal fluid was added to the buffer solution in the ratio 1:4 which
had been adjusted to 39°C. Carbon dioxide was passed through the rumen
liquor:buffer mixture until pH of 7 was reached.
Ruminal liquor-buffer solution (35 mL) was added to the tubes containing
material and flushed with CO, on top of the fluid to create anaerobiosis. Tubes

were closed with stoppers fitted with Bunsen valves which to prevent
accumulation of gas in the tubes. Tubes were incubated for 48 h at 39°C. At 48 h
tubes were centrifuged at 2500 x g for 1S min and supernatant was discarded.
Tubes were again incubated for 48 h at 39°C with 35 mL of acid pepsin. At the
end of incubation the tubes were centrifuged and decanted and dried for 24 h. The
difference in weight loss after incubation was expressed as percentage
disappearance.
In Situ Experiment. Effect of steam explosion on CM and isolated chitin on
ruminal degradation of DM, CP and chitin were estimated. The following were
tested: 1) crab meal, 2) steam exploded CM at low severity, 3) steam exploded
CM at high severity, 4) isolated chitin, 5) steam exploded isolated chitin at low
severity, and 6) steam exploded isolated chitin at high severity. A ruminally-
cannulated steer ( 550 kg, BW) fed as described above was used for the in situ
experiment.
Six gram of samples were placed in polyester bags of 10 x 23 cm size with
59 + 2 um in porosity. Calculated sample size to bag surface area was 15 mg/cm’
(Nocek, 1988 ). Samples were placed in duplicate bags and were closed with 10
cm Bar-Lok” cable ties (Dennison Manufacturing Co., Framingham, MA).
Duplicate bags were tied together with another Bar-Lok” tie. Duplicate bags of six
different materials were attached to a metal chain of ~20 cm in length which was
attached to one end of the polyester thread of 60 cm in length. The bags for the 72
h incubation were tied to the metal chain which was attached to a thicker polyester
thread of 75 cm which acted as the main string to which groups of bags for the
subsequent incubations were attached by 20 cm Bar-Lok* tie.
The incubation periods were 0, 2, 6, 12, 24, 48, 72 h. The bags were
introduced in the rumen in reverse order. Prior to incubation, bags were soaked in

lukewarm water for 15 min (Rodriguez, 1994) and then introduced into the rumen
through the rumen cannula. A total of 84 bags were introduced into the rumen. The
bags for the 0 h were not introduced in the rumen but were soaked in warm water
for 15 min. At 72 h all bags were retrieved, and washed in running tap water.
Bags were separated and washed thoroughly until the wash water was clear. The
bags were then dried in a forced draft owen for 48 h. Bags were weighed and loss
in weight was calculated as percentage DM degradability. Samples were kept for
the analysis for Kjeldahl N (AOAC, 1990) and for chitin estimation. The
procedure for the ADF estimation was used for the estimation of chitin
(Ayangbile, 1989).
Degradability of DM and CP were calculated by using the model of Orskov
and McDonald (1979),
D=A+(Bx kgv)/(kap + kpp),
where,
D = degradability (%); A = fraction A, readily degradable (%); B = fraction

B, degradable (%); kg, = degradation rate constant of degradable B; and k,, = rate
of passage constant (assumed to 5%/h)

The model proposed by Orskov and McDonald (1979) for the estimation of
escape of protein is as follows: Escape = kpb x B/(ka, + kyu) + C, where, B, kas,
and k,, are stated as in above model; C = fraction that is undegraded (at 48 h).
Fraction A was calculated by subtracting the percent remaining at 0 h (after
soaking in warm water ) from 100. Fraction B is calculated by subtracting the sum
of A and C from 100. Fraction C 1s the percent residue left undegraded at 48 h of
incubation. Degradability depends on rate of passage of digesta (k,,), and is
estimated by subjecting the natural log of the residues (expressed as percentage of

the material inserted) at each incubation time minus C fraction to linear regression.
The degradation constant is represented by the slope of this line.
Statistical Analysis: All data are presented as least squares means. Data
were analyzed by analysis of variance using the GLM procedure of SAS (1989).
Tukey’s Studentized Range (HSD) Test was used for comparing the treatment for
different variables. Effects of treatment were compared separately for sources
(crab meal and isolated chitin). Significance of (P < .05) was used throughout
unless otherwise mentioned.
Results and Discussion
The CP averaged 41.0 and 42.2%, respectively for CM and isolated chitin,
while chitin content averaged 14.4 and 93.0, respectively, DM basis (Table 18).
The result shows that isolated chitin (laboratory grade chitin) had 93% chitin and
42.2% CP, which means that the source of this N is the acetylated amino group of
chitin molecule. From the above values the percentage of N of chitin tested was
calculated to be 7.26%. The CP of CM (41.0) was decreased (P < .0001) to 32.6%
and 31.6%, respectively for CM exploded at low and high severities. This loss of
N perhaps was due to deamination of protein to NH; at high temperature. This loss
of N could not be measured. The temperature of the reactor at low severity may be
sufficient for this deamination. This might be the reason for similar values for both
severities. A small decrease in CP was observed for steam explosion of isolated
chitin. Nitrogen in chitin is present in the chitin molecule as acetylated amino
group (-NHCOCH:;) (Mathur and Navrang, 1990). Chitin content in CM was
lowered (P < .05) from 14.4 to 13.5% by steam explosion at low severity, but this
difference was not observed on high severity steam explosion. Although there was

Table 18. Effect of steam explosion on crude protein and chitin of crab meal
and isolated chitin®
Item Crude Protein Chitin?

a
Crab meal 41.04 14.4

Steam exploded crab meal
Low! 32.6° 13.5
High® 31.6° 14.4
SE 50 20
Isolated Chitin 42.2° 93.0
Steam exploded isolated chitin
Low! 39,2° 92.9
High® 40.7° 90.5
SE 0.42 0.84
“Least squares means, avg. of four values
Did not differ (P > .05)
“DM basis
“Means with in a column lacking a common superscript letter (P < .05) (crab meal and
isolated chitin were compared separately)
‘Reactor temperature 193°C, pressure 12.7 kg/cm’ for 3 min 30 sec
®Reactor temperature 226°C, pressure 25.5 kg/cm’ for 3 min 30 sec

a decrease in chitin on steam explosion for isolated chitin, differences were not
significant (P > .05). The same trend was observed for CP of isolated chitin, which
shows that little deamination would have taken place at high severity explosion.
In Vitro Experiment. An increase in percent in vitro DM digestibility was
observed when CM was steam exploded at low and high severity (Figure 11).
About 10% increase (P < .0001) in DM digestibility (75.2%) was observed for CM
steam exploded at high severity compared to unexploded CM (65.9%). Crab meal
exploded at low severity had an intermediate value (71%) which was different (P <
.O5) from unexploded and exploded at high severity. Isolated chitin behaved
differently on steam explosion. Steam explosion had a negative effect on DM
digestibility of isolated chitin. Over 40% reduction (P < .0001) in DM digestibility
was observed for isolated chitin exploded at high severity. Isolated chitin treated
with low severity of explosion had a decrease (P < .001) of 16%, compared
unexploded. Dry matter digestibility of unexploded isolated chitin appears to be
higher, compared to CM.
This implies that chitin is well digested in the digestive tract of ruminants.
Perhaps steam explosion caused structural changes in the chitin which would have
made the molecule inaccessible for the microbial enzymes. In the case of CM, the
increase in the digestibility may not be due to increase in the digestibility of chitin
or protein, but perhaps due to the solubility of the minerals, especially CaCO; in
the rumen liquor:buffer and acid pepsin. Steam explosion would have caused
extensive destructive changes in the chitin-protein structure which is ‘reinforced’
with calcium especially CaCO; and to a lesser extent CaHPO,. It has been reported
that exoskeleton of crustacean contains several chitinous layers, generally
impregnated with mineral salts, such as carbonates and phosphates of calcium
(Mathur and Navrang, 1990; Madhavan, 1992). Johnson and Pennisten (1982)

Ajaaas Yysiy ye papojdxa wieays = Yysip] ‘A}OAOS MO]
159
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reported that crustacean shell contains 30 to 50% mineral matter, DM basis,
mainly CaCO; and 8 to 10% calcium phosphate (of the mineral matter). The total
ash determined for CM in the present study (Chapter III) was 45% which agrees
with previous reports.
In Situ Experiment. Ruminal DM degradability of unexploded and exploded
CM and isolated chitin at two severities at different ruminal incubations are
presented in Figure 12. Ruminal DM degradability of CM, both exploded and
unexploded, were higher until 36 h of incubation, compared to isolated chitin
except for isolated chitin exploded at high severity which was lower than the other
products. Ruminal degradability of DM of steam exploded CM was higher (P <
.O5) at 0 h incubation and this trend continued until 12 h incubation and after 24 h
there were no differences. Low degradability of DM (~10%) was observed for
unexploded and exploded isolated chitin until after 12 h incubation. Exploded
isolated chitin both at low and high severities showed higher degradability (P <
.OS) than unexploded until 12 h incubation. After 12 h the trend changed for
isolated chitin, where degradability increased, but there was a decrease (P < .005)
in DM degradability of steam exploded chitin. At 72 h incubation DM
degradability of CM and isolated chitin were similar.
Steam explosion of CM resulted in higher (P < .05) soluble fraction A of
DM for both severities (Table 19). The higher soluble fraction A may be due to the
physical action of steam explosion which would have broken the mineral bindings.
Soluble A fraction was negligible for exploded and unexploded isolated chitins
(Table 20). Surprisingly, a high level of degradable B fraction of DM was
observed (75.6%) for isolated chitin which was higher than for CM (47.8%). Crab
meal showed a decrease (P < .01) in B fraction of DM both in low and high
severity explosion, while isolated chitin showed a significant decrease (P < .05)
Journal Paper 4, Studies on steam exploded crab meal 160

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‘UlIyd poyejosi popojdxoun = F{D ‘AjoAes Ys Je [eau quid popojdxo wiea}s = EIWD ‘AjOAaS MO] Je [RaW qed popojdxo wea}s = TWO
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Table 19. Effect of steam explosion on fractionation and degradability of dry
matter of crab meal by in situ experiment*
Exploded”
Item Unexploded Low High SE
Dry matter fractions, %
Soluble A’ 17.2° 21.9% 24.6° 48
Degradable B’ 47.8° 41.1° 36.4° 75
Undegradable C 35.0° 37.0° 39.0° 36
kdb” 5.9° 4.9° 55° 63
Degradability, % 42.6° 42.2° 43.5° 1.10
“Least squares mean
’Steam exploded in a 25 L stainless steel, batch steam explosion reactor at reactor temperature
193°C, pressure 12.7 kg/cm’ for 3 min 30 sec (low severity); reactor temperature 226°C, pressure
25.5 kg/cm’ for 3 min 30 sec (high severity)
°“¢Means with in a row lacking a common superscript letter differ (P < .05)
*P< 0]
**Estimated by regression analysis
***Depradability = A + (kax BY/(ka + .05)
Where,
A = fraction A, readily degradable (%)
B = fraction B, degradable (%)
kdb = rate of degradation
.O5 = rate of passage (5%/h)

Table 20. Effect of steam explosion on fractionation and degradability of dry
matter of isolated chitin by in situ experiment*
Exploded”
Soluble A 2.3° 4.2° 2.8° 1.26
Degradable B 75.6° 63.6% 54.0° 3.98
Undegradable C 22.1° 34.24 43.29 3.48
kdb’ 9.5° 8.6° 6.1° 1.05
Degradability, % 51.9° 44.1% 31.6 3.72
Steam exploded in a 25 L stainless steel, batch steam explosion reactor at reactor temperature
25.5 kg/cm? for 3 min 30 sec (high severity)
“¢Means with in a row lacking a common superscript letter differ (P < .05)
*Estimated by regression analysis
**Depradability = A + (ka, x B)/(ka + .05)
Where,
B = fraction B, degradable (“%)
.O5 = rate of passage (5%/h)

only in high severity explosion. There was a decrease of more than 10 percentage
units for degradable fraction B of CM exploded at high severity compared to
unexploded. Undegradable C fraction of DM of CM was increased (P < .05) due
to steam explosion at both severities. Fraction C of DM of isolated chitin was
increased (P < .01) by almost 100% by high severity steam explosion compared to
unexploded. Calculated rate of degradation (ky,) of DM based on regression
calculation was found to be numerically higher for isolated chitin compared to
CM. There was a trend for a decrease in kg, for the exploded meal isolated chitin
compared to unexploded. A similar trend was observed for CM, but these
differences were not significant (P < .05).
Steam explosion did not show any impact on DM degradability of CM
while DM degradability was reduced (P < .005) in isolated chitin due to low and
high severity explosion. Steam exploded isolated chitin at high severity had
significantly lower (P < .05) DM digestibility compared to steam exploded at low
and high severities. The results shows that steam explosion has caused some
structural changes in the protein and chitin molecules which would have made the
microbial enzyme inaccessible for the hydrolytic action. Marchessault (1988)
suggested that a mechanical destructuring of biomass is a key feature of steam
explosion. In the case of carbohydrate polymer the principal chemical
transformation is hydrolysis producing soluble carbohydrate (Marchessault, 1988).
Grethlein et al. (1984) reported that purpose of any pretreatment (e.g. steam
explosion) is to increase the surface area available to the enzyme to act upon. In
the case of cellulose, enzyme cellulase must be able to contact the glycosidic
bonds for cleaving. Cellulose is very porous and has about 600 to 800 m* of
surface area per g of substrate, but approximately 98% of this area is in pores that
are available for smaller molecules (Grethlein and Converse, 1991). There is a

major increase in accessibility to enzymes due to increase of the internal porosity
(Marchessault, 1988). If this is the case, it has to be assumed from the behavior of
DM of iaolated chitin when it was steam exploded at two severities that some
molecular rearrangement which would have lead to a negative response by
lowering the surface area. This would have restricted the accessibility of enzyme.
Digestibility of DM was high for in vitro compared to in situ. This higher
DM digestibility for the in vitro technique, perhaps was due to simply the
solubility of minerals in low acidic environment in abomasum and small intestine
which would have overestimated the DM digestibility, because in the in vitro
method the DM digestibility of the material is estimated only on the basis of loss
in weight of the incubated material. This loss in weight does not give an estimate
of the level of absorption. In the present case, the assumed soluble ash mainly
comprised of Ca is poorly absorbed.
Ruminal CP degradability of steam exploded CM and isolated chitin at
different incubation times are given in Figure 13. Although CM and isolated chitin
contain similar level of CP, their pattern of degradability is different. Crab meal
protein both unexploded and exploded had 50 to 60% degradability by 2 h of
incubation, whereas isolated chitin had only ~10% at 2 to 12 h incubation. After
12 h degradability of CP in isolated chitin increased, but decreased (P < .005)
with steam explosion. Although steam exploded crab meal showed higher (P <
.05) degradability at 2, 6, 12 and 24 h incubation, there were no differences at 48
and 72 h.
Soluble A and degradable B fractions of N of CM did not change when
steam exploded at low severity, but had an increase (P < .001) of 7.2% for soluble
A and a decrease (P < .01) (7.3%), for fraction B on high severity explosion
compared to unexploded (Table 21). The degradable B fraction of CM
Journal Paper 4. Studics on steam exploded crab meal 165

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Table 21. Effect of steam explosion on fractionation and degradability of
crude protein of crab meal by in situ experiment*
Exploded”
Soluble A 33.9° 33.1° 41.1° 96
Degradable B 52.1° 51.4° 44.8° 1.09
Undegradable C 14.0° 15.5° 14.1° 44
kdb" 6.2° 5.1° 6.1° 7)
Escape, % CP” 37.4% 41.2° 34.7° 1.47
*Least squares mean
“*Means with in a row lacking a common superscript letter differ (P < .05)
*DM basis
***F scape = kp, x B/(ka + kpp) + C
Where,
C = fraction C, undegradable (%)
kg, = rate of degradation
k,»= rate of passage (5%/h)

was decreased only by the high severity (P < .1). There was no difference 1n the
undegradable C fraction of N between unexploded and exploded CM. Rate of
degradation also showed no difference (P < .05).
Steam explosion at high severity reduced (P < .05) the soluble A fraction of
N of isolated chitin (Table 22). A decrease in degradable B fraction of N resulted
for isolated chitin exploded at low and high severity explosions, compared to
unexploded. Undegradable C fraction of N of isolated chitin was increased at low
and high severity steam explosions with a significant increase (P < .05) at high
severity compared to unexploded. There was a decrease (P < .0001) for ky, for the
isolated chitin exploded at high severity compared to unexploded isolated chitin.

The escape protein of CM was estimated to be 37.4%. This value was lower
than the value observed for CM (48.4%) in another in situ study conducted
(Chapter V). The higher in situ ruminal degradability of CM N observed in the
present study might be due to better adaptation to the CM diet because of ruminal
microbial adaptation. Steam explosion at both levels of severity did not exert (P >
.05) any effect on ruminal escape value of CP ofCM, whereas there was an
increase (P < .05) in escape of N for explosion at high severity. The difference in
behavior of N of CM and isolated chitin shows that the main structural changes
occurs for acetylated amino group (-NHCOCH;). Focher et al. (1988) reported that
X-ray diffraction of flash treated chitin shows large modification both in
supermolecular structure and in morphology, while NMR analysis did not indicate
any change in primary structure except in the acetyl group content.
In the present study an attempt has been made to fractionate chitin into
three fractions as for DM and CP, soluble fraction A, degradable fraction B and
undegradable fraction C. Chitin content of CM and isolated chitin tested in this

study contained 14.4 and 93.0%, respectively. Chitin content observed in CM

crude protein of isolated chitin by in situ experiment*
Exploded”
Crude protein fractions, %
Soluble A 7.0° 8.7° 3.89 2.56
Degradable B 74.9° 63.34 59.4° 4.40
Undegradable C 18.1° 28.0° 36.8° 2.87
kdb™ 93° 7.0% 4.8° 96
Escape, % CP” 44.7° 55.0% 68.0° 3.57
““Means with in a row lacking a common superscript letter differ (P < .05)
*DM basis
***E scape = ky x B/(ka + kp) + C
Where,
C = fraction C, undegradable (“%)
kg, = rate of degradation
k,,= rate of passage (5%/h)

tested in this study falls in the reported range of 13 to 15% for the crab processing
waste (Ashford et al., 1977; Ayangbile, 1989).
The degradability pattern of chitin in isolated chitin was similar to the

degradability of DM, obviously due to 93% chitin of isolated chitin DM. Ruminal
degradability of chitin of both unexploded and exploded CM and isolated chitin is
presented in Figure 14. For CM, degradability of chitin was lower compared to
that of DM or CP. Steam exploded CM at high severity showed an increase of
degradation at 0 h incubation compared to low severity exploded and unexploded
CM. This trend continued throughout the incubation period until 72 h, where
differences were not significant (P < .05). Chitin degradability of isolated chitin
was higher for low severity at 0, 2, 6, and 12 h incubations. The other differences
were similar to those for DM degradability.
Soluble A fraction of chitin of CM (Table 23) was negligible (0.6%) while
that for isolated chitin was 9.5% (Table 24). Steam explosion of CM at high
severity showed an increase (P < .001) in soluble A fraction of chitin (10.5%),
while CM exploded at low severity did not show any change (P > 05). Degradable
B fraction of chitin was 75% for isolated chitin which was higher than CM
(47.6%). There was a tendency for a small decrease in degradable B fraction of
chitin of CM on steam explosion. A decrease (P < .05) was observed for the B
fraction of chitin on steam explosion, but differences between severities were not
significant. Effect of steam explosion on undegradable fraction C of CM followed

the same trend as that for degradable B fraction. Undegradable C fraction of chitin
in isolated chitin was increased by low and high (25.6 and 34.8%, respectively)
severity steam explosion compared to unexploded isolated chitin (15%). Rate of
degradation of chitin fraction of isolated chitin was found to be more than twice
as that of CM (9.7 and 4.0%, respectively). The effect of steam explosion at high

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chitin of crab meal by in situ experiment*
Exploded”
Chitin fractions, %
Soluble A 0.6° 0.8° 10.5° 1.02
Degradable B 47.6° 44.6° 42.5° 1.92
Undegradable C 51.8° 54.6° 47.0° 2.11
kdb" 4.0° 4.9° 7.0° 1.69
Degradability, % 21.5° 22.3° 34.2° 3.31
25.5 kg/cm? for 3 min 30 sec (high severity)
“¢Means with in a row lacking a common superscript letter differ (P <. 05)
*DM basis
***Degradability = A + (kax B)/(ka + .05)
Where,
.05 = rate of passage (5%/h)

chitin of isolated chitin by in situ experiment*
Exploded”
Chitin fractions, %
Soluble A 9.5° 11.8° 0.8° 3.18
Degradable B 75.0° 62.6° 64.4° 4.60
Undegradable C 15.0° 25.6° 34.8° 2.08
kdb” 9.7° 7.2° 6.7° 9]
Degradability, % 57.7° 46.9°4 36.85 3.50
““*Means with in a row lacking a common superscript letter differ (P < .05)
*DM basis
***Degradability = A + (kax B)/(ka + .05)
Where,
.05 = rate of passage (5%/h)

severity showed different trends for ky, of chitin of CM and isolated chitin, the
former increased while the latter decreased.

Chitin degradability was only 21.5% for the unexploded CM. Steam
explosion of CM at high severity had a degradability of 34.2%, which was higher
(P < .05) than unexploded and exploded at low severity. An opposite trend was
observed for isolated chitin when steam exploded. Chitin degradability was
decreased in steam exploded isolated chitin compared to unexploded (57.7%).
There was a decrease (P < .05) in chitin degradability when isolated chitin was
exploded at low and high severity (46.9 and 36.8%, respectively).
An increase of 60% chitin digestibility was observed for CM when steam
exploded, whereas, degradability of chitin of isolated chitin was lowered due to
steam explosion.. The reason for this diverse result is not clear. Perhaps the higher
degradability of chitin at high severity of steam explosion were due to
morphological modification involving disruption of the structure. These changes
would have resulted in improved enzyme accessibility. Marchessault and St-Pierrie
(1980) reported that the steam explosion treatment process results in a profound
change in enzyme accessibility, compared to the untreated. They suggested that
morphological modification involving disruption into microfibrillar elements of the
cell wall as well as the fiber fracture is responsible for this phenomenon. The
increased enzymatic degradation of cellulose is due to increase in the enzyme
accessible surface due to explosion process. Behavior of chitin in isolated chitin
to steam explosion may partly explained by the chemical and processing changes.
It has been reported that by simply manipulating the chemical and physical
processing a single species source, yielded chitin products of various molecular
weight ranges having significantly different properties (Bough et al., 1978).

Implications
Isolated chitin is well digested (70.9%) in the digestive tract of ruminants

which shows that comparatively poor digestion of chitin in crab meal may not be
due to poor digestibility of chitin but due to high mineralization of the chitin-
protein complex. Although there was an increase of 10% DM digestibility due to
steam explosion, the higher digestibility might be due to higher solubility of Ca.
Furthermore, steam explosion did not improve the escape value of CM protein.
Also there was a loss of over 20% N on steam explosion. The marginal increase in
digestibility of CM may not be justifiable for the loss of N and the extra cost
involved in the process. Therefore, steam explosion does not seem to be a
promising processing method for the better utilization of crab waste for ruminants.
Further studies in intestinal digestion and absorption of nutrients of steam
exploded CM may bring more light to this field.
Literature Cited
AOAC. 1990. Official Methods of Analysis (15" Ed.). Association of Official nalytical
Ashford, N. A., D. Hattis, and A. E. Murray. 1977. Industrial prospects for chitin and
chitosan from prawn shell waste. MIT Sea Grant Report, MIT SG 77-3. MIT
Cambridge.
Bough, W. A., and A. C. Wu. 1977. A study of variables in the chitosan manufacturing
process in relation to molecular weight distribution, chemical characteristics and
waste treatment effectiveness. In: R. A. A. Muzzarelli, and E. R. Pariser (Ed.)

Proceedings of the First International Conference on Chitin/Chitosan . p 88. M.I.T.
Sea Grant Pub. No. MIT-G-78-7.
Brine, C. J., and P. R. Austin. 1981. Chitin isolates: Species variation in residual amino
acid. Comp. Biochem. Physiol. 70:173. In: R. A. A. Muzzarelli, C. Jeuniaux, and
G. W. Goody (Ed.). Chitin in Nature and Technology. p 407. Plenum Press. New
York.
Focher, B., P. L. Beltrame, A. Naggi and G. Torri. 1988. Alkaline N-deacetylation of

chitin enhanced by flash treatment. Structure modification and reaction kinetics
relationship. In: B. Focher, A. Marzetti, V. Crescenzi (Ed.) Steam Explosion
Techniques. Fundamentals and Industrial Applications. p 331. Gordon and Breach
Science Publishers, Philadelphia.
Foody, P. 1982. Steam explosion as a pretreatment for biomass conversion. Final report to
U.S. Department of energy. Contract No. IB-1-9343-1 by Iotech Corp. (Cited by
Donaldson, L. A., K. K. Y. Wong, and K. L. Mackie. 1988. Ultrastructure of
steam-exploded wood. Wood Sci Technol. 22:103).
Grethlein, H. E. and A. O. Converse. 1991. Common aspects of acid prehydrolysis and

steam explosion for pretreating wood. Bioresource Technol. 36:77.
Grethlein, H. E., D. C. Allen, and A. O. Converse. 1984. A comparative study of the

enzymatic hydrolysis of acid pretreated white pine and mixed hard wood.
Biotechnol. Bioeng. 26:1498.
Hackman, R. H. 1984. Cuticle:Biochemistry. In: J. Bereiter-Halm (Ed.) Biology of the

integument, Invertebrates. p 583. Springer - Verlag, Berlin.
Jain, R. K., and W. G. Glasser. 1992. Alternative approaches to isolating biopolymer

fractions from peanut hulls. In: W. G. Glasser (Ed.) Final Report on Value-added
processing of peanut hulls. p 17. Dept. of Wood Science and Forest Products,
Biobased Materials Center, Virginia Polytechnic Institute and State University,
Blacksburg.
Johnson, E. L., and Q. P. Penniston. 1982. Utilization of shellfish waste for chitin and
chitosan production . In: E. Martin, G. J. Flick, C. E. Hebard, and D. R. Ward
(Ed.) Chemistry and Biochemistry of Marine food Products. p 417. AVI
Publishing Co. Westport, Connecticut.
Kamide, K., and K. Okajima. 1987. Cellulose dope, process for preparation and method
for application thereof. U. S. Patent No. 4,634, 470.

Knorr, D. 1991, Recovery and utilization of chitin and chitosan in food processing waste
management. Food Technol. 45:114.
Madhavan. 1992. Chitin, chitosan and their noval application. Popular science lecture
series. Central Institute of Fisheries Technology, ICAR, Kochi, Kerala, India.
Marchessault, R. H. 1988. Steam explosion: A refining process for lignocellulosics. In: B.

Focher, A. Marzetti, V. Crescenzi (Ed.) Steam Explosion Techniques.
Fundamentals and Industrial Applications. p 1. Gordon and Breach Science
Publishers, Philadelphia.
Marchessault, R. H., and St-Pierre. 1980. A new understanding of of the carbohydrate

system. In: L. St. Pierre and G. R. Brown (Ed.) Future Sources of Organic Raw
Material. p 613. Pergamon Press, Elmsford, N.Y.
Mathur, N. K. and C. K. Narang. 1990. Chitin and chitosan, versatile polysaccharides

from marine animals. J. Chem. Education. 67:938.
Morgavi, D. P., M. Sakurada, Y. Tonita, and D. Onodera. 1994. Presence in rumen

bacterial and protozoal populations of enzymes capable of degrading fungal cell
walls. Microbiology. 140:631.
Nocek, J. E. 1988. /# sitt# and other methods to estimate ruminal protein and energy
digestibility: a review. J. Dairy Sci. 71:2051.
Agric. Sci. 92:499,

69:4983.
Cary, NC.

Sciaraffia, F., and A. Marzetti. 1988. Enhancement of wheat straw digestibility by steam
explosion pretreatment. In: B. Focher, A. Marzetti, and V. Crescenzi, (Ed.) Steam
Explosion Techniques. Fundamentals and Industrial applications. p 365. Gordon
and Breach Science publishers. Philadelphia.
forage crops. J. Br. Grassl. Soc. 18:104.
Velez, S. A., J.C. Allen, C. M. Keery, and R. W. Atkinson. 1991. Evaluation of crab and
74:324.

Chapter VI
General Discussion
For rapidly growing animals or high producing dairy cows, the requirement
for metabolizable protein must be met by a combination of microbial protein
synthesized in the rumen and by dietary protein that passes to the small intestine.
In order to increase the quantity of protein that escape ruminal degradation, it is
necessary to use treatments such as physical (heating) or chemical (formaldehyde)
or select feedstuffs which are resistant to microbial degradation, such as corn
gluten meal (CGM), feather meal (FTM), blood meal (BM) and meat and bone
meal. Utilization of these byproducts as a source of escape protein will obviously
reduce the cost of livestock production (Thomas and Beeson, 1977). These
industrial byproducts of both plant and animal origin are limiting in one or other
essential amino acids (AA). Therefore judicial selection of byproducts is needed to
mitigate the deficiencies of individual byproducts as well as microbial protein.
Several studies have been conducted to elicit the complimentary responses by
feeding combinations of the rumen undegradable protein sources such asCGM,
BM and FTM (Stock et al., 1981; Goedeken et al., 1990; Blasi et al., 1991).
Another byproduct of importance is the shell fish waste, a byproduct of the
seafood industry. It has been estimated that approximate global annual production
of crustacean waste (DM basis) is 1.46 Million t and that of US is .1 Million t
(Knorr, 1991). Studies conducted in this laboratory (Ayangbile, 1989; Samuels et
General Discussion 179

al., 1991; 1992; Abazinge et al., 1993; 1994) and elsewhere (Patton et al., 1975;
Velez et al., 1991) have shown that crab processing waste 1s a good protein
supplement for ruminants but lower digestibility has been reported. Lower
digestibility may be attributed to the chitin, an analogue of cellulose, which occurs
as a chitin-protein complex with mineral binding which restrict the accessibility of
enzyme. An attempt was made to explore the feasibility of steam explosion, a
technique of much importance in industries for the fractionation of lignin,
hemicellulose and cellulose.
This study shows that substituting rumen undegradable protein sources for
soybean meal (SBM) did not elicit a profound effect on growth or gain:feed in
growing steers although there was a trend (P < .05) for a positive response for the
supplement based on plant protein and BM and commercial supplement based on
animal protein. Similar results of not having a positive response have been
reported by Adam et al. (1988); Derouen et al. (1988); Coomer et al. (1993), and
Ludden et al. (1995). Feeding low degradable protein to ruminants has often failed
to increase the amount and quality of protein available in the small intestine and
improve animal performance because of inadequate protection of protein from
ruminal degradation (Calsamiglia et al., 1992), decreased microbial protein
synthesis (Windschitl and Stern, 1988), decreased intestinal digestion (Waltz et al.,
1989) and /or AA limitations (Rogers et al., 1989).
In the present study, one of the reasons for not getting a response, perhaps
related to a relatively high escape protein content of SBM. Ludden et al. (1995)
reported that the estimate of ruminal escape of N of SBM was almost twice as that
reported by NRC (1989). Loerch et al. (1983) has reported that a decrease in
ruminal pH associated with feeding diets based on cereal grains may reduce the
protein degradability of SBM. In the study of Ludden et al. (1995), the high level

of corn grain (70%) in the diet would have lowered the pH. In the present study
pH averaged 7.0, although the level of corn grain in the diet averaged 32%. Veen
(1986) suggested that, in general the pH will be higher immediately before
feeding than afterwards, perhaps due to nonavailability of fermentable substrate
remaining in the rumen. Another reason for high pH is due to endogenous
metabolism of bacteria and /or high lysis of bacteria (Wohlt et al., 1976). The time
lag between the sampling of ruminal fluid and the time the steers were fed would
have caused the ruminal pH to be higher in the present study. The ruminal pH of
sheep in the metabolism study was generally lower, 6.4, than the ruminal pH for
SBM, where the level of corn in the diet was only 10%. There are similar reports
of high escape of SBM in low ruminal pH (Loerch et al., 1983; Hussein and
Jordan, 1991).
Ludden et al. (1995) reported 66.0% escape protein for SBM at 6.2 pH.
This shows that the escape protein observed in the in situ study, perhaps would be
a lower than the actual escape in the growth study. The escape of protein of SBM
and and other protein supplements was estimated by dacron bags incubated in
rumen of a steer fed equal proportions of alfalfa and grass hay diet. The probable
reason for the lower escape protein (23.2%) observed in this study compared to
the value (66.1%) reported by Ludden et al. (1995) may be due to the difference in
the ruminal environment attributed to the differences in the diet.
Another reason for not getting a positive response may be due to escape
nature of corn grain protein, zein, which is 58 to 73% undegradable when the diet
contains proportionately high levels of corn (Zinn and Owens, 1983). In the
present study, an average of 32% corn in the diet would have contributed to a
considerable portion of escape protein.

Providing readily available N for the optimum microbial protein synthesis is
an important criterion in the feeding of rumen escape supplements. All the steers
fed different protein supplemented diets had higher levels of ruminal NH; N than
the minimum (5 mg/dL) suggested for the optimum microbial protein synthesis
(Satter and Slyter, 1974). It has been shown that to achieve maximum microbial
activity much higher NH; concentration than required for the maximum microbial
protein production, may be required (Mehrez et al., 1977). Furthermore, there are
reports that some microbes require N in the form of NH; and the feeding of slowly
degraded protein may create an NH; deficiency in the rumen (Klopfenstein et al.,
1976). Therefore, perhaps the performance would have improved if escape protein
supplements were supplemented with soluble N such as urea in the diets.
Steers fed SBM diet would have provided sufficient metabolizable protein
for the optimum feed efficiency for the steers in the present study. The potential of
escape protein supplements has been shown to be highest in young, growing
ruminants, where microbial protein synthesis may not be sufficient for the
optimum requirements for rapid growth (Chalupa, 1975). Oldham and Smith
(1982) suggested that growing cattle weighing more than 200 kg fed diets with
adequate energy intake can meet their protein requirements from the microbial
protein synthesized in the rumen. They concluded that the protein sources that are
rapidly degraded in the rumen will be sufficient to meet the N requirement
through microbial protein synthesis.
Combinations of CGM, BM, and FTM have been found to be more
efficient for growth in steers (Stock et al., 1981; Goedeken, 1990a; Sindt et al.,
1993a; 1993b) than when fed alone. This complementary effect is due to the lysine
content in BM and S AA of CGM. The microbial protein is limiting in methionine
and lysine (Nimrick et al., 1970; Richardson and Hatfield, 1978). A trend for a

positive response in gain:feed for IPA, CS, and ESA may be attributed to the feed
additives included as a premix in the supplement and in the case of IPA and ESA,
there may also have been a complementary effect.
The reason for the poor response in steers fed HESA supplemented diet
compared to other supplements is not known. One reason is perhaps due to low
degradability or low availability of NH; for microbial growth. In the metabolism
studies conducted in sheep, there was a numerical decrease in digestibility of OM
and cellulose and a significant decrease (P < .05) in digestibility of CP, for those
fed the HESA supplemented diet.
The IVDMD of HESA was lower (P < .0001) than for SBM, IPA, CS, and
CM. The overall performance of steers fed HESA supplemented diet in the growth
studies and the various parameters of blood and ruminal fluid of sheep in the
metabolism studies show that lower performance may be due to low digestibility
or low AA profile. Furthermore, incorrect balance of time and temperature during
hydrolysis of FTM will affect the protein digestibility (Morris and Balloun, 1973).
During processing, cystine 1s converted to lanthionine (Robbins et al., 1980)
which will limit the bioavailability of S AA (Robbins et al., 1980) and possibly
other essential AA as well (Papadopoulos et al., 1986). Goedeken et al (1990b)
suggested that protein quality of FTM and BM combination will be reduced if the
BM is added before hydrolysis of FTM due to reduced escape and total tract
protein digestibility. In the present study perhaps the HESA supplement was
processed for too long a period of time which would have caused poor digestibility
of nutrients, especially N.
Conflicting results have been reported for the nutritive value of crab meal
(CM). Improved feed efficiency has been observed in cattle when CM was
included in the diet (Patton et al., 1975; Ayangbile, 1989) whereas negative

responses have been reported (Brundage, 1986; Velez et al., 1991). In the present
study, CM was found to be comparable with SBM in weight gain and gain:feed.
Results in the metabolism study also showed that there were no differences (P >
.05) in digestibilities of nutrients compared to that of SBM. Phosphorus
availability was found to be numerically higher than SBM. The escape protein of
CM was 48.4% when incubated in the rumen of a cannulated steer fed equal
portion of alfalfa and grass hay diet, but, the escape protein of CM was found to
be only 37.4% when incubated after adapting the steer to a CM diet. Bunting et al.
(1988) indicated that ruminal adaptation 1s required to elicit maximum degradation
of crab-craw fish waste CP.
In vitro dry matter digestibility of CM was improved from 65.9 to 75.2% on
steam explosion whereas isolated chitin behaved differently on steam explosion.
Unexploded isolated chitin had 71% digestibility which was reduced on steam
explosion. This shows that chitin is well digested in the digestive tract of
ruminants. A higher IVDMD of steam exploded CM may not be due to increase in
the digestibility of chitin or CP, but may be due to the solubility of the minerals
especially calcium carbonate in the rumen liquor buffer and acid pepsin. Steam
explosion would have caused extensive changes in protein structure in chitin with
heavy mineralization with calcium carbonate.
In situ studies showed that there was no effect on the escape of steam
exploded CM protein . There was a loss of about 20% N on steam explosion.
There was an increase of 60% chitin digestibility i.e., from 21.5 to 34.2%, whereas
chitin content of isolated chitin was decreased on steam explosion. The reason for
this diverse result is not clear, perhaps due to higher degradability of chitin of CM
might be due to morphological modification including disruption of the structures.
Marchessault and St-Pierrie (1980) reported that the steam explosion treatment

results in a profound change in enzyme accessibility compared to the unexploded
material. Decreased chitin degradability of isolated chitin may be due to the result
of the chemical and processing effect of chitin preparation. Bough et al (1978)
indicated that chemical and physical manipulation may change chitin products
with different properties. An increase of 10% ITIVDMD of CM may not be
justifiable for the loss of N and the cost involved in the processing.
In conclusion, substituting SBM with other protein supplements used in this
study did not elicit a significant positive response in weight gain or gain:feed in
growing steers on a high-roughage diet. However, there was a trend for a positive
response for IPA and CS supplements. Crab meal was comparable to SBM or other
commercial products as a protein supplement for steers. Steam explosion
technique does not seem to be a promising processing method for the better
utilization of crab waste for ruminants. Further studies on intestinal digestion and
absorption of nutrients of steam exploded CM in ruminants may be of interest.

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Appendix
213
214
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Appendix Table 3. Effect of steam explosion on degradation of dry matter of crab
meal and isolated chitin by In situ *
Steam Exploded Steam Exploded

Time, h Crab Meal Low High Chitin Low High
%
0 17.2 21.8 24.6 2.4 4.1 2.8
2 34.0 38.7 41.9 4.8 10.0 8.4
6 37.9 40.8 45.0 4.9 10.3 9.0
12 42.8 44.5 46.5 5.6 10.5 9.5
24 56.7 55.3 55.7 46.3 51.5 40.1
48 65.1 63.0 61.2 77.9 65.8 56.8
72 68.1 66.3 63.3 72.1 66.3 64.4
a
Least squares mean, avg. of four values
Appendix 216
Appendix Table 4. Effect of steam explosion on degradation of crude
protein of crab meal and isolated chitin in situ *

%
0 34.0 33.1 41.1 7.0 8.7 3.9
2 52.1 58.4 61.3 9.9 13.3 11.0
6 54.8 62.9 66.0 11.6 15.2 13.7
12 60.5 64.5 69.0 11.6 16.4 15.5
24 72.8 75.8 78.6 49.9 54.4 44.5
48 86.0 84.6 85.9 81.8 72.0 63.3
72 88.7 87.0 88.1 80.1 75.1 72.0
a
Least squares mean, avg. of four values.
Appendix 217
Appendix Table 5. Effect of steam explosion on degradation of chitin of
crab meal and isolated chitin by in situ *

%
0 9 9 10.5 4.5 14.3 8
2 17.8 16.1 25.6 75 16.8 6.4
6 18.6 19.0 26.5 8.7 17.8 7.3
12 21.8 19.7 29.8 8.7 19.0 8.5
24 35.7 34.6 44.7 51.2 58.4 42.0
48 48.2 45.5 53.7 85.3 74.5 62.2
72 54.9 51.1 55.4 81.9 77.3 71.4
a
Least squares mean, avg. of four values
Appendix 218
Vita
T. V. Viswanathan was born in Trichur, Kerala, India on February 5, 1949.

He received his bachelors degree in Veterinary Science in 1971 from Kerala
Veterinary College, Kerala, India. Subsequently, he worked as Veterinary Officer
in a sister organization of Anand Milk Union Limited (AMUL), Gujarat, and as
Veterinary Surgeon in Indo-Swiss Project, Maduppetty, India. He received his
M.V.Sc in Animal Nutrition in 1977. He joined Veterinary Faculty, Kerala
Agricultural University, Mannuthy, Kerala, India in 1977. He entered Virginia
Tech in August, 1992.
219

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EVALUATION OF RUMINAL ESCAPE POTENTIAL OF CRAB

MEAL AND OTHER PROTEIN SUPPLEMENTS AND INFLUENCE

Dissertation submitted to the Faculty of the

Animal and Poultry Sciences

A Dr. D. E. Eversole mafpart 4 Yih

I would like to extend my deepest gratitude and appreciation to Dr. J. P.

ADSEY at oo... cece ccensceeceecesneeeeeesestaeesceseessteeeeceesenaaeeeeeeeesereeetenaneags ii

Dedication ........00..cccccc cc cccccccccessceeeesnececceneeeccseaneceeseneeessesaaeeesesenaaneneoeees vil

Table of Contents 00.00.00... ceceeceeeeeceeneeeeneecenctcnensesseesnsessseeeeeereceseeeeneas Vill

List of Tables 00.0.0... cee ceesecceccceeeseseeessaseaeeeeeseeeeeeeeesteesesteeeseeeeeeeees xii

List Of Figures ...0........ cece ccccceeeceecceceennaaeeeecesceneaneeseseeeaaaeesesaaueesoeeaaess XV

List of Appendix Tables 20.00.0000... cece eeeseceseesnneeceseeesaeeeseseensanaeeeeeees xvii

Chapter I. Introduction ............... ccc cccceecsceeneseeeeaeeaaaeeeeaaeeeeaaenenaneaes ]

Chapter I. Review of Literature.......00.0..000

Evaluation of Protein Supplements ..................:.cccccesesseteteceeeeetees 3

Steam Explosion ...........cccccccccecccsseceessesesesseeeessececessaseseceseeeeseessetess 35

ODjeCHIVES ooo... ccc cccctcscecessseseeeessseeecestaeeceesesstesesesstsateeeeseestsassesesens 39

Chapter III. Journal Paper 1. Effects of Feeding Crab Processing

Table of Contents Vill

ADSUraCt o0.. occ cccccceccececeeencneeeeeentenaeeeeeesecanecesesseeeseecnneeeeeeeeeereees 40

TntrOduction 2.0.0.0... cccccceccccceceeeecnetenceeeeeeceseceeeeeeaaaaaaaeaaaaneaeaaaaeaaaaaas 4]

Experimental Procedures ............ccccccccceceeeseesesenennneneeeececeeeneceeeeeaeanes 43

DISCUSSION ......... ccc ccc ccccceeceeeceseeseseessedeeseeeseteeseteeeeeeseseeseeceeeeeeeeseeenaas 59

Tp lications 20.00.00... eeeeeeccccenceneeeeeceeeeeeeeceeeeteceeseeaeaeaasaeacaesseseseeess 71

Literature Cited ....0..0..ccccccccesscccnceceeceeeeeeeteeeseeetsaeaesassaaeaasasaaaaaas 71

Chapter IV. Journal Paper 2. Effects of Feeding Crab Meal and

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Results o.oo... ccccccccceceeesceesseceeseesesesecseeeedeesetseseeeseescseeeeeeeeeeeenaas 125

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Chapter VI. Journal Paper 4. Effect of Steam Explosion at two levels

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Experimental Procedures .0.........0.cccccccsccessscesseceesseecessseeesseesenaes 152

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Literature Cited ......0.. 0c ccc cccccccccccceccecceccesceccecsecuececcesecencesensusesuess 175

Chapter III (Journal Paper 1)

Composition of protein supplements ..............:::cccccecscceeeees wee eeereneveee

Chemical composition of ingredients used for formulation

Performance of steers fed different protein supplements ... 50

Ruminal pH and blood urea of steers fed different

Ruminal volatile fatty acids of steers fed the different

Ruminal volatile fatty acids of steers fed the different

Chapter IV (Journal Paper 2)

Ingredient composition of experimental diets fed to sheep 84

Chemical composition of experimental diets fed to sheep . eee earewseens 85

10. Apparent digestibility by sheep fed experimental diets ..... 89

11. Nitrogen balance by sheep fed experimental diets ................0.0.....

List of Tables xii

Calcium balance by sheep fed different protem supplements ......... 97

Phosphorus absorption by sheep fed experimental diets ................ 99

14, Ruminal volatile fatty acids of sheep fed the different

15. Blood urea nitrogen, and rumen pH and Ammonia nitrogen

Chapter V (Journal Paper 3)

16. Degradability of dry matter of protein supplements in situ ............ 127

17. Degradability of crude protein of protein supplements in situ ........ 130