Supplementation of guanidinoacetic acid to broiler diets: Effects

on performance, carcass characteristics, meat quality, and energy metabolism

J. Michiels,*†1 L. Maertens,‡ J. Buyse,§ A. Lemme,# M. Rademacher,#

N. A. Dierick,* and S. De Smet*

*Laboratory for Animal Nutrition and Animal Product Quality, Department of Animal Production,
Ghent University, 9090 Melle, Belgium; †Department of Animal Production, Faculty of Biosciences
and Landscape Architecture, University College Ghent, 9000 Ghent, Belgium; ‡Animal Science Unit,
Institute for Agricultural and Fisheries Research, 9090 Melle, Belgium; §Division of Livestock-Nutrition-Quality,
Department of Biosystems, Katholieke Universiteit Leuven, 3001 Leuven, Belgium;
and #Evonik Degussa GmbH, 63457 Hanau-Wolfgang, Germany

ABSTRACT Creatine, (CREA) a central constituent
in energy metabolism, is obtained from dietary animal
protein or de novo synthesis from guanidinoacetic acid
(GAA). Especially in all-vegetable diets, supplemental
CREA or GAA may restore the CREA availability in
tissues, and hence, improve performance. In this study,
768 one-d-old male Ross 308 broilers were assigned to 1
of 4 diets: negative control, all-vegetable corn-soybean-
based; negative control supplemented with either 0.6 or
1.2 g of GAA per kilogram of feed; and positive control
(60, 30, and 30 g/kg of fish meal in the starter, grower,
and finisher diets, respectively). Each treatment was
replicated in 6 pens of 32 birds each. At the end of the
grower period (d 26), 2 birds per pen were euthanized
for metabolic measurements. Four broilers per pen were
selected at slaughter age (d 39) to determine carcass
characteristics and meat quality. Compared with the
negative control, GAA supplementation resulted in an
improved gain:feed ratio (P < 0.05) and ADG (P <
Key words: guanodinoacetic acid, creatine, broiler, metabolism, meat
0.05; + 2.7 and + 2.2% for GAA at 0.6 and 1.2 g/
kg, respectively) throughout the entire period. Breast
meat yield was higher for the GAA diets compared
with that of the negative control birds (P < 0.05; 30.6
vs. 29.4%) and was comparable with that of the posi-
tive control birds (30.2%). With regard to meat qual-
ity, lower ultimate pH values, higher cooking and press
fluid losses, and higher color L* values were observed
for the GAA diets compared with those of the nega-
tive control diet (P < 0.05). These effects were small,
however. The GAA and CREA levels in breast meat
were lower and higher, respectively, in GAA-fed birds
compared with those of the control birds (P < 0.01).
The diets did not affect plasma metabolic traits, except
that plasma insulin-like growth factor I concentrations
were almost twice as high in animals fed 1.2 g/kg of
GAA compared with those of all other treatments. The
GAA included in all-vegetable diets improved animal
performance for the whole rearing period and increased
breast meat yield.
2012 Poultry Science 91:402–412
doi:10.3382/ps.2011-01585

INTRODUCTION is confined to cells that have high but variable energy

Guanidinoacetic acid (GAA), also referred to as gly-
coamine or guanidinoacetate, is a natural precursor of
creatine (CREA) in the vertebrate body. Creatine is
heavily involved in energy metabolism through the cre-
atine and phosphocreatine (PCr) system. The CREA
and PCr system does not occur in all cells; rather, it
©2012 Poultry Science Association Inc.
Received May 3, 2011.
Accepted October 14, 2011.
1 Corresponding author: joris.michiels@hogent.be
demands, particularly muscle cells. This system func-
tions as a backup to the adenosine diphosphate/ade-
nosine triphosphate (ATP)-cycle to store and mobilize
energy when required on short notice. In general, about
1.7% of the CREA and PCr pool is irreversibly con-
verted to creatinine each day and excreted in the urine
(Wyss and Kaddurah-Daouk, 2000). Therefore, CREA
must be continually replaced. The animal’s demand for
CREA can be supplied either directly from animal pro-
tein (e.g., fish or animal by-product meal) in the diet or
by endogenous synthesis. The latter occurs in a 2-step
reaction (Wyss and Kaddurah-Daouk, 2000). Accord-
ing to current evidence, the first step occurs mainly in

402
 GUANIDINOACETIC ACID FOR BROILERS
the kidneys and pancreas whereas the second reaction
takes place in the liver. In the first step, catalyzed by
the enzyme l-arginine:glycine amidinotransferase, l-
arginine reacts with glycine to form l-ornithine and
GAA. Then, GAA is methylated at the amidino group
by S-adenosyl-methionine (SAM) to form CREA in a
reaction that is catalyzed by the enzyme S-adenosyl-
l-methionine:N-guanidinoacetate methyltransferase.
A sodium-linked transporter then transports CREA
from the liver into a variety of tissues against very high
CREA concentration gradients.
The CREA levels were found to be lower in human
vegetarians, who are deprived of exogenous CREA sup-
ply, than in a reference population (Delanghe et al.,
1989; MacCormick et al., 2004). Farm animals fed di-
ets containing reduced amounts of animal protein, or
no animal protein at all, might be deficient in CREA.
Hence, in view of the decreasing amounts of protein
from animal-origin included in animal feeds, particular-
ly in the European Union, supplementation with CREA
or its precursor GAA might restore the CREA load in
tissues. Furthermore, GAA is an immediate precursor
of CREA that requires only a methyl-group transfer
from SAM. Baker (2009) thus hypothesized that di-
etary GAA could spare dietary l-arginine, an essential
amino acid for birds, in the same manner as dietary
CREA. Guanidinoacetic acid might also be favorable
in young fast-growing chicks because of their high need
to supply CREA to growing muscles (Brosnan et al.,
2009) and because the regeneration of ATP from the
CREA and PCr system appears to be of paramount
importance in the cardiac energy management of fast-
growing broilers (Nain et al., 2008).
Creatine as a feed additive shows some drawbacks,
such as instability and high cost, compared with GAA,
which is more stable and less expensive (Baker, 2009).
Guanidinoacetic acid might therefore be more suit-
able for use in animal nutrition. Nevertheless, several
studies including labile methyl-group balance studies
in humans, and estimates of the methylation demand,
indicate that the methylation of GAA to CREA con-
sumes more SAM than all other methylation reactions
combined (Mudd et al., 1980; Stead et al., 2001). It ap-
pears that dietary GAA supplementation considerably
increases the methylation demand (Stead et al., 2006),
which can induce the accumulation of homocysteine in
the blood (Ohuchi et al., 2008; Setoue et al., 2008) or
lead to a deficiency of methionine, and perhaps choline,
folic acid, or vitamin B12 or a combination of these,
depending on the dietary provision of these nutrients.
Creatine supplementation has also been studied in
finisher pigs in relation to their energy status at slaugh-
ter and meat quality (reviewed by James et al., 2002).
In some studies, CREA supplementation delayed the
muscle pH decline postmortem, with a possible positive
effect on water-holding capacity (WHC). In contrast,
Nissen and Young (2006) found a lower pH postmortem
403
and a lighter color of breast meat of Ross 308 broilers
upon CREA and glucose supplementation for 48 h be-
fore slaughter. Stahl et al. (2003) found similar results
when broilers were fed CREA during the entire rearing
period. Therefore, the effects of GAA on meat charac-
teristics should be addressed as well. Recent findings
also highlight the pro-oxidant potential of GAA. Gua-
nidine compounds, like GAA, can induce the forma-
tion of free radicals (review by Hiramatsu, 2003). In
addition, Zugno et al. (2006, 2008) showed that GAA
administration to the rat brain led to a decrease of the
nonenzymatic antioxidant capacity likely due to oxida-
tion of sulfhydryl groups, leading to lower glutathione
levels. These findings warrant further investigation.
Literature contains only limited data on the effects
of CREA or GAA on broiler performance. Halle et al.
(2006) found inconsistent effects on animal performance
and no effect on carcass quality by supplemental CREA
(0.5–10 g/kg), whereas Stahl et al. (2003) found small
but significant improvements in feed conversion after
CREA loading. Ringel et al. (2008a,b) found significant
performance improvements and a higher percentage of
breast meat from dietary GAA supplementation (0.6–
1.2 g/kg). Lemme et al. (2007), feeding diets supple-
mented with 0.6 and 6 g/kg of GAA to colon-fistulated
chickens, found true fecal digestibilities of GAA of 99.4
and 98.9%, respectively. Calculated utilization of di-
gested GAA amounted to 77.1 and 46.4%, respectively.
The aim of the present trial was to investigate how
GAA supplementation in all-vegetable diets affected
animal performance and carcass characteristics, meat
quality, and traits of the energy metabolism, includ-
ing the plasma oxidative status of broilers as compared
with a positive control diet containing fish meal.
MATERIALS AND METHODS
Birds, Diets, and Sampling
The experiment was conducted with the approval of
the Institute for Agricultural and Fisheries Research
Animal Care Protocol Review Committee (Melle, Bel-
gium). One-day-old male broiler (Ross 308; Belgabroed
Hatchery, Merksplas, Belgium) chicks were used in the
trial. Pens contained 32 birds each at a density of 14.3
birds per m2. The floor was covered with wood shav-
ings. Central water heating and infrared bulbs (1/pen)
provided optimal house temperature according to pro-
duction stage. The lighting program was 21L:3D dur-
ing the entire period. The broilers were vaccinated on
their first day of life against Newcastle disease (spray)
and infectious bronchitis (Poulvac IB Primer, Pfizer,
Brussels, Belgium; spray). At 16 d of age, the vaccina-
tion against Newcastle disease was repeated (La Sota,
Clone 30, Intervet, Brussels, Belgium; drinking water).
Paper sheets from the transport hatchery boxes at ar-
rival were examined for Salmonella at the Provincial
404 Michiels et al.
Laboratory of Animal Health (Drongen, Belgium). No
Salmonella was detected. Animals and housing facilities
were inspected twice daily.
The experiment consisted of 4 dietary treatments
replicated in 6 randomized complete blocks with each
replicate (pen) having 32 birds. The dietary treatments
(Table 1) were: 1) negative control (NC), corn-soy-
bean-based; 2) NC supplemented with 0.6 g of GAA
per kg of feed (GAA0.6); 3) NC supplemented with
1.2 g of GAA per kg of feed (GAA1.2); and 4) positive
control (PC), 60.0, 30.0, and 30.0 g/kg of fish meal
at the expense of soybean meal in the starter, grower,
and finisher diets, respectively (rearing periods of 13 d
each). The diets per rearing period were formulated to
be isoenergetic and isonitrogenous according to NRC
requirements (NRC, 1994). Prior to the trial, samples
of stored batches of the main ingredients used (soy-
bean meal 48, fish meal, full-fat soybeans, wheat, and
corn) were analyzed for DM (AOAC, 1990; method
930.15), CP (AOAC, 1990; method 968.06), and amino
acids (Fontaine et al., 2001). These values were intro-
duced in the matrix of a least-cost linear computing
program used to formulate the experimental diets. By
adjusting the PC and NC diets with synthetic amino
acids (lysine, methionine, and threonine), equal dietary
amounts of amino acids were obtained (Table 1). Gua-
nidinoacetic acid was administered by adding the feed
additive CreAMINO (>96% GAA; Evonik Degussa
GmbH, Hanau-Wolfgang, Germany) to the NC diet
by replacing an equal quantity of corn and soybean
meal. Feed as mash and drinking water were provided
ad libitum. Pen weight was recorded at the end of the
starter, grower, and finisher periods (d 13, 26, and 39,
respectively). Feed intake was recorded for each rearing
period: 0 to 13, 14 to 26, and 27 to 39 d. Daily mortal-
ity and cullings were recorded per pen. Corrections for
mortality when calculating zootechnical performances
were done using the number of broiler days (number of
broilers × days alive). Cause of death was determined
by autopsy. Birds that died during the first 3 d were
replaced, and stunted birds were removed at the end of
the starter period.
At the end of the grower period (d 26), 2 birds per
pen with average pen weight were removed for meta-
bolic and oxidative status measurements. Birds were
euthanized by intravenous T61 administration (em-
butramide, 250 mg/mL; mebenzoniumiodide, 50 mg/
mL; tetracaïne hydrochloride, 5 mg/mL; Hoechst,
Brussels, Belgium) and immediately sampled. Blood
samples from the carotid artery were taken in EDTA-
tubes and stored on crushed ice before centrifugation
(2,650 × g for 15 min). Plasma samples were stored at
−20°C until analysis. On d 39, 4 animals per pen with
a weight close to the average weight of the pen were
selected to determine carcass characteristics and breast
meat quality (2 birds each). These birds were tagged,
fasted overnight, weighed, and slaughtered the next day
(d 40). Breast samples for analysis of GAA, CREA,
creatinine, and PCr:ATP were taken immediately af-
ter slaughter, frozen in liquid nitrogen, and stored at
−80°C pending analysis.
Plasma Metabolites and Oxidative
Stress Measures
Plasma metabolites were determined using various
methods. The Idexx VetTest 8008 Chemistry Analyzer
(Idexx Laboratories Inc., Westbrook, ME) with the re-
spective slides (InstruVet nv, Beringen-Paal, Belgium)
was used to quantify glucose, lactate, triglycerides, and
uric acid. Nonesterified fatty acids were determined
with the Wako NEFA-HR(2) ACS-ACOD method ac-
cording to the manufacturer’s guidelines (Sopachem nv,
Eke, Belgium). This method relies upon the acylation
of coenzyme A by fatty acids in the presence of added
acyl-CoA synthetase. The acyl-CoA produced is oxi-
dized by added acyl-CoA oxidase with the generation
of H2O2, which in the presence of peroxidase, permits
the oxidative condensation of 3-methy-N-ethyl-N(β-
hydroxyethyl)-aniline with 4-aminoantipyrine to form
a purple-colored adduct that can be measured spectro-
photometrically at 546 nm. Insulin-like growth factor I
(IGF-I) was measured by radioimmunoassay accord-
ing to the method described by Renaville et al. (1993)
which was validated for avian serum before the experi-
ment. The hormones 3,3′,5-triiodothyronine (T3) and
3,3′,5,5′-tetraiodothryronine (T4) were determined by
competitive radioimmunoassay according to Darras et
al. (1996). In addition, plasma samples were used to
determine traits of oxidative status, including malondi-
aldehyde (MDA) and ferric reducing ability of plasma
(FRAP). The FRAP assay is considered as a measure
of the total antioxidant power, according to the method
described by Benzie and Strain (1996). The MDA con-
centration in plasma was measured using the thiobar-
bituric acid reactive substances (TBARS) method, as
described by Grotto et al. (2007) to assess lipid oxida-
tion.
Analysis of GAA and Energy Compounds
Approximately 10 g of muscle tissue was thoroughly
chopped and transferred into a 250-mL beaker. Then
120 mL of deionized water was added and homogenized
with a rotor-stator mixer. The resulting suspension was
quantitatively transferred into a 500-mL flask and de-
ionized water was added until a total volume of 450
mL. After stirring for 90 min and sonication for 5 min,
deionized water was added until a total volume of 500
mL. Thirty milliliters of the suspension was centrifuged
at 89,000 × g at ambient temperature for 10 min. Then,
an aliquot of the supernatant was filtrated through a
0.45-μm membrane filter and an SPE cartridge. For
determining GAA and creatinine, the filtrate was used
Table 1. Ingredients and composition of experimental diets supplemented with guanidinoacetic acid (GAA)
Experimental diet1

d 0 to 13 d 14 to 26 d 27 to 39

Item NC GAA0.6 GAA1.2 PC NC GAA0.6 GAA1.2 PC NC GAA0.6 GAA1.2 PC

Ingredient, g/kg as-fed                        

 Corn 476.6 476.2 475.8 510.4 559.8 559.4 559 578.1 607.5 607.1 606.7 619.3
  Soybean meal 48 366.4 366.2 366 297.6 285.5 285.3 285.1 250.8 249.7 249.5 249.3 221
  Fish meal — — — 60 — — — 30 — — — 30
  Full-fat soybean 50 50 50 50 50 50 50 50 50 50 50 50
  Animal fat 45.1 45.1 45.1 30.4 43.3 43.3 43.2 35.5 34.4 34.4 34.4 27.7
  Soybean oil 10 10 10 10 10 10 10 10 10 10 10 10
  Dicalcium phosphate 22.3 22.3 22.3 19 25.2 25.2 25.2 21.5 23.8 23.8 23.8 20.2
 Limestone 8.2 8.2 8.2 3.3 5.7 5.7 5.7 4.4 5.5 5.5 5.5 4.2
  Sodium bicarbonate 3.1 3.1 3.1 2.7 2.9 2.9 2.9 2.7 2.1 2.1 2.1 1.8
  Sodium chloride 2.5 2.5 2.5 1.6 2.2 2.2 2.2 1.7 2.2 2.2 2.2 1.8
  l-Lysine-HCl 1.5 1.5 1.5 1.2 1.5 1.5 1.5 1.4 1.4 1.4 1.4 1.1
  dl-Methionine 3.3 3.3 3.3 2.9 2.7 2.7 2.7 2.5 2.5 2.5 2.5 2.3
  l-Threonine 0.6 0.6 0.6 0.5 0.5 0.5 0.5 0.5 0.5 0.5 0.5 0.4
  Ronozyme P 50002 0.2 0.2 0.2 0.2 0.2 0.2 0.2 0.2 0.2 0.2 0.2 0.2
 Clinacox3 0.2 0.2 0.2 0.2 — — — — 0.2 0.2 0.2 0.2
 Sacox4 — — — — 0.5 0.5 0.5 0.5 — — — —
  Trace element/vitamin concentrate5 10 10 10 10 10 10 10 10 10 10 10 10
 GAA6 — 0.6 1.2 — — 0.6 1.2 — — 0.6 1.2 —
Calculated composition, g/kg as-fed                        
 CP 232.3 232.3 232.3 240.3 200.4 200.4 200.4 204.4 187.1 187.1 187.1 193.1
 Ca 10 10 10 10 9.5 9.5 9.5 9.5 9 9 9 9
 Pd 5 5 5 5 4.75 4.75 4.75 4.75 4.5 4.5 4.5 4.5
  ME, MJ/kg 12.9 12.9 12.9 12.9 13.2 13.2 13.2 13.2 13.2 13.2 13.2 13.2
Analyzed composition,7 as-fed                        
  CP, g/kg 234.5 236.3 235.4 240.2 205.3 206.2 207.5 210.4 190.6 192.2 193.6 197.2
  Lysine, g/kg 14.3 14 14 14.4 11.9 11.9 12.1 12.6 11.1 11 10.9 11.3
  Methionine, g/kg 6.6 6.8 6.6 6.8 5.7 5.6 5.6 5.9 5.2 5.3 5.5 5.5
GUANIDINOACETIC ACID FOR BROILERS

  Threonine, g/kg 9.6 9.4 9.5 9.7 8.2 8.2 8.2 8.5 7.7 7.7 7.6 7.8
  Arginine, g/kg 16 15.7 15.7 15.7 13.4 13.2 13.3 13.6 12.3 12.3 12.2 12.5
  Creatine, mg/kg <1 <1 <1 100 <1 <1 <1 46 <1 <1 <1 51
  Creatinine, mg/kg <10 <10 <10 266 <10 <10 <10 120 <10 <10 <10 135
  GAA, mg/kg 1 658 1,240 <1 <1 575 1,196 <1 <1 545 1,190 <1
1NC = negative control; GAA0.6 = negative control supplemented with 0.6 g/kg of GAA; GAA1.2 = negative control supplemented with 1.2 g/kg of GAA; and PC = positive control.
2Ronozyme P 5000, provided 1,000 phytase units per kilogram of diet; DSM Nutritional Products, Deinze, Belgium.
3Clinacox 0.5%, provided 1 mg per kilogram of diet of diclazuryl activity; Janssen Pharmaceutica, Beerse, Belgium.
4Sacox 120, provided 60 mg per kilogram of diet of salinomycin activity; Huvepharma, Antwerp, Belgium.
5Provided (per kilogram of diet): vitamin A (all-trans-retinol), 15,000 IU; vitamin D , 3,000 IU; vitamin E (α-tocopherol), 55 IU; thiamin, 2.0 mg; choline, 600 mg; riboflavin, 5.0 mg; pantothenic
3
acid, 13.5 mg; pyridoxine, 4.0 mg; cyanocobalamin, 0.02 mg; niacin, 15 mg; biotin, 0.20 mg; folic acid, 1.0 mg; I, 1.2 mg; Cu, 20 mg; Se, 0.36 mg; Co, 1.0 mg; Mn, 95.9 mg; Zn, 60 mg; Fe, 49 mg; and
ethoxyquine, 33 mg.
6Guanidinoacetic acid (GAA) was administered by adding the feed additive CreAMINO (>96% GAA; Evonik Degussa GmbH, Hanau-Wolfgang, Germany).
7Values are means of 2 replicates.
405
406 Michiels et al.
directly. For CREA, the filtrate was diluted (1:10,
vol:vol). The GAA and CREA were separated by ion
chromatography (Dionex Series DX500, Dionex Gmbh,
Idstein, Germany) and determined by UV detection at
200 nm (UV/VIS-detector AD25). Samples were inject-
ed with an autoinjecter and the injection volume was
50 μL. The column used was Hypersil Hypercarb 4.6 ×
100 mm, Thermo 35007–104630 (first column, station-
ary phase: porous graphitic carbon, particle size 7 μm)
and Aminopac PA1, Dionex P/N 37022 (second column,
stationary phase: pellicular substrate (2% cross-linked)
agglomerated with a 180-nm MicroBead alkyl quater-
nary ammonium functionalized latex (20% cross-linked,
particle size 10 μm) with pre-column Dionex 37022.
The column temperature was 30°C. Deionized, filtered,
and vacuum-degassed water was used as the mobile
phase at a flow rate of 1.0 mL/min. Quantified CREA
represents both phosphorylated and nonphosphorylated
creatine. Creatinine was separated by HPLC (column:
Nucleosil 5 SA, ET 250/4, Machery-Nagel; stationary
phase: sulfonic acid-modified silica gel, particle size 5
μm) and determined by UV detection at 225 nm (UV/
VIS-detector 151, Gilson). An ammonium dihydrogen
phosphate solution (pH 4.0) was the mobile phase (flow
rate, 1.0 mL/min). Internal standard solutions (GAA,
>99%, Degussa AG; CREA, 99%, Degussa AG; and
creatinine, >99%, Fluka 27910) were prepared to de-
termine response factors. The detection limit for GAA,
CREA, and creatinine in breast meat muscle was 0.4,
22, and 3 mg/kg, respectively. If the compound was not
detected, the detection limit was taken as a set value
for further data processing. The GAA, CREA, and cre-
atinine were quantified in feeds using the same method
following grounding of the feed. The detection limit for
GAA, CREA, and creatinine in feed was 1, 10, and 1
mg/kg, respectively.
The PCr:ATP ratio was determined in a breast meat
sample using 31P nuclear magnetic resonance (NMR)
spectroscopy. Signals arise in the 31P NMR spectrum of
the sample originating from the phosphorous atoms of
ATP and PCr, respectively. The areas of the signals are
directly proportional to the molar concentration of the
analytes. By integrating and comparing the signal ar-
eas, the molar ratio of ATP and PCr can be evaluated.
The samples were prepared and investigated as follows.
About 0.5 to 1.0 g of tissue sample was defrosted by
extraction in a mortar with 1 mL of cold 0.6 mmol/L
perchloric acid for 2 min. The liquid was neutralized
by 1 mL of 1.2 mmol/L Na2CO3 (50% D2O/50% H2O;
pH 7–8), and 0.6 mL of the supernatant solution was
transferred to a 5-mm NMR tube. Standard mixtures of
ATP and PCr were prepared on the basis of stock solu-
tions (0.02 mmol/g) of ATP and PCr in 0.2 mmol/L
carbonate buffer (pH 9.2). The NMR spectrometer
(Bruker Avance 500 MHz, Bruker Biopsin GmbH, Bre-
men, Germany) equipped with a 5-mm broad-band
31P-probe operated at 202.4 MHz for 31P. For each
spectrum, owing to the higher concentrations, only 256
transients were recorded and a relaxation delay of 1 s
with an acquisition time of 0.54 s was used. The areas
of the individual signals were integrated, and signal-to-
noise levels were determined using the NMR Topspin
software version 2.0 from Bruker Biopsin. Phosphoric
acid (85%) was used as an external standard.
Carcass Characteristics and Meat Quality
After chilling for 24 h at 2°C, 2 carcasses per pen
were weighed to determine the slaughter yield (%) and
were cut according to a standardized procedure (Ui-
jttenboogaart and Gerrits, 1982) to determine breast
(without skin), upper legs (thigh), lower legs (tibia and
foot), and wings (expressed as a percentage of carcass
weight). To determine the carcass gross chemical com-
position, all cuts from both birds per pen were pooled.
After freezing, these samples were homogenized in a
cutter. The cutter (Alexanderwerk 10L; Alexanderwerk
AG, Remscheid, Germany) is made out of a horizon-
tal rotating body with a fixed axis fitted with vertical
blades. The samples were processed for approximately
3 min until a homogeneous pasta-like mixture was ob-
tained, then about 300 g was removed and freeze-dried.
Dry matter (ISO, 1973), CP (ISO, 2005), and crude fat
(ISO, 1999) were determined on these samples accord-
ing to standardized ISO methods.
On the carcasses used for meat quality measure-
ments, postmortem pH (t = 0.5, 1, 2, 3, 4, and 24 h),
temperature (°C; t = 2, 3, and 4 h), and conductivity
at 24 h (μS; PQM meter) were determined on the right
side of the breast muscle. Drip loss (%; proportionate
weight loss of a sample hanging in a plastic bag for 48
h at 2°C) and press loss (%; proportionate fluid loss
measured following a modification of the press method
of Grau and Hamm, 1953) were determined the day
after slaughter. After storage at −20°C, thaw loss (%;
proportionate weight loss of a sample before frozen
vacuum storage at −20°C and after overnight thawing
at 4°C) and cooking loss (%; proportionate weight loss
of a sample after cooking in an open plastic bag in a
water bath at 70°C for 40 min followed by cooling in
cold-running tap water for 15 min) were assessed. The
same sample was used for assessing thaw loss and cook-
ing loss. Shear force (N) was determined on cylindrical
cores (diameter, 1.27 cm), taken from cooked samples
by perpendicular shearing to the longitudinal orienta-
tion of the muscle fibers using a Lloyd TA 500 Texture
Analyzer (Lloyd Instruments, Meerbusch, Germany).
Color L*, a*, and b* values were measured on breast
muscle samples 1 d postmortem with a HunterLab
Miniscan XE plus spectrocolorimeter (light source D65,
standard observer 10°, 45°/0° geometry, 1-inch light
surface, white standard) after 30 min blooming. Color
and lipid stability were evaluated after display under
fluorescent light (1,000 lx) for 9 d at 4°C on samples
that had been previously frozen and thawed. Samples
were wrapped in oxygen-permeable polyethylene film.
 GUANIDINOACETIC ACID FOR BROILERS
Color stability was measured by the increase in the
percentage of metmyoglobin calculated from reflectance
values (HunterLab Miniscan XE plus spectrocolorim-
eter, Hunter Associates Laboratory Inc., Reston, VA)
at specific wavelengths according to Krzywicki (1979).
Lipid oxidation was assessed by measuring TBARS us-
ing the distillation method described by Tarladgis et
al. (1960), and it was expressed as micrograms of MDA
per gram of meat.
Statistical Analysis
Data of animal performance (n = 6 per treatment;
pen as the experimental unit), carcass gross chemical
composition (n = 6; on pooled carcasses per pen), car-
cass characteristics, and meat and metabolic traits (n
= 12; animal as the experimental unit) were analyzed
by a linear model with the fixed effect of treatment. If
this analysis indicated significant (P < 0.05) differences
among treatments, the treatment means were compared
using the post hoc Tukey test. Orthogonal contrasts
were applied to explore the effect of treatments.
RESULTS
Diets and Bird Performance
The analyzed GAA concentration in the GAA diets
was close to the intended dose, whereas the control di-
ets contained trace amounts of GAA (≤1 mg/kg; Table
1). The all-vegetable feeds (NC and GAA) were free of
CREA (<10 mg/kg), in contrast to the PC diets that
contained fish meal. The final BW and the ADG in the
starter period of the birds fed with the PC feed were
higher (P < 0.01) than the final BW and the ADG of
the birds fed on the diets with the GAA supplements
and the NC diet (Table 2). Animals fed the PC diet
also showed a higher ADFI compared with the other
treatments. The 2 test feeds (GAA0.6 and GAA1.2)
showed comparable results. In the grower period, ADG
and ADFI were not affected by dietary treatment. Com-
pared with the NC diet, both GAA diets resulted in a
higher BW at d 26 (+ 1.5 and 2.1%, respectively), but
it was still lower than that of animals fed the PC diet.
The final weight of the birds of the control treatment
NC was lower than the final weight of the birds fed
the PC diet (P < 0.05). The gain:feed ratio was higher
for the GAA1.2 compared with the GAA0.6 diet (P <
0.05). At d 39, birds on the GAA diets reached a com-
parable weight to the PC birds due to a slightly higher
growth rate in the finisher period. The final weight of
GAA-fed broilers was higher than that of the NC birds
(P < 0.05). Regarding the overall results (d 0 to 39),
the ADG of GAA-fed birds was 1.8 g/day (GAA0.6)
and 1.5 g/day (GAA1.2) higher than those of the birds
fed the NC diet (P < 0.05) and nonsignificantly differ-
ent from those of the birds fed the PC diet (Table 2).
407
The gain:feed ratio was most favorable in both GAA
diets but only significantly higher than that of the NC
birds (orthogonal contrast I, P < 0.05). Mortality was
low (1.0 to 3.1%). In the finisher period, PC birds had
a higher mortality than GAA-fed birds (P < 0.05).
Plasma Metabolites, Oxidative Stress
Measures, and Energy Compounds
A limited number of plasma metabolites of 26-d old
broilers showed differences between treatments (Table
3). The IGF-I levels in GAA1.2-fed animals were mark-
edly elevated compared with those of the other birds
(P < 0.01). Compared with those from birds in the
GAA treatments, the NC treatment resulted in chick-
ens having higher FRAP values (P < 0.05). Breast
meat of birds supplemented with GAA had reduced
levels of GAA and increased levels of CREA compared
with those of both NC and PC birds (P < 0.001). The
PCr:ATP ratio was significantly higher for birds on the
GAA1.2 diet compared with that of the NC diet (P <
0.05), but it was not different from that of the other
diets. In addition, the orthogonal contrast of GAA-sup-
plemented diets vs. NC was significant for this variable
(P < 0.05). Across diets, breast meat GAA content
was negatively correlated with the CREA content (P
< 0.01; Table 4). The PCr:ATP ratio was positively
related to the CREA content (P < 0.01). However, a
higher breast meat CREA content also resulted in el-
evated creatinine levels (P < 0.01).
Carcass Characteristics and Meat Quality
Dietary supplementation with GAA resulted in a
higher percentage of breast meat in the carcass com-
pared with that from birds of the NC diet (P < 0.05)
and was comparable with that of birds fed the PC diet
(Table 5). The absolute breast yield was higher in the
GAA1.2 diet compared with that from birds in the NC
diet (P < 0.05), whereas the absolute weight of the
lower leg was higher than that in the PC birds (P <
0.05; data not shown). Neither moisture and CP nor
crude fat content of the carcass were affected by the
treatments (Table 5).
The GAA supplementation had only a minor effect
on meat quality (Table 5). The pH values were slightly
lower for the GAA diets compared with those of the
NC (P < 0.05 at 4 h postmortem and P < 0.01 at 24 h
postmortem) and PC treatments. Both press loss (P <
0.01) and cooking loss (P < 0.05) were higher for the
GAA treatments compared with those of the NC treat-
ment but were not different than those of the PC treat-
ment. The color L* value was higher (i.e., paler meat)
for the GAA treatments compared with that of the
NC treatment (P < 0.05). Shear force, metmyoglobin
formation, and lipid stability (MDA) were not affected
by the treatments.
408 Michiels et al.
Table 2. Effect of guanidinoacetic acid (GAA) supplementation on BW, ADG, ADFI, G:F, and mortality in male broilers
Experimental diet1 Significance2

Item NC GAA0.6 GAA1.2 PC SEM I II

d 0 to 13              
  ADG, g/d 28.6b 29.0b 29.1b 31.9a 0.38 0.334 <0.001
  ADFI, g/d 37.6b 39.3ab 39.2ab 41.1a 0.52 0.019 0.009
 G:F 0.76 0.74 0.74 0.78 0.013 0.143 0.012
  Mortality, % 0.5 1.6 1.6 1.0 0.65 0.204 0.519
d 14 to 26              
  ADG, g/d 66.9 68.0 68.5 68.6 0.70 0.139 0.706
  ADFI, g/d 103.6 105.9 103.3 106.3 1.06 0.450 0.209
 G:F 0.65ab 0.65b 0.66a 0.65ab 0.009 0.287 0.249
  Mortality, % 0.5 1.1 0.0 0.5 0.50 0.989 0.989
d 0 to 26              
  ADG, g/d 47.7b 48.5ab 48.8ab 50.2a 0.47 0.140 0.013
  ADFI, g/d 70.5b 72.3ab 71.0b 73.5a 0.61 0.147 0.023
 G:F 0.68 0.67 0.69 0.68 0.010 0.777 0.434
  Mortality, % 1.0 2.6 1.6 1.6 0.76 0.278 0.582
d 27 to 39              
  ADG, g/d 104.1 107.9 106.1 104.1 1.45 0.111 0.120
  ADFI, g/d 191.3 193.4 189.4 190.5 1.17 0.950 0.554
 G:F 0.54 0.56 0.56 0.55 0.011 0.027 0.060
  Mortality, % 0.0 0.0 0.0 1.7 0.58 — 0.025
d 0 to 39              
  Final BW, g 2,639 2,707 2,696 2,706 21.4 0.026 0.865
  ADG, g/d 66.4 68.2 67.9 68.2 0.55 0.026 0.865
  ADFI, g/d 108.9 110.3 108.6 110.5 0.59 0.472 0.145
 G:F 0.61 0.62 0.63 0.62 0.009 0.029 0.315
  Mortality, % 1.0 2.6 1.6 3.1 0.98 0.393 0.394
a,bValues are means of 6 replicates. Values with different superscripts within a row are significantly different at P < 0.05.
1NC = negative control; GAA0.6 = negative control supplemented with 0.6 g/kg of GAA; GAA1.2 = negative control supplemented with 1.2 g/kg
of GAA; and PC = positive control.
2I = orthogonal contrast of GAA-supplemented diets vs. NC; II = orthogonal contrast of GAA-supplemented diets vs. PC.

Table 3. Effect of guanidinoacetic acid (GAA) supplementation on energy metabolites and oxidative status in 26-d-old male broilers
Experimental diet2 Significance3

Item1 NC GAA0.6 GAA1.2 PC SEM I II

Plasma              
  T3 (ng/mL) 0.60 0.63 0.60 0.56 0.062 0.840 0.464
  T4 (ng/mL) 6.90 6.86 7.00 9.10 0.890 0.991 0.067
  Glucose (mmol/L) 12.3 12.2 12.7 12.0 0.35 0.757 0.271
  Lactate (mmol/L) 4.05 3.51 3.74 4.15 0.242 0.180 0.098
  Triglycerides (mmol/L) 0.53 0.36 0.54 0.38 0.079 0.401 0.526
  NEFA (mmol/L) 0.47 0.44 0.38 0.44 0.042 0.324 0.578
  Uric acid (μmol/L) 214 198 158 183 23.2 0.236 0.864
  IGF-I (ng/mL) 27.7b 22.3b 44.6a 25.9b 3.07 0.146 0.061
  MDA (μmol/L) 22.1 17.1 23.5 22.6 2.00 0.494 0.384
  FRAP (μmol Fe2+/L) 0.84 0.53 0.67 0.63 0.084 0.027 0.785
Breast meat              
  GAA (mg/kg of FM4) 8.16a 2.19b 1.40b 5.92a 0.767 <0.001 <0.001
  Creatine (mg/kg of FM) 4,789b 5,322a 5,541a 4,940b 90.0 <0.001 <0.001
  Creatinine (mg/kg of FM) 5.45 5.99 6.07 5.70 0.241 0.056 0.271
 PCr:ATP 2.36b 2.65ab 2.97a 2.60ab 0.160 0.027 0.294
a,bValues are means of 12 replicates. Values with different superscripts within a row are significantly different at P < 0.05.
1T3 = triiodothyronine, T4 = tetraiodothyronine; NEFA = nonesterified fatty acid; IGF-I = insulin-like growth factor I; MDA = malondialdehyde;
FRAP = ferric reducing ability of plasma; and PCr:ATP = phosphocreatine:adenosine triphosphate.
2NC = negative control; GAA0.6 = negative control supplemented with 0.6 g/kg of GAA; GAA1.2 = negative control supplemented with 1.2 g/kg
of GAA; and PC = positive control.
3I = orthogonal contrast of GAA-supplemented diets vs. NC; II = orthogonal contrast of GAA-supplemented diets vs. PC.
4FM = fresh matter.
 GUANIDINOACETIC ACID FOR BROILERS 409
Table 4. Pearson correlation coefficients between metabolic breast traits in male broilers at the end
of the grower period (d 26)1
Item GAA Creatine Creatinine PCr:ATP

GAA   −0.740** −0.198 −0.283

Creatine     0.408** 0.537**
Creatinine       0.170
1GAA = guanidinoacetic acid; PCr:ATP = phosphocreatine:adenosine triphosphate.
**P < 0.01 for Pearson correlation coefficients that are significantly different from zero.

DISCUSSION
Creatine Loading and Effects on Bird
Performance, Carcass Characteristics,
and Meat Quality
Our hypothesis was that dietary GAA could restore
the CREA load in tissues of birds fed all-vegetable diets
and accordingly improve animal performance. Indeed,
the CREA concentration in breast muscle was enhanced
by 11.1 and 15.7% when fed 0.6 and 1.2 g/kg of GAA,
respectively, compared with that of birds fed an all-
vegetable diet. Guanidinoacetic acid is effectively con-
verted into CREA. When considering the entire rear-
ing period, birds fed GAA performed better than birds
fed the all-vegetable diet, mainly because of a better
gain:feed ratio in the finisher period. Related to the PC
treatment, GAA-supplemented birds displayed equal
or better performances only in the finisher period. It
seems that GAA supplementation is most beneficial in
the finisher period, when growth rates are the highest.
Nevertheless, this finding supports the hypothesis that
dietary GAA restores the CREA load in tissues of birds
fed purely vegetable diets and accordingly improves cell
energy management and animal performance. Overall,
feed conversion was most favorable in both GAA diets,
which is consistent with Ringel et al. (2008a,b) and
trials reported in the EFSA opinion on GAA (EFSA,

Table 5. Effect of guanidinoacetic acid (GAA) supplementation on carcass and meat characteristics in male broilers at slaughter (d
39)
Experimental diet1 Significance2

Item NC GAA0.6 GAA1.2 PC SEM I II

Carcass composit3              
  Yield, % 73.1 72.9 73.7 73.2 0.25 0.489 0.813
  Breast meat, % 29.4 30.4 30.7 30.2 0.45 0.033 0.557
  Upper leg, % 26.0 25.7 25.8 25.9 0.29 0.384 0.648
  Lower leg, % 13.3 13.0 13.2 12.6 0.19 0.498 0.039
  Wing, % 10.3 10.4 10.2 10.4 0.12 0.926 0.545
Carcass analysis              
  Moisture, % 66.1 66.7 66.8 65.9 0.34 0.130 0.065
  CP, % 18.5 18.6 18.5 18.6 0.11 0.573 0.710
  Crude fat, % 12.8 12.1 12.3 13.0 0.40 0.195 0.114
Breast meat              
  pH, 0.5 h 6.42 6.31 6.39 6.45 0.054 0.301 0.141
  pH, 24 h 5.88 5.76 5.74 5.81 0.038 0.009 0.192
  T, 2 h, °C 11.1 10.7 11.1 9.1 0.56 0.771 0.010
  T, 3 h, °C 5.9 6.1 6.0 4.7 0.45 0.792 0.014
  T, 4 h, °C 2.9a 2.3ab 2.5ab 1.7b 0.29 0.207 0.054
  Conductivity, μS 7.81 9.49 8.74 8.98 0.586 0.075 0.843
  Drip loss, % 0.8 1.0 1.1 0.9 0.09 0.054 0.188
  Press loss, % 18.3b 20.2ab 21.2a 19.3ab 0.70 0.007 0.108
  Thaw loss, % 3.5 4.4 4.4 4.4 0.49 0.143 0.998
  Cooking loss, % 13.2 14.6 15.4 14.2 0.61 0.022 0.320
  Shear force, N 7.5 8.1 8.6 8.5 0.48 0.150 0.868
 Color              
  L* 51.1 53.0 53.5 52.5 0.66 0.011 0.401
  a* 6.1 6.3 6.9 6.4 0.29 0.178 0.587
  b* 12.4 13.6 14.0 13.3 0.44 0.012 0.355
 Metmyoglobin              
   ∆d 0–9, % 12.0 12.1 12.2 12.5 0.50 0.839 0.613
  Malondialdehyde, μg/g 1.46 1.50 1.38 1.51 0.090 0.920 0.049
a,bValues are means for 6 replicates for carcass analysis and 12 for other variables. Values with different superscripts within a row are significantly
different at P < 0.05.
1NC = negative control; GAA0.6 = negative control supplemented with 0.6 g/kg of GAA; GAA1.2 = negative control supplemented with 1.2 g/kg
of GAA; and PC = positive control.
2I = orthogonal contrast of GAA-supplemented diets vs. NC; II = orthogonal contrast of GAA-supplemented diets vs. PC.
3Carcass yield as a percentage of BW and carcass components expressed as a percentage of carcass weight.
410 Michiels et al.
2009). The PC treatment enhanced growth compared
with the NC diet. Differences in CREA anabolism
can hardly be responsible for this because the dietary
CREA supply through the PC diet was negligible. The
CREA levels in the fish meal and concomitantly PC
diets (Table 1) were unexpectedly low, whereas the cre-
atinine content exceeded that of CREA. The CREA
content in fish meal is heavily variable and transforma-
tion to creatinine during storage and processing can
occur to a considerable extent, as the current trial illus-
trates. One may therefore assume that a higher amino
acid (e.g., arginine) provision by the PC diet must have
caused the growth promotion. Profiles of analyzed es-
sential amino acids (Table 1) were similar for all diets,
but total CP content was consistently highest for the
PC diet in all rearing periods.
Ringel et al. (2008a) also addressed the question of
breast meat as a percentage of carcass weight. They
found that this percentage linearly increased with GAA
inclusion level. In the present study, the absolute breast
yield was significantly higher in the GAA1.2 and the
positive control diets compared with that of the NC
treatment. Creatine supplementation in human sports
nutrition is known to increase the lean body mass, par-
ticularly in men, and has been ascribed mainly to an
increase in water accretion rather than protein accre-
tion (review by Williams and Branch, 1998). Our data
do not suggest a similar effect. Effects of GAA feeding
on meat quality were similar or slightly different from
previous reports either supplementing CREA (Stahl et
al., 2003) or GAA (Ringel et al., 2008b). Ringel et al.
(2008b) found no effects on meat quality except that
redness (a*) appeared to be lower in GAA-fed broil-
ers. In the current trial, particularly lightness (L*) and
yellowness (b*) were ultimately higher. Breast muscle
of broilers contains primarily fast-twitch (type IIB)
muscle fibers prone to rapid glycolytic metabolism. In
these fibers, large quantities of lactic acid are generated
postmortem, which may result in a rapid reduction in
muscle pH and a decreased WHC, which ultimately
decreases the retail value of fresh poultry. Several stud-
ies have focused on the ability of CREA to influence
pork and poultry meat quality (e.g., Stahl et al., 2003).
Here, marginally lower pH values postmortem and low-
er WHC due to higher drip, higher press, and greater
cooking loss may be considered for the GAA treatments
when compared with those of the NC treatment. Thus,
CREA loading resulted in the opposite effects than ex-
pected. These effects are small, however, and are of no
concern regarding retail value.
Intermediate Metabolism
and Oxidative Status
Current data on GAA, CREA, and creatinine con-
tent and the PCr:ATP ratio in breast meat of birds
fed GAA within the dose-range tested show the same
trend as seen in previous trials (EFSA, 2009). Supple-
menting GAA to all-vegetable diets results in a dose-
related increase of CREA concentrations in breast meat
and enhanced PCr:ATP ratios. It should be noted that
CREA concentrations in breast meat represent both
phosphorylated and nonphosphorylated creatine in our
study. Guanidinoacetic acid feeding caused a strong de-
crease in GAA content. The Pearson correlation coef-
ficients shown in Table 4 confirm these relationships in
the current trial. Because the conversion of GAA into
CREA is irreversible, GAA in muscle tissue must have
originated through the circulatory system. Surprising-
ly, in GAA-supplemented birds, the GAA content in
breast meat is much lower than in nonsupplemented
birds. This has 2 possible explanations. First, several
studies indicate that CREA downregulates the renal
and pancreatic l-arginine:glycine amidinotransferase
expression by a feedback mechanism (e.g., Edison et
al., 2007), which might result in lower circulatory levels
of GAA. Second, limitations in the GAA bioconversion
upon an increased GAA availability from a dietary ori-
gin may induce an effective elimination through urine
and ultimately result in lower deposition in breast
meat. Whether this excreted GAA stems from endog-
enous synthesis or has a dietary origin remains unclear.
Treatment comparisons do not indicate that increased
CREA in breast meat leads to higher creatinine con-
tents, although the positive Pearson correlation coef-
ficient between these 2 variables indicates the opposite.
EFSA (2009) confirms that there is a GAA dose-related
rise in creatinine content in breast meat that becomes
apparent at levels above 1.5 g of GAA per kilogram of
feed. Nonetheless, the most interesting feature is that
PCr:ATP ratios in GAA birds were the highest, + 12.3
and + 25.9%, compared with those of the NC treatment
birds for GAA0.6 and GAA1.2, respectively. This indi-
cates that the buffering capacity for ATP hydrolysis by
PCr is increased and supports the idea that creatine-
loaded muscles have the capacity for increased growth
or work, which might be beneficial for both skeletal
muscle growth and for contractile activity of supply
organs, such as heart. Another benefit might arise from
the fact that intramuscular PCr can attract water into
the muscle cell and increase the cell volume (Hultman
et al., 1996). Häussinger (1996) found that a superhy-
drated muscle may trigger protein synthesis, minimize
protein breakdown, and increase glycogen synthesis, as
partly illustrated by Young et al. (2007). The GAA1.2-
supplemented birds showed remarkably elevated IGF-I
circulatory concentrations, which might support muscle
growth as well. This corroborates recent evidence show-
ing that the anabolic effects of CREA supplementation
in humans might also be mediated by upregulation of
muscle IGF-I expression (Burke et al., 2008; Deldicque
et al., 2005). Remarkably, in the current trial, only the
GAA1.2 treatment yielded higher IGF-I levels, whereas
in both GAA0.6- and GAA1.2-treated birds, CREA was
significantly increased. Opposite to this substantial in-
crease in circulatory IGF-I levels, the increase in growth
 GUANIDINOACETIC ACID FOR BROILERS
performance was not very marked. Spencer et al. (1991)
demonstrated that the autocrine/paracrine action of
IGF-I for muscle growth was more important than the
role of circulatory IGF-I. Other explanations are that
excess IGF-I may compromise glucose homeostasis or
may decrease growth hormone release due to inhibitory
feedback (Scanes, 2009); though our results on plasma
metabolites do not substantiate the former. According
to literature, the effect of IGF-I treatment on growth
in chickens remains equivocal (Scanes, 2009). Hence,
it is not surprising that the increase in growth perfor-
mance was limited. Other metabolic traits showed only
numerical differences. The FRAP values, correspond-
ing to the total antioxidant capacity of plasma, were
reduced by GAA feeding. It might reflect a decrease
of glutathione levels, as shown by Zugno et al. (2006,
2008). Implications for animal health and performance
are to be established.
Conclusions
Guanidinoacetic acid supplementation in the diet
(0.6 and 1.2 g/kg) markedly increased the CREA con-
centration in breast meat of 26-d-old broilers. The data
suggest that supplementing GAA in all-vegetable di-
ets improves performance and carcass characteristics
in terms of the gain:feed ratio and the breast meat
yield; however, it slightly reduced water-holding capac-
ity, although this did not differ from that of the positive
control. With the current trial design, effects were most
pronounced in the finisher period. The PCr:ATP ratios
in the breast meat of GAA-fed birds were markedly
increased compared with those of the NC-treatment
birds. This indicates that the buffering capacity for
ATP hydrolysis by PCr is increased and supports the
idea that creatine-loaded muscles have the capacity for
increased growth or work, which might be beneficial for
both skeletal muscle growth and for contractile activ-
ity of supply organs, such as the heart. Elevated IGF-I
circulatory concentrations, specifically for the GAA1.2-
treated birds, may also be involved in the anabolic ef-
fects.
ACKNOWLEDGMENTS
The authors are grateful to E. Turtelboom (Ghent
University, Melle, Belgium), S. Lescouhier (Ghent Uni-
versity), A. Vermeulen (Institute for Agricultural and
Fisheries Research, Melle, Belgium), and M. Laseure
(University College Ghent, Belgium) for their excellent
and skillful technical support. We acknowledge the as-
sistance of M. Levenson (Institute for Agricultural and
Fisheries Research, Merelbeke, Belgium) in revising the
language of the manuscript.
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