Ó 2022 The Authors. Published by Elsevier Inc. on behalf of Poultry Science Association Inc.

This is an open access article under the CC BY-

NC-ND license (http://creativecommons.org/licenses/by-nc-nd/4.0/).

Titration of dietary isoleucine and evaluation of

branched-chain amino acid levels in female Cobb
500 broilers during a 22- to 42-day finisher period
C. W. Maynard,* G. J. Mullenix,* C. J. Maynard,* S. C. Wells-Crafton,* J. T. Lee,y
S. K. Rao,z L. D. Butler,z S. K. Orlowski,* and M. T. Kidd*,1
*
Center of Excellence for Poultry Science, University of Arkansas, Fayetteville, AR, 72701,
USA; yCJ America-Bio, Downers Grove, IL, 60515, USA; and zCobb-Vantress, Inc., Siloam
Springs, AR, 72701, USA

Primary Audience: Nutritionists and Researchers

SUMMARY
Recent research into the branched-chain amino acids has indicated that broiler responses to
dietary isoleucine may vary from classic data. Current isoleucine data has focused on male
broilers leaving a gap in the literature for female data. An experiment was conducted to deter-
mine the isoleucine requirements for a 22 to 42 day finisher period in Cobb MV £ 500 female
broilers. A basal diet was formulated to contain dietary isoleucine, valine, and leucine to
lysine ratios of 56, 78, and 135, respectively, and graded amounts of L-isoleucine were added
to achieve isoleucine to lysine ratios of 56, 59, 62, 65, 68, 71, and 74. An additional external
control diet was formulated with isoleucine, valine, and leucine to lysine ratios of 65, 72, and
135, respectively, to which aliquots of L-isoleucine, L-valine, and L-leucine were added to
create diets containing isoleucine, valine, and leucine ratios ranging from 65 to 71, 72 to 81,
and 135 to 175, respectively. Digestible lysine was maintained at 1.10 percent. Linear
responses were observed for day 42 BW, BW gain, feed conversion, fat pad yield, and breast
yield. No other significant responses were observed for the isoleucine titrations. Comparison
among the external controls generated a significant decrease in fat pad yield when all 3
branched-chain amino acids were increased compared to birds fed the 65 isoleucine to lysine
diet. The linear response observed for both feed conversion and breast meat yield indicate that
optimal isoleucine levels may be beyond the 74 isoleucine to lysine ratio tested in this study.

Key words: female broilers, breast yield, branched-chain amino acids, isoleucine
2022 J. Appl. Poult. Res. 31:100245
https://doi.org/10.1016/j.japr.2022.100245

DESCRIPTION OF PROBLEM genetic progress and determine if current rear-

Several methods can be employed in the
determination of amino acid requirements, but
the core experimental design is a dose-response
study (Lewis, 1992). Dose-response studies are
continually conducted in order to account for
1
Corresponding author. mkidd@uark.edu
ing practices (i.e., sex separate) influence
optimal requirement recommendations
(Lewis, 1992). Recent studies investigating iso-
leucine requirements have been conducted
using male Ross broilers and have left a void in
the literature concerning Cobb requirements
(Brown, 2021; Duarte et al., 2015;
Schedle et al., 2019; Wise et al., 2021). These
2 JAPR: Research Report
research papers, albeit conducted with Ross
broilers, indicate that the isoleucine require-
ments for Cobb broilers may exceed isoleucine
recommendations currently suggested by the
primary breeder for Cobb 500 broilers (Cobb-
Vantress 2018). Additionally, published isoleu-
cine requirements for female broilers are lim-
ited to two papers (Hale et al., 2004; Hale et al.,
2004) and are dated, having been published
over a decade ago.
Classic experiments investigating the
branched-chain amino acids have reported that
the requirements of isoleucine can be shifted
when excess leucine and valine are fed
(D’Mello and Lewis, 1970; Farran and
Thomas, 1990). Recent evaluation of the
branched-chain amino acids has indicated that
performance and carcass trait responses to indi-
vidual branched-chain amino acid titrations
may not be reflective of what is observed when
all three are manipulated (Kidd et al., 2021;
Maynard et al., 2021). These observations indi-
cate that formulating to multiple requirements
in order to allow for an optimal outcome in
commercial practice may not result in an addi-
tive response, but inadvertently reduce perfor-
mance. Maynard et al. (2021) found that
increasing isoleucine to lysine ratios from 66 to
70 in a diet containing a valine to lysine ratio of
78, resulted in an approximate one percentage
point decrease in carcass and total breast (Pec-
toralis major + P. minor) yield. Furthermore, a
meta-analysis by Maynard et al. (2021)
reported a negative quadratic response of breast
meat yield when isoleucine was increased from
60 to 70 isoleucine to lysine at a leucine level
below 140 leucine to lysine, whereas a positive
linear response was observed for the same iso-
leucine range at a leucine level above 140 leu-
cine to lysine.
Despite this apparent intertwined nature of the
branched-chain amino acids, determination of
isoleucine requirements through single amino
acid titration studies allow for a starting point for
use in larger factorial type or central composite
studies and indicate the sensitivity of broilers to
the tested amino acid. Therefore, a study was
conducted to determine the optimal isoleucine to
lysine ratios for performance and carcass traits
for female Cobb MV £ 500 broilers. Addition-
ally, external controls containing varying
branched-chain amino acid supplementation
were fed to determine if responses shift when the
levels of dietary branched-chain amino acids are
altered.
MATERIALS AND METHODS
All procedures utilized in the present study
were approved by the Institutional Animal Care
and Use Committee of the University of Arkan-
sas (Fayetteville, AR; Protocol number 21124).
Bird Husbandry
A total of 1,152 female Cobb 500 broiler
chicks were obtained from a commercial hatch-
ery (Siloam Springs, AR) where they received
Marek’s vaccinations at day of hatch and were
transported to the University of Arkansas
Broiler Research Farm. Upon arrival, chicks
were placed into 96 pens at 12 birds per pen.
Each pen was equipped with a hanging feeder,
section of continuous nipple drinker line (5 nip-
ples per pen), and built-up, top-dressed litter
composed of pine shavings. A common starter
diet was offered as crumbles from day 0 to 10,
and a common grower was offered as pellets
from day 11 to 21. Initial temperature set points
were set 32.2˚C and gradually reduced to
18.3˚C at the conclusion of the experiment.
Lighting schedules were set at 24L:0D for day
0, 23L:1D from day 1 to 6, and 18L:6D from
day 7 to 42 with light intensities initially set at
5 foot candle for day of placement, 2.5 foot can-
dle from day 1 to 6, 2.0 foot candle from day 7
to 13, 1.5 foot candle from day 14 to 20, 1.0
foot candle from day 21 to 27, and 0.5 foot can-
dle from 28 to 42. Water flow rates were set at
21 mL per minute and increased by 7 mL per
minute every week ending at 56 mL per minute.
Light intensities were verified at bird level
using a light meter (LT300, Extech Instruments,
Waltham, MA) and water flow rates were veri-
fied using the equipment and methods estab-
lished by Miles et al. (2004).
Experimental Diets
Prior to formulation, samples of all intact pro-
tein containing ingredients were submitted for
MAYNARD ET AL: ISOLEUCINE TITRATION 3

Table 1. Analyzed crude protein and amino acid contents of feed ingredients used in experimental diets.
Soybean Meat and Blood
Item, % as-is Corn1 meal1 DDGS2 bone meal1 meal1
Crude protein 7.25 48.06 28.44 58.19 92.44
Arginine 0.38 3.23 1.11 4.18 7.97
Cystine 0.18 0.74 0.45 0.72 0.51
Glycine 0.31 1.95 1.13 7.06 4.62
Histidine 0.20 1.18 0.74 1.29 7.32
Isoleucine 0.30 2.33 1.04 2.06 0.21
Leucine 0.83 3.88 3.30 4.21 13.06
Lysine 0.26 2.77 1.01 3.43 8.36
Methionine 0.22 0.72 0.55 1.10 0.62
Phenylalanine 0.32 2.28 1.38 1.83 6.45
Proline 0.57 2.02 2.44 4.06 3.35
Serine 0.36 2.48 1.31 2.73 4.19
Threonine 0.28 1.91 1.14 2.23 2.89
Tryptophan 0.07 0.49 0.25 0.35 1.64
Tyrosine 0.32 1.89 0.90 1.68 1.97
Valine 0.41 2.38 1.42 2.73 8.02
1
Analyzed by Novus Analytical Services (St. Charles, MO).
2
Dried distiller’s grains with solubles. Analyzed by Eurofins Scientific Inc. (Des Moines, IA).

total amino acid analysis (Table 1) (Eurofins Sci-
entific Inc., Des Moines, IA; Novus Analytical
Services, St. Charles, MO; method 994.12;
982.30; 988.15; (AOAC 2006)). An isoleucine
deficient diet containing corn, soybean meal, and
dried distiller’s grains with solubles was formu-
lated to 56, 78, and 135 isoleucine, valine, and
leucine to lysine, respectively. The common iso-
leucine deficient diet was batched and mixed
with graded amounts of L-isoleucine to achieve
isoleucine to lysine ratios of 56, 59, 62, 65, 68,
71, and 74 (Table 2). A second basal diet was for-
mulated in order to evaluate the interactive
effects of the branched-chain amino acids. Diet
ingredients were maintained from the titration
diets to limit the effects of the using two basal
diets. Isoleucine, valine, and leucine to lysine
ratios were formulated to 65, 72, and 135, respec-
tively. Aliquots of L-isoleucine, -valine, and -leu-
cine were added to create five external control
(C) diets which resulted in isoleucine, valine, and
leucine to lysine ratios of: 65, 72, and 135 (C1);
65, 78, and 175 (C2); 71, 78, and 175 (C3); 65,
82, and 175 (C4); and 71, 82, and 175 (C5),
respectively (Table 3). The aforementioned
external controls were included to test the effects
of practical adjustments in isoleucine and valine
when dietary leucine levels were in excess in
accordance with Maynard (2021). Additions of
all amino acids were added at the expense of
washed builder’s sand.
All diets were mixed in a vertical screw mixer,
pelleted at 65.5˚C, and bagged. Representative
samples were collected during bagging and sub-
mitted for analysis (Eurofins Scientific Inc., Des
Moines, IA). The common diets were analyzed
for total amino acids (method 982.30; 988.15;
994.12; (AOAC 2006)), titration diets analyzed
for free isoleucine (method 999.13;
(AOAC, 2006)), and controls analyzed for free
isoleucine, valine, and leucine (method 999.13
(AOAC, 2006)).
Measurements
Live Performance. Initial and ending pen
weights were recorded for the experimental
period. Feed intake was recorded for the experi-
mental period. Mortality was collected twice
daily, and weights were recorded. Individual
BW gain was recorded by subtracting initial
from final pen weights and dividing by number
of birds. Feed conversion ratio, representing g
of feed intake to g of BW gain, was calculated
by dividing pen feed intake by the summation
of pen BW gain and recorded mortality weight
for each pen. Feed intake was adjusted for mor-
tality using bird days as outlined by
Greenwood et al. (2004).
Carcass Traits. Following the final pen
weigh, 4 birds per pen were randomly selected
and tagged for determination of carcass traits.
4 JAPR: Research Report

Table 2. Composition of isoleucine titration diets fed to Cobb MV £ 500 females from 22 to 42 days post-hatch.
Item, % as-fed 56 59 62 65 68 71 74
Corn 67.38 67.38 67.38 67.38 67.38 67.38 67.38
DDGS1 15.00 15.00 15.00 15.00 15.00 15.00 15.00
Soybean meal 10.21 10.21 10.21 10.21 10.21 10.21 10.21
Meat and bone meal 2.00 2.00 2.00 2.00 2.00 2.00 2.00
Limestone 1.14 1.14 1.14 1.14 1.14 1.14 1.14
Blood cells 0.76 0.76 0.76 0.76 0.76 0.76 0.76
Filler2 0.25 0.22 0.18 0.15 0.12 0.08 0.05
L-lysine-HCl 0.59 0.59 0.59 0.59 0.59 0.59 0.59
Poultry fat 0.50 0.50 0.50 0.50 0.50 0.50 0.50
Sodium bicarbonate 0.18 0.18 0.18 0.18 0.18 0.18 0.18
L-arginine 0.35 0.35 0.35 0.35 0.35 0.35 0.35
Dicalcium phosphate 0.34 0.34 0.34 0.34 0.34 0.34 0.34
DL-methionine 0.29 0.29 0.29 0.29 0.29 0.29 0.29
L-threonine 0.22 0.22 0.22 0.22 0.22 0.22 0.22
L-isoleucine 0.05 0.08 0.11 0.15 0.18 0.21 0.25
Mineral premix3 0.08 0.08 0.08 0.08 0.08 0.08 0.08
L-valine 0.14 0.14 0.14 0.14 0.14 0.14 0.14
Potassium carbonate 0.21 0.21 0.21 0.21 0.21 0.21 0.21
Choline chloride, 60% 0.05 0.05 0.05 0.05 0.05 0.05 0.05
Vitamin premix4 0.05 0.05 0.05 0.05 0.05 0.05 0.05
L-tryptophan 0.05 0.05 0.05 0.05 0.05 0.05 0.05
Phytase5 0.01 0.01 0.01 0.01 0.01 0.01 0.01
Salt 0.16 0.16 0.16 0.16 0.16 0.16 0.16
Calculated composition, % unless noted otherwise
AME, kcal/kg 3,085 3,085 3,085 3,085 3,085 3,085 3,085
CP 17.92 17.92 17.92 17.92 17.92 17.92 17.92
Ca 0.84 0.84 0.84 0.84 0.84 0.84 0.84
Available P 0.41 0.41 0.41 0.41 0.41 0.41 0.41
Na 0.17 0.17 0.17 0.17 0.17 0.17 0.17
Cl 0.31 0.31 0.31 0.31 0.31 0.31 0.31
K 0.73 0.73 0.73 0.73 0.73 0.73 0.73
Digestible lysine 1.10 1.10 1.10 1.10 1.10 1.10 1.10
Digestible methionine 0.59 0.59 0.59 0.59 0.59 0.59 0.59
Digestible TSAA 0.84 0.84 0.84 0.84 0.84 0.84 0.84
Digestible threonine 0.74 0.74 0.74 0.74 0.74 0.74 0.74
Digestible valine 0.86 0.86 0.86 0.86 0.86 0.86 0.86
Digestible isoleucine 0.62 0.65 0.69 0.72 0.75 0.79 0.82
Digestible leucine 1.49 1.49 1.49 1.49 1.49 1.49 1.49
Digestible arginine 1.14 1.14 1.14 1.14 1.14 1.14 1.14
Analyzed composition6, %
Free isoleucine 0.04 0.07 0.10 0.12 0.16 0.20 0.29
1
Dried distiller’s grains with soluble.
2
Washed builder’s sand.
3
The mineral premix contributed (per kg of diet): manganese, 180.0 mg; zinc, 108.0 mg; copper, 5.1 mg; iodide, 3.5 mg; sele-
nium, 0.30 mg.
4
The vitamin premix contributed (per kg of diet): vitamin A, 15,432 IU; vitamin D3, 11,023 ICU; vitamin E, 110 IU; niacin,
77.16 mg; d-pantothenic acid, 19.84 mg; riboflavin, 13.23 mg; pyridoxine, 5.51 mg; thiamine, 3.09 mg; menadione, 3.00 mg;
folic acid, 1.76 mg; biotin, 0.17 mg; vitamin B12, 0.03 mg.
5
Supplied 200 FTU per kg of complete diet
6
Basal diet found to contain total amino acid levels of (% as-is): lysine, 1.31; methionine, 0.56; TSAA, 0.83; threonine, 0.78;
valine, 0.97; isoleucine, 0.64; arginine, 1.18; tryptophan, 0.23; leucine, 1.65; histidine, 0.49; phenylalanine, 0.80; tyrosine,
0.47; glycine, 0.79; serine, 0.80; proline, 1.19.
MAYNARD ET AL: ISOLEUCINE TITRATION 5

Table 3. Composition of external control diets (C) fed to Cobb MV £ 500 females from 22 to 42 days posthatch con-
taining various branched-chain amino acid supplementation.
Item, % as-fed C1 C2 C3 C4 C5
Corn 67.55 67.55 67.55 67.55 67.55
DDGS1 15.00 15.00 15.00 15.00 15.00
Soybean meal 9.43 9.43 9.43 9.43 9.43
Meat and bone meal 2.00 2.00 2.00 2.00 2.00
Limestone 1.15 1.15 1.15 1.15 1.15
Blood cells 0.96 0.96 0.96 0.96 0.96
Filler2 0.65 0.14 0.07 0.09 0.03
L-lysine-HCl 0.60 0.60 0.60 0.60 0.60
Poultry fat 0.50 0.50 0.50 0.50 0.50
Sodium bicarbonate 0.41 0.41 0.41 0.41 0.41
L-arginine 0.37 0.37 0.37 0.37 0.37
Dicalcium phosphate 0.34 0.34 0.34 0.34 0.34
DL-methionine 0.30 0.30 0.30 0.30 0.30
L-threonine 0.23 0.23 0.23 0.23 0.23
L-isoleucine 0.16 0.16 0.23 0.16 0.23
Mineral premix3 0.08 0.08 0.08 0.08 0.08
L-valine 0.07 0.14 0.14 0.19 0.19
Potassium carbonate 0.05 0.05 0.05 0.05 0.05
Choline chloride, 60% 0.05 0.05 0.05 0.05 0.05
Vitamin premix4 0.05 0.05 0.05 0.05 0.05
L-tryptophan 0.05 0.05 0.05 0.05 0.05
Phytase5 0.01 0.01 0.01 0.01 0.01
L-leucine 0.00 0.44 0.44 0.44 0.44
Calculated composition, % unless noted otherwise
AME, kcal/kg 3,085 3,085 3,085 3,085 3,085
CP 17.80 17.80 17.80 17.80 17.80
Ca 0.84 0.84 0.84 0.84 0.84
Available P 0.41 0.41 0.41 0.41 0.41
Na 0.17 0.17 0.17 0.17 0.17
Cl 0.21 0.21 0.21 0.21 0.21
K 0.63 0.63 0.63 0.63 0.63
Digestible lysine 1.10 1.10 1.10 1.10 1.10
Digestible methionine 0.60 0.60 0.60 0.60 0.60
Digestible TSAA 0.84 0.84 0.84 0.84 0.84
Digestible threonine 0.74 0.74 0.74 0.74 0.74
Digestible valine 0.79 0.86 0.86 0.90 0.90
Digestible isoleucine 0.72 0.72 0.79 0.72 0.79
Digestible leucine 1.49 1.93 1.93 1.93 1.93
Digestible arginine 1.14 1.14 1.14 1.14 1.14
Analyzed composition6, %
Free valine 0.07 0.13 0.13 0.17 0.17
Free isoleucine 0.12 0.13 0.18 0.13 0.19
Free leucine 0.00 0.36 0.39 0.38 0.38
1
Dried distiller’s grains with solubles
2
Washed builder’s sand
3
The mineral premix contributed (per kg of diet): manganese, 180.0 mg; zinc, 108.0 mg; copper, 5.1 mg; iodide, 3.5 mg; sele-
nium, 0.30 mg.
4
The vitamin premix contributed (per kg of diet): vitamin A, 15,432 IU; vitamin D3, 11,023 ICU; vitamin E, 110 IU; niacin,
77.16 mg; d-pantothenic acid, 19.84 mg; riboflavin, 13.23 mg; pyridoxine, 5.51 mg; thiamine, 3.09 mg; menadione, 3.00 mg;
folic acid, 1.76 mg; biotin, 0.17 mg; vitamin B12, 0.03 mg.
5
Supplied 200 FTU per kg of complete diet
6
Basal diet found to contain total amino acid levels of (% as-is): lysine, 1.26; methionine, 0.54; TSAA, 0.80; threonine, 0.76;
valine, 0.90; isoleucine, 0.69; arginine, 1.11; tryptophan, 0.22; leucine, 1.63; histidine, 0.47; phenylalanine, 0.78; tyrosine,
0.45; glycine, 0.76; serine, 0.77; proline, 1.17.
6 JAPR: Research Report
On day 43, 384 broilers were transported to the
University of Arkansas Pilot Processing Plant
following an overnight feed withdrawal (10 h).
Broilers were individually weighed, hung on
shackles, electrically stunned, and exsangui-
nated via a jugular vein cut with a knife. Fol-
lowing a 2 min bleed, broilers were scalded
(58.3˚C) and defeathered (Gent-L-Flex, Food-
craft, New Holland, PA) for 30 s. Heads, necks,
and feed were removed. Carcasses were rehung
and eviscerated on an automatic evisceration
machine (Baader Linco 1396, BAADER
LINCO, Inc., Kansas City, KS). Fat pads were
collected according to Waldroup et al. (1990).
Hot carcass and fat pad weights were recorded,
and carcasses were submerged in plastic con-
tainers filled with ice water for a 4-h chill. Car-
casses were then deboned on a debone line to
obtain parts weights, which included: breast
(Pectoralis major), tenders (P. minor), wings,
and legs (bone-in skin-on drumstick and thigh).
Carcass and part yields were calculated using
the weight of various cuts divided by day 43
fasted live BW taken immediately prior to
slaughter.
Following deboning, breast fillets were evalu-
ated for the incidence and severity of woody
breast. A single individual scored breast fillets via
tactile evaluation on a scale of 0 to 2. Breast fillets
with a score of 0 exhibited no hardness in the cau-
dle region of the breast fillet, breast fillets with a
score of 1 exhibited hardness in the cranial and
caudle region of the breast fillet but remained
flexible in the mid region of the fillet, and breast
fillets with a score of 2 exhibited stiffness
throughout the fillet including the mid region.
Statistics
Pen was considered the experimental unit,
and treatments were assigned to pens in a
randomized complete block design with pen
location serving as the blocking factor. All
treatments were represented by eight repli-
cate pens of 12 birds. Titration data were
analyzed by the PROC GLIMMIX procedure
of SAS 9.4 (SAS Institute, 2012) to assess
the effects of dietary isoleucine. Titration
responses were assessed for linear and qua-
dratic effects. Preplanned contrasts were con-
ducted between the 64 isoleucine to lysine
titration diet and the external control diets
using the PROC GLIMMIX procedure of
SAS 9.4 (SAS Institute, 2012). Statistical
significance was considered at P ≤ 0.05.
RESULTS AND DISCUSSION
Analyzed total amino acid levels for the
titration and external control basal diets were in
close agreement with calculated total levels
(total levels not shown). While analyzed free
levels of the branched-chain amino acids were
lower than formulated values, trends in increas-
ing inclusion agreed with formulated values
(Tables 2 and 3). Average weights at d 42 were
2.538 kg with an average mortality of 4.51%
for the experimental period (i.e., 22−42 d).
Mortality was not influenced by dietary treat-
ment (P > 0.05; data not shown).
At the beginning of the experimental period,
initial BW exceeded target weights by 6% with
a flock CV of 2.97 (Cobb-Vantress, 2018). The
final BW of the broilers fed the 71 and 74 iso-
leucine to lysine titration diet (i.e., 2 highest
isoleucine levels) were slightly below target
BW published by the primary breeder (Cobb-
Vantress, 2018). These observations align with
the linear responses of 42 d BW and BW gain
reported herein, indicating that that isoleucine
requirements may be beyond the levels tested
in the current study. Likewise, the lack of a
response in feed intake but a significant
response in feed conversion indicated that
increasing isoleucine level primarily led to an
increased utilization of ingested feed as
opposed to a direct effect on feed intake as is
seen with valine (Maynard et al., 2021).
No quadratic responses (P > 0.05) were
observed for any recorded measurement for live
performance, carcass traits, or woody breast.
Live performance responses are presented in
Table 4. Day 42 BW (P = 0.037) and BW gain
(P = 0.044) linearly increased as isoleucine to
lysine levels increased from 56 to 74. Similarly,
feed conversion linearly decreased (P = 0.001)
as dietary isoleucine increased. Increasing die-
tary isoleucine had no influence (P > 0.05) on
feed intake. Carcass characteristics and woody
breast data are displayed in Tables 5 and 6. No
measured breast fillets exhibited a woody breast
MAYNARD ET AL: ISOLEUCINE TITRATION 7

Table 4. Influence of digestible isoleucine and branched-chain amino acid level on the live performance of Cobb
MV £ 500 female broilers.
Treatment1
Feed
Ile Val Leu BW, kg BW gain, kg Feed intake, kg conversion, g:g
Titration2
T1 56 2.487 1.422 3.092 2.221
T2 59 2.539 1.487 3.189 2.149
T3 62 2.569 1.520 3.165 2.126
T4 65 2.595 1.504 3.130 2.125
T5 68 2.545 1.498 3.099 2.115
T6 71 2.641 1.568 3.226 2.088
T7 74 2.612 1.544 3.095 2.033
Controls3
C1 65 72 135 2.544 1.500 3.150 2.153
C2 65 78 175 2.531 1.454 3.069 2.164
C3 71 78 175 2.424 1.405 3.012 2.158
C4 65 81 175 2.447 1.422 3.023 2.188
C5 71 81 175 2.495 1.436 3.035 2.122
SEM 0.0692 0.0667 0.0814 0.0468
P-value
Titration
Linear 0.037 0.044 0.999 0.001
Quadratic 0.636 0.443 0.525 0.544
Controls
T4 vs. C1 0.825 0.975 0.873 0.714
T4 vs. C2 0.781 0.630 0.682 0.411
T4 vs. C3 0.252 0.335 0.296 0.682
T4 vs. C4 0.362 0.385 0.323 0.379
T4 vs. C5 0.422 0.458 0.442 0.952
1
Values shown represent isoleucine (Ile), valine (Val), and leucine (Leu) to lysine ratio.
2
T = titration diet
3
C = control diet

score of 2, and therefore that category was
removed from Table 6. Differences in carcass
traits were limited to fat pad (P = 0.048) and
breast yield (P = 0.002). Both measurements lin-
early improved, resulting in an approximate
9.4% reduction in fat pad yield and a 7.0%
increase in breast yield when isoleucine to lysine
ratios were increased from 56 to 74. No differen-
ces in woody breast were observed (P > 0.05),
and overall incidence was low with average
scores ranging from 0.000 to 0.125.
Breast yield has been shown to be sensitive
to dietary isoleucine levels (Hale et al., 2004;
Kidd et al., 2004; Mejia et al., 2011) and the lin-
ear increases in breast yield followed the trends
in live performance. The lack of linear
decreases in parts beyond the breast indicate
the specificity of the effects of isoleucine, in
that the primary response is to stimulate P.
major growth and subsequently carcass yield.
Furthermore, the linear reduction in fat pad
yield coincide with the increase in feed utiliza-
tion and its conversion to lean muscle.
Only one study has been published evaluat-
ing the isoleucine requirements of female
broilers for growth performance and carcass
traits (Hale et al., 2004). Hale et al. (2004)
observed significant quadratic effects on all live
performance measurements as well as total
breast, wings, and drumsticks. Based on these
responses, Hale et al. (2004) reported require-
ment estimates of 60 to 65 isoleucine to lysine.
These requirement values were lower than the
68 isoleucine to lysine requirement derived
from male studies conducted from 2000 to
2020 summarized by Maynard et al. (2021).
These results indicated that female require-
ments for growth were likely lower than males
but recently published data indicates that the
requirement for Ross 708 male ranges between
66 and 73 for a similar growout age to those
tested herein (Brown et al., 2021; Wise et al.,
8 JAPR: Research Report

Table 5. Influence of digestible isoleucine and branched-chain amino acid level on the carcass and part yields of
Cobb MV £ 500 female broilers processed on day 43.1
Treatment2
Ile Val Leu Carcass, % Fat pad, % Breast, % Tender, % Wing, % Leg, %
Titration3
T1 56 74.34 2.02 18.91 4.07 8.05 22.92
T2 59 74.47 2.09 19.14 4.13 7.93 22.89
T3 62 74.35 2.04 19.41 4.20 7.94 22.59
T4 65 74.61 2.01 19.48 4.25 7.97 22.77
T5 68 74.40 1.90 19.24 4.24 8.05 22.75
T6 71 74.82 2.03 20.38 4.30 7.92 22.27
T7 74 75.00 1.83 20.24 4.19 7.95 22.56
Controls4
C1 65 72 135 74.55 2.04 19.42 4.13 7.92 22.70
C2 65 78 175 74.81 2.03 20.13 4.14 8.02 22.34
C3 71 78 175 75.06 2.01 19.78 4.30 8.10 22.43
C4 65 81 175 74.72 2.02 19.40 4.26 7.94 22.66
C5 71 81 175 74.95 1.78 19.56 4.20 8.07 22.83
SEM 0.266 0.077 0.380 0.074 0.080 0.234
P-value
Titration
Linear 0.061 0.048 0.002 0.059 0.577 0.067
Quadratic 0.410 0.349 0.601 0.112 0.869 0.878
Controls
T4 vs. C1 0.884 0.767 0.899 0.079 0.668 0.843
T4 vs. C2 0.693 0.826 0.170 0.073 0.726 0.234
T4 vs. C3 0.351 0.989 0.673 0.573 0.327 0.378
T4 vs. C4 0.790 0.885 0.866 0.945 0.759 0.787
T4 vs. C5 0.440 0.020 0.861 0.511 0.385 0.832
1
Average BW (kg) for treatment groups taken immediately prior to slaughter were: T1, 2.524; T2, 2.570; T3, 2.570; T4,
2.621; T5, 2.508; T6, 2.665; T7, 2.601; C1, 2.567; C2, 2.611; C3, 2.516; C4, 2.575; C5, 2.544.
2
Values shown represent isoleucine (Ile), valine (Val), and leucine (Leu) to lysine ratio.
3
T = titration diet.
4
C = control diet.

2021). The perplexing contradiction of the cur-
rent data indicates that females may now have a
higher requirement than males. Similarly,
Kidd et al. (2021) found that female Indian
River broilers were more responsive to
branched-chain amino acids than their male
counterparts.
Varying branched-chain amino acid sup-
plementation in the external controls resulted
in minimal differences in measured response
variables when compared with the 65 isoleu-
cine to lysine titration diet. An approximate
11% decrease in fat pad yield was observed
when broilers were fed the external control
diet containing increased supplementation of
all three branched-chain amino acids when
compared with broilers fed the 65 isoleucine
to lysine titration diet. No other significant
differences were observed as a result of
varying branched-chain amino acid supple-
mentation.
The external controls were implemented to
determine if varying branched-chain amino
acid levels would result in different responses
to those observed in the titration, similar to the
external control used by Maynard et al. (2021)
when evaluating valine. The C1 diet imple-
mented a lower valine level to test the hypothe-
sis of Maynard et al. (2020) that the valine
requirement is lower when leucine levels are
reduced. No differences were observed as a
result of this reduction in dietary valine, but
this observation would be disingenuous to
make without noting that the only difference in
responses observed when broilers were fed the
external controls was the reduction in fat pad
yield in broilers fed increased levels of all 3
branched-chain amino acids. Previous research
MAYNARD ET AL: ISOLEUCINE TITRATION 9

Table 6. Influence of digestible isoleucine and range of isoleucine and valine levels will result
branched-chain amino acid level on the incidence and in an “optimal response” or slightly reduce per-
severity of woody breast present in Cobb MV £ 500
female broilers processed on day 43. formance in most cases (i.e., approximate 50 g
Treatment1 Distribution2, %
reduction in BW gain or 2 point increase in
feed conversion). These wide ranges of accept-
Ile Val Leu Average Score 0 Score 1 able levels of isoleucine and valine, which
3
Titration show that performance can be supported
T1 56 0.031 96.88 3.13 with traditionally “unbalanced” supplementa-
T2 59 0.094 90.63 9.38
T3 62 0.000 100.00 0.00
tion of isoleucine and valine, are perplexing
T4 65 0.031 96.88 3.13 as they disagree with classic research by
T5 68 0.031 96.88 3.13 Mendonca and Jensen (1989) but have been
T6 71 0.125 87.50 12.50 previously observed in modern broilers by
T7 74 0.094 90.63 9.38 Maynard et al. (2021).
Controls4
C1 65 72 135 0.063 93.75 6.25
C2 65 78 175 0.063 93.75 6.25
C3 71 78 175 0.156 84.38 15.63 CONCLUSIONS AND
C4 65 81 175 0.031 96.88 3.13 APPLICATIONS
C5 71 81 175 0.000 100.00 0.00
SEM 0.0511 5.114 5.114
1. A dietary isoleucine to lysine ratio above 74
P-value
Titration may be required in order to maximize live
Linear 0.269 0.269 0.269 performance and breast yield in female
Quadratic 0.355 0.355 0.355 Cobb 500 broilers.
Controls 2. Branched-chain amino acid antagonism
T4 vs. C1 0.554 0.554 0.554
poses a complex problem that may require
T4 vs. C2 0.554 0.554 0.554
T4 vs. C3 0.273 0.273 0.273 large scale factorial studies to quantify
T4 vs. C4 1.000 1.000 1.000 responses as opposed to simple testing of
T4 vs. C5 0.334 0.334 0.334 diets containing various supplementation to
1
Values shown represent isoleucine (Ile), valine (Val), and develop formulation strategies.
leucine (Leu) to lysine ratio. 3. Further determination of female require-
2
Breast fillets with a score of 0 exhibited no hardness in the ments at various growout ages is needed in
caudal region, whereas fillets with a score of 1 exhibited order to assess differences between the sexes
hardness in the cranial and caudal regions but remained due to differences in growth patterns.
flexible in the middle.
3
T = titration diet.
4
C = control diet
DISCLOSURES
All authors declare that they have no conflict
in female broilers has shown that fatty acid syn- of interest.
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