Leg health and growth performance of broiler chickens
supplemented with grape seed extract
Salud de piernas y comportamiento productivo de pollos suplementados con extracto de semilla de uva
Saúde das pernas e comportamento produtivo de frangos suplementados com extrato de semente de uva
Leodan T Rodríguez-Ortega1 ; Alejandro Rodríguez-Ortega1 ; Arturo Pro-Martínez2 ; Eliseo Sosa-Montes3 ;
Filogonio J Hernández-Guzmán1 ;Hector Leyva-Jimenez4* .
1Universidad Politécnica de Francisco I. Madero, Tepatepec, Hidalgo, km 2 carretera Tepatepec-San Juan Tepa, México.
2Colegio de Postgraduados, Campus Montecillo, km 36.5 carretera México-Texcoco, Montecillo, Texcoco, Estado de México.
3Universidad Autónoma Chapingo, Departamento de Zootecnia, km 38.5 carretera México-Texcoco, Chapingo, Estado de México.
4United Animal Health, 322 S Main St., Sheridan, Indiana, United States.
To cite this article:
Rodríguez-Ortega LT, Rodríguez-Ortega A, Pro-Martínez A, Sosa-Montes E, Hernández-Guzmán FJ, Leyva-Jimenez H. Leg
health and growth performance of broiler chickens supplemented with grape seed extract. Rev Colomb Cienc Pecu 2024;
37(2):61–72. https://doi.org/10.17533/udea.rccp.v37n2a1
Abstract
Background: Active bone remodeling processes can be altered by the presence of reactive oxygen species and, therefore,
cause leg problems in broiler chickens. The antioxidant activity present in grape seed extract (GSE) could be a viable alternative
to high inclusion levels of vitamin E (VE) as a nutritional strategy to improve the antioxidant capacity of birds and, thus,
prevent leg abnormalities. Objective: To evaluate the effect of partial substitution of VE with grape GSE on leg health and
performance of broiler chickens. Methods: Four hundred and twenty newly-hatched Ross 308 male broiler chickens were
distributed into three treatments: 1) Control-AL, a diet containing 40 IU/kg of VE and fed ad libitum (CAL); 2) Control-FR,
the CAL diet but offered through a feed restriction program (CFR); and 3) grape seed extract-AL, a diet containing 10 mg/kg
of GSE + 10 IU/kg of VE fed ad libitum (GSE-AL). The trial was conducted for 47 days. Feed intake and body weight of the
chickens were recorded weekly to evaluate performance. At day 43, gait score (GS), valgus/varus angulation (AngV), foot burn
(FB), and hock burn (HB) lesions were evaluated. At day 47, tibia-breaking strength (TBS) and gastrocnemius tendon-breaking
strength (GTBS) were evaluated. Results: Birds in the GSE-AL treatment showed reduced (p≤0.05) GS compared to the other
treatments. No statistical differences (p>0.05) were found in AngV, HB, FB, TBS, TGBS, and growth performance among
treatments. Conclusions: These results suggest that GSE may partially replace VE in broiler diets without negative effects on
growth performance or leg health. Further research is required to evaluate the potential of grape seed extract to replace VE or
other ingredients with antioxidant activity under different rearing conditions and feeding programs.
Keywords: breaking strength; broiler; gait score; gastrocnemius; grape seed extract; leg health, nutrient restriction;
performance; tendon; tibia; vitamin E.
Received: January 22, 2023. Accepted: August 29, 2023
*Corresponding author. 4310 State Road 38 West, Sheridan, IN, US, 46069. Email: ing.leyva531@gmail.com
This work is licensed under a Creative Commons Attribution-NonCommercial-ShareAlike 4.0 International License, which permits unrestricted reuse,
distribution, and reproduction in any medium, provided the original work is properly cited.
eISSN: 2256-2958 Rev Colomb Cienc Pecu 2024; 37(2, Apr-Jun):61–72
https://doi.org/10.17533/udea.rccp.v37n2a1
© 2024 Universidad de Antioquia. Publicado por Universidad de Antioquia, Colombia.

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https://doi.org/10.17533/udea.rccp.v37n2a1Rev Colomb Cienc Pecu 2024; 37(2, Apr-Jun):61–72Broiler supplementation with grape seed
Resumen
Antecedentes: Los procesos activos de remodelación ósea pueden verse alterados por la presencia de especies reactivas de
oxígeno y, por lo tanto, causar problemas en las patas de los pollos de engorde. La actividad antioxidante presente en el extracto
de semilla de uva (GSE) podría ser una alternativa viable a la inclusión de altos niveles de vitamina E (VE) como estrategia
nutricional para mejorar la capacidad antioxidante de las aves y así prevenir anomalías en las patas. Objetivo: Evaluar el efecto
de la sustitución parcial de VE por GSE en la salud podal y el comportamiento productivo de pollos de engorde. Métodos:
Cuatrocientos veinte pollos de engorde machos Ross 308 se distribuyeron en tres tratamientos: 1) Control-AL, dieta con 40 UI/
kg de VE ofrecida ad libitum (CAL); 2) Control-FR, la dieta CAL ofrecida bajo restricción alimenticia (CFR); y 3) extracto
de semilla de uva-AL; dieta con 10 mg/kg de GSE + 10 UI/kg de VE ofrecida ad libitum (GSE-AL). El experimento se llevó
a cabo durante 47 días. El consumo de alimento y el peso corporal de los pollos se registraron semanalmente para evaluar
su comportamiento productivo. En el día 43 se evaluaron capacidad de caminar (GS), angulación en valgo/varo (AngV),
quemadura de pie (FB) y quemadura de corvejón (HB). En el día 47, se evaluaron resistencia a la ruptura de la tibia (TBS) y
resistencia a la ruptura del tendón del gastrocnemio (GTBS). Resultados: Las aves del tratamiento GSE-AL tuvieron menor
GS (p≤0,05) en comparación con los otros tratamientos. No hubo diferencias estadísticas (p>0,05) en AngV, HB, FB, TBS,
TGBS y comportamiento productivo entre tratamientos. Conclusiones: Estos resultados sugieren que GSE puede reemplazar
parcialmente a VE en la dieta de pollos de engorde, sin efectos negativos sobre el comportamiento productivo o la salud podal.
Se requiere más investigación para evaluar el potencial del extracto de semilla de uva para reemplazar VE u otros ingredientes
con actividad antioxidante en diferentes condiciones de crianza y programas de alimentación.
Palabras clave: comportamiento productivo; extracto de semilla de uva; gastrocnemio; marcha; pollo de engorda;
resistencia a la ruptura; restricción de nutrientes; salud podal; tendón; tibia; vitamina E.
Resumo
Antecedentes: Os processos ativos de remodelação óssea podem ser interrompidos pela presença de espécies reativas de
oxigênio e, portanto, causar problemas nas pernas dos frangos de corte. A atividade antioxidante presente no extrato de semente
de uva pode ser uma alternativa viável ao uso de altos níveis de vitamina E (VE) como estratégia nutricional para melhorar
a capacidade antioxidante das aves e, assim, prevenir anormalidades nas pernas. Objetivo: Avaliar o efeito da substituição
parcial de VE por extrato de semente de uva (GSE) na saúde dos pés e no desempenho produtivo de frangos de corte. Métodos:
Quatrocentos e vinte frangos de corte machos Ross 308 foram divididos em três tratamentos: 1) Controle-AL, dieta contendo
40 UI/kg de VE e alimentação ad libitum (CAL); 2) Controle-FR, a dieta CAL mas oferecida através de um sistema alimentar
restrito (CFR); e 3) extrato de semente de uva-AL; uma dieta contendo 10 mg/kg GSE + 10 UI/kg VE com comida oferecida
ad libitum (GSE-AL). O experimento foi realizado por 47 dias. O consumo de ração e o peso corporal das galinhas foram
registrados semanalmente para avaliar o desempenho produtivo. No dia 43, foram avaliados a capacidade de marcha (GS),
angulação valgo/varo (AngV), queimadura do pé (FB) e queimadura do jarrete (HB). No dia 47, a resistência à ruptura da
tíbia (TBS) e a resistência à ruptura do tendão do gastrocnêmio (GTBS) foram avaliadas. Resultados: As aves do tratamento
GSE-AL apresentaram menor (p≤0,05) capacidade de marcha GS em comparação com os outros tratamentos.Não houve
diferenças estatísticas (p>0,05) em AngV, HB, FB, TBS, TGBS e desempenho produtivo entre os tratamentos. Conclusões:
Esses resultados sugerem que o GSE pode substituir parcialmente o VE em dietas de frangos de corte sem efeitos negativos no
desempenho produtivo ou na saúde dos pés. Mais pesquisas são necessárias para avaliar o potencial do extrato de semente de
uva para substituir VE ou outros ingredientes com atividade antioxidante em diferentes condições de criação e programas de
alimentação.
Palavras-chave: comportamento produtivo; extrato de semente de uva; frango de corte; força de quebra; gastrocnêmio;
marcha; restrição de nutrientes; saúde dos pés; tendão; tíbia; vitamina E.

63Rev Colomb Cienc Pecu 2024; 37(2, Apr-Jun):61–72
https://doi.org/10.17533/udea.rccp.v37n2a1Broiler supplementation with grape seed
Introduction
Leg abnormalities lead to decreased growth
performance and negatively impact the welfare
of commercial poultry (Danbury et al., 2000).
Birds with severe lameness have limited access
to feeders and drinkers, causing emaciation and
dehydration (Talaty et al., 2010). The economic
impact due to lameness could account for up to
30% of the total industry losses (Julian, 1995).
According to Waldenstedt (2006), the most
common leg problems in commercial broiler
facilities are tibial dyschondroplasia, rickets, and
angular bone deformities such as valgus/varus
deviations (inward curved/outward curved). The
bone is a dynamic tissue subject to a continuous
process of resorption and formation (Raisz, 2005).
During this process, osteoclasts remove old bone,
whereas osteoblasts form new bone (Mueller and
Russell, 2003). A recent study showed that reactive
oxygen species (ROS) such as H2O2 are related to
apoptosis of stem cells in the bone marrow (Chen
et al., 2016). The ROS are particularly involved
in the homeostasis of mineral tissue by bone
resorption (Wauquier et al., 2009). Moreover, ROS
could inhibit cartilage formation (chondrogenesis;
Zakani et al., 2005). Additionally, high altitude and
ad libitum feeding are predisposing factors that
increase ROS and mortality by ascites (Rodríguez-
Ortega et al., 2014).
Vitamin E (VE), a fat-soluble vitamin, has
multiple functions for optimal growth and health
of broiler chickens. A VE deficiency can cause
nutritional encephalomalacia, exudative diathesis,
and muscular dystrophy (Ames, 1956). The VE
is widely used in poultry diets as an antioxidant
because it can neutralize ROS during propagation
of free radicals due to its peroxyl radical-scavenging
activity (Rizvi et al., 2104; Selvam et al., 2017). A
strategy to increase the antioxidant effect of VE is to
supplement it in high levels. However, the addition
of high levels of VE can increase the cost of the
diet (Kennedy et al., 1992). More economically
feasible alternatives such as grape seed extract
(GSE) could be employed as a nutritional strategy
to improve the birds' antioxidant capacity without
the need to use high levels of VE. The GSE has
antioxidant activity due to its high content of
polyphenolic compounds (Brenes et al., 2010),
such as procyanidins, catechins, epicatechins,
gallocatechins, and epigallocatechins (Chamorro
et al., 2013). However, limited information is
available evaluating the effect of GSE when it is
fed to broiler chickens. An experiment conducted
in rats reported that grape proanthocyanidins can
increase the quality and strength of jaw bones
during growth (Masaru et al., 2004). Another
experiment showed that rats fed high-fructose
diets and supplemented with GSE had increased
activity of hepatic superoxide dismutase, catalase,
and suppressed lipid peroxidation when compared
to rats fed a high-fructose diet alone (Suwannaphet
et al., 2010).
To better understand the potential leg health
and performance benefits of supplementing GSE
to broiler diets, the present study evaluated the
effect of partial substitution of VE by GSE on
gait score (GS), valgus/varus angulation (AngV),
foot burn (FB), and hock burn (HB) lesions, tibia
breaking strength (TBS), and gastrocnemius
tendon breaking strength (GTBS), as well as
growth performance of broiler chickens.
Materials and Methods
Ethical considerations
The experimental procedures followed the
standards for ethics, biosafety, and animal well-
being of the Mexican Official Standard (NOM-
062-ZOO-1999; 2001) for the use of animals in
research. Euthanasia procedures were performed
according to the Mexican Official Norm (NOM-
033-SAG/ZOO-2014, 2015).
Poultry facilities and management
The experiment was carried out at the poultry
research unit of Colegio de Postgraduados,
Campus Montecillo, Estado de México, MX
(98º 48' 27'' W and 19º 48' 23" N), located at an
altitude of 2,278 m above sea level (Barometric
Pressure: 581.1 mmHg, partial pressure of oxygen
(PO2): 122 mm Hg; (Vázquez-Garcia and Pérez-
Padilla, 2000) to increase reactive oxygen species
(ROS) production. Four hundred and twenty
(420) newly hatched male broiler chickens (Ross

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https://doi.org/10.17533/udea.rccp.v37n2a1Rev Colomb Cienc Pecu 2024; 37(2, Apr-Jun):61–72Broiler supplementation with grape seed
308) were used in this 47-day study. Twenty-one
(21) groups of chicks with similar starting weight
(45.59 g ± 0.42; p>0.05) were distributed into
three experimental treatments using a completely
randomized design, with seven replicates per
treatment and 20 chickens per replicate pen. The
birds were housed in floor pens of three m2 with
new wood-shavings litter. Each pen was equipped
with a bell drinker and a hanging cylindrical feeder
(111 and 121 cm circumference, respectively).
Water was offered ad libitum throughout the trial.
The lighting program was 23 h of light and one
hour of darkness during the experimental period.
The broiler house was set for 32-33 °C initial
temperature and it was gradually reduced after two
days (~1 to 2 °C per day) to 24 °C, according to
the birds’ comfort. The facilities used a curtain-
ventilation system.
Dietary treatments
All diets were formulated to meet or exceed
Ross nutritional requirements for broilers.
Chickens were randomly allocated to one of
three dietary treatments; 1) Control: basal diet
containing 40 IU/kg of VE (DL-α-tocopherol
acetate) and fed ad libitum (CAL); 2) Control
fed restricted (CFR): basal diet containing 40
IU/kg of VE offered through a feed restriction
program (feed was offered 16 h per day starting
from day 14 until the end of the experiment); and
3) Grape seed extract treatment: a diet containing
10 mg/kg of Procyanidins equivalent to 30 IU/
kg VE (according to recommended conversion
provided by the product manufacturer) + 10 IU/kg
of VE and fed ad libitum (GSE-AL). The feeding
program was divided into two phases: 1) a starter
(0 to 21 days) diet containing 3,025 kcal/kg ME
and 22% CP and 2) a grower/finisher (21 to 47
days) diet containing 3,100 kcal/kg ME and 19%
CP (Table 1). Birds were fed diets in mash form.
The antioxidant activity test (AOA) was evaluated
in all diets.
Antioxidant activity of diets
The antioxidant activity (AOA) of each diet
was measured as % inhibition of the in vitro 1,
1-diphenyl-2 picrylhydrazyl (DPPH) radical,
according to Rodriguez-Ortega et al. (2017)
with minor modifications. A sample of 500 g
of feed was grounded in a commercial blender
(Oster, Owosso, MI, USA). A gram of feed was
mixed with 10 mL of methanol (Sigma-Aldrich,
St Louis, MO, USA) and incubated at 30 °C for
30 min. The mixture was vortexed for 20 seconds
and then centrifuged at 1,342 × g for 10 min. The
methanol extract was filtered using Whatman
(Cytiva, Marlborough, MA, USA) 4 filters. Two
hundred μL of the extract was taken. Three mL of
methanolic DPPH solution was added (0.11 mM)
and stirred for 10 seconds. The extract was kept in
a dark room for 20 minutes and then analyzed in
a spectrophotometer (Thermo-Scientific, 10SVIS
model, Waltham, MA, USA) at 515 nm. The
antioxidant activity of the diets was calculated
using the following equation:
AOA (% DPPH inhibition) = [(absorbance of
DPPH - absorbance of samples) / absorbance of
DPPH]*100
Performance evaluation
Body weight (BW), BW gain (BWG), feed
intake (FI), and feed conversion ratio (FCR)
were calculated by phase. Mortality data from the
present study is not presented in this paper as it has
been previously reported in a separate publication
(Rodríguez-Ortega et al., 2017).
Gait score, valgus/varus angulation, hock, and
footpads burn lesion
On day 43, three birds per pen were randomly
selected (21 birds/treatment) to evaluate gait score
(GS), valgus/varus angulation (AngV), foot burns
(FB) and hocks burn (HB) lesions. Gait score was
evaluated according to the methodology described
by Kestin et al. (1992) and later modified by
Garner et al. (2002). Six score categories (0 to 5)
were used. A score of 0 was given to broilers with
normal locomotion and a 5 score to completely
lame chickens unable to walk or stand. Two
experienced observers viewed and scored each
bird individually; when the evaluators did not
reach a consensus, they evaluated another bird.

65Rev Colomb Cienc Pecu 2024; 37(2, Apr-Jun):61–72
https://doi.org/10.17533/udea.rccp.v37n2a1Broiler supplementation with grape seed
Table 1. Composition of the experimental diets.
Ingredient (%) Starter 0-21 d Grower/Finisher 21-47 d
Control GSE Control GSE
Yellow corn 53.38 53.38 61.67 61.67
Dehulled soybean meal 39.69 39.69 31.27 31.27
Soybean oil 2.64 2.64 2.80 2.80
Calcium carbonate 1.66 1.66 1.34 1.34
Dicalcium phosphate 1.68 1.68 1.57 1.57
L-Lysine HCl 0.028 0.028 0.077 0.077
L-Threonine 0.066 0.066 0.085 0.085
DL-Methionine 0.122 0.122 0.088 0.088
Mineral premix1 0.100 0.100 0.100 0.100
Vitamin premix2 0.050 0.050 0.050 0.050
Selenium 0.005 0.005 0.005 0.005
Vitamin E3 0.007 0.002 0.007 0.002
Grape seed extract3 0.000 0.001 0.000 0.001
Choline chloride 0.213 0.213 0.213 0.213
Coccidiostat4 0.050 0.050 0.050 0.050
Pigment (xanthophylls) 0.000 0.000 0.370 0.370
Salt (NaCl) 0.300 0.300 0.300 0.300
Calculated composition (%)
ME (kcal/kg) 3,025.00 3,025.00 3,100.00 3,100.00
CP 22.00 22.00 19.00 19.00
Arginine 1.50 1.50 1.50 1.50
Lysine 1.28 1.28 1.09 1.09
Methionine 0.48 0.48 0.41 0.41
Methionine + Cystine 0.88 0.88 0.75 0.75
Threonine 0.90 0.90 0.80 0.80
Calcium 1.00 1.00 0.85 0.85
Available phosphorus 0.45 0.45 0.42 0.42
Sodium 0.16 0.16 0.16 0.16
Chloride 0.22 0.22 0.22 0.22
Vitamin E (IU) 40.00 10.00 40.00 10.00
Procyanidins (mg)3 0.00 10.00 0.00 10.00
Choline (mg) 1,600.00 1,600.00 1,600.00 1,600.00
1Mineral premix (per kg of premix): Zn 100 g; Fe 50 g; Cu 10 g; Mn 100 g; I 1 g.
2Vitamin premix (per kg of premix): vitamin A 24,000,000 IU; vitamin D3 8,000,000 IU; pyridoxine 8 g; thiamine 6 g; riboflavin
16 g; niacin100 g; cyanocobalamin 60 mg; menadione 10 g; calcium pantothenate 28 g; folic acid 3 g.
3Vitamin E (DL-α-tocopherol acetate); GSE (Grape seed extract; Procyanidins) are estimated values based on the product
manufacturer recommendations.
4Coccidiostat, Olistimax®, PiSA Agropecuaria, Guadalajara, Jalisco, Mexico.

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https://doi.org/10.17533/udea.rccp.v37n2a1Rev Colomb Cienc Pecu 2024; 37(2, Apr-Jun):61–72Broiler supplementation with grape seed
Valgus/varus angulation (VAng) measured the
angle size of the tibia-metatarsus according to the
protocol described by Vargas-Galicia et al. (2017).
A four-scale lesion score was assigned from 0 to
3, where 0 represented birds with no angulation;
Score 1, chickens with slight angulation; Score 2,
birds with obvious angulation; and Score 3, birds
with severe angulation. Foot and hock burns were
measured following the methodology of Su et al.
(1999). Both legs were evaluated on a scale of 0
to 3, where: 0 represented undamaged feet and
hocks; 1, bird with minor damage; 2, presence of
obvious damage; and 3, chicken with extensive
burns and inflammation.
Tibia and gastrocnemius tendon breaking strength
At the end of the experiment, 14 birds per treatment
(2 birds/pen) were euthanized by cervical
dislocation to evaluate tibia-breaking strength
(TBS) and gastrocnemius tendon-breaking
strength (GTBS). Both legs were frozen at -20
°C and stored (3 months) until TBS and GTBS
assessments were performed. The TBS and GTBS
were measured in Newtons (N) using a Vernier
force plate (Vernier Software & Technology,
Beaverton, OR, USA). The tendon and tibia
were dissected the same day they were thawed,
as described by Rodríguez-Ortega et al. (2022).
All measurements were conducted at the animal
nutrition laboratory of Universidad Autónoma
Chapingo, Mexico.
Statistical analysis
Growth performance (BW, BWG, FI, FCR),
gastrocnemius tendon-breaking strength (GTBS),
and tibia breaking strength (TBS) data were
analyzed with one-way ANOVA using the PROC
MIXED procedure with orthogonal contrasts
using SAS software, version 9.0 (SAS Institute
Inc., Cary, NC, USA; 2011). Gait score (GS),
valgus/varus angulation (AngV), foot burns (FB),
and hocks burns (HB) were analyzed by contrasts
using the GLIMMIX procedure (SAS Institute
Inc., 2011). Contrasts were analyzed as follows:
CAL vs. CFR, CAL vs. GSE-AL; CFR vs. GSE-
AL. A statistical significance was considered when
p-value was ≤0.05.
Results
The effects of partial substitution of VE by
GSE on performance variables and leg health was
evaluated. The GSE diets had lower AOA than the
control diets (Table 2). No significant differences
(p>0.05) in cumulative BWG, FI, and FCR were
observed among treatments (Table 3).
Table 2. Antioxidant activity of the experimental diets.
% inhibition DPPH1
Diet Starter Grower/Finisher
Control 14 17
Grape seed extract 11 11
1The antioxidant activity (AOA) of each diet was measured
as % inhibition of in vitro 1, 1-diphenyl-2 picrylhydrazyl
radical (DPPH).
Birds in the GSE-AL treatment showed the
lowest (p≤0.05) GS (worsen walking capacity)
with 57% of the birds being classified in Category
3 in comparison with the control treatments. The
Control-AL treatment had the highest (p≤0.05) %
of birds with a score of 0 (Table 4). There were no
significant differences (p>0.05) in FB, HB, AngV
(Table 5), TBS, and GTBS (Table 6).
Discussion
In the literature, proper GSE levels to be included
in the diets for broilers have not been conclusive.
In this study, no differences in performance were
observed in chickens fed GSE ad libitum. Our
results agree with Brenes et al. (2010), who found
no significant differences in body weight gain
(BWG), feed intake (FI), and FCR among broiler-
fed diets GSE at 0.6, 1.8, and 3.6 g/kg of diet. In
contrast, Hughes et al. (2005) observed a decrease
in FI and growth of broiler chickens fed diets that
included 30 g/kg GSE, where the GSE product
contained 90.2% total extractable polyphenols. In
the present study, the GSE product had 85% total
extractable polyphenols added at 10 mg/kg of diet;
in general, high dietary supplementation of GSE is
related to reduced performance (Chamorro et al.,
2013); therefore, the low inclusion of GSE in this
experiment could explain the performance results.

Table 3. Body weight gain (BWG), feed intake (FI), and feed conversion ratio (FCR) of broilers1.
Treatment¥ CAL CFR GSE-AL SEM ANOVA CAL vs.
CFR
CAL vs.
GSE-AL
CFR vs.
GSE-AL
0-21 d
BWG (g) 835 828 813 16 0.611 0.942 0.593 0.790
FI (g) 1,266a 1,205b 1,223ab 12 0.006 0.005 0.052 0.554
FCR 1.53 1.47 1.50 0.02 0.249 0.220 0.667 0.667
21-47 d
BWG (g) 2,525 2,496 2,579 48 0.346 0.860 0.613 0.323
FI (g) 4,764 4,724 4,730 80 0.927 0.931 0.950 0.998
FCR 1.89 1.89 1.84 0.03 0.457 1.00 0.521 0.521
0-47 d2
BWG (g) 3,360 3,324 3,392 47 0.603 0.811 0.888 0.535
FI (g) 6,030 5,929 5,953 80 0.657 0.655 0.779 0.976
FCR 1.79 1.79 1.76 0.02 0.544 0.931 0.756 0.931
Different superscripts letters (a, b) within the same row are statistically different at p≤0.05.
¥Control-AL: basal diet containing 40 IU/kg of vitamin E (VE) (dl-α-tocopheryl acetate) and fed ad libitum (CAL); Control-FR:
CAL diet containing 40 IU/kg of VE offered through a feed restriction program (CFR: feed was offered 16 h per day starting from
day 14 until the end of the experiment); and grape seed extract (GSE)-AL: a diet containing 10 mg/kg of GSE (equivalent to 30
IU/kg VE) + 10 IU/kg of VE and fed ad libitum (GSE-AL).
1Contrasts with p≤0.05 are statistically different; SEM: pooled standard error of the mean.
2Cumulative 0-47 d performance data was previously published (Rodríguez-Ortega et al., 2017).
Table 4. Gait score (GS) of broilers at 43 days1.
Treatment¥ Gait score (%)
0 1 2 3 4 5
CAL 5 23 40 32 0 0
CFR 10 38 47 5 0 0
GSE-AL 0 0 43 57 0 0
p-value
ANOVA <0.001
CAL vs. CFR 0.042
CAL vs. GSE-AL 0.069
CFR vs. GSE-AL 0.002
¥Control-AL: basal diet containing 40 IU/kg vitamin E (VE) (dl-α-tocopheryl acetate) and fed ad libitum (CAL); Control-FR: CAL
diet containing 40 IU/kg VE offered through a feed restriction program (CFR: feed was offered 16 h per day starting from day 14
until the end of the experiment); and grape seed extract (GSE)-AL: a diet containing 10 mg/kg GSE (equivalent to 30 IU/kg VE)
+ 10 IU/kg VE and fed ad libitum (GSE-AL).
1Contrasts with p≤0.05 are statistically different.

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Table 5. Hocks burn (HB), foot burn (FB), and valgus/varus angulation (AngV) frequency of chickens at 43 days1.
Treatment¥ HB (%) FB (%) AngV (%)
0 1 2 3 0 1 2 3 0 1 2 3
CAL 52 48 0 0 86 14 0 0 5 76 14 5
CFR 71 24 5 0 100 0 0 0 5 81 14 0
GSE-AL 67 33 0 0 100 0 0 0 5 71 24 0
p-value
ANOVA 0.718 1.000 0.798
CAL vs. CFR 0.466 0.983 0.686
CAL vs. GSE-AL 0.517 0.983 0.796
CFR vs. GSE-AL 0.933 1.000 0.506
¥ Control-AL: basal diet containing 40 IU/kg vitamin E (VE) (dl-α-tocopheryl acetate) and fed ad libitum (CAL); Control-FR:
CAL diet containing 40 IU/kg VE offered through a feed restriction program (CFR: feed was offered 16 h per day starting from
day 14 until the end of the experiment); and grape seed extract (GSE)-AL: a diet containing 10 mg/kg GSE (equivalent to 30 IU/
kg VE) + 10 IU/kg VE and fed ad libitum (GSE-AL).
1Contrasts with p≤0.05 are statistically different.
Table 6. Tibia (TBS) and gastrocnemius tendon (GTBS) breaking strength at 47 days1.
Treatment¥ GTBS (N) TBS (N)
CAL 168 404
CFR 156 420
GSE-AL 184 421
SEM 10 20
p-value
ANOVA 0.139 0.799
CAL vs. CFR 0.672 0.836
CAL vs. GSE-AL 0.451 0.824
CFR vs. GSE-AL 0.120 0.999
¥Control-AL: basal diet containing 40 IU/kg vitamin E (VE) (dl-α-tocopheryl acetate) and fed ad libitum (CAL); Control-FR: CAL
diet containing 40 IU/kg VE offered through a feed restriction program (CFR: feed was offered 16 h per day starting from day 14
until the end of the experiment); and grape seed extract (GSE)-AL: a diet containing 10 mg/kg GSE (equivalent to 30 IU/kg VE)
+ 10 IU/kg VE and fed ad libitum (GSE-AL).
1Contrasts with p≤0.05 are statistically different; SEM: pooled standard error of the mean.
The broilers fed GSE in combination with
VE had the lowest GS, which could be due to
the number of free radicals released by high
metabolic rate (fed ad libitum diets) and the high
altitude where the experiment was conducted
(Kalmar et al., 2013). Rodríguez-Ortega et al.
(2017) evaluated glutathione peroxidase activity,
nitric oxide concentration, and lipid peroxidation
[malondialdehyde (MDA) concentration] in
plasma, lungs, heart and liver, observing that
ad libitum diets decreased lung, heart, and liver
antioxidant activity in broiler chickens raised
at 2,278 m of altitude (PO2 of 122 mm Hg).
High altitudes generate tissue hypoxia (Maiti et
al., 2006) and decrease electron transport, thus
generating free radicals (Poyton et al., 2009). Free
radicals affect the formation of bone and cartilage
(McAlindon et al., 1996), affecting locomotion.
Broilers raised at high altitudes might benefit from
high inclusion levels of GSE to effectively prevent
oxidative stress when fed ad libitum diets.

69Rev Colomb Cienc Pecu 2024; 37(2, Apr-Jun):61–72
https://doi.org/10.17533/udea.rccp.v37n2a1Broiler supplementation with grape seed
High bird density, litter type, moisture, and
broiler weight can influence the frequency and
severity of HB and FB (Tasistro et al., 2004;
Almeida et al., 2010). The present study showed
no significant difference between treatments for
HB and FB. The lack of effect of GSE on HB
and FB could have been partially due to low
moisture content of the litter. Future trials should
consider the use of higher bird density to introduce
a bigger challenge to the litter, thus, increasing
the opportunity to observe the GSE effect on the
reduction of HB and FB lesions. Angular bone
deformity is one of the most common leg defects
in broilers (Julian, 1984). In the present study,
no significant difference was observed for AngV
among treatments. One possible explanation for
the results observed in AngV could be birds’ age
(higher bone maturity at 43 days). These problems
are related to lack of maturity of the tissue and
BW because the tissue becomes stronger and
more resistant with age (Julian, 1998). Applegate
and Lilburn (2002) observed that bone deformity
problems in broilers are related to constant increase
in live weight and immature skeletal tissue.
Feeding ingredients with antioxidant activity
can reduce cartilage loss and progression of
diseases due to osteoarthritis (Mobasheri et al.,
2012). Sanchez et al. (2013) observed that
chickens fed 80 IU/kg VE and vitamin C at 1 g/kg
had greater strength to the gastrocnemius tendon
rupture compared to birds fed VE 40 IU/kg only.
Moreover, Feresin et al. (2013) found that VE
improves bone quality, decreases bone resorption,
and increases bone formation, but does not
restore bone density. One possible reason for the
beneficial effects of VE on bone health is because it
suppresses inflammatory mediators (prostaglandin
E2, PGE2, tumor-α necrosis factor, TNF-α,
interleukin 1 (IL-1)) and reduces free radicals that
stimulate bone resorption (Garret et al., 1990). We
hypothesize that the GSE mode of action could be
similar to VE, but further research is needed to find
the specific effects of GSE on bone remodeling
processes and interaction with other tissues.
In conclusion, GSE may partially replace
vitamin E without negative effects on growth
performance and overall leg health of broilers.
Further research is required to evaluate the potential
of the GSE to replace VE or other ingredients
with antioxidant activity under different rearing
conditions and feeding programs.
Declarations
Conflict of interest
The authors declare they have no conflicts of
interest with regard to the work presented in this
report.
Funding
The author Rodríguez-Ortega L. T. expresses
his gratitude to Consejo Nacional de Ciencia y
Tecnología (CONACyT-México) for the scholar-
ship granted to carry out his Ph. D. studies.
Author contributions
Leodan T. Rodríguez-Ortega: experimental
design, data, and sample collection, supervised
the health status of the chickens, and manuscript
writing. Alejandro Rodríguez-Ortega: experimen-
tal design conceptualization. Arturo Pro-Mar-
tínez: conceptualization of the study. Filogonio J.
Hernández-Guzmán: writing, review, and editing.
Eliseo Sosa-Montes: laboratory analysis. Hec-
tor Leyva-Jimenez: manuscript writing, and final
editing.
Use of artificial intelligence (AI)
No AI or AI-assisted technologies were used
during the preparation of this work.
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