Effect of Dietary Grape Pomace and Vitamin E on Growth Performance,

Nutrient Digestibility, and Susceptibility to Meat Lipid Oxidation in Chickens

I. Goñi,* A. Brenes,†1 C. Centeno,† A. Viveros,‡ F. Saura-Calixto,† A. Rebolé,‡ I. Arija,‡ and R. Estevez†

*Departamento de Nutrición I, Facultad de Farmacia, Universidad Complutense de Madrid, Ciudad Universitaria,

Madrid 28040, Spain; †Departamento de Metabolismo y Nutrición, Instituto del Frı́o, CSIC, Avda. Ramiro de Maeztu s/n,
Ciudad Universitaria, Madrid 28040, Spain; and ‡Departamento de Producción Animal, Facultad de Veterinaria,
Universidad Complutense de Madrid, Ciudad Universitaria, Madrid 28040, Spain

ABSTRACT Grape pomace (GP) is a source of poly-
phenols with powerful antioxidant capacity. An experi-
ment was conducted to investigate the effect of inclusion
of GP at levels of 5, 15, and 30 g/kg and α-tocopheryl
acetate (200 mg/kg) in a corn-soybean basal diet on
growth performance, protein and amino acid digestibili-
ties; antioxidant activity of diet, serum and excreta, lipid
oxidation of breast and thigh meats during refrigerated
storage, and liver vitamin E concentration. Growth per-
formance and protein and amino acid digestibilities were
not affected among the different treatments. Total intake
and digestibility of extractable polyphenols in the birds
fed the GP diet were increased compared with birds fed
supplemented and unsupplemented vitamin E diets. An-
tioxidant activity in vitamin E and GP diets and excreta
exhibited higher scavenging free radical capacity than the
control diet using 3-ethylbenzthiazoline-6-sulfonic acid
and ferric reducing antioxidant power methods. Lipid
oxidation (malondialdehyde concentration) in breast and
Key words: grape pomace, chick, lipid oxidation
thigh meats was lower in the birds fed the supplemented
vitamin E diet than the control diet after 1, 4, and 7 d of
refrigerated storage. Results showed a linear reduction
of lipid oxidation in breast and thigh meats at 4 and 7 d
with increasing content of GP in the diet. Oxidative stabil-
ity in breast and thigh meats at 1, 4, and 7 d of storage
was equivalent or less effective in GP diets compared
with the vitamin E diet. A linear increase was observed
in liver α-tocopherol concentration with increasing con-
tent of GP in the diet, but it was inferior to the supple-
mented vitamin E diet. In conclusion, the results showed
that a dietary inclusion rate up to 30 g/kg of GP did not
impair chickens growth performance and protein and
amino acids digestibilities and increased antioxidant ac-
tivity in diet and excreta. Grape pomace and vitamin E
diets reduced the lipid oxidation of meat during refriger-
ated storage and increased liver α-tocopherol concentra-
tion, although these effects were greater, in some cases,
by adding vitamin E to the diet.
2007 Poultry Science 86:508–516

INTRODUCTION
Grape pomace (GP) is the residue left after juice extrac-
tion by pressing grapes in the wine industry. In Spain
alone, over 250 million kilograms of this by-product (con-
stituted by seeds, skin, and stem) are used every year
either as animal feed (with low nutritional value) or for
ethanol production by fermentation and distillation (low-
level benefit). This material is under-exploited, and most
of it is generally disposed in open areas, leading to serious
environmental problems (Botella et al., 2005). Recent in-
vestigations have stressed the importance of this by-prod-
uct from wine processing as plant material particularly
rich in a wide range of polyphenols (Bonilla et al., 1999;
©2007 Poultry Science Association Inc.
Received June 27, 2006.
Accepted October 26, 2006.
1
Corresponding author: abrenes@if.csic.es
Alonso et al., 2002; Torres et al., 2002). Grape skins and
seeds are rich source of flavonoids, including monomeric
phenolic compounds such as (+)-catechins, (-)-epica-
techin, and (-)-epicatechin-3-O-gallate and dimeric, tri-
meric, and tetrameric procyanidins. Studies have shown
flavonoids have the capacity to act as powerful antioxi-
dants by scavenging free radicals and terminating oxida-
tive reactions (Gonzalez-Paramás et al., 2004). Flavanols
and flavanol oligomers and polymers (proanthocyani-
dins) have been proven to possess powerful antioxidant
properties (Yilmaz and Toledo, 2004). The application of
GP compounds in food technology has also demonstrated
a potent edible oil antioxidant capacity and an inhibitor
of the oxidation of fish lipids, frozen fish muscle, and
cooked, cold stored turkey meat (Wanasundara and Shah-
idi, 1994; Pazos et al., 2005; Mielnick et al., 2006). In experi-
ments with rats, the inclusion of grape flavonoids causes
a diminution of tissue lipid peroxidation in kidney, liver,
and lung (Preuss et al., 2001; Rodrigo et al., 2005) and

508
 GRAPE POMACE IN CHICKEN DIETS
considerable antioxidant activity within the large intes-
tine and feces derived from excreted extractable poly-
phenols (EP) and nonextractable polyphenols (Goñi and
Serrano, 2005).
Poultry meat is relatively rich in polyunsaturated fatty
acids and is, therefore, readily susceptible to oxidative
deterioration (Kanner, 1994). Increasing the unsaturation
degree of the muscle membrane by dietary manipulation
increases the susceptibility of chicken meat to oxidative
deterioration during storage (Enberg et al., 1996), and as
a consequence, flavor and nutritional value are decreased.
Synthetic antioxidants such as butylated hydroxytoluene
and butylated hydroxyanisole have long been used to
control lipid oxidation in stored meat and meat products,
but concern over their use (Imaida et al., 1983; Okada et
al., 1990), has created a need and prompted research for
alternative antioxidants, particularly from natural
sources. The oxidative stability of poultry meat depends
largely on the contained α-tocopherol present in cell
membrane phospholipids, which in turn is dependent on
the level of α-tocopheryl acetate added to the diet (Wen
et al., 1997). Dietary supplementation with this antioxi-
dant has been shown to increase vitamin E in muscle
tissues, improving the oxidative stability of meat during
storage (Carreras et al., 2004). Apart from α-tocopherol,
research has shown that dietary supplementation of es-
sential oils from rosemary, sage, and oregano to broilers
could improve the oxidative stability of chicken and tur-
key meat during refrigerated or long-term storage (Lopez-
Bote et al., 1998; Botsoglou et al., 2002; Botsoglou et al.,
2003a). Evidence is also available on the antioxidative
effect of added tea catechins on susceptibility of chicken
meat to lipid oxidation (Tang et al., 2000, 2001).
Wine by-products represent sources of antioxidants
that have been relatively unexploited to date, but they
are subjects of increased industrial interest. No evidence
is available on the potential antioxidant properties of GP
when added in poultry diets. The objective of this study
was to evaluate the effect of dietary GP and vitamin E
on broiler chicken performance, nutrient digestibility, an-
tioxidant activity of diet, serum, and excreta. The suscepti-
bility to oxidation of breast and thigh refrigerated meats
and liver α-tocopherol concentration was also de-
termined.
MATERIALS AND METHODS
Test Product
Red GP (peels and seeds; Vitis vinifera var. Cencibel)
was obtained from a winery (Vinı́cola de Castilla S.A.,
Manzanares, Ciudad Real, Spain). Proximate composition
of GP is shown in Table 1. Grape pomace was used as a
source of dietary fiber and polyphenols in the chicken
diets. The α-tocopheryl acetate used in the diets was do-
nated by DSM Nutritional Products Iberia S.A., Alcalá de
Henares, Madrid, Spain.
509
1
Table 1. Proximate composition of grape pomace
Item DM (g/kg)
Protein 137.9 ± 1.2
Soluble sugars 20.7 ± 0.3
Fat 102.6 ± 6.4
Fiber 325.0 ± 8.5
Ash 24.1 ± 0.3
Extractable polyphenols 48.6 ± 2.7
Nonextractable polyphenols 160.4 ± 7.4
Data are the mean of 4 determinations ± SD.
1
Birds and Diets
A total of 120, one-day-old male broiler chicks (Cobb
strain) were obtained from a commercial hatchery. The
birds were housed in electrically heated stainless steel
starter battery brooders in an environmentally controlled
room with 23 h of constant overhead fluorescent lighting
during 3 wk. Diets in mash form and water were provided
for ad libitum consumption. Chicks were allocated to 20
cages, each cage containing 6 chicks, to receive 5 dietary
treatments with 4 replicates of each treatment. Celite (Cel-
ite Corp., Lompoc, CA), a source of acid insoluble ash
(AIA), was added at 10 g/kg to all diets as an indigestible
marker. All diets were formulated to meet or exceed the
minimum NRC (1994) requirements for broiler chickens.
At the end of the experimental period, birds were
weighed, and feed consumption was recorded for feed
efficiency computation. All housing and handling were
approved by the University Complutense of Madrid Ani-
mal Care and Ethics Committee in compliance with the
Ministry of Agriculture, Fishery and Food for the Care
and Use of Animals for Scientific Purposes. Ingredients
and nutrient composition of diets are shown in Table
2. Experimental diets were as follows: 1) control corn-
soybean diet + 30 g/kg of cellulose (CS), 2) CS + vitamin
E (200 mg/kg of α-tocopheryl acetate), 3) CS + 5 g/kg of
GP, 4) CS + 15 g/kg of GP, 5) CS + 30 g/kg of GP. The
cellulose was substituted by GP in the experimental diets.
Collection of Samples and Measurements
At 21 d of age, 8 birds were randomly selected from
each treatment (2 per replicate). Serum was prepared
from blood obtained by cardiac puncture for subsequent
determination of antioxidant activity. The blood samples
were allowed to clot in polypropylene tubes for 2 h at
room temperature at 1,500 × g for 10 min, and the superna-
tant was removed. All samples were stored at −20°C until
assayed. After killing the chicks by cervical dislocation,
liver was removed, cleaned from adhering tissue, and
frozen at −20°C. The ileum was quickly dissected out
and the content expressed by gentle manipulation into a
plastic container and stored at −20°C. Digesta were pooled
from 2 birds of each replicate within the same treatment.
Ileal contents were freeze-dried and ground (1-mm
screen) and subsequently analyzed for CP, amino acids,
and celite. Clean stainless steel collection trays were also
510 GOÑI ET AL.
Table 2. Ingredients and nutrient composition of experimental diets (g/kg as fed)

Control Control Control Control

Item Control + vitamin E + 5 GP1 + 15 GP + 30 GP

Ingredients
Corn (8.1% CP) 430.7 430.7 432.6 436.4 442.2
Soybean (48% CP) 404.4 404.4 402.6 398.9 393.4
Sunflower oil 85.0 85.0 85.0 85.0 85.0
Cellulose 30.0 30.0 25.0 15.0 —
GP (13.2% CP) — — 5.0 15.0 30.0
Dicalcium phosphate 19.3 19.3 19.3 19.3 19.3
Calcium carbonate 10.5 10.5 10.4 10.3 10.0
Salt 3.0 3.0 3.0 3.0 3.0
Vitamin-mineral premix2 5.0 5.0 5.0 5.0 5.0
DL-Met 2.1 2.1 2.1 2.1 2.1
Celite3 10.0 10.0 10.0 10.0 10.0
Analyzed composition
CP 219.0 228.0 221.0 227.3 223.0
Total polyphenols 2.6 2.8 NA4 NA 4.1
Vitamin E (mg/kg) 95.3 250.5 83.7 74.2 86.8
Calculated composition
AME5(kcal/kg) 3,101 3,101 3,105 3,111 3,122
Met + Cys 9.0 9.0 9.0 9.0 9.0
Ca 10.0 10.0 10.0 10.0 10.0
Available P 4.5 4.5 4.5 4.5 4.5
1
GP = grape pomace.
2
Vitamin and mineral mix supplied the following per kilogram of diet: vitamin A, 8,250 IU; cholecalciferol,
1,000 IU; vitamin E, 11 IU; vitamin K, 1.1 mg; vitamin B12, 12.5 ␮g; riboflavin, 5.5 mg; Ca panthotenate, 11mg;
niacin, 53.3 mg; choline chloride, 1,020 mg; folic acid, 0.75 mg; biotin, 0.25 mg; delquin, 125 mg; DL-Met, 500
mg; amprol, 1 g; Mn, 55 mg; Zn, 50 mg; Fe, 80 mg; Cu, 5 mg; Se, 0.1 mg; I, 0.18 mg; NaCl, 2,500 mg.
3
Celite Corp, Lompoc, CA.
4
NA = not analyzed.
5
Calculated value (FEDNA, 2003).

placed under each cage, and excreta from the birds were
collected for 48 h. A subsample of excreta was collected
in polyethylene bags and freeze-dried for subsequent de-
termination of celite, extractable polyphenols, and antiox-
idant activity. Eight birds (2 per replicate with the live
weight closest to the particular replicate average) per
treatment were slaughtered, and carcasses were immedi-
ately trimmed for breast and thigh meat. These tissues
were individually sliced and sampled for lipid oxidation
studies. Tissue samples, breast excluding skin and thigh
with skin, were wrapped in transparent O2-permeable
polyvinyl chloride film (13,500 cm3/m2 per d), frozen,
and stored at −20°C until required. After thawed, the raw
meat samples were placed in a nonilluminated refriger-
ated cabinet at 4°C, and the progress of lipid oxidation
was determined after 1, 4, and 7 d of storage.
Chemical Analysis
Dry matter (930.15), CP (976.05), crude fiber (978.10),
and ash (942.05) were analyzed according to the methods
of the Association of Official Analytical Chemists (1995).
Crude fat was determined by extraction in petroleum
ether following acidification with a 4 N HCl solution
(Wiseman et al., 1992). Analysis of soluble sugars was
carried out by using anthrone and thiourea as a reagent
following the conditions described by Southgate (1976).
The AIA contents of diet, ileal digesta, and excreta were
measured after ashing the samples and treating the ash
with boiling 4 M HCl (Siriwan et al., 1993). Amino acids
in the diets and the ileal contents were analyzed (994.12)
following AOAC (1995) procedures and separated using
a Beckman model 6300 autoanalyzer (Beckman Coulter,
Monheim, Germany). Determination of the amino acid
(AA) Trp was not possible under the conditions of analy-
sis used. The extent of lipid oxidation was determined
by measuring the TBA-reacting substances at 1, 4, and 7
d of storage and was expressed as micrograms of malon-
dialdehyde (MDA) per gram of muscle using the proce-
dure described by Salih et al. (1987). Ten grams of ground
meat was homogenized with 35 mL of 3.86% perchloric
acid in an Ultra-Turrax (IKA Works Inc., Wilmington,
NC) at 21,280 × g for 1 min. Butylated hydroxyanisole
was added before homogenization at a level of 125 ␮g/mg
of fat. The blended sample was filtered through Whatman
number 2V filter (Whatman International Ltd., Maid-
stone, UK) into 50-mL Erlenmeyer flasks. Five milliliters
of the filtrate was mixed with 5 mL of 0.02 M TBA in
distilled water in capped test tubes. Tubes were incubated
at room temperature in the dark for 15 to 17 h or heated
in boiling water for 30 min. The absorbance was deter-
mined at 531 nm against a blank containing 5 mL of
distilled water and 5 mL of 0.02 M TBA solution.
Polyphenols were extracted in diet and excreta by shak-
ing at room temperature with methanol water (50:50 vol/
vol, 50 mL/g sample during 60 min, at room temperature
and with constant shaking). After centrifugation (15 min,
3,000 × g) supernatants were combined and used to mea-
sure the antioxidant capacity by the 2, 2-azinobis (3-ethi-
lenzotiazolin)-6-sulfonate (ABTS; Fluka Chemicals, Ma-
drid, Spain) method.
 GRAPE POMACE IN CHICKEN DIETS 511
Extractable polyphenols were determined in methanol, Table 3. Performance of broiler chicks (0 to 21 d) fed diets containing
grape pomace1 (GP) and vitamin E
acetone, and water extracts obtained from GP, diet, and
excreta by the Folin-Ciocalteau procedure (Montreau, Weight Feed Feed
1972) using gallic acid as a standard. gain consumption efficiency
Treatments (g) (g) (g:g)
Residues from the extract were treated with 5-mL/L
of HCl-butanol during 3 h at 100°C (Reed et al., 1982). Control 656 856 1.31
Control + vitamin E 649 839 1.29
Nonextracted polyphenols were calculated from the ab- Control + 5 GP 655 862 1.32
sorbance at 550 nm of the anthocyanidin solutions. Con- Control + 15 GP 651 855 1.31
densed tannins from Mediterranean carob pod (Ceratonia Control + 30 GP 673 869 1.29
Pooled SEM 39 47 0.03
siliqua L.) supplied by Nestlé S.A. (Vevey, Switzerland) Statistical significance2
were treated under the same conditions to obtain stan- Control vs. vitamin E NS NS NS
dard curves. GP vs. no GP NS NS NS
The ABTS assay was determined in extracted samples Vitamin E vs. GP NS NS NS
Type of response3
(GP, diet, and excreta) and serum. The antioxidant activ- Linear NS NS NS
ity was estimated following the procedure described by Quadratic NS NS NS
Re et al. (1999) with some modifications. The ABTS radical 1
Data are means of 4 pens of 6 chicks.
cation (ABTS+ⴢ) was produced by reacting 7 mM ABTS 2
Probability value of contrast; NS = P > 0.05.
stock solution with 2.45 mM potassium persulfate and 3
Due to percentage GP in diet.
allowing the mixture to stand in the dark at room temper-
ature for 12-16 h before use. The ABTS+ⴢ solution was
diluted with methanol to an absorbance of 0.70 ± 0.02 at concentration in feed/AIA concentration in ileal digesta
658 nm. After addition of 100 ␮L of extracted samples or or excreta) × (CP and AA concentration in ileal digesta
Trolox standard (6-hydroxy-2, 5, 7, 8-tetramethylchro- and EP concentration in excreta/CP and AA concentra-
man-2-carboxylic acid; Sigma-Aldrich Co, St Louis, MO) tion in feed)]. Data were subjected to ANOVA using the
to 3.9 mL of diluted ABTS+ⴢ solution, absorbance readings GLM procedures of SAS (SAS Institute, 2001), and single
were taken every 20 s using a Beckman DU-640 (Beckman df linear contrasts were used to separate treatments. Lin-
Instruments Inc, Fullerton, CA). The reaction was moni- ear and quadratic effects were also analyzed. Significant
tored for 6 min. The percentage inhibition of absorbance differences among treatment means were determined at
vs. time was plotted, and the area below the curve (0 to P < 0.05 by Duncan’s multiple-range test.
6 min.) was calculated. Methanolic solutions of known
Trolox concentrations were used for calibration of the RESULTS
measurement of EP antioxidant activity.
The ABTS determination on serum was similar to the Growth Performance
method previously indicated, but 10 ␮L of serum was
added to 3 mL of ABTS+ solution and an aqueous solution The addition of increasing concentration of GP in the
of Trolox was used for calibration of the measurement of chicken diets did not impair growth performance (BW,
antioxidant activity. feed consumption, and feed efficiency) compared with
The ferric antioxidant power (FRAP) of the samples those birds fed the unsupplemented and supplemented
was estimated according to the procedure previously de- vitamin E diets (Table 3).
scribed (Benzie and Strain, 1996; Pulido et al., 2000).
Briefly, FRAP reagent was mixed with distilled water and Apparent Digestibilities of Protein
either the sample or appropriate reagent blank. Readings
at 30 min were selected for calculation of FRAP values.
and Amino Acids
Reduction power activities were as micromolars of Trolox The inclusion of graded concentrations of GP did not
equivalents per gram of DM. affect the apparent ileal digestibility of CP and essential
α-Tocopherol content of diets and liver was determined and nonessential amino acids. Statistical analysis also
by the method of Butriss and Diplok (1984), which in- demonstrated a reduction of Arg (P < 0.05), Leu (P < 0.05),
cludes saponification with saturated KOH in the presence Phe (P < 0.01), Glu (P < 0.01), Pro (P < 0.01), Tyr (P <
of pyrogallol; for liver, 1 mL of 25% liver homogenate 0.01), His (P < 0.05), and Cys (P < 0.01) digestibilities in
was used. The α-tocopherol was then extracted with hex-
birds fed GP diets compared with those fed the vitamin
ane and measured by normal-phase HPLC using a Hyper-
E diet. Likewise, a quadratic effect (P < 0.05) was observed
sil Si 100 (5 ␮m) column and a mobile phase of hexane-
in Lys, Thr, Glu, and Ser with increasing dietary GP (Ta-
isopropanol (98:2 vol/vol) and detected by fluorescence
ble 4).
using a HPLC system (Hewlett-Packard 1100, Agilent
Technologies GmbH, Waldbronn, Germany).
Antioxidant Activity in Diets,
Calculations and Statistical Analysis Excreta, and Serum
Apparent ileal CP, AA, and EP digestibilities were cal- Total intake and digestibility of EP in the birds fed the
culated using the following formula: 100% ( [100% × (AIA GP diet were significantly increased (P < 0.05) up to 1.6
512 GOÑI ET AL.

and 2.3 times, respectively, compared with birds fed the

70.08
73.69
71.07
69.24
67.96
3.47

0.01
Cys

NS
NS

NS
NS
supplemented and unsupplemented vitamin E diets (Ta-
ble 5).

88.60
89.96
88.65
88.71
88.09
1.40

0.01
Antioxidant activity in vitamin E and GP diets exhib-
Tyr

NS
NS

NS
NS
ited significantly higher scavenging free radical capacity
than control diets using ABTS (3.4 and 6.6 times, respec-
84.00
86.55
86.77
85.65
84.57
1.72

0.05
tively) and FRAP (1.4 and 1.5 times, respectively) meth-
Ser

NS
NS

NS
NS
ods. Similarly, the birds fed vitamin E and GP diets exhib-
ited significantly higher scavenging free radical capacity
86.49ab
85.02b

84.31b
84.18b
87.59a

in excreta than those fed control diets using ABTS (1.2

Pro

1.67

0.01
NS
NS

NS
NS
Table 4. Apparent ileal digestibility (%) of protein and essential and nonessential amino acids of broiler chicks (0 to 21 d) fed grape pomace1 (GP) and vitamin E

and 1.2 times, respectively) and FRAP (1.2 and 1.2 times,
respectively) methods.
82.35b
84.68b

82.10b
81.30b
89.41a

The dietary treatment did not affect the antioxidant

2.65

0.01
Gly

NS
NS

NS
NS

activity measured on serum (Table 6). Animals fed diets

containing GP or vitamin E showed more elevated values
90.23

87.68
91.30

89.62
89.63
1.57

0.05
0.01

than the control group, but the difference was not sig-
Glu

NS
NS

NS

nificant.
84.97

86.32
86.30

85.81
84.48
1.74
Asp

MDA Concentration
NS
NS

NS
NS
NS

The extent of lipid oxidation, as measured by MDA

86.85

88.47
88.74

87.46
87.28
1.83
Ala

NS
NS

NS
NS
NS

formation, in breast and thigh meats was significantly

lower (P < 0.05) in the supplemented vitamin E diet, in
a range of 25 to 58%, than the control group after 1, 4,
87.82

87.95
89.61

87.01
84.75
2.12

0.05
His

NS
NS

NS
NS

and 7 d of refrigerated storage. The inclusion of GP in the

%

diets significantly reduced MDA values in breast samples

after 4 (P < 0.05, up to 33%) and 7 d (P < 0.001, up to
86.12

87.26
87.50

86.00
84.99
2.18
Val

NS
NS

NS
NS
NS

47%) of refrigerated storage and in thigh samples (P <

0.001, up to 30%) at 7 d compared with samples obtained
from birds fed the control diet. Malondialdehyde values
81.34

83.82
83.36

82.25
81.26
2.33

0.05
Thr

NS
NS

NS
NS

of thigh meat samples from birds fed GP diets at 1, 4,

and 7 d were significantly increased (67, 53, and 32%,
81.84

82.44
83.65

81.81
80.73
2.28

0.01

respectively) compared with samples obtained from birds

Phe

NS
NS

NS
NS

fed the vitamin E diet. A linear response was observed

in breast and thigh meats (P < 0.01) at 4 and 7 d, respec-
91.66

93.18
93.19

92.64
92.79
1.47
Met

tively, with increasing content of GP in the diet. Likewise,

NS
NS

NS
NS
NS

a quadratic response was also observed in breast meat

(P < 0.001) at 7 d of refrigerated storage (Table 6).
88.49

91.15
90.47

89.11
88.09
2.01

0.05
Lys

NS
NS

NS
NS

Liver α-Tocopherol
88.41

89.84
90.33

88.78
88.84
1.40

0.05
Leu

NS
NS

NS
NS

The inclusion of vitamin E in the diet increased P <

0.001) liver α-tocopherol concentration (47%) compared
with the control diet. Liver α-tocopherol concentration
87.83

88.85
89.14

88.06
87.10
1.73

NS
NS

NS
NS
NS
Ile

Data are means of 8 chicks for each treatment.

was increased (P < 0.01; up to 33%) in birds fed GP diets

Probability value of contrast; NS = P > 0.05.

compared with those fed control diet. Likewise, the inclu-

sion of GP in the diets reduced (P < 0.001) liver α-tocoph-
91.51

92.75
93.44

92.49
91.66
1.17

0.05
Arg

NS
NS

NS
NS

erol concentration (20%) compared with the vitamin E

diet. A linear response (P < 0.01) was observed in liver
Due to percentage GP in diet.
85.03

85.26
85.38

84.73
84.50
1.41

α-tocopherol concentration at 21 d of age with increasing

NS
NS
NS

NS
NS
CP

content of GP in the diet (Table 6).

Control vs. vitamin E
Statistical significance2

DISCUSSION
Control + vitamin E

Vitamin E vs. GP
Type of response3
Control + 15 GP
Control + 30 GP

The performance of chicks for each experimental group

Control + 5 GP

GP vs. no GP
Pooled SEM

indicated that GP exerted no growth-promoting effect

Treatments

Quadratic

when administered up to 30 g/kg. There are few refer-

Control

Linear

ences in the literature in relation to the use of grape by-

1

products in chicken feed. Hughes et al. (2005) and Lau

 GRAPE POMACE IN CHICKEN DIETS 513
Table 5. Total intake, digestibility, and antioxidant activity of extractable polyphenols in diets, excreta, and serum of broiler chicks fed diets
containing grape pomace (GP) and vitamin E

Antioxidant activity (␮mol of Trolox equivalent/g)

1 2
Total Digestibility Diet Diet2 Excreta1 Excreta1 Serum3
Treatment intake1 (g) (%) (ABTS method) (FRAP method) (ABTS method) (FRAP method) (ABTS method)

Control 2.23b 14.3b 1.87 8.63 63.2b 75.7b 370

Control + vitamin E 2.35b 16.0b 6.29 11.96 75.1 88.6a 390
Control + 30 GP 3.57a 32.8a 12.26 12.95 75.8a 89.9a 391
Pooled SEM 0.11 5.96 0.33 0.69 7.54 6.89 33.51

Means in columns with no common superscript differ significantly (P < 0.05).

a,b

1
Data are means of 4 pens of 6 chicks each.
2
Data are the mean of 3 determinations. ABTS = 2, 2-azinobis (3-ethilenzotiazolin)-6-sulfonate; FRAP = ferric antioxidant power.
3
Data are means of 8 chicks.

and King (2003) reported growth depression in chickens
fed diets containing grape seed extract. The poor response
obtained from the former studies could be justified, be-
cause grape seed extract was a pure form containing
90.2% of total phenolics, expressed as a gallic acid equiva-
lent by the Folin method, and incorporated in the diet at
30 g/kg. In the current experiment, grape seed pomace
contained 4.86% of total polyphenols by the Folin method.
Thus, the total EP in the diet containing the highest pro-
portion of GP were 0.41%. Similarly, the concentration of
condensed tannins present in the higher concentration of
GP diet could be relatively low to produce a growth
depression effect. The effect of polyphenols has also been
studied in chickens using ingredients like sorghum and
faba bean. In general, relatively high dietary concentra-
tions of polyphenols by the addition of these ingredients
reduced performance in chickens as well as other live-
stock (Gualtieri and Rapaccini, 1990; Jansman et al., 1989;
Nyachotti et al., 1997).
Polyphenolic compounds are also known for their abil-
ity to interact with different molecules such as proteins.
In the current experiment, apparent ileal digestibility of
protein and essential and nonessential amino acids were
not affected. This lack of effect could be attributed to the
low content of polyphenols in the experimental diets to
cause detrimental effect. Polyphenols bind to proteins
due to the interaction of their reactive hydroxyl groups
with the carbonyl group of protein. As a result of this
complexation, protein and AA digestibility were reduced
by the inclusion of sorghum and faba bean polyphenols
in chicken and pig diets (Rostagno et al., 1973; Jansman et
al., 1989; Ortiz et al., 1993). Inhibition of different digestive
enzymes by tannins has also been reported (Reddy and
Pierson, 1985).
There are many references in the literature to the com-
position and antioxidant properties of grape polyphenols
(Gonzalez-Paramás et al., 2004; Yilmaz and Toledo, 2004),
but there have been very few studies on the digestibility
and intestinal degradation of polyphenols and other ma-
jor grape constituents. Available data on the absorption
and metabolism of polyphenols suggest negligible bio-
availability of polymeric proanthocyanidins (Dèprez et

Table 6. Effect of refrigerated storage on lipid oxidation of breast and thigh meats and liver α-tocopherol content
of broiler chicks fed diets containing grape pomace1 (GP) and vitamin E

Malondialdehyde concentration α-Tocopherol

(mg/kg of meat) (␮g/g of tissue)
d1 d4 d7 d 21

Treatments Breast Thigh Breast Thigh Breast Thigh Liver

a a a a a a
Control 0.13 0.13 0.30 0.30 0.47 0.43 18.19c
Control + vitamin E 0.09b 0.09b 0.24b 0.19c 0.32c 0.28d 26.66a
Control + 5 GP 0.10ab 0.14a 0.29a 0.29a 0.38b 0.37b 21.39bc
Control + 15 GP 0.13a 0.15a 0.21b 0.27ab 0.25dc 0.35bc 23.56ab
Control + 30 GP 0.11ab 0.14a 0.20b 0.23b 0.27d 0.30cd 24.25ab
Pooled SEM 0.029 0.034 0.043 0.038 0.031 0.433 3.03
Statistical significance2
Control vs. vitamin E 0.05 0.05 0.05 0.05 0.05 0.001 0.001
GP vs. no GP NS NS 0.05 NS 0.001 0.001 0.01
Vitamin E vs. GP NS 0.001 NS 0.001 NS 0.001 0.001
Type of response3
Linear NS NS 0.001 0.01 0.001 0.01 0.01
Quadratic NS NS NS NS 0.001 NS NS

Means in columns with no common superscript differ significantly (P < 0.05).

a–d
1
Data are means of 7 chicks for each treatment.
2
Probability value of contrast; NS = P > 0.05.
3
Due to percentage GP in diet.
514 GOÑI ET AL.
al., 2000). In the current experiment, the digestibility of
EP was 32.8%; this means that a higher amount of EP
was available in the intestinal tract of the animal-fed diet
containing GP that could be partially absorbed in the
small intestine. However, the antioxidant capacity of se-
rum from GP-fed birds did not show significant differ-
ences compared with the other dietary groups. The digest-
ibility of nonextracted polyphenols present in the diets
has also been determined (Brenes et al., unpublished
data), but negative digestibility values were obtained.
Possible interferences between polyphenols with a high
degree of polymerization and polyphenols associated
with high molecular weight compounds and other com-
pounds like amino acids and sugars, together with limita-
tions in the extraction techniques, make the determination
difficult. The increase in the antioxidant activity of EP in
the excreta (20%) in the highest grape pomace-fed birds
compared with those fed the control diet suggests that
part of polyphenols are degraded by intestinal microflora.
Goñi et al. (2005) reported that intestinal bacteria showed
a high capacity to degrade EP in rats. Dèprez et al. (2000)
and Ward et al. (2004) also reported that major polyphe-
nolic constituents of GP (polymeric proanthocyanidins)
were degraded by human colonic microflora into smaller
compounds including phenolic acids that could be ab-
sorbed and metabolized. Özkan et al. (2004), Dolara et al.
(2005), and Papadopoulou et al. (2005) also demonstrated
that phenolic grape extracts and red wine polyphenols
influenced intestinal microflora, decreasing the number of
Propionibacteria, Bacteroides, and Clostridia and increasing
Lactobacilli and Bifidobacteria numbers. Similarly, dietary
fiber associated with polyphenols in GP could play an
additional mechanism to stimulate the intestinal fermen-
tation and to influence the production of particular micro-
bial metabolites. Such findings on the effectiveness of
polyphenolic compounds may be beneficial as alterna-
tives to the dietary antimicrobial growth promoters,
which are currently banned within the European Union.
Future studies must be done to determine the nature
and extent of specific components of polyphenols with
potential antimicrobial properties.
Nutritional interest in polyphenolic compounds has
increased greatly in light of their antioxidant capacity
(Scalbert and Williamson, 2000). The relative contribution
of EP to the total antioxidant activity in excreta, obtained
by the FRAP and ABTS methods, depends on the diet.
The influence of different factors on the effectiveness of
antioxidants in complex heterogeneous foods and biologi-
cal systems cannot be evaluated using only 1 assay. The 2
systems chosen to evaluate the antioxidant activity (FRAP
and ABTS) measure the total reduction power and the free
radical-scavenging activity. In the current experiment, the
vitamin E and the GP diets exhibited the highest antioxi-
dant activity in comparison with the control diet using
both methods. The antioxidant compounds present in
grape have already been identified as phenolic acids (ben-
zoic and hydroxycinnamic acids), stilbene derivatives,
flavan-3-ols (catechin and epicatechin), flavonols (querce-
tin and and myricetin), and anthocyanidins (Caillet et al.,
2006). The antioxidant activity caused by the presence of
these compounds could have additional effects, sparing
other antioxidants and protecting molecules from oxida-
tive damage during digestion and preserving the intesti-
nal epithelium from potential oxidative damage caused
by dietary factors or bacterial metabolism (Scalbert and
Williamson, 2000; Goñi and Serrano, 2005).
Using the MDA content and the digestibility of EP as an
index of absorption of the dietary grape seed constituents
indicates that the antioxidant compounds occurring in
this wine by-product could be distributed, retained, and
remained functional in muscle and liver. This research
carried out in chickens lends support to observations pre-
viously reported by Tang et al. (2000) on the significant
effect of tea catechin in chicken meat quality and in the
comparable effectiveness of this dietary polyphenol as
compared with dietary α-tocopheryl acetate. Similarly,
this study confirms in vitro observations that the addition
of wine polyphenols to various food systems (fish lipids,
frozen fish, and turkey meat) inhibits lipid oxidation (Lau
and King, 2003; Pazos et al., 2005; Mielnik et al., 2006)
and those that provide an enhancement of the antioxidant
defense potential in kidney and liver of rats by flavonol-
rich red wine (Fremont et al., 2000; Rodrigo et al., 2002,
2005). In the current experiment, the liver concentration
of vitamin E was similar to those reported by Villaverde
et al. (2004) in chickens using a high polyunsaturated
fatty acid diet. A reduction in α-tocopherol deposition in
chicks fed unsaturated diets was also reported by Surai
and Sparks (2000) and Sijben et al. (2002). The increase
in liver vitamin E content by the inclusion of GP could
be due to the sparing effect of GP on vitamin E in the
intestine. Less vitamin E would be destroyed through
oxidation, resulting in greater amounts of absorbed vita-
min E. It has been reported that polyphenols [quercetin,
(-)-epicatechin and (+)-catechin], owing to their 1-electron
reduction potentials, may spare vitamin E to delay lipid
oxidation and to regenerate tocopherol in rat and human
models (Frank, 2005). Hence, addition of feedstuffs rich
in these bioactive compounds may prove beneficial for
the enhancement of vitamin E status and for the reduction
in lipid oxidation of the tissues.
Results in this study also confirm that dietary GP and
vitamin E can delay lipid oxidation in breast and thigh
chicken meats and reduce the potential risk induced by
lipid oxidation products. Similar results have been re-
ported using tea catechins by Tang et al. (2000, 2001) and
vitamin E in chickens by Maraschiello et al. (1999) and De
Winne and Dirinck (1996). Dietary GP supplementation at
the level of 15 and 30 g/kg was effective in delaying lipid
oxidation compared with 200 mg/kg α-tocopheryl acetate
diet in breast (4 and 7 d of storage) and thigh meat (7
d of storage). The greater efficiency in preventing lipid
peroxidation in breast meat compared with thigh meat
by dietary GP could be justified by the greater susceptibil-
ity of thigh meat to oxidation attributed to the higher
absolute content of polyunsaturated fatty acids and the
large amount of prooxidative agents originating from
myoglobin and other Fe-containing proteins in thigh mus-
 GRAPE POMACE IN CHICKEN DIETS
cle tissues compared with breast muscle tissues (Wen et
al., 1997; Higgins et al., 1998; Botsoglou et al., 2003b).
In conclusion, the results presented in this study
showed that increasing the concentration of GP up to 30
g/kg did not impair performance and protein and AA
digestibilities. An increase in the antioxidative activity of
broiler diet, excreta, and meat as a result of the dietary
administration of GP and vitamin E was also reported.
The better oxidative stability of meat samples receiving
the diet supplemented with the higher concentration of
GP was accompanied by an increase in the concentration
of vitamin E in the liver. Therefore, GP could be consid-
ered as a good alternative to α-tocopheryl acetate supple-
mentation of feeds and to improve vitamin E status. Anti-
oxidant constituents (mainly flavonoids), present in GP,
might be considered responsible for this mechanism and
on the sparing effect of vitamin E in liver. Currently, work
is in progress with the aim to assess the effects of dietary
concentrations of polyphenols by the addition of GP or
their isolated bioactive ingredient on performance, on
sparing effect of vitamin E, and to study the susceptibility
to lipid oxidation of breast and thigh tissues of broiler
chickens during a 42-d growth period.
ACKNOWLEDGMENTS
We thank the Ministerio de Educación y Ciencia for
financial support of this investigation (project AGL2006-
10312/GAN).
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