Effects of grape pomace supplementation to laying hen diet on performance, egg quality, egg lipid peroxidation and some biochemical parameters

Abstract

This study was carried out to determine the effects of dried grape pomace supplementation in a corn-soy-based diet on performance, egg quality, plasma and egg lipid peroxidation, and some biochemical parameters of laying hens. In this study, 96 moulted 80-week-old Bovans laying hens were distributed into three groups with eight replicates. The hens were fed a supplemented diet with 0% (control group), 4% and 6% grape pomace (experimental groups) for 12 weeks. In the study, the effects of grape pomace supplementation in a corn-soy-based diet on performance, internal and external egg quality, serum total cholesterol, total protein, glucose and triglyceride levels, and plasma and egg yolk malondialdehyde (MDA) levels were determined. The addition of grape pomace to laying hen diet did not significantly affect live weight, feed intake, egg production and feed efficiency (P > 0.05). In all treatment groups, supplementation did not significantly affect albumen index, Haugh unit, egg yolk index, yolk colour, eggshell ratio, eggshell thickness, egg-specific gravity, total cholesterol, total protein and triglyceride compared to the control group (P > 0.05). In the study, 4% grape pomace supplementation to diet significantly increased egg weight (P < 0.001), and both 4% and 6% grape pomace supplementation levels significantly increased liver weight (P < 0.05) and liver weight ratio (P < 0.01). Grape pomace addition to laying hens diet significantly decreased plasma MDA and serum glucose levels (P < 0.001). Also, grape pomace addition significantly decreased egg yolk MDA levels on day 1 (incubation on 0 minute; P < 0.001), and day 15 [incubation on 0, 30, 60 and 90 (P < 0.05) minutes]. The results of this study concluded that performance, egg quality and serum total cholesterol, total protein and triglyceride levels were not negatively affected. Plasma and egg yolk MDA, and serum glucose levels were reduced by 4% and 6% supplementation to laying hen diets. It was concluded that grape pomace supplementation has the potential to extend shelf life.

Keywords:

• biochemical parameters
• egg quality
• grape pomace
• malondialdehyde
• laying hen
• performance

1. Introduction

Grape pomace is produced as a by-product during the production of molasses, grape juice, vinegar, dried fruit pulp and wine, in particular (Nerantzis & Tataridis 2006; Sayago-Ayerdi et al. 2009). It contains a high amount of polyphenolic compounds as well as 25–35% crude cellulose, 4–10% hemicellulose, 5–6% pectin, 8–14% crude protein, 4–10% crude fat and 30–45% non-nitrogen substance (Karakaya et al. 2001; Jayaprakasha et al. 2003; Özkan et al. 2004; Kara 2012; Zheng et al. 2012). Karakaya et al. (2001) reported that the amounts of phenolic compounds in dried and red grapes are 3.99 mg/g and 2.21 mg/g, respectively. Pomace produced from Emir grapes and Kalecik black grapes included a total phenolic compound content of 68.77 mg/g and 96.25 mg/g, respectively (Özkan et al. 2004). The polyphenol content of the grape pomace and seed include some flavonoids such as catechin, epicatechin, procyanidin and antocyanidin; some phenolic acids such as gallic and ellagic acid, and some stilbens such as resveratrol and piseid (Jayaprakasha et al. 2003; Shi et al. 2003; Yilmaz & Toledo 2006).

In recent years, consumers have become interested in functional eggs (those containing high selenium, high polyunsaturated fatty acids (PUFAs) and herbal antioxidants etc.; Sahin et al. 2008; Jung et al. 2011; Ghasemi et al. 2014; Zhang & Kim 2014). There has been much information about the positive effects of functional eggs on human health. These effects are including cardioprotective, antioxidant, anti-ageing, anticancer, immunomodulatory etc. PUFAs are found in the lipids of egg yolk, and these fatty acids are susceptible to lipid oxidation depending on storage conditions and storage duration (Sahin et al. 2008, 2010; Jung et al. 2011; Razmaite et al. 2014). Antioxidant supplements improve animal origin food quality regarding colour, oxidative stability, tenderness and storage properties. Therefore, antioxidants of a kind of natural polyphenols (catechins, resveratrol), carotenoids and tocopherols have been used (Biswas et al. 2000; Uuganbayar et al. 2005; Sahin et al. 2008, 2010; Jung et al. 2011).

Polyphenols, mainly condensed tannins (proanthocyanidins), of these ingredients, may have negative effects on animal performance by reducing fat and protein absorption associated with the complexes they form with macromolecules (e.g. protein and fat) and enzymes (e.g. protease and lipase) in the digestive tract (Roy & Schneeman 1981; Griffiths 1986; Manach et al. 2004). Silici et al. (2011) indicated that supplementing quail diets with grounded grape seed did not change egg performance, feed consumption, egg weight and egg quality measured by Haugh unit, egg-specific gravity, eggshell thickness and yolk colour; however, it increased feed conversion efficiency. Studies carried out on broilers show that grape pomace and grape seed extract supplementation to diet had limited effects on performance parameters and protein and amino acid digestibility of broilers (Goñi et al. 2007; Brenes et al. 2008, 2010). However, there is evidence that grape pomace supplementation decreases fat digestibility in broilers (Goñi et al. 2007; Brenes et al. 2008). In addition, Brenes et al. (2008) reported that red grape pomace (15, 30 and 60 g/kg) and α-tocopheryl acetate (200 mg/kg) inclusion of broiler diet reduced lipid peroxidation in breast meat stored at +4°C. It has been stated that there were no significant differences in MDA concentration between red grape pomace supplementation compared with α-tocopheryl acetate supplementation (Brenes et al. 2008). Sayago-Ayerdi et al. (2009) found that meat Thiobarbituric acid reactive substances (TBARS) concentration of broilers fed with 3% and 6% concentrated grape pomace significantly decreased at six months of storage in raw and cooked samples.

It appears that there is very little research on the possible influence of feeding laying hens with grape pomace on performance and egg quality, and lipid peroxidation. This study was, therefore, conducted to determine the effects of supplementing laying hen diets with dried grape pomace on performance, egg quality, plasma and egg yolk lipid peroxidation, and some biochemical parameters.

2. Materials and methods

2.1. Experimental design and animal management

Animal care procedures for the experiment were conducted under a research protocol approved (09-08/48) by the Local Ethics Committee for Animal Experiments of Erciyes University, Kayseri-Turkiye. In this study, a total of 80-week-old 96 Bovans laying hens which had moulted were used. The chickens were assigned to three groups, 32 hens each as eight replicates. The live weights of all animals were recorded at the beginning and end of the study. The chickens were fed with basal diet (control group) and an isocaloric and isonitrogenic diet including 4% and 6% dried grape pomace (experimental groups) for 12 weeks. The regional origin of the dried grape pomace used in the present study was Cappadocia in the Nevşehir province of Turkiye. It was obtained after drying and grinding of the wet grape pomace, a by-product of separating the grape must from Dimrit grapes which are used in wine production. The hens were exposed to 17 hours of light including daylight in the coop. Feed and water were provided to the animals ad libitum. The ingredient and nutrient composition of the diet, and the nutrient and phenolic substance composition of grape pomace used in this study were presented in Tables 1 and 2 (Kara & Kocaoglu-Guclu 2012), respectively. The dry matter, crude protein, crude cellulose, diethyl ether extract, crude fiber and crude ash compositions of the diets were analysed according to the Association of Official Analytical Chemists (Association of Official Analytical Chemists 1990) methods. Neutral detergent fiber and acid detergent fiber contents of grape pomace were determined according to Van-Soest et al. (1991). The phenolic substances composition of grape pomace were analysed by using the methods of Al-Farsi et al. (2005) and Mattila et al. (2006).

Table 1. The ingredient and nutrient composition of diet used in the study.

		Experimental groups
	Control group	4% GP	6% GP
Ingredients [% of DM]
Maize	56.00	52.08	50.40
Soybean meal (with 48% CP)	17.10	15.90	15.00
Wheat	10.40	9.77	9.35
Limestone	9.05	8.42	8.26
Sunflower meal (with 36% CP)	3.10	2.77	2.79
Corn gluten (with 60% CP)	1.00	0.90	0.80
Full fat soy	–	2.00	3.00
Dried grape pomace	–	4.00	6.00
Dicalcium phosphate	2.30	2.14	2.30
Vegetable oil	0.50	1.47	1.55
Sodium chloride	0.25	0.25	0.25
Vitamin-mineral Premix*	0.20	0.20	0.20
Enzyme**	0.10	0.10	0.10
Calculated composition
Dry matter, %	90.18	90.13	90.18
Crude protein, %	15.52	15.61	15.56
Calcium, %	3.86	3.62	3.60
Phosphorus (available), %	0.48	0.45	0.48
Lysine, %	0.73	0.74	0.65
Methionine + cystine, %	0.55	0.59	0.57
Metabolizable energy, kcal/kg	2727.13	2745.87	2722.00
Analyzed values
Dry matter, %	91.52	91.34	91.49
Crude protein, %	16.18	16.24	16.19
Crude fiber, %	5.86	6.20	6.34
Diethyl ether extract, %	3.23	3.35	3.56
Crude ash, %	10.80	10.86	10.77

*One kilogram of vitamin-mineral premix: Vitamin A, 6000000 IU; vitamin D₃, 1200000 IU; vitamin E, 15000 mg; vitamin K₃, 2500 mg; vitamin B₁, 1250 mg; vitamin B₂, 2500 mg; vitamin B₆, 2000 mg; vitamin B₁₂, 15 mg; folic acid, 325 mg; choline chlorite, 150000 mg; calcium pantothenate, 150000 mg; D-biotin, 25 mg; Fe, 40000 mg; Mg, 40000 mg; Zn, 40000 mg; I, 300 mg; Cu, 5000 mg; Se, 125 mg; Co, 150 mg; antioxidant, 25 mg.

**One kilogram of enzyme premix (Kavimix® Safizyme GP60/Turkey): Endo-1.3(4)-β glucanase 1420000 IU, endo-1.4- xylanase 600000 IU, cellulase 10200 IU.

Table 2. Nutrients and phenolic substances composition of grape pomace (Kara & Kocaoglu-Guclu 2012).

Parameters	Concentration
Dry matter	916.00 g/kg
Crude protein	117.20 g/kg
Diethyl ether extract	55.40 g/kg
Crude ash	76.50 g/kg
Crude fiber	266.30 g/kg
Neutral detergent fiber	466.00 g/kg
Acid detergent fiber	318.90 g/kg
Flavonoids
Catechin	1.10 mg/kg
Epicatechin	5.30 mg/kg
Gallocatechin	0.50 mg/kg
Epigallocatechin	4.30 mg/kg
Phenolic acids
Gallic acid	393.90 mg/kg
Caffeic acid	3.90 mg/kg
p-cumaric acid	0.00 mg/kg
Total phenol	733.37 mg gallic acid equivalent/100 g

2.2. Performance and egg quality

The egg production was recorded daily. Feed conversion efficiency was calculated by measuring the feed consumption and egg weights of the animals every second week. The egg-specific gravity (g/cm³) of all eggs was calculated, using the Archimedes (Thompson & Hamilton 1982; Hempe et al. 1988) method every month. The eggs were weighed (g) in a precision scale after being kept at room temperature for 24 hours. The egg albumen height, yolk height, yolk diameter, albumen length and width (mm × 10⁻²) of all eggs were measured and used to calculate the Haugh unit, yolk index and albumen index. Haugh unit, an indicator of egg albumen quality, was calculated using the following formula: Haugh unit = 100 × log (H + 7.57 − 1.7 × W ^0.37), where H = albumen height (mm) and W = egg weight (g) (Eisen et al. 1962). The colour evaluation of the egg yolks were performed by using the Roche colour scale. The eggshell weights were recorded once the membranes of the eggshells of the broken eggs were removed. These parameters were used to calculate the ratio of the eggshell weight (%). The eggshell thickness (mm × 10⁻²) was measured using a micrometer (Mitutoyo, Dial Caliper Gage, Japan) by averaging the samples of three different locations on the eggshell from which the eggshell membranes had been removed (Wells 1968).

2.3. Egg yolk MDA

In the last week of the study, the malondialdehyde (MDA) levels of egg yolks were analysed in 40 shell eggs, of which 20 samples were analysed straight away on the first day of collection, while the other half was kept at +4°C for 15 days. To determine the lipid oxidation (shelf-life) in egg yolk during storage we stored shell eggs. MDA concentrations were determined according to the modified distillation (Kara & Kocaoglu-Guclu 2012) method described by Kornbrust and Mavis (1980). Approximately 1.0 g of egg yolk samples were diluted in 9 mL of KCl (1.15%). A measure of 0.1 mL of these diluted were homogenized in 5 mL of 80 mM Tris-maleate buffer (pH 7.4), 0.2 mL of 5 mM iron sulphate and 0.2 mL of 2 mM ascorbic acid at 3000 rpm for 10 s in capped tubes on a vortex (Velp, Italy). Homogenates were incubated at 37°C in a shaking water bath (15 rpm; Memmert, Germany) for incubation duration on 0, 30, 60 and 90 minutes. After incubation, 2 mL of TCA-TBA-HCl mixture (150 g of trichloroacetic acid and 3.75 g of thiobarbituric acid were dissolved in 1 L of 0.25 N HCl) was added to homogenates. The tubes were incubated at 100°C and then cooled. The tubes were centrifuged at 2200 rpm (+4°C) for 20 minutes in a cooled centrifuge (Nüve NF800R, Turkey). The absorbance values of upper supernatants were measured on a spectrophotometer (Shimadzu 1208 UV/VIS, Japan) at 535 nm against a blank. The concentration of MDA in analysed samples (nmol/mg, yolk) was calculated using the following formulation: (MDA, nmol/mg = 6.4102 × 1000 × 3 × absorbance value)/(100 × mg/mL samples).

2.4. Blood biochemical parameters

Cervical dislocation was performed in 12 chickens (will not be used in production) in each group. The blood samples collected into tubes with heparin and silicon were centrifuged at 3000 rpm for 10 minutes. The MDA concentrations of the plasma samples were measured by the method described by Moreno et al. (2003). For the determination of plasma MDA levels, 0.5 mL plasma and 0.025 mL butylatedhidroxytoluen [glacial acetic acid, 1% (w/v)] was stirred, and then 1 mL 0.8% (w/v) 2-thiobarbituric acid solution was added. The mixture was heated at 95°C in a water bath for 30 minutes. Chromogen was extracted with 3 mL n-butanol and centrifugation at 4000 rpm for 10 minute. The absorbance of the separated organic phase was measured on a spectrophotometer (Shimadzu 1208 UV/VIS, Japan) at 535 nm. The absorbance of standard solutions was prepared with 1,1,3,3-tetraethoxypropan and the concentration ranged from 1 to 20 µM/L were measured and standard curve was plotted. The MDA levels of samples were determined via the absorbance values comparison with standard curve.

Serum samples were also analysed for glucose, total cholesterol, total protein and triglyceride levels, using a Shimadzu 1208 UV/VIS (Japan) brand spectrophotometer and commercial kits (Biolabo, France).

2.5. Statistical analysis

The statistical analysis of data was performed using SPSS 15.0 software. The statistical significance between groups was determined by one-way analysis of variance (ANOVA) analysis. ‘Duncan's Multiple Range Test’, one of the multiple comparison tests, was used when the difference between groups was found to be statistically significant. The measure of statistical significance was a P value below 0.05 (P < 0.05). The data were presented on the basis of mean ± standard error of mean.

3. Results

In this study, grape pomace addition to laying hen diet did not change the live weight, egg production, feed intake and feed conversion efficiency (P > 0.05); however, it increased the liver weight (P < 0.05) and liver weight ratio (P < 0.01; Table 3). The egg weight was significantly higher (P < 0.001) in the group fed with diet containing 4% grape pomace than in the control group (Table 4). There was no significant difference among the groups in terms of albumen index, Haugh unit, yolk index, eggshell ratio, eggshell thickness, egg-specific gravity, serum total cholesterol, total protein and triglyceride levels (P > 0.05; Table 4).

Table 3. The effects of grape pomace supplementation to laying hen diet on performance.

		Experimental groups
Parameters	Control group	4% GP	6% GP	SD	P value
Initial live weight (g)	1521.00 ± 53.20	1541.37 ± 30.10	1526.19 ± 39.92	173.45	0.85
Final live weight (g)	1505.45 ± 52.27	1555.43 ± 37.74	1535.57 ± 50.53	186.15	0.74
Liver weight (g)	26.59 ± 1.15^b	31.66 ± 1.93^a	33.03 ± 1.43^a	5.89	<0.03
Liver weight ratio (%)*	1.76 ± 0.06^b	2.02 ± 0.09^a	2.15 ± 0.09^a	0.33	<0.01
Feed consumption (g)	114.36 ± 2.34	117.83 ± 2.08	115.10 ± 1.64	3.47	0.43
Egg production (%)	78.90 ± 2.43	83.56 ± 1.13	78.38 ± 2.33	4.18	0.22
Feed efficiency**	2.25 ± 0.06	2.12 ± 0.05	2.28 ± 0.10	0.07	0.13

Note: Means with different superscript letters in the same row differ at P < 0.05 (Duncan test).

GP, Grape pomace; SD, standard deviation.

*(weight of liver/live weight) ×100; **kg feed/kg egg.

Table 4. Effects of grape pomace supplementation to laying hen diet on internal and external egg quality.

		Experimental groups
Parameters	Control group	4% GP	6% GP	SD	P value
Egg weight (g)	64.57 ± 0.24^b	66.38 ± 0.23^a	65.06 ± 0.30^b	0.96	<0.001
Egg albumen index (%)	8.45 ± 0.33	9.72 ± 1.42	9.29 ± 0.33	1.20	0.30
Haugh unit	82.71 ± 1.35	82.70 ± 1.12	85.87 ± 1.11	2.87	0.41
Egg yolk index (%)	41.41 ± 0.38	40.34 ± 0.38	40.73 ± 0.42	0.79	0.62
Egg yolk colour	10.95 ± 0.15	11.13 ± 0.09	11.02 ± 0.11	0.07	0.85
Egg shell ratio (%)	9.75 ± 0.12	9.39 ± 0.17	9.35 ± 0.12	0.13	0.54
Egg shell thickness (mm × 10^–2)	34.45 ± 0.004	33.91 ± 0.004	33.96 ± 0.004	0.36	0.72
Egg specific gravity (g/cm^3)	1.0852 ± 0.008	1.0846 ± 0.007	1.0858 ± 0.007	0.001	0.67

Note: Means with different superscript letters in the same row differ at P < 0.05 (Duncan test).

GP, Grape Pomace; SD, standard deviation.

It was found that the plasma MDA and serum glucose levels decreased significantly with the addition of 4% and 6% grape pomace (P < 0.001; Table 5). The addition of grape pomace as a supplement to laying hen diets significantly reduced the yolk MDA concentration of both eggs analysed on the first day of collection (P < 0.001 at 0 minute incubation) and eggs stored at +4°C for 15 days (P < 0.001 at 0, P < 0.05 at 30, 60 and 90 minute incubations; Table 6).

Table 5. Effects of grape pomace supplementation of laying hen diet on plasma biochemical parameters.

		Experimental groups
Parameters	Control group	4% GP	6% GP	SD	P value
Glucose (mg/dL)	248.71 ± 11.36^a	199.68 ± 8.08^b	206.69 ± 7.40^b	37.71	<0.001
Total cholesterol (mg/dL)	130.28 ± 9.27	144.09 ± 10.26	133.25 ± 6.36	30.17	0.51
Triglyceride (mg/dL)	603.20 ± 112.21	615.14 ± 122.31	618.21 ± 150.14	398.89	0.99
Total protein (g/dL)	4.65 ± 0.46	5.28 ± 0.46	4.58 ± 0.40	1.51	0.48
Malondialdehyde (μM/L)	19.00 ± 0.42^a	16.31 ± 0.58^b	17.52 ± 0.30^b	1.87	<0.001

Note: Means with different superscript letters in the same row differ at P < 0.05 (Duncan test).

GP, Grape Pomace; SD, standard deviation.

Table 6. Effects of grape pomace supplementation of laying hen diet on egg yolk MDA levels.

		Egg yolk MDA level (nmol/mg)
		Incubation times
		0th min	30th min	60th min	90th min
		Day 1
Control group		0.119 ± 0.038^a	0.094 ± 0.012	0.257 ± 0.049	0.264 ± 0.070
Experimental groups	4% GP	0.054 ± 0.008^b	0.084 ± 0.011	0.143 ± 0.022	0.263 ± 0.070
	6% GP	0.044 ± 0.008^b	0.087 ± 0.014	0.116 ± 0.016	0.240 ± 0.050
SD		0.105	0.053	0.219	0.284
P value		<0.001	0.85	0.09	0.95
		Day 15
Control group		0.152 ± 0.027^a	0.335 ± 0.088^a	0.403 ± 0.106^a	0.806 ± 0.174^a
Experimental groups	4% GP	0.051 ± 0.007^b	0.123 ± 0.011^b	0.181 ± 0.051^b	0.410 ± 0.079^b
	6% GP	0.055 ± 0.010^b	0.159 ± 0.045^b	0.171 ± 0.042^b	0.421 ± 0.081^b
SD		0.081	0.241	0.284	0.504
P value		<0.001	<0.02	<0.05	<0.05

Note: Means with different superscript letters in the same row differ at P < 0.05 (Duncan test).

GP, Grape Pomace.

4. Discussion

The number of studies concerning the impact of grape pomace and seed on poultry especially laying hens is limited. Research indicates that when supplemented by 3%, grape pomace has no adverse effects on broilers (Goñi et al. 2007; Brenes et al. 2008; Sayago-Ayerdi et al. 2009); however, inclusion over 6% may reduce feed intake (Sayago-Ayerdi et al. 2009) and fat digestion (Brenes et al. 2008). On the other hand, Wang et al. (2008) reported that procyanidin extract of grape seed supplemented at 5, 10, 20, 40 and 80 mg/kg diet improved the low performance of broilers associated with oxidative stress that was caused by parasite infestation. In this study, including 4% and 6% grape pomace supplementation to laying hen diets did not have any significant impact on egg production and feed intake. This result is further supported by the findings of the study carried out by Silici et al. (2011) in which 1.0% and 1.5% grape pomace were added to the diet of breeder quails. A similar result was reported by Kara and Kocaoglu-Guclu (2012) who found that supplementing grape pomace at 2% did not show any significant effect on the average egg production, egg weight and feed conversion efficiency of moulted laying hens. The fact that supplementing grape pomace at 4% increased the egg weight in this study can be attributed to the augmentative effect of grape pomace on probiotic bacteria count in the intestines (Viveros et al. 2011). The results in the present study suggest that including grape pomace into laying hen diets at 4% and 6% increased the liver weight and liver weight ratio. On the contrary, Gladine et al. (2007b) indicated that the inclusion of plant (rosemary, grape, citrus and marigold) extracts rich in polyphenols (5 g/kg of diet) in the diet of male Wistar rats did not alter the liver weight. Our findings on the fact that including 4% and 6% grape pomace into laying hen diets did not have any significant effects on egg quality are in line with those reported by Kara and Kocaoglu-Guclu (2012) who found that supplementing laying hen diets with 2% grape pomace did not alter eggshell thickness, eggshell weight, rate of eggshell weight, albumen index and egg-specific gravity. These results are also supported by Silici et al. (2011), who demonstrated that the addition of grounded grape seed into breeder quail diets at 0.5, 1.0 and 1.5% did not affect Haugh unit, egg-specific gravity, eggshell thickness and yolk colour. On the other hand, a study conducted by Ozgan (2008) reported an increment in albumen height and albumen index with the addition of 2% grape pomace to laying hen diets.

Recently, the antioxidant effects of grape pomace, seed and their extracts have been the subject of much research (Karakaya et al. 2001; Jayaprakasha et al. 2003; Shi et al. 2003; Özkan et al. 2004). It has been widely emphasized that these products have a high potential to increase the shelf life of animal products by reducing lipid peroxidation (Ozgan 2008; Brenes et al. 2008, 2010). In this study, grape pomace supplementation significantly reduced the plasma MDA concentration, which is a similar finding to that reported by Wang et al. (2008). In addition, Gladine et al. (2007a) found that the plasma MDA concentration of rats decreased when fed with plant extracts rich in polyphenols. However, reduced MDA concentration in eggs stored at 4°C at days 1 and 15 is a similar finding to those reporting that the shelf-life of meats increased with the addition of grape pomace (Goñi et al. 2007; Brenes et al. 2008; Sayago-Ayerdi et al. 2009) and grape seed extract (Smet et al. 2008) due to the reduced lipid peroxidation in broiler meats. In a previous study, we demonstrated that supplementation of grape pomace in laying hens at 2% significantly decreased the egg yolk MDA concentrations in fresh eggs (daily) at 0, 30 and 60-minute incubations; however, it had no significant effects on eggs stored at +4°C at days 15 and 30 (Kara & Kocaoglu-Guclu 2012). The antioxidant characteristics of grape and grape products (e.g. grape juice, wine and molasses), and their by-products (e.g. grape pomace and seed) may be associated with the attribute of the phenolic compounds they contain to scavenge free radicals, to form complexes with metal ions and to prevent or reduce the development of singlet oxygen (Rice-Evans et al. 1995; Yilmaz & Toledo 2006; Surai 2014).

No change of serum triglyceride and total cholesterol levels in this study are in agreement with that reporting that including plant extracts rich in polyphenols such as rosemary, grape, citrus and marigold at 5 g/kg diet in rats (Gladine et al. 2007a). This is further reinforced by the findings of Yamakoshi et al. (2002) who also claimed that supplementing the diets of Fischer rats with proanthocyanidin-rich extracts from grape seeds at 0.02, 0.2 and 2% of diet did not alter the total protein levels. The reduced serum glucose level with the addition of 4% and 6% grape pomace in this study shows that grape pomace plays a protective role in the β-cell functions of the pancreas due to the antioxidative effect of proanthocyanidins in grape pomace (El-Alfy et al. 2005). Moreover, research indicates that some phenolic compounds in grape seed may have impacts on inhibiting sodium-dependent glucose transport, increasing insulin resistance and reducing glucose absorption in the digestive tract (Shanmuganayagam et al. 2007). Additionally, Ozgan (2008) stated that plasma glucose level was not affected by the inclusion of 1.0% grape seed oil in laying hen diets.

The impact of grape pomace, seed and extracts used in animal studies on performance, egg quality and lipid peroxidation can vary due to several factors. These factors are (1) the different rates of total polyphenols they contain based on the type of the grape and the soil structure where they were grown; (2) the variation in the processing techniques of these products used, e.g. the production of wine, vinegar and grape juice; (3) the supplementation rate of these products into diet; and (4) other feed compounds. The lower phenolic substance content of the grape pomace used in this study (total phenolic substance level of 733.37 mg GAE/100 g; catechin, epicatechin, gallocatechin and epigallocatechin levels of 1.1, 5.3, 0.5 and 4.3 mg/kg, respectively; and gallic acid, caffeic acid and p-cumaric acid levels of 393.9, 3.9 and 0 mg/kg, respectively) than in other studies (Mcdougall & Stewart 2005; Yilmaz & Toledo 2006; Goñi et al. 2007; Gladine et al. 2007a, 2007b; Brenes et al. 2008; Brenes et al. 2010) may reflect the limited effect on lipid peroxidation in egg yolk and other biochemical parameters.

5. Conclusion

It can be concluded that supplementing laying hen diets with grape pomace at 4% and 6% does not cause any adverse effects on performance and egg quality; however, it may improve the egg shelf life by reducing the MDA concentration in egg yolk. Further studies are required to elaborate the impacts of grape pomace and similar products on animal health, digestive activities, immune system and egg shelf life.