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The effect of vitamin E on laying performance and egg quality in laying hens fed corn dried distillers grains with solubles
Research Note The effect of vitamin E on laying performance and egg quality in laying hens fed corn dried distillers grains with solubles Wen Jiang, Licong Zhang, and Anshan Shan1 Institute of Animal Nutrition, Northeast Agricultural University, Harbin, 150030, P. R. China cantly improved egg production and yolk percentage (P < 0.05). Increasing the dietary levels of vitamin E caused a decrease in cholesterol and an increase in the α-tocopherol concentration of the egg yolk and serum (P < 0.05). Diets supplemented with DDGS decreased the proportion of saturated fatty acids (P < 0.05) and increased the proportion of unsaturated fatty acids in egg yolk (P < 0.05). Supplementation with high levels of vitamin E decreased malondialdehyde and increased glutathione peroxidase and total superoxide dismutase concentrations of the egg yolk and serum (P < 0.05). In conclusion, our results showed that DDGS was successfully fed to laying hens at levels up to 10% without adverse effects on laying performance. Additionally, vitamin E supplementation improved egg production and egg quality and provided health benefits to laying hens.
Key words: corn dried distillers grains with solubles, vitamin E, laying performance, egg quality, egg fatty acid composition 2013 Poultry Science 92:2956–2964 http://dx.doi.org/10.3382/ps.2013-03228
INTRODUCTION Corn dried distillers grains with solubles (DDGS) is a coproduct of ethanol production. During 2010, the US ethanol industry produced 33.4 million metric tons of distillers coproducts (Hoffmann and Baker, 2011), encouraging the use of DDGS in poultry diets. Dried distillers grains with solubles is not a new ingredient for poultry, and has been available for many decades as a by-product of the brewing industry. Dried distillers grains with solubles is recognized as a valuable source of energy, protein, amino acids, water-soluble vitamins, linoleic acid, xanthophyll, and minerals for poultry diets (Scott, 1965; Jensen, 1978, 1981; Parsons and Baker, 1983; Wang et al., 2007; Salim et al., 2010; Cozannet et al., 2010). Recently, Roberts et al. (2007) reported that adding 10% DDGS to laying hen diets did not negative©2013 Poultry Science Association Inc. Received April 8, 2013. Accepted August 4, 2013. 1 Corresponding author: [email protected]
ly affect egg production and egg quality parameters. Lumpkins et al. (2005) and Swiątkiewicz and Koreleski (2006) reported that supplementation with up to 15% DDGS could be used in diets, whereas inclusion of 20% negatively affected laying rate and egg weight. Deniz et al. (2013) observed that corn DDGS up to 15% did not affect egg production and egg quality and reduced feed cost. No differences in hen daily egg production were observed among dietary treatments even at 25% DDGS (Masa’deh et al., 2011). However, Cromwell et al. (1993), Spiehs et al. (2002), Pineda et al. (2008), and Corzo et al. (2009) reported that the use of DDGS in poultry diets had been low, which may be due to the high percentage of crude fiber, unpalatable, sulfur content, high variability in nutrient (especially lysine), and high proportion of polyunsaturated fatty acid of the DDGS. Vitamin E is an indispensable cellular antioxidant (Puthpongsiriporn et al., 2001; Brigelius-Flohé et al., 2002) and may help the immune response in chickens by protecting lymphocytes, macrophages, and other cells against oxidative damage (Franchini et al., 2002; Leeson, 2007). Additionally, Ajuyah et al. (1993)
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ABSTRACT The objective of the present study was to investigate the effect of vitamin E on laying performance, egg quality, egg fatty acid composition, antioxidant capacity, and several biochemical parameters of laying hens fed corn distillers dried grains with solubles (DDGS) during the laying period (40 to 63 wk of age). A total of 360 Hy-Line Variety Brown hens were randomly assigned to 6 groups, consisting of 6 replicates with 10 hens each. Hens were allocated to diets 1 through 6 in a 3 × 2 factorial design. The dietary treatments included 3 levels of DDGS (0, 10, and 20%) and 2 levels of vitamin E (0 and 200 mg/ kg). The results indicated that yolk color and eggshell thickness increased with increasing DDGS (P < 0.05). However, increasing DDGS to 20% in laying hen diets significantly reduced feed conversion (P < 0.05). Supplementation with 200 mg/kg of vitamin E signifi-
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RESEARCH NOTE
MATERIALS AND METHODS Experimental Materials The DDGS was purchased from Fuel Alcohol Limited of Jilin in China. Vitamin E acetate (50% purity) was purchased from Biotechnology Limited in Zhejiang, China. The source of vitamin E used in this study was d-α-tocopherol acetate. The basal level of vitamin E in the diet without additional vitamin E was 10 mg/kg.
Birds and Experimental Design A total of 360 Hy-Line Variety Brown hens were randomly assigned into 6 treatment groups. Each treatment group was composed of 6 replicates with 10 hens each. The hens were allocated to diets 1 through 6 and fed during wk 40 through 63 in a 3 × 2 factorial design. The dietary treatments included 3 levels of DDGS (0, 10, and 20%) and 2 levels of vitamin E (0 and 200 mg/ kg). The diets were a corn-soybean meal basal feed with or without DDGS at 10 and 20%, formulated to meet the NRC (1994) nutrient requirement for laying hens. The compositions of the diets were listed in Table 1.
Management During the laying period, 2 hens were housed in a metal cage (48 × 34 × 39 cm), with a replicate plot consisting of 10 hens in 5 adjoining cages. Hens were maintained on a 16L:8D photoperiod throughout the study. All hens were housed in an environmentally controlled house with the temperature maintained at approximately 20°C. The experimental protocol was approved by the Northeast Agricultural University Ethics Committee on the Use and Care of Animals. These guidelines are similar to those of the US National Institutes of Health.
Data and Sample Collection Laying Performance. Egg production was recorded daily. Feed consumption was recorded weekly by calculating the average daily feed consumption of the 10 hens in each replicate. Egg weight was measured using all eggs produced during 2 consecutive days every week. Egg mass was calculated as laying rate × egg weight, whereas feed conversion (feed consumption/egg mass) was calculated every week throughout the experimental period. Egg Quality. Egg quality was measured on 4 eggs from each replicate the last day of each of the 4-wk periods, and the data were pooled for analysis. Eggshell breaking strength was measured using an eggshell strength tester II (Fujihira Industry Co. Ltd., Tokyo, Japan) after the eggs were broken on a NFN-382 meaTable 1. Composition of experimental diets1 Item Ingredient (%) Corn Soybean meal Fish meal DDGS Sodium chloride Dicalcium phosphate Limestone dl-Methionine l-Lysine∙HCl Choline chloride Vitamin premix2 Mineral premix3 Total Calculated composition ME (MJ/kg) CP (%) Crude fiber (%) Methionine (%) Lysine (%) Calcium (%) Available phosphorus (%) Total phosphorus (%) Analyzed composition (%) DM CP Crude fiber Crude fat Calcium Total phosphorus
0% DDGS
10% DDGS
20% DDGS
62.40 25.50 1.00 0.00 0.30 1.50 8.80 0.08 0 0.10 0.02 0.30 100.00
58.90 19.00 1.00 10.00 0.30 1.42 8.80 0.07 0.09 0.10 0.02 0.30 100.00
56.40 11.50 1.00 20.00 0.30 1.33 8.80 0.06 0.19 0.10 0.02 0.30 100.00
11.03 16.40 3.65 0.34 0.87 3.82 0.42 0.63 89.48 18.42 3.73 4.29 3.68 0.64
10.91 16.34 3.80 0.34 0.88 3.81 0.44 0.65 89.40 17.76 3.82 4.87 3.72 0.61
10.89 16.38 3.86 0.35 0.89 3.80 0.44 0.65 89.37 17.65 3.97 5.04 3.70 0.63
1DDGS: dried distillers grains with solubles. Analyzed composition of DDGS used in the experiment analyzed according to AOAC International (1990) was as follows (%): DM, 89.19; CP, 32.9; crude fat, 8.3; crude fiber, 11.2; crude ash, 5.06; calcium, 0.15; phosphorus, 0.74; lysine, 0.87; methionine, 0.61; cysteine, 0.53; threonine, 1.08; tryptophan, 0.23; histidine, 0.77; isoleucine, 1.02; leucine, 2.95; phenylalanine, 1.62; valine, 1.44 and arginine, 1.09. Fatty acid composition of DDGS (%): C16:0, 11.52; C16:1–7, 0.13; C18:0, 1.62; C18:1n-9, 21.78; C18:2n-6, 53.37; C18:3n-3, 0.54; C18:3n-6, 0.19; C20:0, 0.08; C20:1n-9, 0.28 and C22:0, 0.14. 2The vitamin premix contained (per kilogram of diet): vitamin A, 12,000 IU; vitamin D3, 2,400 IU; vitamin E, 10 IU; vitamin K3, 2.5 mg; vitamin B12, 0.015 mg; vitamin B1, 3 mg; vitamin B2, 7 mg; vitamin B6, 4 mg; folic acid, 1 mg; choline, 1,000 mg; nicotinic acid, 30 mg and pantothenic acid, 10 mg. 3The mineral premix contained (per kilogram of diet): manganese, 60 mg; zinc, 80 mg; iron, 60 mg; copper, 8 mg; iodine, 0.35 mg; and selenium, 0.3 mg.
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and Cherian et al. (1996) reported that the increase in yolk vitamin E concentration and the incorporation of tocopherols in hen diets prevents lipid oxidative deterioration. Bollengier-Lee et al. (1998) and Ciftci et al. (2005) also showed that vitamin E supplementation significantly increased egg production in laying hens exposed to heat stress. There was little information in the literature on the influence of vitamin E in diets containing 10 or 20% DDGS in broilers. Therefore, the hypothesis of the study was that vitamin E could improve performance and other parameters of laying hens fed high levels of DDGS, which contain a high concentration of polyunsaturated fatty acids that are susceptible to oxidation. We investigated the effects of vitamin E in diets containing different levels of DDGS on the laying performance, egg quality, egg fatty acid composition, antioxidant capacity, and biochemical parameters of laying hens from 40 to 63 wk of age.
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high-density lipoprotein (HDL) cholesterol, aspartate aminotransferase (AST), alanine aminotransferase (ALT), P, and Ca levels in serum were determined using reagent kits (Biosion Bio-Technology and Science Inc., Beijing, China) following the manufacturer’s directions and using a fully automatic biochemical analyzer.
Statistical Analysis Data were analyzed using the GLM procedure of SPSS 20.0 for 2-way ANOVA to assess the main effects of DDGS and vitamin E and their interactions. Significant differences between treatments were determined at P < 0.05 using Duncan’s new multiple range tests. The values given in the tables are means and pooled SEM.
RESULTS AND DISCUSSION Laying Performance There were no significant interaction effects between DDGS and vitamin E levels for any production parameter (Table 2). The effects of DDGS supplementation on feed intake, egg weight, egg production, and egg mass were not significant. Our results agreed with Masa’deh et al. (2011), who found that increasing levels of DDGS from 0 to 25% for White Leghorn-type hens did not have negative effects on feed intake, egg weight, and egg production. Feeding 20% DDGS in the diets yielded the worst feed conversion compared with the 0% DDGS (P < 0.05), consistent with previous reports (Shalash et al., 2010; Ghazalah et al., 2011). Deniz et al. (2013) reported that laying performance declined with 20% DDGS may be attributed to the low palatability and high concentration of fibrous constituents, so the hens were not fully able to meet their energy and amino acid requirements. Moreover, Creswell (2006) reported that 20% DDGS contained a low level of starch, meaning that the hens relied solely on converting part of dietary amino acids to glucose through the gluconeogenesis pathway to maintain normal glucose concentrations in the blood and relying increasingly on fatty acid oxidation to supply energy. These changes could have influenced poultry metabolism over time, and ultimately might have affected laying performance. We also found that diets supplemented with vitamin E improved egg production. This result was consistent with previous reports (Scheideler and Froning, 1996; Ciftci et al., 2005). Bollengier-Lee et al. (1998) reported that dietary supplementation with vitamin E improved egg production by facilitating the release of vitellogenin from the liver and by increasing its concentration into blood. However, egg weight, feed intake, egg mass, and feed conversion were not significantly influenced by vitamin E, consistent with previous reports (Scheideler et al., 2010; Irandoust et al., 2012).
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surement stand. Eggs were weighed and broken, yolk and albumen were separated and weighed as indicated by Grobas et al. (2001), and their relative proportions (% egg weight) were determined. Egg yolk color score was evaluated using a Roche yolk color fan. Eggshell thickness was measured using micrometers to the nearest 0.01 mm at the equator. The Haugh unit was determined as described by Haugh (1937). The α-tocopherol concentration in the yolk and serum and the cholesterol in the yolk were tested using reagent kits (JianCheng Bioengineering Institute, Nanjing, China) and an UV-visible spectrophotometer UV-2401PC (Shimadzu Co., Kyoto, Japan). The CP content of the egg yolk was determined by the block digestion method using a copper catalyst and steam distillation into boric acid (AOAC International, 2005) and a nitrogen analyzer (Foss, KT-2300, Foss Co. Ltd., Beijing, China). A factor of 6.25 was used to calculate protein content from the nitrogen values. The crude fat content of the egg yolk was determined using the diethyl ether extraction-submersion method (AOAC International, 2005). Egg Fatty Acid Composition. For fatty acid analysis, fat from egg yolks was extracted with chloroform/ methanol (2:1, vol/vol; Folch et al., 1957). The fatty acid methyl esters were formed using BF3 and methanolic KOH and separated on a gas chromatograph (GC-2010, Shimadzu Co.) equipped with a flame ionization detector. A volume of 1 μL of sample was injected into the chamber and split (split ratio, 100:1) with an SP-2560 capillary column (100 m × 0.25 mm × 0.20 μm, Supelco Inc.) using nitrogen as the carrier gas. An initial oven temperature of 140°C was maintained for 5 min and then increased by 4°C min−1 to a final temperature of 240°C. The injector and detector temperatures were 260°C. The carrier gas was delivered at a flow rate of 1.04 mL min−1, with a linear velocity of 20 cm/s. Fatty acid identification was achieved by comparison of their retention times with Supelco 37 component FAME standard mix (Sigma-Aldrich Co.). Results were expressed as area percentages of each fatty acid methyl ester relative to the total detected. Antioxidant Capacity. Total superoxide dismutase (T-SOD) activity in the yolk and serum were determined by the xanthine oxidase method. Malondialdehyde (MDA) content was determined by the sulfur generation barbituric acid method (TAB). Glutathione peroxidase (GSH-Px) activity was determined by the 5,5′-dithio bis-(2-nitrobenzoic acid) color method. Test reagent kits (JianCheng Bioengineering Institute, Nanjing, China) and an UV-visible spectrophotometer UV-2401PC (Shimadzu Co., Kyoto, Japan) were used. Biochemical Parameters. At the end of the experimental period, the hens were fasted for 12 h and the venous blood from the wings was collected. Serum was separated and stored at −20°C for analysis. Cholesterol (CHO), low-density lipoprotein (LDL) cholesterol,
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RESEARCH NOTE Table 2. Effect of vitamin E on laying performance of laying hens fed dried distillers grains with solubles
(DDGS)1
Laying performance VE (mg/kg)
Item DDGS (%) 0 10 20
Egg weight (g)
Egg production (%)
Feed conversion
Egg mass (g/hen per d)
99.6 101 101 102 102 102
63.3 63.1 64.3 63.7 62.7 63.0
80.7 82.8 82.4 84.6 79.0 80.7
1.95 1.93 1.91 1.90 2.06 2.00
51.1 52.3 52.9 53.9 49.5 50.8
100 102 102
63.2 64.0 62.8
81.8ab 83.5a 79.9b
1.94b 1.91b 2.03a
51.7ab 53.4a 50.2b
101 101
63.4 63.3
80.7b 82.7a
1.97 1.94
51.2 52.3
0.40 0.53 0.90 0.508
0.32 0.81 0.85 0.310
0.019 0.047 0.97 0.494
0.015 0.32 0.88 0.017
0.009 0.15 0.99 0.394
a,bMeans 1VE:
with different superscripts within each column are significantly different (P < 0.05). vitamin E; DDGS × VE: interaction of DDGS and VE. Data are means of 6 replicates of 10 hens each.
Egg Quality The results shown in Table 3 indicated that no significant effects on yolk and albumen percentages were observed by the addition of DDGS to the diets of laying hens. However, egg yolk color and eggshell thickness were significantly increased by including DDGS in laying hen diets (P < 0.05). These results were consistent with the findings reported by Shalash et al. (2010). Egg yolk color was linearly increased with increasing levels of DDGS. This observation was expected because corn grain contains about 20 ppm of xanthophyll (NRC, 1994), and the xanthophyll content in DDGS is 3 times as concentrated due to the removal of starch in the fermentation process. Eggshell breaking strength and Haugh units were not significantly influenced by DDGS, consistent with previous reports (Deniz et al., 2013). Neither DDGS nor vitamin E had a significant effect on CP and crude fat. Our results indicated that yolk color, eggshell breaking strength, eggshell thickness, and Haugh units were not significantly influenced by dietary vitamin E levels. These results were in agreement with Irandoust et al. (2012), who found that supplementation with 250 mg/ kg of vitamin E had no significant effect on egg quality. The hens fed diets containing 200 mg/kg of vitamin E had an increased yolk percentage and a reduced albumen percentage than hens fed 0 mg/kg of vitamin E (P < 0.05). These results were consistent with the findings reported by Ciftci et al. (2005). Diets supplemented with high levels of vitamin E led to an increase in α-tocopherol concentration in the yolk (P < 0.05). Vitamin E can be stored in tissues as a fat-soluble vitamin, so α-tocopherol concentration in plasma and tissues were enhanced. This result was similar to those
reported previously (Mori et al., 2003; Wang et al., 2012). There was a significant difference in CHO in the egg yolk, in agreement with Sahin et al. (2006), who reported that dietary supplementation with higher levels of vitamin E decreased CHO concentration in Japanese quail. However, α-tocopherol and CHO concentrations were not significantly influenced by DDGS.
Yolk Fatty Acid Composition The dietary DDGS clearly affected the fatty acid composition of eggs (Table 4). The proportions of palmitic acid (C16:0) and stearic acid (C18:0) in egg yolks decreased (P < 0.05) with increased levels of DDGS in the diets, which was attributed to a decrease in saturated fatty acids (P < 0.05). In contrast, the proportion of linoleic (18:2n-6) and γ-linolenic (C18:3n-6) acids in yolk linearly increased as DDGS levels increased (P < 0.05). We also found that polyunsaturated fatty acids (PUFA), unsaturated fatty acids, and the ratio of unsaturated fatty acids to saturated fatty acids linearly increased as DDGS increased (P < 0.05). These results were consistent with previous reports (Schilling et al., 2010; Wang et al., 2012). Dietary fat, as a nutritional factor, could deposit in animal tissues and affect fatty acid profiles of adipose tissues (Wiseman and Agunbiade, 1998). Distillers dried grains with solubles contain approximately 10% fat, and are rich in PUFA, particularly linoleic acid (C18:2). Consequently, diets containing high levels of DDGS changed the egg fatty acid composition. Supplementation of the diet with 200 mg/kg of vitamin E significantly decreased stearic acids (C18:0) levels, in agreement with Wang et al. (2012). The evidence clearly indicated that saturated fatty acid intake levels
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Main effect means for DDGS 0% DDGS 10% DDGS 20% DDGS Main effect means for VE 0 mg/kg of VE 200 mg/kg of VE P-value DDGS VE DDGS × VE SEM
0 200 0 200 0 200
Feed intake (g/hen per d)
1VE:
0 200 0 200 0 200
0.36 0.36 0.37 0.37 0.39 0.38 0.36b 0.37b 0.39a 0.37 0.37 0.001 0.80 0.76 0.003
6.74 6.81 7.63 7.61 8.34 8.36 6.78c 7.62b 8.35a 7.57 7.59 0.000 0.89 0.98 0.093
Eggshell thickness (mm)
0.96 0.35 0.99 0.041
3.49 3.57
3.52 3.52 3.54
3.47 3.56 3.48 3.56 3.51 3.58
Eggshell breaking strength (kg/cm2)
0.71 0.20 0.98 0.403
80.1 81.2
81.0 80.8 80.2
80.5 81.4 80.4 81.3 79.6 80.8
Haugh units
0.45 0.028 0.27 0.226
59.3a 58.3b
30.0b 31.0a 0.38 0.034 0.34 0.236
58.7 58.6 59.2
59.6 57.8 59.2 57.9 59.2 59.2
Albumen weight (%)
30.8 30.7 30.0
29.9 31.7 30.1 31.3 30.0 30.1
Yolk weight (%)
with different superscripts within each column are significantly different (P < 0.05). vitamin E; DDGS × VE: interaction of DDGS and vitamin E. Data are means of 6 replicates, with 4 samples per replicate.
a–cMeans
DDGS (%) 0 10 20 Main effect means for DDGS 0% DDGS 10% DDGS 20% DDGS Main effect means for VE 0 mg/kg of VE 200 mg/kg of VE P-value DDGS VE DDGS × VE SEM
Yolk color
0.73 0.94 0.84 0.213
53.7 53.7
53.5 53.9 53.8
53.5 53.4 53.7 54.1 53.9 53.7
Crude fat (%)
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Item
VE (mg/kg)
Egg quality
Table 3. Effect of vitamin E on egg quality of laying hens fed dried distillers grains with solubles (DDGS)1
0.88 0.000 0.87 0.049
16.0a 14.8b
2.52b 3.60a 32.0 32.0 0.46 0.89 0.56 0.138
15.2 15.6 15.4
3.04 3.09 3.04
31.8 32.2 32.0
0.76 0.009 0.99 0.211
15.8 14.6 16.1 15.0 16.0 14.8
2.47 3.61 2.54 3.64 2.54 3.55
31.7 31.9 32.1 32.3 32.2 31.7
Cholesterol (mg/g)
α-Tocopherol (µg/g)
CP (%)
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47.9 48.8 0.92 0.44 0.99 0.534
35.5 34.3 0.011 0.057 0.53 0.288
MUFA
48.3 48.1 48.6
0.010 0.001 0.99 0.056
36.2a 34.5b 34.0b
0.27 0.94 0.98 0.080
0.024 0.16 0.47 0.263
0.80 0.42 0.99 0.499
43.9 44.7
7.94a 7.55b
47.8 48.8 47.7 48.5 48.2 49.0
4.06 4.07
27.2 26.5
44.1 44.0 44.7
43.6 44.6 43.6 44.3 44.3 45.2
C18:1n-9
7.94a 7.79a 7.50b
8.13 7.75 7.99 7.59 7.69 7.31
C18:0
36.9 35.5 35.4 33.7 34.1 33.9
4.19 4.12 3.88
27.9a 26.4b 26.2b
SFA
4.20 4.18 4.09 4.15 3.88 3.86
C16:1-7
28.5 27.4 27.1 25.7 26.1 26.3
C16:0
0.002 0.64 0.42 0.276
11.5 11.3
10.4b 10.9b 13.0a
10.0 10.8 11.3 10.6 13.4 12.5
0.001 0.63 0.35 0.284
12.9 12.6
11.6b 12.2b 14.3a
11.2 12.1 12.7 11.8 14.7 13.9
0.25 0.23
0.22 0.24 0.25
0.23 0.21 0.26 0.23 0.26 0.24
C18:3n-3
0.079 0.73 0.53 0.004
0.26 0.26
0.27 0.27 0.25
0.26 0.27 0.28 0.27 0.25 0.25
C20:0
0.014 0.52 0.57 0.431
60.8 61.3
59.9b 60.3b 62.9a
59.0 60.8 60.4 60.2 63.0 62.9
UFA
0.049 0.28 0.88 0.16 0.80 0.99 0.006 0.007 Egg fatty acid composition
0.23 0.23
0.21b 0.24ab 0.25a
0.20 0.21 0.24 0.24 0.25 0.24
C18:3n-6
PUFA
C18:2n-6
Egg fatty acid composition
1.66c 1.75b 1.85a 1.72 1.79 0.000 0.055 0.42 0.018
11.5 11.5 0.041 0.89 0.15 0.217
0.056 0.24 0.67 0.002
11.1b 11.1b 12.3a
0.85 0.23 0.76 0.002
0.99 0.59 0.93 0.003
0.07 0.08
1.60 1.72 1.71 1.79 1.85 1.86
0.03 0.03
0.05 0.05
0.08 0.08 0.07
11.4 10.9 10.5 11.7 12.7 11.9
0.03 0.03 0.03
0.05 0.05 0.05
0.08 0.08 0.07 0.08 0.06 0.07
0.057 0.65 0.76 0.014
0.74 0.75
0.71 0.74 0.79
0.64 0.78 0.81 0.67 0.77 0.81
C22:6n-3
U/S
0.03 0.03 0.04 0.03 0.03 0.03
0.05 0.05 0.05 0.05 0.05 0.05
C22:0
n-6/n-3
C20:5n-3
C20:4n-6
1VE:
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with different superscripts within each column are significantly different (P < 0.05). vitamin E; DDGS × VE: interaction of DDGS and vitamin E; SFA: saturated fatty acids; MUFA: monounsaturated fatty acids; PUFA: polyunsaturated fatty acids; UFA: unsaturated fatty acids; U/S: ratio of unsaturated fatty acids to saturated fatty acids. The n-6 to n-3 was calculated as follows: (18:2n-6 + 18:3n-6 + 20:4n-6)/(18:3n-3 + 20:5n-3 + 22:6n-3), unsaturated to saturated fatty acid ratio calculated as follows: (16:1n-7 + 18:1n-9 + 18:2n-6 + 18:3n-6 + 18:3n-3 + 20:4n-6 + 20:5n-3 + 22:6n-3)/(16:0 + 18:0 + 20:0 + 22:0). Data are means of 6 replicates, with 4 samples per replicate.
a–cMeans
DDGS (%) 0 0 200 10 0 200 20 0 200 Main effect means for DDGS 0% DDGS 10% DDGS 20% DDGS Main effect means for VE 0 mg/kg of VE 200 mg/kg of VE P-value DDGS VE DDGS × VE SEM
DDGS (%) 0 0 200 10 0 200 20 0 200 Main effect means for DDGS 0% DDGS 10% DDGS 20% DDGS Main effect means for VE 0 mg/kg of VE 200 mg/kg of VE P-value DDGS VE DDGS × VE SEM
Item
VE (mg/kg)
Table 4. Effect of vitamin E on egg fatty acid composition of laying hens fed dried distillers grains with solubles (DDGS)1
RESEARCH NOTE
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need to be reduced, and replacing them with PUFA has potential benefits (Givens, 2005). However, a high concentration of PUFA is an indicator of increased susceptibility to oxidation (Faustman, 1994). Therefore, the use of DDGS in poultry diets has been low to avoid any adverse effects on the quality of animal products.
Antioxidant Capacity
Biochemical Parameters The AST, ALT, Ca, HDL, LDL, CHO, and α-tocopherol concentrations in serum were not signifi-
Table 5. Effect of vitamin E on antioxidant capacity of laying hens fed dried distillers grains with solubles (DDGS)1 Antioxidant capacity In egg yolk Item DDGS (%) 0 0 0 Main effect means for DDGS 0% DDGS 10% DDGS 20% DDGS Main effect means for VE 0 mg/kg of VE 200 mg/kg of VE P-value DDGS VE DDGS × VE SEM a,bMeans
VE (mg/kg)
MDA (nmol/mg of protein)
0 200 0 200 0 200
1.56 1.46 1.61 1.47 1.55 1.45
55.9 59.9 59.0 62.7 55.8 62.0
184 189 184 189 185 194
3.03 2.83 3.06 2.89 3.06 2.90
662 686 637 659 656 671
287 300 278 293 273 295
1.51 1.54 1.50
57.9 60.9 58.9
186 187 189
2.93 2.99 2.98
674 648 664
293 286 284
1.57a 1.46b
56.9b 61.5a
184b 190a
3.05a 2.88b
652b 672a
279b 296a
0.82 0.044 0.96 0.027
GSH-Px (U)
In serum
0.44 0.022 0.85 0.954
T-SOD (U/mg of protein)
0.57 0.022 0.78 1.278
MDA (nmol/mL)
0.82 0.044 0.91 0.040
GSH-Px (U)
0.18 0.046 0.92 4.926
T-SOD (U/mL)
0.49 0.023 0.85 3.408
with different superscripts within each column are significantly different (P < 0.05). vitamin E; DDGS × VE: interaction of DDGS and vitamin E. MDA: malondialdehyde; GSH-Px: glutathione peroxidase; T-SOD: total superoxide dismutase. Data are means of 6 replicates, with 4 samples per replicate. 1VE:
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Table 5 showed significant effects of 200 mg/kg of vitamin E on the specific activities of MDA, GSH-Px, and T-SOD of the egg yolk and serum (P < 0.05), similar to reports by Lin et al. (2005) and Song (2013). According to antioxidant theory, when the concentrations of antioxidants decrease, lipid peroxidation increases in the tissues and plasma, leading to damage of cell membranes (Gallo-Torres, 1980; McDowell, 1989). Vitamin E is the major fat-soluble antioxidant, which breaks the chain reaction of lipid peroxidation. By its free radical quenching activity, it breaks chain propagation and thus terminates free radical attack at an early stage, such an effect of vitamin E is on polyunsaturated fatty acids of biomembranes (McDowell, 1989). However, our results did not show significant effects of DDGS supplementation on MDA, T-SOD, and GSH-Px. Consequently, dietary supplementation with higher levels of vitamin E enhanced antioxidant capacity of egg and laying hens.
cantly influenced by DDGS (Table 6). However, increasing DDGS to 10 or 20% in laying hen diets significantly (P < 0.05) increased P content in serum compared with 0% DDGS. Dietary supplementation with 200 mg/kg of vitamin E significantly decreased CHO (P < 0.05). The decrease of CHO in serum was correlated to decreased CHO in egg yolk. Meanwhile, dietary supplementation with 200 mg/kg of vitamin E significantly increased the α-tocopherol concentration in serum as well as yolk. These results were consistent with the results of Sahin et al. (2002). High α-tocopherol concentration may increase the oxidative stability and provide a source of tocopherols that is useful for human nutrition and health. The AST, ALT, Ca, HDL cholesterol, LDL cholesterol, and P were not significantly influenced by vitamin E, consistent with previous reports (Nadia et al., 2008). In conclusion, DDGS and vitamin E did not have adverse effects on liver functions (AST and ALT) or blood constituents. The results showed an improvement due to dietary supplementation of 200 mg/kg of vitamin E independent of the presence of DDGS, when the vitamin E level in the basal diet was 10 mg/kg. This was well above the NRC requirement. Scheideler and Froning (1996) reported that diet supplemented with 50 mg/kg of vitamin E had greater egg production than diet with 27 mg/kg of vitamin E. Ciftci et al. (2005) showed that supplementation with 125 mg/kg of vitamin E significantly increased egg production in laying hens exposed to heat stress. Also, Lin et al. (2005) reported dietary supplementation of 40, 80, 120, and 160 mg/kg of vitamin E enhanced the oxidative stability in the brain and liver of Taiwan native chicks. These results suggest that
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RESEARCH NOTE Table 6. Effect of vitamin E on biochemical parameters of laying hens fed dried distillers grains with solubles
(DDGS)1
Biochemical parameter VE (mg/kg)
Item
0 200 0 200 0 200
ALT (U/L)
CHO P Ca (mmol/L) (mmol/L) (mmol/L)
HDL cholesterol (mmol/L)
LDL cholesterol (mmol/L)
α-Tocopherol (µg/mL)
164 171 158 160 170 178
22.5 24.3 21.3 22.0 22.8 23.3
2.89 2.63 3.02 2.71 2.82 2.51
2.26 2.17 2.72 2.71 2.86 2.76
6.35 6.98 6.60 7.03 6.60 6.95
0.07 0.06 0.08 0.07 0.07 0.07
0.55 0.55 0.53 0.55 0.54 0.54
8.01 8.70 8.51 9.86 8.71 9.99
168 157 174
23.4 21.5 23.1
2.76 2.86 2.67
2.21b 2.72a 2.81a
6.66 6.81 6.77
0.07 0.08 0.07
0.55 0.54 0.54
8.35 9.18 9.35
161 172
22.0 23.3
2.91a 2.62b
2.61 2.54
6.51 6.99
0.07 0.07
0.54 0.55
8.41b 9.51a
0.35 0.012 0.98 0.055
0.022 0.72 0.98 0.089
0.95 0.27 0.96 0.209
0.65 0.41 0.98 0.004
0.68 0.44 0.93 0.004
0.079 0.006 0.73 0.185
0.38 0.27 0.90 4.819
0.11 0.10 0.76 0.376
a,bMeans
with different superscripts within each column are significantly different (P < 0.05). vitamin E; AST: aspartate aminotransferase; ALT: alanine aminotransferase; CHO: cholesterol; HDL = high-density lipoprotein; LDL = lowdensity lipoprotein; DDGS × VE: interaction of DDGS and vitamin E. Data are means of 6 replicates, with 4 samples per replicate. 1VE:
the vitamin E requirement in some circumstances may be higher than that of NRC.
Conclusions The results showed that supplementation with 10% DDGS did not negatively affect laying performance and egg quality parameters. However, supplementation with 20% DDGS yielded the worst feed conversion. Diets containing high levels of DDGS changed the egg fatty acid composition. Additionally, dietary supplementation with 200 mg/kg of vitamin E improved egg production and egg quality, and enhanced the antioxidant capacity of laying hens. However, there was no interaction between DDGS and vitamin E, which suggested that vitamin E improved performance of layers due to vitamin E independent of the presence of DDGS.
ACKNOWLEDGMENTS This work was supported by the National Natural Science Foundation of China (no. 31272453). We thank Lijun Xu, Chongpeng Bi, Baojing Cheng, and Zhongyu Li at Northeast Agricultural University for technical assistance.
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DDGS (%) 0 10 20 Main effect means for DDGS 0% DDGS 10% DDGS 20% DDGS Main effect means for VE 0 mg/kg of VE 200 mg/kg of VE P-value DDGS VE DDGS × VE SEM
AST (U/L)
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Gallo-Torres, D. C. 1980. Absorption, blood transport and metabolism of vitamin E. Pages 170–267 in A Comprehensive Treatise. L. J. Machlin, ed. Marcel Dekker, New York, NY. Ghazalah, A. A., M. O. Abd-Elsamee, and E. S. Moustafa. 2011. Use of distillers dried grains with solubles (DDGS) as replacement for soybean meal in laying hen diets. Int. J. Poult. Sci. 10:505–513. Givens, D. I. 2005. The role of animal nutrition in improving the nutritive value of animal-derived foods in relation to chronic disease. Proc. Nutr. Soc. 64:395–402. http://dx.doi.org/10.1079/ PNS2005448. Grobas, S., J. Mendez, R. Lazaro, C. Blas, and G. G. Mateos. 2001. Influence of source and percentage of fat added to diet on performance and fatty acid composition of egg yolks of two strains of laying hens. Poult. Sci. 80:1171–1179. Haugh, R. R. 1937. The Haugh unit for measuring egg quality. US Egg Poultry Magazine. 43:552–573. Hoffmann, L. A., and A. Baker. 2011. Estimating the substitution of distillers’ grains for corn and soybean meal in the U.S. feed complex. A report from the Economic Research Service, United States Department of Agriculture. Accessed Nov. 28, 2011. http://www.ers.usda.gov/Publications/FDS/2011/09Sep/FDS11I01/FDS11I01.pdf. Irandoust, H., A. H. Samie, H. R. Rahmani, M. A. Edriss, and G. G. Mateos. 2012. Influence of source of fat and supplementation of the diet with vitamin E and C on performance and egg quality of laying hens from forty four to fifty six weeks of age. Anim. Feed Sci. Technol. 177:75–85. Jensen, L. S. 1978. Distillers feeds as sources of unidentified factors for laying hens. Proc. Distillers Res. Conf. 33:17–22. Louisville, KY. Jensen, L. S. 1981. Value of distillers dried grains with solubles in poultry feeds. Proc. Distillers Res. Conf. 36:87–93. Cincinnati, OH. Leeson, S. 2007. Vitamin requirements: Is there basis for re-evaluating dietary specifications. World’s Poult. Sci. J. 63:255–266. Lin, Y. F., H. L. Tasi, Y. C. Lee, and S. J. Chang. 2005. Maternal vitamin E supplementation affects the antioxidant capability and oxidative status of hatching chicks. J. Nutr. 135:2457–2461. Lumpkins, B. S., A. B. Batal, and N. M. Dale. 2005. Use of distillers dried grains plus solubles in laying hen diets. J. Appl. Poult. Res. 14:25–31. Masa’deh, M. K., S. E. Purdum, and K. J. Hanford. 2011. Dried distillers grain with solubles in laying hen diets. Poult. Sci. 90:1960–1966. McDowell, L. R. 1989. Vitamins in Animal Nutrition: Comparative Aspects to Human Nutrition. Academic Press, London, UK. Mori, A. V., C. X. Mendonca Jr., C. R. M. Almeida, and M. C. G. Pita. 2003. Supplementing hen diets with vitamins A and E affects egg yolk retinol and α-tocopherol levels. J. Appl. Poult. Res. 12:106–114. Nadia, R. L., R. A. Hassan, E. M. Qota, and H. M. Fayek. 2008. Effect of natural antioxidant on oxidative stability of eggs and productive and reproductive performance of laying hens. Int. J. Poult. Sci. 7:134–150. NRC. 1994. Nutrient Requirements of Poultry. 9th rev. ed. Natl. Acad. Press, Washington, DC. Parsons, C. M., and D. H. Baker. 1983. Distillers dried grains with solubles as a protein source for the chick. Poult. Sci. 62:2445– 2451. Pineda, L., S. Roberts, B. Kerr, R. Kwakkel, M. Verstegen, and K. Bregendahl. 2008. Maximum dietary content of dried distiller’s grains with soluble in diets for laying hens: Effects on nitrogen
Key words: corn dried distillers grains with solubles, vitamin E, laying performance, egg quality, egg fatty acid composition 2013 Poultry Science 92:2956–2964 http://dx.doi.org/10.3382/ps.2013-03228
INTRODUCTION Corn dried distillers grains with solubles (DDGS) is a coproduct of ethanol production. During 2010, the US ethanol industry produced 33.4 million metric tons of distillers coproducts (Hoffmann and Baker, 2011), encouraging the use of DDGS in poultry diets. Dried distillers grains with solubles is not a new ingredient for poultry, and has been available for many decades as a by-product of the brewing industry. Dried distillers grains with solubles is recognized as a valuable source of energy, protein, amino acids, water-soluble vitamins, linoleic acid, xanthophyll, and minerals for poultry diets (Scott, 1965; Jensen, 1978, 1981; Parsons and Baker, 1983; Wang et al., 2007; Salim et al., 2010; Cozannet et al., 2010). Recently, Roberts et al. (2007) reported that adding 10% DDGS to laying hen diets did not negative©2013 Poultry Science Association Inc. Received April 8, 2013. Accepted August 4, 2013. 1 Corresponding author: [email protected]
ly affect egg production and egg quality parameters. Lumpkins et al. (2005) and Swiątkiewicz and Koreleski (2006) reported that supplementation with up to 15% DDGS could be used in diets, whereas inclusion of 20% negatively affected laying rate and egg weight. Deniz et al. (2013) observed that corn DDGS up to 15% did not affect egg production and egg quality and reduced feed cost. No differences in hen daily egg production were observed among dietary treatments even at 25% DDGS (Masa’deh et al., 2011). However, Cromwell et al. (1993), Spiehs et al. (2002), Pineda et al. (2008), and Corzo et al. (2009) reported that the use of DDGS in poultry diets had been low, which may be due to the high percentage of crude fiber, unpalatable, sulfur content, high variability in nutrient (especially lysine), and high proportion of polyunsaturated fatty acid of the DDGS. Vitamin E is an indispensable cellular antioxidant (Puthpongsiriporn et al., 2001; Brigelius-Flohé et al., 2002) and may help the immune response in chickens by protecting lymphocytes, macrophages, and other cells against oxidative damage (Franchini et al., 2002; Leeson, 2007). Additionally, Ajuyah et al. (1993)
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ABSTRACT The objective of the present study was to investigate the effect of vitamin E on laying performance, egg quality, egg fatty acid composition, antioxidant capacity, and several biochemical parameters of laying hens fed corn distillers dried grains with solubles (DDGS) during the laying period (40 to 63 wk of age). A total of 360 Hy-Line Variety Brown hens were randomly assigned to 6 groups, consisting of 6 replicates with 10 hens each. Hens were allocated to diets 1 through 6 in a 3 × 2 factorial design. The dietary treatments included 3 levels of DDGS (0, 10, and 20%) and 2 levels of vitamin E (0 and 200 mg/ kg). The results indicated that yolk color and eggshell thickness increased with increasing DDGS (P < 0.05). However, increasing DDGS to 20% in laying hen diets significantly reduced feed conversion (P < 0.05). Supplementation with 200 mg/kg of vitamin E signifi-
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MATERIALS AND METHODS Experimental Materials The DDGS was purchased from Fuel Alcohol Limited of Jilin in China. Vitamin E acetate (50% purity) was purchased from Biotechnology Limited in Zhejiang, China. The source of vitamin E used in this study was d-α-tocopherol acetate. The basal level of vitamin E in the diet without additional vitamin E was 10 mg/kg.
Birds and Experimental Design A total of 360 Hy-Line Variety Brown hens were randomly assigned into 6 treatment groups. Each treatment group was composed of 6 replicates with 10 hens each. The hens were allocated to diets 1 through 6 and fed during wk 40 through 63 in a 3 × 2 factorial design. The dietary treatments included 3 levels of DDGS (0, 10, and 20%) and 2 levels of vitamin E (0 and 200 mg/ kg). The diets were a corn-soybean meal basal feed with or without DDGS at 10 and 20%, formulated to meet the NRC (1994) nutrient requirement for laying hens. The compositions of the diets were listed in Table 1.
Management During the laying period, 2 hens were housed in a metal cage (48 × 34 × 39 cm), with a replicate plot consisting of 10 hens in 5 adjoining cages. Hens were maintained on a 16L:8D photoperiod throughout the study. All hens were housed in an environmentally controlled house with the temperature maintained at approximately 20°C. The experimental protocol was approved by the Northeast Agricultural University Ethics Committee on the Use and Care of Animals. These guidelines are similar to those of the US National Institutes of Health.
Data and Sample Collection Laying Performance. Egg production was recorded daily. Feed consumption was recorded weekly by calculating the average daily feed consumption of the 10 hens in each replicate. Egg weight was measured using all eggs produced during 2 consecutive days every week. Egg mass was calculated as laying rate × egg weight, whereas feed conversion (feed consumption/egg mass) was calculated every week throughout the experimental period. Egg Quality. Egg quality was measured on 4 eggs from each replicate the last day of each of the 4-wk periods, and the data were pooled for analysis. Eggshell breaking strength was measured using an eggshell strength tester II (Fujihira Industry Co. Ltd., Tokyo, Japan) after the eggs were broken on a NFN-382 meaTable 1. Composition of experimental diets1 Item Ingredient (%) Corn Soybean meal Fish meal DDGS Sodium chloride Dicalcium phosphate Limestone dl-Methionine l-Lysine∙HCl Choline chloride Vitamin premix2 Mineral premix3 Total Calculated composition ME (MJ/kg) CP (%) Crude fiber (%) Methionine (%) Lysine (%) Calcium (%) Available phosphorus (%) Total phosphorus (%) Analyzed composition (%) DM CP Crude fiber Crude fat Calcium Total phosphorus
0% DDGS
10% DDGS
20% DDGS
62.40 25.50 1.00 0.00 0.30 1.50 8.80 0.08 0 0.10 0.02 0.30 100.00
58.90 19.00 1.00 10.00 0.30 1.42 8.80 0.07 0.09 0.10 0.02 0.30 100.00
56.40 11.50 1.00 20.00 0.30 1.33 8.80 0.06 0.19 0.10 0.02 0.30 100.00
11.03 16.40 3.65 0.34 0.87 3.82 0.42 0.63 89.48 18.42 3.73 4.29 3.68 0.64
10.91 16.34 3.80 0.34 0.88 3.81 0.44 0.65 89.40 17.76 3.82 4.87 3.72 0.61
10.89 16.38 3.86 0.35 0.89 3.80 0.44 0.65 89.37 17.65 3.97 5.04 3.70 0.63
1DDGS: dried distillers grains with solubles. Analyzed composition of DDGS used in the experiment analyzed according to AOAC International (1990) was as follows (%): DM, 89.19; CP, 32.9; crude fat, 8.3; crude fiber, 11.2; crude ash, 5.06; calcium, 0.15; phosphorus, 0.74; lysine, 0.87; methionine, 0.61; cysteine, 0.53; threonine, 1.08; tryptophan, 0.23; histidine, 0.77; isoleucine, 1.02; leucine, 2.95; phenylalanine, 1.62; valine, 1.44 and arginine, 1.09. Fatty acid composition of DDGS (%): C16:0, 11.52; C16:1–7, 0.13; C18:0, 1.62; C18:1n-9, 21.78; C18:2n-6, 53.37; C18:3n-3, 0.54; C18:3n-6, 0.19; C20:0, 0.08; C20:1n-9, 0.28 and C22:0, 0.14. 2The vitamin premix contained (per kilogram of diet): vitamin A, 12,000 IU; vitamin D3, 2,400 IU; vitamin E, 10 IU; vitamin K3, 2.5 mg; vitamin B12, 0.015 mg; vitamin B1, 3 mg; vitamin B2, 7 mg; vitamin B6, 4 mg; folic acid, 1 mg; choline, 1,000 mg; nicotinic acid, 30 mg and pantothenic acid, 10 mg. 3The mineral premix contained (per kilogram of diet): manganese, 60 mg; zinc, 80 mg; iron, 60 mg; copper, 8 mg; iodine, 0.35 mg; and selenium, 0.3 mg.
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and Cherian et al. (1996) reported that the increase in yolk vitamin E concentration and the incorporation of tocopherols in hen diets prevents lipid oxidative deterioration. Bollengier-Lee et al. (1998) and Ciftci et al. (2005) also showed that vitamin E supplementation significantly increased egg production in laying hens exposed to heat stress. There was little information in the literature on the influence of vitamin E in diets containing 10 or 20% DDGS in broilers. Therefore, the hypothesis of the study was that vitamin E could improve performance and other parameters of laying hens fed high levels of DDGS, which contain a high concentration of polyunsaturated fatty acids that are susceptible to oxidation. We investigated the effects of vitamin E in diets containing different levels of DDGS on the laying performance, egg quality, egg fatty acid composition, antioxidant capacity, and biochemical parameters of laying hens from 40 to 63 wk of age.
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high-density lipoprotein (HDL) cholesterol, aspartate aminotransferase (AST), alanine aminotransferase (ALT), P, and Ca levels in serum were determined using reagent kits (Biosion Bio-Technology and Science Inc., Beijing, China) following the manufacturer’s directions and using a fully automatic biochemical analyzer.
Statistical Analysis Data were analyzed using the GLM procedure of SPSS 20.0 for 2-way ANOVA to assess the main effects of DDGS and vitamin E and their interactions. Significant differences between treatments were determined at P < 0.05 using Duncan’s new multiple range tests. The values given in the tables are means and pooled SEM.
RESULTS AND DISCUSSION Laying Performance There were no significant interaction effects between DDGS and vitamin E levels for any production parameter (Table 2). The effects of DDGS supplementation on feed intake, egg weight, egg production, and egg mass were not significant. Our results agreed with Masa’deh et al. (2011), who found that increasing levels of DDGS from 0 to 25% for White Leghorn-type hens did not have negative effects on feed intake, egg weight, and egg production. Feeding 20% DDGS in the diets yielded the worst feed conversion compared with the 0% DDGS (P < 0.05), consistent with previous reports (Shalash et al., 2010; Ghazalah et al., 2011). Deniz et al. (2013) reported that laying performance declined with 20% DDGS may be attributed to the low palatability and high concentration of fibrous constituents, so the hens were not fully able to meet their energy and amino acid requirements. Moreover, Creswell (2006) reported that 20% DDGS contained a low level of starch, meaning that the hens relied solely on converting part of dietary amino acids to glucose through the gluconeogenesis pathway to maintain normal glucose concentrations in the blood and relying increasingly on fatty acid oxidation to supply energy. These changes could have influenced poultry metabolism over time, and ultimately might have affected laying performance. We also found that diets supplemented with vitamin E improved egg production. This result was consistent with previous reports (Scheideler and Froning, 1996; Ciftci et al., 2005). Bollengier-Lee et al. (1998) reported that dietary supplementation with vitamin E improved egg production by facilitating the release of vitellogenin from the liver and by increasing its concentration into blood. However, egg weight, feed intake, egg mass, and feed conversion were not significantly influenced by vitamin E, consistent with previous reports (Scheideler et al., 2010; Irandoust et al., 2012).
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surement stand. Eggs were weighed and broken, yolk and albumen were separated and weighed as indicated by Grobas et al. (2001), and their relative proportions (% egg weight) were determined. Egg yolk color score was evaluated using a Roche yolk color fan. Eggshell thickness was measured using micrometers to the nearest 0.01 mm at the equator. The Haugh unit was determined as described by Haugh (1937). The α-tocopherol concentration in the yolk and serum and the cholesterol in the yolk were tested using reagent kits (JianCheng Bioengineering Institute, Nanjing, China) and an UV-visible spectrophotometer UV-2401PC (Shimadzu Co., Kyoto, Japan). The CP content of the egg yolk was determined by the block digestion method using a copper catalyst and steam distillation into boric acid (AOAC International, 2005) and a nitrogen analyzer (Foss, KT-2300, Foss Co. Ltd., Beijing, China). A factor of 6.25 was used to calculate protein content from the nitrogen values. The crude fat content of the egg yolk was determined using the diethyl ether extraction-submersion method (AOAC International, 2005). Egg Fatty Acid Composition. For fatty acid analysis, fat from egg yolks was extracted with chloroform/ methanol (2:1, vol/vol; Folch et al., 1957). The fatty acid methyl esters were formed using BF3 and methanolic KOH and separated on a gas chromatograph (GC-2010, Shimadzu Co.) equipped with a flame ionization detector. A volume of 1 μL of sample was injected into the chamber and split (split ratio, 100:1) with an SP-2560 capillary column (100 m × 0.25 mm × 0.20 μm, Supelco Inc.) using nitrogen as the carrier gas. An initial oven temperature of 140°C was maintained for 5 min and then increased by 4°C min−1 to a final temperature of 240°C. The injector and detector temperatures were 260°C. The carrier gas was delivered at a flow rate of 1.04 mL min−1, with a linear velocity of 20 cm/s. Fatty acid identification was achieved by comparison of their retention times with Supelco 37 component FAME standard mix (Sigma-Aldrich Co.). Results were expressed as area percentages of each fatty acid methyl ester relative to the total detected. Antioxidant Capacity. Total superoxide dismutase (T-SOD) activity in the yolk and serum were determined by the xanthine oxidase method. Malondialdehyde (MDA) content was determined by the sulfur generation barbituric acid method (TAB). Glutathione peroxidase (GSH-Px) activity was determined by the 5,5′-dithio bis-(2-nitrobenzoic acid) color method. Test reagent kits (JianCheng Bioengineering Institute, Nanjing, China) and an UV-visible spectrophotometer UV-2401PC (Shimadzu Co., Kyoto, Japan) were used. Biochemical Parameters. At the end of the experimental period, the hens were fasted for 12 h and the venous blood from the wings was collected. Serum was separated and stored at −20°C for analysis. Cholesterol (CHO), low-density lipoprotein (LDL) cholesterol,
2959
RESEARCH NOTE Table 2. Effect of vitamin E on laying performance of laying hens fed dried distillers grains with solubles
(DDGS)1
Laying performance VE (mg/kg)
Item DDGS (%) 0 10 20
Egg weight (g)
Egg production (%)
Feed conversion
Egg mass (g/hen per d)
99.6 101 101 102 102 102
63.3 63.1 64.3 63.7 62.7 63.0
80.7 82.8 82.4 84.6 79.0 80.7
1.95 1.93 1.91 1.90 2.06 2.00
51.1 52.3 52.9 53.9 49.5 50.8
100 102 102
63.2 64.0 62.8
81.8ab 83.5a 79.9b
1.94b 1.91b 2.03a
51.7ab 53.4a 50.2b
101 101
63.4 63.3
80.7b 82.7a
1.97 1.94
51.2 52.3
0.40 0.53 0.90 0.508
0.32 0.81 0.85 0.310
0.019 0.047 0.97 0.494
0.015 0.32 0.88 0.017
0.009 0.15 0.99 0.394
a,bMeans 1VE:
with different superscripts within each column are significantly different (P < 0.05). vitamin E; DDGS × VE: interaction of DDGS and VE. Data are means of 6 replicates of 10 hens each.
Egg Quality The results shown in Table 3 indicated that no significant effects on yolk and albumen percentages were observed by the addition of DDGS to the diets of laying hens. However, egg yolk color and eggshell thickness were significantly increased by including DDGS in laying hen diets (P < 0.05). These results were consistent with the findings reported by Shalash et al. (2010). Egg yolk color was linearly increased with increasing levels of DDGS. This observation was expected because corn grain contains about 20 ppm of xanthophyll (NRC, 1994), and the xanthophyll content in DDGS is 3 times as concentrated due to the removal of starch in the fermentation process. Eggshell breaking strength and Haugh units were not significantly influenced by DDGS, consistent with previous reports (Deniz et al., 2013). Neither DDGS nor vitamin E had a significant effect on CP and crude fat. Our results indicated that yolk color, eggshell breaking strength, eggshell thickness, and Haugh units were not significantly influenced by dietary vitamin E levels. These results were in agreement with Irandoust et al. (2012), who found that supplementation with 250 mg/ kg of vitamin E had no significant effect on egg quality. The hens fed diets containing 200 mg/kg of vitamin E had an increased yolk percentage and a reduced albumen percentage than hens fed 0 mg/kg of vitamin E (P < 0.05). These results were consistent with the findings reported by Ciftci et al. (2005). Diets supplemented with high levels of vitamin E led to an increase in α-tocopherol concentration in the yolk (P < 0.05). Vitamin E can be stored in tissues as a fat-soluble vitamin, so α-tocopherol concentration in plasma and tissues were enhanced. This result was similar to those
reported previously (Mori et al., 2003; Wang et al., 2012). There was a significant difference in CHO in the egg yolk, in agreement with Sahin et al. (2006), who reported that dietary supplementation with higher levels of vitamin E decreased CHO concentration in Japanese quail. However, α-tocopherol and CHO concentrations were not significantly influenced by DDGS.
Yolk Fatty Acid Composition The dietary DDGS clearly affected the fatty acid composition of eggs (Table 4). The proportions of palmitic acid (C16:0) and stearic acid (C18:0) in egg yolks decreased (P < 0.05) with increased levels of DDGS in the diets, which was attributed to a decrease in saturated fatty acids (P < 0.05). In contrast, the proportion of linoleic (18:2n-6) and γ-linolenic (C18:3n-6) acids in yolk linearly increased as DDGS levels increased (P < 0.05). We also found that polyunsaturated fatty acids (PUFA), unsaturated fatty acids, and the ratio of unsaturated fatty acids to saturated fatty acids linearly increased as DDGS increased (P < 0.05). These results were consistent with previous reports (Schilling et al., 2010; Wang et al., 2012). Dietary fat, as a nutritional factor, could deposit in animal tissues and affect fatty acid profiles of adipose tissues (Wiseman and Agunbiade, 1998). Distillers dried grains with solubles contain approximately 10% fat, and are rich in PUFA, particularly linoleic acid (C18:2). Consequently, diets containing high levels of DDGS changed the egg fatty acid composition. Supplementation of the diet with 200 mg/kg of vitamin E significantly decreased stearic acids (C18:0) levels, in agreement with Wang et al. (2012). The evidence clearly indicated that saturated fatty acid intake levels
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Main effect means for DDGS 0% DDGS 10% DDGS 20% DDGS Main effect means for VE 0 mg/kg of VE 200 mg/kg of VE P-value DDGS VE DDGS × VE SEM
0 200 0 200 0 200
Feed intake (g/hen per d)
1VE:
0 200 0 200 0 200
0.36 0.36 0.37 0.37 0.39 0.38 0.36b 0.37b 0.39a 0.37 0.37 0.001 0.80 0.76 0.003
6.74 6.81 7.63 7.61 8.34 8.36 6.78c 7.62b 8.35a 7.57 7.59 0.000 0.89 0.98 0.093
Eggshell thickness (mm)
0.96 0.35 0.99 0.041
3.49 3.57
3.52 3.52 3.54
3.47 3.56 3.48 3.56 3.51 3.58
Eggshell breaking strength (kg/cm2)
0.71 0.20 0.98 0.403
80.1 81.2
81.0 80.8 80.2
80.5 81.4 80.4 81.3 79.6 80.8
Haugh units
0.45 0.028 0.27 0.226
59.3a 58.3b
30.0b 31.0a 0.38 0.034 0.34 0.236
58.7 58.6 59.2
59.6 57.8 59.2 57.9 59.2 59.2
Albumen weight (%)
30.8 30.7 30.0
29.9 31.7 30.1 31.3 30.0 30.1
Yolk weight (%)
with different superscripts within each column are significantly different (P < 0.05). vitamin E; DDGS × VE: interaction of DDGS and vitamin E. Data are means of 6 replicates, with 4 samples per replicate.
a–cMeans
DDGS (%) 0 10 20 Main effect means for DDGS 0% DDGS 10% DDGS 20% DDGS Main effect means for VE 0 mg/kg of VE 200 mg/kg of VE P-value DDGS VE DDGS × VE SEM
Yolk color
0.73 0.94 0.84 0.213
53.7 53.7
53.5 53.9 53.8
53.5 53.4 53.7 54.1 53.9 53.7
Crude fat (%)
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Item
VE (mg/kg)
Egg quality
Table 3. Effect of vitamin E on egg quality of laying hens fed dried distillers grains with solubles (DDGS)1
0.88 0.000 0.87 0.049
16.0a 14.8b
2.52b 3.60a 32.0 32.0 0.46 0.89 0.56 0.138
15.2 15.6 15.4
3.04 3.09 3.04
31.8 32.2 32.0
0.76 0.009 0.99 0.211
15.8 14.6 16.1 15.0 16.0 14.8
2.47 3.61 2.54 3.64 2.54 3.55
31.7 31.9 32.1 32.3 32.2 31.7
Cholesterol (mg/g)
α-Tocopherol (µg/g)
CP (%)
2960 Jiang et al.
47.9 48.8 0.92 0.44 0.99 0.534
35.5 34.3 0.011 0.057 0.53 0.288
MUFA
48.3 48.1 48.6
0.010 0.001 0.99 0.056
36.2a 34.5b 34.0b
0.27 0.94 0.98 0.080
0.024 0.16 0.47 0.263
0.80 0.42 0.99 0.499
43.9 44.7
7.94a 7.55b
47.8 48.8 47.7 48.5 48.2 49.0
4.06 4.07
27.2 26.5
44.1 44.0 44.7
43.6 44.6 43.6 44.3 44.3 45.2
C18:1n-9
7.94a 7.79a 7.50b
8.13 7.75 7.99 7.59 7.69 7.31
C18:0
36.9 35.5 35.4 33.7 34.1 33.9
4.19 4.12 3.88
27.9a 26.4b 26.2b
SFA
4.20 4.18 4.09 4.15 3.88 3.86
C16:1-7
28.5 27.4 27.1 25.7 26.1 26.3
C16:0
0.002 0.64 0.42 0.276
11.5 11.3
10.4b 10.9b 13.0a
10.0 10.8 11.3 10.6 13.4 12.5
0.001 0.63 0.35 0.284
12.9 12.6
11.6b 12.2b 14.3a
11.2 12.1 12.7 11.8 14.7 13.9
0.25 0.23
0.22 0.24 0.25
0.23 0.21 0.26 0.23 0.26 0.24
C18:3n-3
0.079 0.73 0.53 0.004
0.26 0.26
0.27 0.27 0.25
0.26 0.27 0.28 0.27 0.25 0.25
C20:0
0.014 0.52 0.57 0.431
60.8 61.3
59.9b 60.3b 62.9a
59.0 60.8 60.4 60.2 63.0 62.9
UFA
0.049 0.28 0.88 0.16 0.80 0.99 0.006 0.007 Egg fatty acid composition
0.23 0.23
0.21b 0.24ab 0.25a
0.20 0.21 0.24 0.24 0.25 0.24
C18:3n-6
PUFA
C18:2n-6
Egg fatty acid composition
1.66c 1.75b 1.85a 1.72 1.79 0.000 0.055 0.42 0.018
11.5 11.5 0.041 0.89 0.15 0.217
0.056 0.24 0.67 0.002
11.1b 11.1b 12.3a
0.85 0.23 0.76 0.002
0.99 0.59 0.93 0.003
0.07 0.08
1.60 1.72 1.71 1.79 1.85 1.86
0.03 0.03
0.05 0.05
0.08 0.08 0.07
11.4 10.9 10.5 11.7 12.7 11.9
0.03 0.03 0.03
0.05 0.05 0.05
0.08 0.08 0.07 0.08 0.06 0.07
0.057 0.65 0.76 0.014
0.74 0.75
0.71 0.74 0.79
0.64 0.78 0.81 0.67 0.77 0.81
C22:6n-3
U/S
0.03 0.03 0.04 0.03 0.03 0.03
0.05 0.05 0.05 0.05 0.05 0.05
C22:0
n-6/n-3
C20:5n-3
C20:4n-6
1VE:
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with different superscripts within each column are significantly different (P < 0.05). vitamin E; DDGS × VE: interaction of DDGS and vitamin E; SFA: saturated fatty acids; MUFA: monounsaturated fatty acids; PUFA: polyunsaturated fatty acids; UFA: unsaturated fatty acids; U/S: ratio of unsaturated fatty acids to saturated fatty acids. The n-6 to n-3 was calculated as follows: (18:2n-6 + 18:3n-6 + 20:4n-6)/(18:3n-3 + 20:5n-3 + 22:6n-3), unsaturated to saturated fatty acid ratio calculated as follows: (16:1n-7 + 18:1n-9 + 18:2n-6 + 18:3n-6 + 18:3n-3 + 20:4n-6 + 20:5n-3 + 22:6n-3)/(16:0 + 18:0 + 20:0 + 22:0). Data are means of 6 replicates, with 4 samples per replicate.
a–cMeans
DDGS (%) 0 0 200 10 0 200 20 0 200 Main effect means for DDGS 0% DDGS 10% DDGS 20% DDGS Main effect means for VE 0 mg/kg of VE 200 mg/kg of VE P-value DDGS VE DDGS × VE SEM
DDGS (%) 0 0 200 10 0 200 20 0 200 Main effect means for DDGS 0% DDGS 10% DDGS 20% DDGS Main effect means for VE 0 mg/kg of VE 200 mg/kg of VE P-value DDGS VE DDGS × VE SEM
Item
VE (mg/kg)
Table 4. Effect of vitamin E on egg fatty acid composition of laying hens fed dried distillers grains with solubles (DDGS)1
RESEARCH NOTE
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Jiang et al.
need to be reduced, and replacing them with PUFA has potential benefits (Givens, 2005). However, a high concentration of PUFA is an indicator of increased susceptibility to oxidation (Faustman, 1994). Therefore, the use of DDGS in poultry diets has been low to avoid any adverse effects on the quality of animal products.
Antioxidant Capacity
Biochemical Parameters The AST, ALT, Ca, HDL, LDL, CHO, and α-tocopherol concentrations in serum were not signifi-
Table 5. Effect of vitamin E on antioxidant capacity of laying hens fed dried distillers grains with solubles (DDGS)1 Antioxidant capacity In egg yolk Item DDGS (%) 0 0 0 Main effect means for DDGS 0% DDGS 10% DDGS 20% DDGS Main effect means for VE 0 mg/kg of VE 200 mg/kg of VE P-value DDGS VE DDGS × VE SEM a,bMeans
VE (mg/kg)
MDA (nmol/mg of protein)
0 200 0 200 0 200
1.56 1.46 1.61 1.47 1.55 1.45
55.9 59.9 59.0 62.7 55.8 62.0
184 189 184 189 185 194
3.03 2.83 3.06 2.89 3.06 2.90
662 686 637 659 656 671
287 300 278 293 273 295
1.51 1.54 1.50
57.9 60.9 58.9
186 187 189
2.93 2.99 2.98
674 648 664
293 286 284
1.57a 1.46b
56.9b 61.5a
184b 190a
3.05a 2.88b
652b 672a
279b 296a
0.82 0.044 0.96 0.027
GSH-Px (U)
In serum
0.44 0.022 0.85 0.954
T-SOD (U/mg of protein)
0.57 0.022 0.78 1.278
MDA (nmol/mL)
0.82 0.044 0.91 0.040
GSH-Px (U)
0.18 0.046 0.92 4.926
T-SOD (U/mL)
0.49 0.023 0.85 3.408
with different superscripts within each column are significantly different (P < 0.05). vitamin E; DDGS × VE: interaction of DDGS and vitamin E. MDA: malondialdehyde; GSH-Px: glutathione peroxidase; T-SOD: total superoxide dismutase. Data are means of 6 replicates, with 4 samples per replicate. 1VE:
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Table 5 showed significant effects of 200 mg/kg of vitamin E on the specific activities of MDA, GSH-Px, and T-SOD of the egg yolk and serum (P < 0.05), similar to reports by Lin et al. (2005) and Song (2013). According to antioxidant theory, when the concentrations of antioxidants decrease, lipid peroxidation increases in the tissues and plasma, leading to damage of cell membranes (Gallo-Torres, 1980; McDowell, 1989). Vitamin E is the major fat-soluble antioxidant, which breaks the chain reaction of lipid peroxidation. By its free radical quenching activity, it breaks chain propagation and thus terminates free radical attack at an early stage, such an effect of vitamin E is on polyunsaturated fatty acids of biomembranes (McDowell, 1989). However, our results did not show significant effects of DDGS supplementation on MDA, T-SOD, and GSH-Px. Consequently, dietary supplementation with higher levels of vitamin E enhanced antioxidant capacity of egg and laying hens.
cantly influenced by DDGS (Table 6). However, increasing DDGS to 10 or 20% in laying hen diets significantly (P < 0.05) increased P content in serum compared with 0% DDGS. Dietary supplementation with 200 mg/kg of vitamin E significantly decreased CHO (P < 0.05). The decrease of CHO in serum was correlated to decreased CHO in egg yolk. Meanwhile, dietary supplementation with 200 mg/kg of vitamin E significantly increased the α-tocopherol concentration in serum as well as yolk. These results were consistent with the results of Sahin et al. (2002). High α-tocopherol concentration may increase the oxidative stability and provide a source of tocopherols that is useful for human nutrition and health. The AST, ALT, Ca, HDL cholesterol, LDL cholesterol, and P were not significantly influenced by vitamin E, consistent with previous reports (Nadia et al., 2008). In conclusion, DDGS and vitamin E did not have adverse effects on liver functions (AST and ALT) or blood constituents. The results showed an improvement due to dietary supplementation of 200 mg/kg of vitamin E independent of the presence of DDGS, when the vitamin E level in the basal diet was 10 mg/kg. This was well above the NRC requirement. Scheideler and Froning (1996) reported that diet supplemented with 50 mg/kg of vitamin E had greater egg production than diet with 27 mg/kg of vitamin E. Ciftci et al. (2005) showed that supplementation with 125 mg/kg of vitamin E significantly increased egg production in laying hens exposed to heat stress. Also, Lin et al. (2005) reported dietary supplementation of 40, 80, 120, and 160 mg/kg of vitamin E enhanced the oxidative stability in the brain and liver of Taiwan native chicks. These results suggest that
2963
RESEARCH NOTE Table 6. Effect of vitamin E on biochemical parameters of laying hens fed dried distillers grains with solubles
(DDGS)1
Biochemical parameter VE (mg/kg)
Item
0 200 0 200 0 200
ALT (U/L)
CHO P Ca (mmol/L) (mmol/L) (mmol/L)
HDL cholesterol (mmol/L)
LDL cholesterol (mmol/L)
α-Tocopherol (µg/mL)
164 171 158 160 170 178
22.5 24.3 21.3 22.0 22.8 23.3
2.89 2.63 3.02 2.71 2.82 2.51
2.26 2.17 2.72 2.71 2.86 2.76
6.35 6.98 6.60 7.03 6.60 6.95
0.07 0.06 0.08 0.07 0.07 0.07
0.55 0.55 0.53 0.55 0.54 0.54
8.01 8.70 8.51 9.86 8.71 9.99
168 157 174
23.4 21.5 23.1
2.76 2.86 2.67
2.21b 2.72a 2.81a
6.66 6.81 6.77
0.07 0.08 0.07
0.55 0.54 0.54
8.35 9.18 9.35
161 172
22.0 23.3
2.91a 2.62b
2.61 2.54
6.51 6.99
0.07 0.07
0.54 0.55
8.41b 9.51a
0.35 0.012 0.98 0.055
0.022 0.72 0.98 0.089
0.95 0.27 0.96 0.209
0.65 0.41 0.98 0.004
0.68 0.44 0.93 0.004
0.079 0.006 0.73 0.185
0.38 0.27 0.90 4.819
0.11 0.10 0.76 0.376
a,bMeans
with different superscripts within each column are significantly different (P < 0.05). vitamin E; AST: aspartate aminotransferase; ALT: alanine aminotransferase; CHO: cholesterol; HDL = high-density lipoprotein; LDL = lowdensity lipoprotein; DDGS × VE: interaction of DDGS and vitamin E. Data are means of 6 replicates, with 4 samples per replicate. 1VE:
the vitamin E requirement in some circumstances may be higher than that of NRC.
Conclusions The results showed that supplementation with 10% DDGS did not negatively affect laying performance and egg quality parameters. However, supplementation with 20% DDGS yielded the worst feed conversion. Diets containing high levels of DDGS changed the egg fatty acid composition. Additionally, dietary supplementation with 200 mg/kg of vitamin E improved egg production and egg quality, and enhanced the antioxidant capacity of laying hens. However, there was no interaction between DDGS and vitamin E, which suggested that vitamin E improved performance of layers due to vitamin E independent of the presence of DDGS.
ACKNOWLEDGMENTS This work was supported by the National Natural Science Foundation of China (no. 31272453). We thank Lijun Xu, Chongpeng Bi, Baojing Cheng, and Zhongyu Li at Northeast Agricultural University for technical assistance.
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Bollengier-Lee, S., M. A. Mitchell, D. B. Utomo, P. E. Williams, and C. C. Whitehead. 1998. Influence of high dietary vitamin E supplementation on egg production and plasma characteristics in hens subjected to heat stress. Br. Poult. Sci. 39:106–112. Brigelius-Flohé, R., F. J. Kelly, J. T. Salonen, J. Neuzil, J. M. Zingg, and A. Azzi. 2002. The European perspective on vitamin E: Current knowledge and future research. Am. J. Clin. Nutr. 76:703–716. Cherian, G., F. W. Wolfe, and J. S. Sim. 1996. Dietary oils with added tocopherols: Effects on egg or tissue tocopherols, fatty acids, and oxidative stability. Poult. Sci. 75:423–431. Ciftci, M., O. N. Ertas, and T. Guler. 2005. Effects of vitamin E and vitamin C dietary supplementation on egg production and egg quality of laying hens exposed to a chronic heat stress. Revue Méd. Vét. 156:107–111. Corzo, A., M. W. Schilling, R. E. Loar, V. Jackson, S. Kin, and V. Radhakrisnan. 2009. The effect of feeding distillers dried grains with solubles on broiler meat quality. Poult. Sci. 88:432–439. Cozannet, P., M. Lessire, J. P. Metayer, C. Gady, Y. Primot, P. A. Geraert, L. Le. Tutour, F. Skiba, and J. Noblet. 2010. Nutritive value of wheat and maize distillers dried grains with solubles for poultry. INRA Prod. Anim. 23:405–414. Creswell, D. C. 2006. DDGS: Benefits and limitations. Asian Poultry Magazine 22–24. Cromwell, G. L., K. L. Herkelman, and T. S. Stahly. 1993. Physical, chemical, and nutritional characteristics of distillers dried grains with solubles for chicks and pigs. J. Anim. Sci. 71:679–686. Deniz, G., H. Gencoglu, S. S. Gezen, I. I. Turkmen, A. Orman, and C. Kara. 2013. Effects of feeding corn distillers dried grains with solubles with and without enzyme cocktail supplementation to laying hens on performance, egg quality, selected manure parameters, and feed cost. Livest. Sci. 152:174–181. Faustman, C. 1994. Postmortem changes in muscle foods. Pages 63–78 in Muscle Foods. D. M. Kinsman, A. W. Kotula, and B. C. Breidenstein, ed. Chapman and Hall, New York, NY. Folch, J., M. Lees, and G. H. Sloane Stanley. 1957. A simple method for the isolation and purification of total lipids from animal tissues. J. Biol. Chem. 226:497–509. Franchini, A., F. Sirri, N. Tallarico, G. Minelli, N. Iaffaldano, and A. Meluzzi. 2002. Oxidative stability and sensory and functional properties of eggs from laying hens fed supranutritional doses of vitamins E and C. Poult. Sci. 81:1744–1750.
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DDGS (%) 0 10 20 Main effect means for DDGS 0% DDGS 10% DDGS 20% DDGS Main effect means for VE 0 mg/kg of VE 200 mg/kg of VE P-value DDGS VE DDGS × VE SEM
AST (U/L)
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