2010 Poultry Science Association, Inc. Supplemental vitamin E and selenium effects on egg production, egg quality, and egg deposition of α-tocopherol and selenium S. E. Scheideler,* 1 P. Weber,* and D. Monsalve * Department of Animal Science, University of Nebraska, 1035 NE 43rd St., Lincoln 68583-0908; and Hills Pet Nutrition, Topeka, KS 66617 Primary Audience: Egg Producers and Egg Processors, Poultry Nutritionists SUMMARY Diets were fed to laying hens for 12 wk during hot summer weather (34 to 35 C high temperatures daily) to investigate the effects of feeding higher levels of the dietary antioxidants d l-α-tocopherol and selenium (from 2 sources, inorganic or organic) on egg production and egg quality. High basal levels of selenium were present in the corn-soybean meal diets (0.25 ppm), resulting in selenium treatment levels of 0.55 or 0.75 ppm; supplemented α-tocopherol treatments were 50, 100, or 150 IU/kg. Increasing dietary selenium had a positive effect on egg production and egg mass (g of egg/d) as well as stored egg vitelline membrane strength. Vitamin E supplementation did not affect egg production except during wk 7 of the trial, during a particularly hot period in which hens fed the higher levels of vitamin E (100 or 150 ppm) did not have a decline in egg production. Yolk vitelline membrane strength improved with vitamin E supplementation in fresh eggs and eggs stored for 2 wk. Vitamin E supplementation also affected egg ph, improving albumen ph by decreasing ph of freshly laid eggs. Yolk α-tocopherol increased linearly with vitamin E supplementation. Yolk selenium content also increased with dietary Se supplementation and was deposited more efficiently when feeding the organic source (Sel-Plex) compared with the inorganic source of selenium. In summary, vitamin E and selenium can be supplemented to a laying hen ration to improve the vitelline membrane strength of fresh and aged eggs while also increasing the levels of these nutrients in the egg yolk. Key words: vitelline membrane strength, α-tocopherol, selenium, egg quality 2010 J. Appl. Poult. Res. 19 :354 360 doi: 10.3382/japr.2010-00198 DESCRIPTION OF PROBLEM Physiological processes and production performance of laying hens are affected by extreme temperature conditions (cold or hot). According to the Hy-Line W-36 Commercial Management Guide [1], hens should be housed at or near 21 to 28 C. At temperatures above 28 C (e.g., in the Midwest region of the United States), diet and management adjustments must be made to maintain good performance, productivity, and overall quality of eggs produced when feed intake decreases because of high temperatures [1]. Egg variables that can be negatively affected by high ambient temperatures (>30 C) include egg production, egg weight, Haugh units, eggshell 1 Corresponding author: sscheideler@unl.edu
Scheideler et al.: VITAMIN E AND SELENIUM 355 quality, and yolk vitelline membrane strength [2 4]. Studies in the literature on vitelline membrane strength as affected by house temperature, nutrient intake, and egg age are limited. Kirunda and McKee [5] reported a decrease in vitelline membrane strength in aged eggs compared with fresh eggs. Several management and dietary modifications are available to help alleviate the negative effects of high environmental temperature on laying hen production variables. Typically, breeders will recommend increased nutrient density for diets during heat stress because of decreased feed intake to maintain essential nutrient intake recommendations [1]. Vitamin E is known to be a lipid-soluble component of biological membranes and is considered a major antioxidant, protecting cells and tissues from oxidative damage induced by free radicals [6] during heat stress. Tocopherols are incorporated into the liposomal membranes to limit oxidation of phospholipids, particularly phospholipids high in polyunsaturated fatty acids, such as those found in the egg yolk. Selenium is also known as a highly effective antioxidant. A very important metabolic role of selenium is its function at the active site of the enzyme glutathione peroxidase, which protects cells from damage caused by free radicals and lipid peroxides [7]. Glutathione peroxidase destroys peroxides before they have a change to damage the liposomal membranes. Information is limited concerning the potential benefits of vitamin E and selenium supplementation for hens on egg quality measurements, such as vitelline membrane strength and oxidation during heat stress. Kirunda et al. [8] reported an improvement in feed intake, egg production, Haugh units, egg weights, vitelline membrane strength, and egg functional properties such as foam stability, angel cake volume, and emulsification capacity when hens were fed high levels of vitamin E (60 IU/kg) compared with 20 IU/kg or the NRC [9] recommended level of 10 IU/kg. The objective of this research was to investigate vitamin E and selenium supplementation from 2 dietary sources (organic vs. inorganic) in laying hen diets for improved egg quality properties during heat stress conditions. MATERIALS AND METHODS Single Comb White Leghorn hens, Bovan White [10], 24 wk old, were obtained from a commercial laying hen operation and transported to the Animal Science Department at the University of Nebraska Lincoln. This age of hen was selected for the trial because the hens were already reaching peak egg production (>90% hen-day egg production). The hens were acclimated for 2 wk, from 24 to 26 wk of age, before the beginning of the trial. During this time, they were fed the basal ration with no supplemental α-tocopherol or selenium. Animal care for this experiment complied with procedures approved by the University of Nebraska Institutional Animal Care and Use Committee. Two hundred eighty-eight 26-wk-old laying hens were randomly assigned to 48 cages in a single laying hen unit, with 6 hens per cage. The unit was a stacked-deck Big Dutchman [11] cage unit (44 52.8 cm) with 3 tiers, equipped with an automated manure belt, nipple waterers, and feed troughs. Hens had access to 387 cm 2 /hen. Cages were blocked by side, north and south, each side with a total of 24 cages, in an augmented 3 2 2 factorial arrangement of 3 levels of supplemental d l-α-tocopherol (vitamin E [12]; calculated: 50, 100, or 150 IU/kg; actual: 49, 119, 170 IU/kg), 2 levels ary selenium (calculated: 0.25 or 0.50 ppm; actual: 0.55 or 0.75 ppm), and 2 supplemental sources of selenium, inorganic (Na 2 SeO 3 ) or organic (Sel-Plex [13]), added to a corn-soybean meal basal diet (basal level of selenium was 0.25 ppm). Each treatment was assigned to 4 replicate cages. Birds were fed the dietary treatments for 12 wk (until phase 2 of egg production). A basal diet was formulated and the dietary treatments varying in α-tocopherol and selenium were added to the basal diets (Table 1). Diets were formulated to meet nutrient requirements as recommended by the NRC [9] and the breeder company (Hendrix Poultry Breeders [10]), based on a feed consumption rate of 95 to 100 g/ hen per day at temperatures above 30 C. Selenium and α-tocopherol E were weighed, added to a small bowl with 4 kg of basal diet, and mixed for 20 min to create a premix. The premix was added to 100 kg of basal diet and mixed again
356 Table 1. Diet composition and nutrient content of the standard corn-soybean diet Item Amount, % Ingredient Corn 58.70 Soybean meal 26.11 Corn oil 3.41 Calcium carbonate 9.28 Dicalcium phosphate 1.56 Salt 0.49 d l-methionine 0.22 Lysine 0.15 Vitamin premix 1 0.08 Mineral premix 2 0.08 Calculated nutrient composition ME, kcal/kg 2,900 Protein, % 18.25 Methionine, % 0.47 Lysine, % 0.95 Calcium, % 3.95 Available phosphorus, % 0.38 Total phosphorus, % 0.60 1 Vitamin premix minimum guaranteed analysis: vitamin A, 6,000,000 IU/lb (2,727,272 IU/kg); vitamin D, 2,600,000 IU/ lb (1,181,818 IU/kg); vitamin E, 8,000 IU/lb (3,636 IU/kg), vitamin K, 1,200 IU/lb (545 IU/kg); vitamin B 12, 10 mg/lb (4.5 mg/kg); riboflavin, 25,000 mg/lb (11,363 mg/kg); niacin, 25,000 mg/lb (11,363 mg/kg); d-pantothenic acid, 8,500 mg/lb (3,863 mg/kg); folic acid, 200 mg/lb (91 mg/kg). 2 Mineral premix minimum guaranteed analysis: Mn, 88 mg; Cu, 66 mg; Fe, 8.5 mg; Zn, 88 mg; Se, 0.30 mg/kg. for 20 min in a 100-kg mixer. Diet samples were collected from each batch, mixed, and pooled for analysis of α-tocopherol and selenium. A total of 3 batches were mixed during the trial. Hens were weighed on d 1 of the trial and then every 4 wk. Hen average BW was calculated to determine BW change. Feed intake was recorded on a daily basis and calculated per hen per day. Egg production was recorded daily and calculated on a hen per day basis. Egg weights were recorded for 1-d egg production on a weekly basis. Egg mass was calculated as a function of egg weight percentage of egg production to estimate grams of egg produced per day. Two eggs were used per cage to measure egg components, percentage of dry shell, albumen, and yolk on a monthly basis. Haugh unit values [14], a mathematical relationship between egg weight and albumen height, were measured every 4 wk on the same 2 eggs per replicate cage used for other egg measurements. Pooled samples (n = 6 per pool 4 replicates for each treatment group) JAPR: Research Report were analyzed for α-tocopherol content of the yolk [15] and yolk selenium content [16]. Fresh eggs and aged eggs (stored at 5 C for 2 wk) were used to measure vitelline membrane strength (6 eggs per replicate cage) by an extrusion cell on an Instron universal testing machine [17]. Production data were analyzed using the repeated-measures MIXED procedure of SAS software [18] for a randomized complete block design. Blocking was implemented to reduce the effect of temperature stratification in the cage unit. Block was considered a random effect, and dietary treatments were considered fixed effects. The main effects of time (wk) and dietary treatments (vitamin E, selenium level, and selenium source) were tested, as well as 2- and 3-way interaction effects. No 3-way interaction effects were significant. A significant time treatment interaction effect was significant only during wk 7 of the trial for egg production. Average values for the variables were generated and were subsequently analyzed separately to determine differences between treatment means using the least squares difference. RESULTS AND DISCUSSION There was not an overall effect of vitamin E on egg production; however, when monitoring the egg production time interactions, we found a considerable decrease in egg production attributable to hot weather during wk 7. Week 7 was during August, when high temperatures rose to an average of 34 to 35 C for a 4-d period. Hens fed 50 IU/kg of vitamin E had lower egg production (87.5%) compared with hens fed 150 and 100 IU/kg of vitamin E (92.4 and 92.1%, respectively) during wk 7 only (P < 0.03). Vitamin E, selenium level, and selenium source had no significant effects on feed intake, feed efficiency, egg weight, or hen BW (Table 2) during the entire trial. Level ary selenium did significantly affect rate of egg production (P < 0.03) and egg mass (P < 0.02). Hens fed diets with 0.75 ppm of selenium laid 1.68% more eggs and the egg mass increased by 1.23 g compared with those fed diets with 0.55 ppm of selenium (Table 2). Hens consuming the lowest level of vitamin E in the diet (50 IU/kg) had less α-tocopherol in the yolk (121.74 µg/g) compared with hens fed vitamin E levels of 100 and 150 IU/kg, which
Scheideler et al.: VITAMIN E AND SELENIUM 357 Table 2. Effect ary treatments on hen production measurements Item Vitamin E, IU/kg Se, mg/kg Se source Egg Feed production, intake, % g/hen per day Egg weight, g Egg mass, g/d Hen BW, kg Diet 1 50 0.55 Inorganic 92.94 93.05 56.15 52.28 1.50 2 50 0.55 Organic 92.21 95.80 57.72 53.21 1.51 3 50 0.75 Inorganic 93.99 97.86 58.95 55.44 1.57 4 50 0.75 Organic 93.45 100.45 57.95 54.19 1.59 5 100 0.55 Inorganic 91.17 97.02 59.24 54.13 1.55 6 100 0.55 Organic 93.85 98.89 58.30 54.78 1.53 7 100 0.75 Inorganic 94.69 98.10 58.04 55.00 1.54 8 100 0.75 Organic 92.38 100.17 59.35 54.89 1.59 9 150 0.55 Inorganic 92.38 97.66 58.41 53.93 1.53 10 150 0.55 Organic 91.93 96.37 58.20 53.57 1.54 11 150 0.75 Inorganic 94.14 95.35 57.30 53.99 1.55 12 150 0.75 Organic 95.48 101.86 58.06 55.53 1.54 SEM 1.31 2.26 0.53 1.009 0.02 Main effect Vitamin E, IU/kg 50 93.15 96.87 57.70 53.79 1.54 100 93.06 98.60 58.72 54.72 1.55 150 93.50 97.88 57.99 54.29 1.54 SEM 0.80 1.16 0.38 0.71 0.01 Se, mg/kg 0.55 92.41 b 96.50 57.99 53.65 b 1.52 0.75 94.06 a 99.08 58.29 54.88 a 1.56 SEM 0.73 0.95 0.33 0.66 0.01 Se source Inorganic 93.21 96.57 57.99 54.12 1.53 Organic 93.26 99.01 58.29 54.41 1.55 SEM 0.73 0.95 0.33 0.66 0.01 Contrast, P-value Vitamin E, IU/kg 0.848 0.582 0.093 0.345 0.943 Se, mg/kg 0.030 0.079 0.451 0.02 0.076 Se source 0.950 0.094 0.446 0.568 0.461 Vitamin E Se source 0.741 0.960 0.998 0.842 0.877 Vitamin E Se level 0.502 0.503 0.068 0.406 0.339 Se source Se level 0.453 0.367 0.918 0.668 0.742 a,b Means with no common superscript differ significantly (P < 005). showed significant differences (P < 0.001), with 260.84 and 373.40 μg/g of α-tocopherol, respectively (Table 3). Hens fed the lower level of selenium in the diet (0.55 ppm) had higher α-tocopherol content in the yolk (273.21 µg/g) compared with hens fed 0.75 ppm of selenium (230.77 µg/g), resulting in a significant interaction between vitamin E and selenium levels (P < 0.04) on egg yolk deposition of antioxidants (Table 3). Supplementing dietary selenium can be a less expensive antioxidant option in commercial poultry diets than adding high levels of vitamin E. In this trial, selenium deposition in the egg yolk was significantly higher (P < 0.001) for the organic selenium source (0.75 μg/g) than the inorganic source (0.60 µg/g; Table 3). Hens fed the higher level of selenium (0.75 ppm) in the diet had significantly higher (P < 0.001) selenium deposition in the egg yolk compared with hens fed the lower level ary selenium (0.55 ppm; Table 3). Dietary selenium added to the hen diet, whether as an inorganic or an organic source, will accumulate more in the egg yolk than in the egg albumen according to Paton et al. [19], who also reported selenium deposition in the egg yolk to be significantly higher for
358 JAPR: Research Report Table 3. Effect ary treatments on egg variables Item Vitamin E, IU/kg Se, mg/kg Se source Yolk ph Albumen ph VMSF, 1 kg/mm VMSA, 2 kg/mm Yolk Se, μg/g Yolk, α-tocopherol, μg/g Diet 1 50 0.55 Inorganic 6.12 9.05 0.43 0.44 0.56 122.39 2 50 0.55 Organic 6.07 8.87 0.34 0.42 0.69 129.00 3 50 0.75 Inorganic 6.15 8.86 0.42 0.39 0.64 114.03 4 50 0.75 Organic 6.06 9.08 0.44 0.44 0.81 121.54 5 100 0.55 Inorganic 6.09 8.96 0.45 0.43 0.58 298.31 6 100 0.55 Organic 6.05 9.04 0.46 0.41 0.62 238.97 7 100 0.75 Inorganic 6.07 9.01 0.47 0.50 0.62 269.32 8 100 0.75 Organic 6.07 9.04 0.48 0.48 0.83 236.77 9 150 0.55 Inorganic 6.18 8.91 0.53 0.45 0.57 443.75 10 150 0.55 Organic 6.13 8.71 0.49 0.45 0.76 406.85 11 150 0.75 Inorganic 6.10 9.10 0.49 0.47 0.63 310.98 12 150 0.75 Organic 6.13 8.60 0.48 0.50 0.79 332.00 SEM 0.02 0.06 0.02 0.02 0.07 47.69 Main effect Vitamin E, IU/kg 50 6.10 b 8.97 a 0.41 c 0.42 0.67 121.74 c 100 6.07 b 9.01 a 0.46 b 0.45 0.66 260.84 b 150 6.14 a 8.83 b 0.50 a 0.47 0.69 373.40 a SEM 0.017 0.03 0.01 0.01 0.05 21.08 Se, mg/kg 0.55 6.11 8.92 0.45 0.43 0.63 b 273.21 a 0.75 6.10 8.95 0.47 0.46 0.72 a 230.77 b SEM 0.016 0.02 0.008 0.01 0.05 18.61 Se source Inorganic 6.12 a 8.98 a 0.46 0.45 0.60 b 259.80 Organic 6.08 b 8.89 b 0.45 0.45 0.75 a 244.19 SEM 0.016 0.02 0.008 0.01 0.05 18.61 Contrast, P-value Vitamin E, IU/kg 0.003 0.001 0.001 0.074 0.761 0.001 Se, g/kg 0.359 0.518 0.181 0.072 0.001 0.04 Se source 0.008 0.002 0.241 0.758 0.001 0.435 Vitamin E Se source 0.139 0.202 0.271 0.554 0.667 0.536 Vitamin E Se level 0.235 0.946 0.090 0.123 0.422 0.104 Se source Se level 0.324 0.827 0.069 0.355 0.273 0.475 a c Means with no common superscript differ significantly (P < 0.05). 1 Vitelline membrane strength measured in fresh eggs. 2 Vitelline membrane strength measured in aged eggs. the organic selenium source (0.75 µg/g) than the inorganic source (0.60 µg/g). Hens fed inorganic selenium had higher yolk ph (6.12; P < 0.008) and albumen ph (8.98; P < 0.002) than those fed organic selenium (6.08 and 8.89; Table 3). An increase in ph is a sign of oxidation caused by aging of the egg. As the egg ages and air enters the pores, oxidation of the albumen and yolk can increase, resulting in increased ph. In this study, lower ph in fresh eggs caused by selenium would be an indication of reduced oxidation between the time the egg was laid and the time the ph had been determined. This could have been due to improved vitelline membrane integrity, improved shell membrane strength, or both. We did not test shell membrane strength in this study but did find improved vitelline membrane strength with the addition of vitamin E (Table 3). Addition of vitamin E at 150 IU/kg increased yolk ph by 0.07 units (P <
Scheideler et al.: VITAMIN E AND SELENIUM 359 0.003) and reduced albumen ph by 0.018 (P < 0.001) units compared with hens fed 50 and 100 IU/kg of vitamin E (Table 3). Addition of 150 IU/kg of vitamin E in the diet significantly (P < 0.001) improved vitelline membrane strength of fresh eggs by 0.05 and 0.08 kg/mm, in contrast to 100 and 50 IU/kg of vitamin E in the diet (Table 3). Vitamin E had no significant effect on vitelline membrane strength of aged eggs. The dietary level of selenium approached significance (P < 0.07) with the effects on vitelline membrane strength of stored eggs, with an improvement from 0.43 to 0.46 kg/mm as selenium supplementation increased in the diet from 0.55 to 0.75 mg/kg. This improvement in vitelline membrane strength could help reduce losses in a breaker plant separating yolk from albumen and could improve the shelf life of carton eggs such that the whole egg yolk would not break upon cracking and preparing the egg for consumption. Based on the improvement in rate of egg production and egg quality in this study, we support selenium supplementation of laying hen diets. In addition, others [7, 19] have reported the beneficial effects of selenium supplementation on feed consumption, BW, BW gain, and the prevention of selenium deficiency symptoms and mortality in poultry. The NRC [9] cautions that high levels ary selenium (5 to 20 ppm) can be toxic; however, in this trial, significant benefits were observed by increasing dietary selenium well above the basal diet level of 0.25 ppm. Furthermore, the US Food and Drug Administration limits commercial feed production practices to a maximum of 0.3 ppm of selenium added to complete feeds. The full benefits of increasing selenium may not be fully realized by the poultry producer given these limitations. CONCLUSIONS AND APPLICATIONS 1. Vitamin E and selenium had no beneficial effects on feed intake or egg production variables during this study. Selenium supplementation did increase egg mass at the higher levels fed (0.75 ppm), which would exceed US Food and Drug Administration-regulated selenium levels in the diet. 2. Vitamin E and selenium supplementation both resulted in improved vitelline membrane strength in fresh and stored eggs, respectively. 3. One of the most striking findings in this trial was the competing effect of increased dietary selenium on vitamin E metabolism and transfer to the egg yolk. As vitamin E level increased, egg yolk vitamin E concentration increased linearly, but as selenium level increased, vitamin E concentration in the egg yolk decreased. REFERENCES AND NOTES 1. Hy-Line. 2010. Hy-Line Variety W-36 Commercial Management Guide, 2009 2011. Hy-Line Int., West Des Moines, IA. http://www.hyline.com Accessed June 7, 2010. 2. Smith, A. J. 1974. Changes in the average weight and shell thickness of eggs produced by hens exposed to high environmental temperatures A review. Trop. Anim. Health Prod. 6:237 244. 3. Bell, D. D., W. D. Weaver, and M. O. North, 2002. Commercial Chicken Meat and Egg Production. 5th ed. Kluwer Academic Publishers, Norwell, MA. 4. Stadelman, W. J., and O. J. Cotterill, 1995. Egg Science and Technology. 4th ed. Food Products Press, New York, NY. 5. Kirunda, D. F., and S. R. McKee. 2000. Relating quality characteristics of aged eggs and fresh eggs to vitelline membrane strength as determined by a texture analyzer. Poult. Sci. 79:1189 1193. 6. Gallo-Torres, H. E. 1972. Vitamin E in animal nutrition. Int. J. Vitam. Nutr. Res. 42:312 323. 7. Combs, G. F., and S. B. Combs. 1986. The Role of Selenium in Nutrition. Academic Press, Orlando, Toronto, Canada. 8. Kirunda, D. F., S. E. Scheideler, and S. R. McKee. 2001. The efficacy of vitamin E (d l-α-tocopheryl acetate) supplementation in hen diets to alleviate egg quality deterioration associated with high temperature exposure. Poult. Sci. 80:1378 1383. 9. NRC. 1994. Nutrient Requirements of Poultry. 9th rev. ed. Natl. Acad. Press, Washington, DC. 10. Bovans White, Hendrix Poultry Breeders, Nutreco Company, Boxmeer, the Netherlands. 11. Big Dutchman Inc., Holland, MI. 12. Vitamin E 50% (α-tocopherol acetate), ADM Animal Health and Nutrition, Des Moines, IA. 13. Sel-Plex 2000 (150 g/tonne = 0.3 ppm), Alltech, Nicholasville, KY. 14. Haugh, R. 1937. A new method for determining the quality of an egg. US Poult. Mag. 39:27 49. 15. All diets were analyzed for protein [AOAC. 1996. Protein (Crude) in Animal Feed and Pet Food, Kjeldahl Method. Method 4.2.02. Assoc. Off. Anal. Chem., Gaithersburg, MD] and α-tocoperol (Gaál, T., M. Miklós, R. C.
360 JAPR: Research Report Noble, J. Dixon, and B. K. Speake. 1995. Development of antioxidant capacity in tissues of the chick embryo. Comp. Biochem. Physiol. 112:711 716). 16. Diets and yolks were analyzed for selenium at Agriculture and Agri-Food Canada, Dairy and Swine Nutrition and Metabolism Centre, Lennoxville, Quebec, Canada. 17. An Instron universal testing machine (4500 series, Instron, Norwood, MA), equipped with a back-extrusion food cell, a 2-kg tension load, and a crosshead speed of 1 mm/ min, was used to measure vitelline membrane strength on fresh and aged (2 wk, 5 C) eggs [8]. 18. SAS Institute. 2002. SAS for Linear Models, User s Guide. 4th ed. SAS Inst. Inc., Cary, NC. 19. Paton, N. D., A. H. Cantor, A. J. Pescatore, M. J. Ford, and C. A. Smith. 2002. The effect ary selenium source and level on the uptake of selenium by developing chick embryos. Poult. Sci. 81:1548 1554.