The Effect of Vitamin E on Egg Production, Hatchability and Humoral Immune Response of Chickens. ROBERT P. TENGERDY Department of Microbiology AND

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1 778 H. S. ROSTAGNO, J. C. ROGLER AND W. R. FEATHERSTON sorghum vulgare for the growing rat. Nutrition Reports International, 1: Rostagno, H. S., W. R. Featherston and J. C. Rogler, Studies on the nutritional value of bird resistant sorghum grain for chicks. 1. Growth studies. Poultry Sci. 52: Soares, J. H. Jr., D. Miller, N. Fitz and M. Sanders, Some factors affecting the biological availability of amino acids in fish protein. Poultry Sci. 50: Steel, R. G. D., and J. H. Torrie, Principles and Procedures of Statistics, Ed. 1. McGraw Hill Company, New York, New York. Stephenson, E. L., J. O. York, D. B. Bragg and C. A. Ivy, The amino acid content and availability of different strains of grain sorghum to the chick. Poultry Sci. 50: Vohra, P., F. H. Kratzer and M. A. Joslyn, 19. The growth depressing and toxic effects of tannins to chicks. Poultry Sci. 45: The Effect of Vitamin E on Egg Production, Hatchability and Humoral Immune Response of Chickens THE requirement for vitamin E in practical chicken diets is established on an empirical basis, but the real need for various life functions is little known. Although vitamin E as an antioxidant (Tappel, 1968) and perhaps cell membrane regulatory agent (Lucy, 1972) may affect many metabolic functions, its mode of action is practically unknown and theories about it are contradictory (Legge, 1971). In a previous research, Tengerdy and Heinzerling (1972) and Tengerdy et al. (1972) observed recently that the humoral immune responseof chickensagamst sheep red blood cells was increased by 20-25%, measured by hemagglutination and antibody plaque forming cell tests, if the ROBERT P. TENGERDY Department of Microbiology AND CHERYL F. NOCKELS Department of Animal Sciences, Colorado State University, Fort Collins, Colorado (Received for publication July 31, 1972) ABSTRACT Vitamin E supplementation ( mg./kg.) to a control chicken diet significantly (P < 0.01) increased the humoral immune response of 2 and 4 week old chicks. The same supplementation had no effect on the hatchability of chicken embryos. Hatching at higher altitude was not significantly influenced by vitamin E supplementation. The altitude of hatching had no effect on the immune response of chicks. The immune response of hens and chicks maintained on afish-oilbase vitamin E depleting diet was not impaired but the egg production and hatchability was drastically reduced. POULTRY SCIENCE 52: ,1973 practical diet was supplemented with mg. vitamin E/kg. diet. When the chicks were subjected to 4800 m. simulated high altitude (hypoxia) for 3 days simultaneously with immunization the increase in the above responses was 30-40%. Hypoxia alone also stimulated the immune response of young chicks, as reported by Tengerdy (1970). On the other hand, hypoxia significantly reduced the hatchability of chicken embryos (Tengerdy et al., 1969). These observations suggest that vitamin E and hypoxia may act synergistically as antioxidants or metabolic redox potential regulatory agents. In certain cellular developmental processes such as in immuno-

2 VITAMIN E EFFECTS 779 poietic development this antioxidant effect is apparently stimulatory, yet in the overall embryonic development such an effect may be deleterious. The main purpose of the present investigation was to find out how vitamin E affects the hatchability of chicken embryos and the humoral immune response of chicks and hens, and to find out what may be an optimal dietary vitamin E level for these functions. High altitude induced hypoxia was used as an experimental parameter in some of the experiments because it may contribute to our knowledge on the assumed antioxidant role of vitamin E in these functions. MATERIALS AND METHODS Chickens and their care: Since two main nutritional experiments were conducted, one with normal or excessive vitamin E levels, the other with a vitamin E depleted diet, the two are described here separately. In the first experiment, two hundred and sixteen one-year-old Colorado State University Single Comb White Leghorn station stock hens were equally divided into three groups. There were two hens per cage and water and feed were supplied ad libitum. All three groups of hens were fed the same diet (Table 1) which differed only in the amount of DL-a-tocopherol added per kg.: group 1, 6.6 mg., group 2, mg., group 3, mg. The hens were artificially inseminated and eggs were collected for incubation five weeks after the hens had been on their respective diets. Two hundred eggs from each treatment group of hens were incubated in the Colorado State University Animal Sciences Department incubator (1500 m. altitude), and 200 eggs from each group were incubated at the University of Wyoming, Laramie, incubator (2340 m. altitude). Approximately 65 eggs from each treat- TABLE 1.Composition of the basal diet Ingredient % in Ration Ground yellow corn Soybean meal (44% protein) Dehydrated alfalfa 2.50 Ground limestone 4.50 Dicalcium phosphate 2.70 Salt (iodized) 0.50 Trace mineral mix* 0.05 Vitamin mix** 0.58 ' * Trace mineral mix supplies: Mn, 50 p.p.m.; Fe, 50 p.p.m.; Cu, 5 p.p.m.; Co, 0.5 p.p.m.; I 2, 1.5 p.p.m.; and Zn, 50 p.p.m. ** Vitamin mix supplies per kg. of ration: Vitamin A, 2643 units; vitamin D3, 743 units; vitamin E, 6.6 units; niacin, 26 mg.; choline, 330 mg.; pantothenic acid, 2.2 mg.; riboflavin, 2.2 mg.; menadione, 0.2 mg.; and vitamin B12, 4.4 meg. ment group were incubated in a hypobaric altitude chamber (3000 m. simulated altitude) with corresponding controls incubated in the Colorado State University incubator. For the simulated high altitude exposure a hypobaric chamber, described in detail in a previous publication, was used (Tengerdy et al., 1969). The eggs were rotated daily by 180 while the chamber was brought to ambient pressure briefly. The temperature and humidity were maintained approximately as in the commercial incubator, but the fluctuations were much greater due to constant drawing of air through the chamber. The eggs were in the hypobaric chamber between the days of incubation. Chicks were placed on the standard Colorado State University chick starter diet which was supplemented at the same three levels of vitamin E as the diet of the dams (Dorr and Nockels, 1971). The chicks were placed on the same vitamin E level as their dams had received and housed on raised wire floors in electrically heated battery brooders. In the second experiment the effect of vitamin E deficiency on the immune response was tested. One hundred and

3 780 R. P. TENGERDY AND C. F. NOCKELS TABLE 2.Composition of diets for hens and chicks Milo Soybean meal (44% protein) Soybean oil Ground limestone Dicalcium phosphate Salt (iodized) Methionine Coccidiostat Trace mineral mix 1 Vitamin mix Hen Chick Trace mineral mix is the same as shown in Table 1. 2 Vitamin mix supplies per kg. of ration: Vitamin A, 8800 units; vitamin D3, 1210 units; vitamin E, 44 units; niacin, 31.5 mg.; pantothenic acid, 5.28 mg.; riboflavin, 4.4 mg.; menadione, 2.9 mg.; choline, 1540 mg.; folic acid, 8.8 mg.; thiamine, 2.9 mg.; pyridoxine, 5.5 mg.; vitamin B12, 10.3 meg. 3 Vitamin mix supplies per kg. of ration: Vitamin A, 00 units; vitamin D 3, 880 units; vitamin E, 44 units; niacin, 21.5 mg.; pantothenic acid, 5.5 mg.; riboflavin, 4.4 mg.; menadione, 2.9 mg.; choline, 440 mg.; folic acid 1.5 mg.; thiamine 2.3 mg.; pyridoxine, 6.6 mg.; vitamin B12, 10.3 meg. ninety-two Single Comb White Leghorn hens, 32 weeks old, were placed on the 10% soybean oil diet shown in Table 2 for two weeks. There were two hens per cage, and the hens received 16 hours of light daily and feed and water ad libitum. Egg production was determined for each cage of hens in the two-week pretreatment period. After the pretreatment, 96 hens were placed on a 10% fish oil ration. This fish oil ration was identical to the soybean oil ration except that the fish oil was substituted for the soybean oil and the vitamin E was omitted. This fish oil was used to deplete the hens' reserves of vitamin E as this oil had produced typical vitamin E deficiency symptoms when fed to chicks (National Research Council, 1971). Egg production records were maintained on both control and vitamin E depleted hens. The hens were artificially inseminated and eggs were collected for hatching one, two, three, and four weeks after the fish oil ration was fed. Hatched chicks from control hens were placed on a 10% soybean oil ration chick starter (Table 2) and chicks from depleted hens were placed on the 10% fish oil chick starter ration. The fish oil ration was identical to the soybean oil ration except the fish oil replaced the soybean oil and vitamin E was omitted from the ration. The chicks were housed in electrically heated batteries on raised wire floors and received feed and water ad libitum. Immunization procedures and tests: Twentyfive 2 and 4 week old chicks and hens after 5 weeks on their respective diets were immunized intravenously with 0.1 or 0.5 ml. 20% sheep red blood cell (SRBC) respectively. The humoral immune response was measured 4 and 7 days after immunization by the micro hemagglutination (HA) test (Herbert, 1967). Since a higher response was always obtained on the seventh day, only these results are reported. Five chicks or hens per group were used in the HA test, and the assays were done in triplicate. The significance of the results was calculated from the t test and given as the probability (P) value (Snedecor and Cochran, 1967). RESULTS Effect of altitude and high dietary vitamin E level on hatchability and immune response of chickens: The purpose of this experiment was to find out whether or not high dietary vitamin E level, which is stimulatory for the humoral immune response, may affect adversely hatchability at control (1500 m.) or high altitudes due to a possible enhanced antioxidant effect. The results showed that excessive levels of vitamin E had no significant effect on the hatchability either at control altitude (1500 m.) or at a higher natural (2340 m.) or simulated altitude (3000 m.) (Table 3).

4 VITAMIN E EFFECTS 781 At 2340 m. the hatchability was greatly reduced in all dietary treatment groups. Even the relatively moderate increase of 840 m. in altitude resulted in a significant (P<0.01), greater than 20% decrease in hatchability. A greater decrease resulted in the altitude chamber (3000 m.) even though the eggs were there only between the days of incubation. This decrease, however, was probably partly due to the inadequacy of the altitude chamber as an egg incubator. Although the differences between the control and vitamin E supplemented groups were not significant, it appears that excess vitamin E may further reduce hatchability at high altitudes. This suggests that the antioxidant effect of vitamin E may aggravate the hypoxia caused oxygen insufficiency for the developing chicken embryo. Twenty-five chicks from each hatched group (dietary and/or altitude treatment) were immunized at age 2 or 4 weeks with SRBC and the HA response was measured 7 days later. The results are shown in Table 4. The HA titers were significantly higher (P<0.01) in the vitamin E supple- TABEE 3.Effect of dietary vitamin E level on the hatchability of chicken embryos at different altitudes Altitude (m.) 1500 (Ft. Collins Station 1 ) 2340 (Laramie Station 1 ) 3000 (altitude chamber 2 ) Vitamin E supplement mg./kg %of fertile eggs % of eggs hatched' 79A 76A 76A 57B 55B 56B 22C 20C 21C i Two hundred eggs were in each treatment group. 8 Sixty-five eggs were in each treatment group. The eggs were in the altitude chamber from the days of incubation only, because full time incubation in the chamber resulted in no hatching at all. «The differences between altitude treatments (different capital letters) were highly significant (P <0.01); the differences between vitamin E treatments (same capital letters) were not significant. TABLE 4.Effect of dietary vitamin E level on the primary immune response of chicks against sheep red Mood cell Hatching Altitude (m.) 1500 (Ft. Collins Station) 2340 (Laramie Station) Vitamin E supplement mg./kg Hemagglutination Ioga titer 2 week old 1 chicks mean±s.d. 5.8±0.21A 2 6.8±0.22B 8.2±0.50C 6.0±0.22A 6.8±0.22B 8.1±0.19C 4 week old 1 chicks mean±s.d A 6.7±0.31B 9.3±0.40C 6.3±0.22A 6.9±0.31B 9.3±0.42C 1 Twenty-five chicks were in each group. 2 The differences between vitamin E groups (different capital letters) were significant (P<0.01), but the differences between altitude groups (same capital letters) were insignificant. mented groups, but the altitude of hatching apparently did not affect the humoral immune response of the chicks. Effect of vitamin E deficiency on hatchability, egg production, and immune development of chicks: The purpose of this experiment was to determine the progressive deterioration in egg production, hatchability, and immune response of chicks with increasing vitamin E depletion. The results of the percent egg production, percent fertile eggs, and hatchability of fertile eggs from the hens fed the control and vitamin E depleting rations are shown in Table 5. Pretreatment egg production results showed that there was no difference between the control and experimental hens. However, egg production was markedly reduced while the number of embryos dead at 7 days incubation was greatly increased after the hens had ingested the fish oil ration for two weeks. Percent hatch of fertile eggs from fish oil fed hens was much reduced after just one week on the diet and after 2 weeks it approached zero. The chicks hatched from eggs of hens maintained on the fish oil ration for 1 or 2 weeks were immunized at age 7 days with SRBC, and the humoral immune response was followed by the HA test. The chicks

5 782 R. P. TENGERDY AND C. F. NOCKELS TABLE 5.Egg production, fertility and hatchability of eggs from hens fed a control and vitamin E depleting ration Weeks %Egg production Eggs from control hens Eggs set Eggs from vitamin E depleted hens % eggs fertile % dead at 1 days incubation % dead, pipped and hatched are computed from fertile eggs. 2 Two week pretreatment period. 3 Hatched fertile eggs. were maintained on the same vitamin E level as their dams (Table 2). At the time of the immunization, the chicks showed typical symptoms of vitamin E deficiency. For comparison the vitamin E depleted hens were also immunized with SRBC after 4 weeks on the fish oil diet. The HA response of both chicks and hens is shown in Table 6. There was no significant change in the humoral immune response of either hens or chicks.this experiment thus shows that the same degree of vitamin E deficiency that drastically reduces hatchability and egg production has no effect on the humoral immune response of either hens or chicks. DISCUSSION The vitamin E requirement for chickens is usually estimated on the basis of hatching and egg production (National Research Council, 1971), but if other life functions are also considered this requirement may change. One of the important functions of higher forms of animal life is the immune response which is mainly responsible for disease resistance, and surveillance of cancer, and therefore, should % Pipped % HFE« have a practical importance for the poultry grower. In the present paper the effect of vitamin E on two different life functions, hatchability and immune response, was compared, because it appeared that the vitamin E requirement for these two functions was different and perhaps the mode of action of vitamin E was opposite. The "normal" dietary level of vitamin E, about mg./kg. diet, used in many commercial chicken feed rations seems to be adequate for supporting embryonic development under normal or high altitude TABLE 6.Effect of vitamin E deficiency on the primary immune response of chicks and hens against sheep red blood cell Diet Vitamin E depleted Control Hemagglutination log* titer Hens' means±s.d. 5.8± ±2.1 1st hatch* means±s.d. 2.64± ±1.03 Chicks 2nd hatch! means+s.d. 3.06± ± Five hens were immunized after 4 weeks on the depleting fish-oil diet. 2 Eggs collected from hens maintained on the depleting diet for 1 week; 25 chicks were immunized. 8 Eggs collected from hens maintained on the depleting diet for 2 weeks; 25 chicks were immunized. All chicks were immunized at age 7 days, with 0.1 ml. of 20 v.% SRBC suspension; 5 chicks were used per HA assay. The observed differences in HA titers are not significant.

6 VITAMIN E EFFECTS 783 hatching conditions. An excessive vitamin E level does not improve hatchability, and at high altitudes may even counteract it. On the other hand, the optimal vitamin E level for the immune response of chickens was at least at or above mg./kg. diet, far above the vitamin E level of commercial chicken rations. The depletion of the vitamin E level, provided by the fish oil depleting diet, drastically reduced egg production and hatchability, while the immune response of chicks or hens was not significantly impaired. Vitamin E apparently does not play a direct role in immune development since both the vitamin E deficient chicks and the vitamin E supplemented chicks responded to SRBC immunization, but the level of vitamin E appears to be important in regulating the amount of antibody produced. This view is also supported by the fact that hypoxia during hatching did not affect the immune response of the 2 or 4 week old chicks, and there was no difference whether the eggs came from vitamin E supplemented or control hens. If one compares this observation with the earlier observed synergistic effect of hypoxia and vitamin E in enhancing the immune response of chicks, when the hypoxic and antigenic stimuli were applied simultaneously (Tengerdy et ah, 1972), then it appears that vitamin E and hypoxia must act directly on antibody biosynthesis by an instantaneous regulation of protein biosynthesis, probably connected to the antioxidant or redox regulatory role of these factors. In summary the results of these experiments indicate that although the presently used dietary vitamin E level (15-30 mg./ kg.) in chicken feed rations is adequate for hatching and egg production, for an optimal immune response, which is important for an increased disease resistance, a much higher mg./kg. vitamin E level can be recommended. ACKNOWLEDGMENTS We thank Mr. J. W. Fitch for performing the chamber experiments, and Mr. R. G. Comp for supervising the hatching studies. We also thank Linda Wick, Allen Mitchell and Robert Moore for useful technical help. We also wish to thank Hoffmann-LaRoche, Inc. for their contribution of vitamin E. REFERENCES Dorr, P. E., and C. F. Nockels, Effect of aging and tissue ascorbic acid in the domestic hen. Poultry Sci. 50: Herbert, W. Y., Passive hemagglutination. In: D. M. Weir ed. Handbook of Experimental Immunology, Blackwell, Sci. Publ., Oxford, England Legge, R. F., Resolving the vitamin E controversy. Can. Res. Devi. Sept.-Oct. p Lucy, T. A., Functional and structural aspects of biological membranes. In: Proceedings of a Conference on Vitamin E and its Role in Cellular Metabolism. Annals New York Acad. Sci. (In press). National Research Council, Nutrient requirements of domestic animals. No. 1. Nutrient requirements for poultry. Snedecor, G. W., and W. G. Cochran, Statistical Methods. 6th Ed. Iowa State University Press, Ames, Iowa. Tappel, A., Will antioxidant nutrients slow aging processes? Geriatrics, 23: October Tengerdy, R. P., Immune response at high altitude. Int. Arch. Allergy, 9:426^434. Tengerdy, R. P., J. W. Fitch and R. E. Moreng, Hatchability of chicken embryos under simulated high altitude conditions. Poultry Sci. 48: Tengerdy, R. P., and R. H. Heinzerling, The effect of vitamin E on the immune response of hypoxic and normal chicken. Annals New York Acad. Sci. (In press) Tengerdy, R. P., R. H. Heinzerling and C. F. Nockels, The effect of vitamin E on the immune response of hypoxic and normal chicken. Infect. Immun. 5: