METABOLISM AND NUTRITION. Effect of Dietary Supplemental Levels of Vitamin A on the Egg Production and Immune Responses of Heat-Stressed Laying Hens

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1 METABOLISM AND NUTRITION Effect of Dietary Supplemental Levels of Vitamin A on the Egg Production and Immune Responses of Heat-Stressed Laying Hens H. Lin, 1 L. F. Wang, J. L. Song, Y. M. Xie, and Q. M. Yang Department of Animal Science, Shandong Agricultural University, Taian, Shandong , P. R. China ABSTRACT Two experiments were conducted to evaluate birds were exposed to a high temperature (31.5 C) 15 the effect of vitamin A supplementation of a commer- cial layer diet on the laying performance and immune function of heat-stressed hens. In Experiment 1, two different levels of vitamin A supplementation (3,000 and 9,000 IU/kg) were used to investigate the laying performance and antibody titer against Newcastle disease virus (NDV) of heat-stressed hens. Results showed that the high level of vitamin A supplementation (9,000 IU/kg) had a beneficial effect on the feed intake and laying rate of heat-stressed hens (P < 0.05), compared with the control group (3,000 IU/kg). The antibody titers were not influenced by the level of vitamin A (P > 0.05). In Experiment 2, the effect of four levels of vitamin A (3,000, 6,000, 9,000, and 12,000 IU/kg) on the antibody titer to NDV and T lymphocyte proportion was studied. The experimental d after NDV vaccination (Treatment 1) or immediately (Treatment 2). The results showed that the egg weight was increased (P < 0.01) by the high levels of vitamin A supplementation (6,000 and 9,000 IU/kg), but feed intake, laying rate, and body weight loss were not (P > 0.05). In Treatment 1, vitamin A had no significant effect on antibody titers against NDV in normal or hot environments but increased (P < 0.01) the proportion of α-naphthyl acetate esterase (ANAE)-positive cells. Vitamin A supplementation had a significant effect on NDV antibody titer and ANAE-positive cell proportion in Treatment 2 (P < 0.01). The results of the present study suggested that vitamin A supplementation in commercial layer diets to layer chickens under heat stress was beneficial to laying performance and immune function. (Key words: vitamin A, immunity, laying performance, heat stress) 2002 Poultry Science 81: INTRODUCTION The harmful effects of high temperature on the laying performance of laying hens have been well studied. Feed intake (Marsden and Morris, 1987), egg production, egg weight, and shell quality (de Andrade et al., 1977; Deaton et al., 1981) are decreased in heat-stressed birds. High environmental temperature not only has an adverse effect on laying performance but also can impede disease resistance. Environmental stress may depress the immune function of birds by impeding production of antibodies and effective cell-mediated immunity (Zulkifli et al., 1994). The phagocytic potential of chicken macrophages is decreased during heat exposure (Miller and Qureshi, 1992). Guo et al. (1998) reported that high temperature resulted in restraint of the development of immune organs of broilers. The dietary characteristics (level of nutrients or the type of ingredients) can modulate the susceptibility of birds to infectious challenges (Klasing, 1988). It is not known if the harmful effect of heat stress on the immune function of birds can be overcome by nutritional methods. Because vitamin A supports the differentiation of epithelial cells (Brody, 1993), it may have an effect on the immune function of birds. It was reported that vitamin A deficiency results in decreased immune response (Friedman and Sklan, 1989a), depressed response to mitogenic stimulation (Davis and Sell, 1983), disturbed immunoglobulin metabolism (Davis and Sell, 1989), depressed T lymphocyte responses and antibody production (Friedman and Sklan, 1989a,b; Sklan et al., 1994), and increased susceptibility to E. coli infection (Friedman et al., 1991). Excess vitamin A intake also has a detrimental effect on the immune function of birds (Friedman et al., 1991). Although the NRC (1994) has provided a recommendation for adequate levels of dietary vitamin A, adequate levels for maximal immune responses of chicken need to be evaluated further (Sklan et al., 1994). It has been reported that vitamin supplementation (vitamins A, D, E, and B complex) during episodic heat stress is beneficial at both the induction and maturation 2002 Poultry Science Association, Inc. Received for publication April 25, Accepted for publication November 5, To whom correspondence should be addressed: hailin@sdau.edu.cn. Abbreviation Key: ANAE = α-naphthyl acetate esterase; HA-HI = hemagglutinin assay- microhemagglutination-inhibition; HS = heat stress; L:D = light:dark; NDV = Newcastle disease virus. 458

2 EFFECT OF VITAMIN A ON PERFORMANCE OF HEAT-STRESSED HENS 459 stages of the antibody synthesis response (Ferket and Qureshi, 1992). That means vitamin A might play a role in the beneficial effect. As the immune system also needs to respond to infection or immune vaccination in high environmental temperature and low feed intake, the requirement of vitamin A might be changed in heat-stressed hens, and an investigation of the adequate supplemental level of vitamin A under these conditions is needed. However, vitamin A, being a fat-soluble vitamin, can be stored in liver of birds, and storage pools may affect requirements. The objectives of the present study were to evaluate the effects of dietary supplemental level of vitamin A on the immune function and laying performance of heatstressed hens fed a common commercial diet (corn-soybean). The immune function was estimated by immune vaccination before, during and at the meanwhile of heat exposure, respectively. Experiment 1 MATERIALS AND METHODS Birds and Treatments. Forty commercial Hy-Line 2 laying hens, 56 wk of age, with a laying rate of 85%, were obtained from a commercial layer farm. The commercial layer diet contained 7,000 IU vitamin A/kg of diet. The hens were divided into two groups, each group having 20 hens, and were maintained in one temperature-controlled chamber. The chicks had been vaccinated against Newcastle disease with LoSota vaccine 3 by eye drop at 7, 30, and 60 d of age, and with NDV vaccine 3 (containing mineral oil as adjuvant) by i.m. inoculation at 120 d of age. The hens were housed in individual cages ( cm) in the chamber. Experimental birds were fed the control or experimental diet 2 wk before the formal experiment was conducted. The basal diet was a typical commercial layer diet formulated according to the recommendation of NRC (1994) (Table 1). The supplemental levels of vitamin A (BASF, 4 500,000 IU/g, retinyl acetate) were 3,000 (control group) and 9,000 IU/kg diet, respectively. The vitamin A was first added to the vitamin-premix and then mixed with other dietary components. When the experiment was conducted, the room temperature was elevated rapidly to 31.5 C and maintained during the entire 35-d experiment. All the birds had free access to feed and water during the experiment. The light regimen was 16L:8D. Fifteen days after heat exposure, all birds were vaccinated against Newcastle disease virus (NDV) (live attenuated La Sota strain 3 ) by eye drop (Day 0). The egg production, egg weight and feed intake of each bird in both groups were recorded daily. The body weights of experimental birds were recorded at the beginning and end of the experiment. 2 Hy-Line Indian River Company, West Des Moines, IA. 3 Qilu Animal Health Company, Shandong , P. R. China. 4 BASF Aktiengesellschaft, Germany. Ingredient TABLE 1. Composition of basal the diet 1 Amount Corn Soybean meal 17.9 Fish meal 2.0 Shell meal CaHPO Salt 0.3 DL-Methionine 0.14 L-Lysine 0.02 Vitamin premix Mineral premix Choline chloride 0.2 Calculated analysis ME, Kcal/kg 2,809 CP, % 16.0 Ca, % 3.5 Available phosphorus, % 0.30 Methionine % 0.35 Lysine % The basal diet was fed during the entire experiment. 2 Oyster shell, contained 33.4% Ca. 3 The vitamin premix provided the following per kilogram of complete diet: vitamin D, 300 IU; vitamin E, 5 IU; vitamin K 0.5 mg; thiamine, 0.75 mg; riboflavin, 2.5 mg; pantothenic acid, 2.0 mg; pyridoxine, 2.5 mg; niacin, 10.0 mg; vitamin B 12, mg; biotin, 0.1 mg; folic acid, 250 mg. 4 The mineral premix provided the following per kilogram of complete diet: manganese, 20 mg; zinc, 35 mg; iron, 45 mg; iodine, 0.35 mg; copper, 8.0 mg; selenium, 0.06 mg. Assay of Serum Antibody Titers Blood samples were taken via wing vein from each bird before vaccination (Day 1) and at 7 and 14 d after vaccination (Days 7 and 14). Sera were separated and stored at 20 C until analysis. Serum antibody titers were determined by means of hemagglutinin assay-microhemagglutination-inhibition (HA-HI) test. The titers were expressed as log 2 of the highest dilution giving total agglutination (Liu, 1999). Statistical Analyses A single factorial model was used for statistical analysis of laying performance of experimental birds. The main effect of vitamin A supplemental level was estimated by ANOVA (SAS, 1989). A 2 3 factorial statistical model was used for antibody titer with individual hens serving as replicates. The main effects of vitamin A supplemental level and time were analyzed by ANOVA (SAS, 1989). The interaction of vitamin A and time was also estimated. Experiment 2 Birds and Diets. Two hundred female 1-d-old Hy- Line 2 chicks were obtained. The chicks were vaccinated against Newcastle disease (live attenuated La Sota strain 3 ) by eye drop at 7, 30, and 60 d of age. The birds were reared under normal conditions according to recommendations to 25 wk of age. The chicks were fed a diet containing 8,000 IU of vitamin A/kg until 6 wk of age, followed by a diet containing 7,000 IU of vitamin A/kg until

3 460 LIN ET AL. FIGURE 1. Design of Experiment wk of age, and then received a diet containing 10,000 vitamin A IU/kg until 25 wk of age. One hundred twenty-eight laying hens, laying at 90%, were selected from the birds above and were assigned, at 25 wk of age, to 8 groups with 16 birds per group. The chickens were housed in two temperature-controlled chambers and each chamber had four groups. The hens were housed in individual cages ( cm) in the chambers. Two weeks before heat exposure, birds were fed the experimental diets formulated on the basis of NRC recommendations (1994), except for the level of vitamin A (Table 1). The rearing facilities and light regimens were kept the same in the two chambers as that in Experiment 1. There were four supplementation levels of vitamin A: 3,000 (control group), 6,000, 9,000, and 12,000 IU/kg diet. The vitamin A was mixed in the vitamin premix before being added to other ingredients. Heat-Stress Treatment and Immunizations In Experiment 2, two treatments were designed to estimate the effects of heat stress on the immune function. One treatment exposed birds to high temperature (31.5 C) 15 d after NDV vaccination (Treatment 1), and another exposed the birds to high temperature (31.5 C) immediately after administering the vaccine (Treatment 2; Figure 1). In the two treatments, the experimental birds were immunized with NDV vaccine (live attenuated La Sota strain, the same as in Experiment 1) by eye drop. The experiment ran 35 d, and the heat exposure lasted 3 wk in the two treatments. The temperatures of both chambers were elevated to the setting value (31.5 C) rapidly (within 2 h) after heat exposure began and then was maintained at a constant 31.5 C. Egg production, egg weight, and feed intake of every bird in both groups were recorded daily during heat exposure period in both treatments. The body weights of experimental birds were recorded at the beginning and end of heat exposure. Blood Sampling and Hemagglutinin Assay In Treatment 1, the blood samples were taken 1 d before (Day 1) and at 7 and 14 d (Days 7 and 14) after vaccination against NDV and at 7, 14, and 21 d after heat exposure (Days 22, 29, and 35 after vaccination), respectively. Blood sampling in Treatment 2 was the day before, and at 7, 14, and 21 d (Days 1, 7, 14, and 21) after heat exposure and vaccination against NDV, respectively. Two milliliters of blood sample was taken via wing vein from every experimental bird at each time, and the serum and anticoagulant plasma were prepared, respectively. Determination of Serum Antibody Titers Serum antibody titers were determined by means of HA-HI test (Liu, 1999). The titers were expressed as log 2 of the highest dilution giving total agglutination. Peripheral T-Lymphocyte Count (ANAE-Positive Cells) Peripheral blood T-lymphocyte counts were determined by the histochemical demonstration of α-naphthyl

4 EFFECT OF VITAMIN A ON PERFORMANCE OF HEAT-STRESSED HENS 461 TABLE 2. The laying performance of hens in Experiment 1 Feed intake Egg yield Egg weight Feed efficiency Vitamin (IU) (g/h d) (%) (g/egg) (kg feed:kg egg) 3, ± 5.95 b ± 7.89 b ± ± , ± 8.17 a ± 7.85 a ± ± 0.30 a,b Means with different superscripts in the same column differed significantly (P < 0.05). acetate esterase (ANAE) according to Mueller et al. (1975). The lymphocytes with reddish-brown products were regarded as ANAE-positive cells (ANAE-PC). In each sample, 200 lymphocytes were counted, and the positive frequency was calculated. Statistical Analyses Laying performance data were collected for 2 wk after hens were transferred into the chambers, and the effect of chamber was analyzed. As no significant effect (P > 0.05) was observed, the effect of chamber was not considered in the statistical analyses of the experiment. Laying performances of hens were analyzed as 4 2 factorial model by ANOVA, and the effects of vitamin A supplementation and treatment were estimated with individual birds serving as replicates. In Treatment 1, the immunity data obtained were analyzed in a 4 2or4 3 factorial model with the individual hens serving as replicates to estimate the effects of vitamin A and time under normal and heat-stress conditions, respectively. Immunity data in Treatment 2 were analyzed in a 4 3 factorial model with the individual hens serving as replicates. The main effects of vitamin A supplemental level and time on the immune parameters were estimated by ANOVA and treatment means were compared using Duncan s multiple-range test (Duncan, 1955). The interaction of vitamin A and time was estimated at the same time. To estimate the effects of heat stress, the NDV antibody titer and ANAE-positive cells from Treatments 1 and 2 were compared further with a 2 4 model at Days 7 and 14, respectively. The main effects of different heat treatments, vitamin A supplemental level and their interaction were estimated. Statements of statistical significance were based on P < 0.05, unless otherwise indicated. Experiment 1 RESULTS The high level of vitamin A supplementation (9,000 IU/kg) had a beneficial effect on laying performance of hens exposed to high temperature environment; feed intake and laying rate were elevated significantly (P < 0.05), compared to control group (3,000 IU/kg). However, no significant effects on egg weight and feed efficiency were observed (P > 0.05; Table 2). Antibody titers were not influenced by different vitamin A supplemental levels (P > 0.05), but were increased significantly after NDV vaccination (P < 0.05); however, no significant change of antibody titer was observed on Day 7 or 14 (Table 3). Experiment 2 The results showed that neither the vitamin A supplemental levels nor the treatment regimens had a significant effect on feed intake, laying rate, and body weight loss of experimental hens (P > 0.05). Egg weight, however, was significantly affected by different levels of vitamin A supplementation and by different temperature and vaccine treatments (P < 0.01). In Treatment 1, egg weight increased along with the supplemental level of vitamin A, but the highest egg weight was observed in 9,000 IU vitamin A/kg in Treatment 2. The group that received 3,000 IU vitamin A/kg had the lower egg weight of the two treatments. Feed efficiency was not influenced by the supplementation of vitamin A, but was decreased significantly by Treatment 2 (P < 0.05) (Table 4). The supplemental levels of vitamin A had no significant advantageous effect on antibody titers against NDV in the normal environment (Day 7 and 14) or in the hot environment (Days 22, 29, and 36) in Treatment 1 (vaccination first and heat exposure later). However, there was a significant positive effect of vitamin A supplementation (P < 0.05) in Treatment 2 (heat exposure immediately after vaccination), the NDV antibody titer was increased significantly by 12,000 IU/kg supplemental level of vitamin A, compared to 3,000 IU. The levels of antibody titer changed significantly at different times in the two treatments. In Treatment 1, they increased along with the time before heat exposure (from Days 1 to 14), decreased at the 14th d after heat exposure (Day 29) and then rebounded at 21 d (Day 36) (P < 0.01). In Treatment 2, the level of antibody titers increased from Days 1 through 14 but declined at Day 21 (Table 5). Different heat treatments had no significant effect (P > 0.05) on antibody titers at Days 7 and 14. The vitamin A supplementation had a significant effect on ANAE-positive cells proportions in both periods of Treatment 1 (P < 0.01) and Treatment 2 (P < 0.01). The proportion of ANAE-positive cells was elevated along with increasing supplemental levels of vitamin A and the highest response was found with 12,000 IU of vitamin A /kg. The trends of the ANAE-positive cell proportion over time were different in the two treatments. In Treatment 1, the ANAE-positive cell proportion response peaked at Day 14 postvaccination, decreased 15 d after heat exposure (Day 29), and rebounded on 22 d (Day 36). However, the ANAE-positive cell proportion began to

5 462 LIN ET AL. TABLE 3. The effects of supplemental vitamin A levels on antibody titers of Newcastle disease virus (NDV) in Experiment 1 (log 2 ) Supplemental vitamin A levels Time 3,000 9,000 Mean Significance Day ± ± b Vitamin A: NS Day ± ± a Day ± ± a Time* Mean a,b Means with different superscripts in the same column differed significantly (P < 0.05). 1 Means do not include the basal value from Day 1. *P < decrease at 21d (Day 21) after vaccination in Treatment 2. Different heat treatments had a significant effect on ANAE-positive cell proportions; higher (P < 0.01) proportions were observed at Day 7 in Treatment 1 than in Treatment 2, but no significant difference (P > 0.05) was found on Day 14 in the two treatments. Laying Performance DISCUSSION Generally, vitamin A serves three functions: (1) to support the differentiation of epithelial cells, (2) to support the viability of the reproductive system, and (3) is used in the visual cycle (Brody, 1993). Because of these essential functions, vitamin A is usually added to a commercial layer diet. Coskun et al. (1998) reported that vitamin A levels above the NRC recommendation of 3,000 IU/kg (4,000, 12,000 and 24,000 IU/kg) had no significant effect on the laying performance of hens under normal conditions. However, when the birds suffered stress, the situation might change. For heat-stressed broilers, vitamin supplementation of drinking water (vitamins A, D, E, and B complex) have been reported to be beneficial for the performance and immune function (Ferket and Qureshi, 1992), and vitamin A might play a role in the effect. The results of the present study showed that the supplementation of vitamin A was advantageous to the laying performance of heat-stressed hens. However, this beneficial effect was observed in different indices in the two experiments of the present study: feed intake and laying-rate in Experiment 1, egg weight in Experiment 2. For the different responses of the hens, the possible reason might be that the susceptibility of laying performance was different in stressed hens at different laying stages. The ages of experimental birds used in the two experiments were different. The hens used in Experiment 1 were 56 wk of age and in the late laying period, and had relative lower TABLE 4. Laying performance of hens in Experiment 2 Supplemental vitamin A levels (IU/kg diet) 3,000 6,000 9,000 12,000 Mean Significance 1 Feed intake, g Treatment ± ± ± ± T NS Treatment ± ± ± ± VA NS Mean T VA NS Laying rate, % Treatment ± ± ± ± T NS Treatment ± ± ± ± VA NS Mean T VA NS Egg weight, g Treatment ± 0.97 c ± 0.33 bc ± 0.21 b ± 0.68 a m T** Treatment ± 1.76 c ± 0.71 b ± 1.51 a ± 0.93 b n VA** Mean z y x x T VA** Feed efficiency, g/g Treatment ± ± ± ± m T* Treatment ± ± ± ± n VA NS Mean T VA NS Body weight loss, g Treatment ± ± ± ± T NS Treatment ± ± ± ± VA NS Means T VA NS a-c Means with different superscripts in the same row differ significantly (P < 0.05). m-n Means with different superscripts in the same rank differ significantly (P < 0.05). 1 VA = vitamin A; T = treatment. *P < **P < 0.01.

6 EFFECT OF VITAMIN A ON PERFORMANCE OF HEAT-STRESSED HENS 463 TABLE 5. The effects of heat stress on antibody titers of Newcastle disease virus (NDV) from hens in Experiment 2 (log 2 ) Supplemental vitamin A levels, IU/kg Item 3,000 6,000 9,000 12,000 Means Significance 1 Treatment 1 2 Time Day ± ± ± ± c Time** Day ± ± ± ± b VA NS Day ± ± ± ± a Time VA NS Mean Day ± ± ± ± b Time** Day ± ± ± ± c VA NS Day ± ± ± ± a Time VA NS Mean Treatment 2 2 Time Day ± ± ± ± c Time** Day ± ± ± ± b VA* Day ± ± ± ± a Time VA NS Day ± ± ± ± b Mean y 4.08 xy 3.96 xy 4.50 x a-c Means with different superscripts in the same column differed significantly (P < 0.05). x,y Means with different superscripts in the same row differed significantly (P < 0.05). 1 VA = vitamin A. 2 There were no significant effects of treatment and its interaction with vitamin A (P > 0.05). 3 Means do not include the basal value of Day 1. *P < **P < laying rate than the hens used in Experiment 2 that were 25 wk of age. Immune Function The effects of stress on immune function have been well documented (Siegel, 1987). However, there were different reports about the effect of heat stress. Thaxton and Siegel (1970, 1972, 1973) reported a suppressive effect of heatstress on antibody production. Regnier et al. (1980) could not observe any immuosuppressive effects of heat stress. Donker et al. (1990) found a significant effect of heat-stress on antibody titers (from 1 to 14 d postimmunization) only in one of the three experiments conducted and suggested antibody production was not consistently affected by heat stress. In Experiment 2 of the present study, the hens TABLE 6. Effects of heat stress on ANAE 1 -positive cell proportions in blood from hens in Experiment 2 (%) Supplemental vitamin A levels, IU/kg Time 3,000 6,000 9,000 12,000 Means Significance 2 Treatment 1 3 Day ± 4.29 b ± 4.91 ab ± 4.10 ab ± 5.12 a n Time** Day ± 3.28 b ± 3.57 ab ± 3.56 a ± 4.37 a m VA** Mean z yz y x Time VA NS Day ± 3.73 b ± 3.56 b ± 4.37 ab ± 5.16 a m Day ± 4.50 b ± 4.52 ab ± 3.13 ab ± 3.38 a n Time** Day ± 3.80 b ± 4.24 b ± 4.69 ab ± 4.58 a m VA** Mean z y x w Time VA NS Treatment 2 3 Day ± 3.02 b ± 2.99 ab ± 3.83 ab ± 3.17 a o Time** Day ± 2.71 c ± 3.59 bc ± 2.91 ab ± 2.91 a m VA** Day ± 4.14 c ± 4.31 bc ± 5.02 ab ± 3.01 a n Time VA NS Mean z yz xy x a-c, w-z Means with different superscripts in the same row differed significantly (P < 0.05). m-o Means with different superscripts in the same column differed significantly (P < 0.05). 1 ANAE = α-naphthyl acetate esterase. 2 VA = Vitamin A. 3 There were significant differences between the two treatments at Day 7 (P < 0.01) but not at Day 14 (P > 0.05). There was no significant interaction between treatment and vitamin A supplementation (P > 0.05). *P < **P < 0.01.

7 464 LIN ET AL. remained in normal environment (20 C) for 2 wk after vaccination in Treatment 1 or exposed to hot environment (31.5 C) for 3 wk in Treatment 2. The results show that changes of antibody titer (Days 7 and 14) over time had similar trends in the two treatments (P > 0.05) and suggested that antibody production was not influenced by heat stress. Many studies have indicated that vitamin A deficiency is associated with the increasing susceptibility to infections. Sijtsma et al. (1989a,b) reported that the birds marginally vitamin A deficient had higher severity of disease following experimental NDV infection. Davis and Sell (1983) reported that a deficiency of vitamin A had a detrimental effect on growth and development of lymphoid tissues and vitamin A-deficient broilers had lower relative bursa and thymus weights. On the other hand, a high dietary level of vitamin A has detrimental affect on immune function (Friedman et al., 1991). A recent study conducted by Coskun et al. (1998) indicated that vitamin A supplementation (4,000, 12,000, and 24,000 IU/kg) was not required to obtain high immune responses in laying hens feeding diets based on wheat, corn, soybean meal, or sunflower oil meal. In Treatment 1 of the present study, the experimental hens were housed under normal thermal conditions 2 wk after NDV vaccination, which showed that supplemental vitamin A level had no significant effect on the NDV antibody titer but did increase ANAE-positive cell proportion during this period. Hens in the present study were fed a commercial layer diet supplemented with 10,000 IU vitamin A/kg 2 wk before the vaccination, and vitamin A could be stored in the liver for months when the diet contained high level of vitamin A (Aye et al., 2000). These results suggest that dietary vitamin A level has an effect on immune function during the initial period of vaccination, even if the liver did have a high level of vitamin A stored. As the proportion of ANAE-positive cells increased, the ANAE-negative cells (mainly B lymphocytes) decreased in proportion. This result suggested that immune function induced by B lymphocytes might be decreased accordingly. When hens were exposed to high environmental temperature, feed intake is suppressed (Marsden and Morris, 1987), which means the nutrient intake is decreased. As micronutrients (vitamin A, E, and C; Klasing, 1998), play an important role in immune function, supplementation of these nutrients might be helpful for the immune function of heat-stressed birds. Ferket and Qureshi (1992) reported that vitamin supplementation (vitamins A, D, E, and B complex) was helpful to the antibody synthesis response during episodic heat stress. Results of the present experiments showed that the effect of vitamin A supplementation on antibody titers was affected by the times of heat stress and vaccination. Significant effects of vitamin A supplementation on antibody titer were only observed in Treatment 2 of Experiment 2. In Experiment 1 and Treatment 1 of Experiment 2, the hens were vaccinated during or before heat exposure, there was a 14-d interval between heat-stress time and vaccination time. However, the heat exposure and vaccination were at the same day in Treatment 2 of Experiment 2. The result of the present study suggested that the hens that suffered heat-stress immediately after vaccination need higher dietary vitamin A intakes to obtain maximal level of antibody production. Vitamin A deficiency has been shown to depress T- cell proliferation responses (Friedman and Sklan, 1989a,b; Friedman et al., 1991; Sklan et al., 1994). Sklan et al. (1994) reported that antibody production and T-cell proliferation were depressed in chicks fed with no added dietary vitamin A, compared with the chicks receiving vitamin A, and that antibody production and T-cell proliferation increased with dietary vitamin A up to 6,660 µg/kg. The results of Experiment 2 in the present study indicated that peripheral T lymphocyte proportion (ANAE-PC) was increased along with dietary levels of vitamin A supplementation (from 3,000 to 12,000 IU/kg). In the experiment, a similar response to vitamin A was observed in Treatments 1 and 2. These results suggested that the supplementation of vitamin A (more than NRC recommendation) was helpful to the peripheral T-cell proportion of hens and the effect was not changed by different heatstress time and vaccination time. In conclusion, the supplementation of vitamin A might be advantageous for the laying performance of stressed hens. 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