THE citric acid concentration of tissues

Transcription

1 Dietary Vitamin Levels and the Response of Blood Citric Acid Concentrations to Stressors 1 C. H. HILL with the technical assistance of VIRGINIA C. BAKER Department of Poultry Science, North Carolina State College, Raleigh, North Carolina THE citric acid concentration of tissues has been found to increase in response to such stressors as Salmonella infections (Berry et al., 1954; Gilfillan et al., 1956), starvation, (Hill et al., 1960), cold and heat, (Hill et al., 1961). This phenomenon is evidently under hormonal control since the administration of ACTH also results in an increase in the blood concentration of this metabolite (Hill et al., 1961). Factors which influence the response of tissue citric acid concentration to stressors are of interest since it has been shown that the administration of citric acid or of compounds which block the tricarboxylic acid cycle and thus cause an increase in the tissue concentration of citric acid, render chicks and mice more susceptible to Salmonella infections (Berry and Mitchell, 1953; Gilfillan et al., 1956). The protein concentration of the diet has been shown to influence the response of blood citric acid concentration to various stressors (Hill, 1961). Another comparison which is of interest in this regard is the vitamin supplementation of the diet, since it has been shown (Hill and Garren, 1955) that the addition of vitamins at high levels increased the resistance of chicks to Salmonella gallinarum infection. This report presents the results of studies on the response of blood citric acid concentration to stres- 1 Contribution from the Poultry Science Department, North Carolina Agricultural Experiment Station, Raleigh, North Carolina. Published with the approval of the Director of Research as Paper No of the Journal Series. (Received for publication November 21, 1960) sors as affected by vitamin supplementation to the diet. EXPERIMENTAL White Plymouth Rock chicks obtained from a commercial hatchery were used in these studies. They were fed the experimental diets ad libitum from one day of age. The chicks were five weeks old when the series of experiments started and nine weeks old when they ended. The diet used throughout these studies is presented in Table 1. The vitamin supplement used in the normal vitamin supplement supplies at least the minimum requirement for chicks as established by the National Research Council (1954). The diet of those chicks receiving the high level of vitamins was supplemented ten-fold with the vitamin mix. In addition, ascorbic acid was added at 0.1% since previous work (Hill and Garren, 1955) had indicated that this was an essential vitamin for maximum resistance to S. gallinarum infection. Since the response of blood citric acid to the stressors heat, cold, or starvation has been found to be affected by dietary manipulation of protein levels, these stressors were used in the present studies. The chicks were subjected to cold treatment (38 F.) for fourteen hours, to heat treatment (110 F.) for five hours, and starvation for twenty-five hours. In addition, a study was conducted in which ACTH (Upjohn) was administered intravenously. Citric acid concentration of the blood was determined by the method of Ettinger et 1311

2 1312 C. H. HILL AND V. C. BAKER TABLE 1. Experimental diet and vitamin mix Ingredient Basal diet Soybean oil meal (50% protein) Yellow corn Defluorinated rock phosphate Salt Methionine Choline chloride MnS0 4 H 2 0 Vitamin mix % mg./lb. 100 mg./lb. 1.0 al. (1952). The detailed procedures for carrying out these studies have been published (Hill, 1960). RESULTS AND DISCUSSION The first study (Table 2) was designed to determine whether or not vitamin supplementation of the diet would influence the response of blood citric acid concentration to ACTH. Previous work (Hill, 1960) indicated that the effect of ACTH on blood citric acid concentration was manifested at approximately six hours after the first injection was given. In this study at the normal level of vitamin supplementation no effect of ACTH on citric acid concentration was observed. At the high level of vitamin supplementation, however, the blood citric acid increased significantly at four hours after the first injection and returned to normal two hours later. These results indicate that the level of vitamin supplementation does affect the response of blood citric acid concentration to ACTH. In fact, these results, taken by themselves, would indicate that the citric acid response to ACTH was dependent upon the vitamin supplementation. However, previous work (Hill, 1960), using a diet with the same basal level of vitamin supplementation, but which differed by the inclusion of cerelose instead Vitamin Vitamin mix Thiamine Riboflavin Ca Pantothenate Niacin Folic Acid Pyridoxine HC1 Menadione Alpha tocopherol acetate Biotin Vitamin B12 Vitamin A Vitamin D Supplies/lb. diet 8.4 mg mg mg mg mg. 40 meg. 4 meg. 1,400 I.U. 90 I.C.U. of yellow corn, indicated that a citric acid response to ACTH was obtained in six hours. The results obtained in this experiment are therefore interpreted to indicate that the citric acid response to ACTH is more rapid under conditions of high dietary vitamin supplementation. The second study (Table 3) was designed to determine the effect of vitamin supplementation on the blood citric acid response to cold. In both groups the citricacid concentration increased when exposed to the cold temperature and this increase was greater than observed in the control chicks from which feed had been removed for the same length of time. While the citric acid concentration was greater in those chicks receiving the high TABLE 2. Vitamin level and citric acid response to ACTH 1 Vitamin level Hours after first ACTH injection citric acid /xg./ml. + S. E. 2 Normal ±3.1 High ± ±4.9* Total of 3 units ACTH given in 4 injections at 20 minute intervals. 2 Mean of 5 chicks per treatment. 3 Ten-fold normal vitamin supplementation plus 0.1% ascorbic acid. * Significantly higher (P<.05) than at 2 hours.

3 BLOOD CITRIC ACID 1313 TABLE 3. Vitamin level and citric acid response to cold 1 Hours on experiment Vitamin level Cold Control citric acid /xg/ml. + S.E. 3 Normal High Chicks exposed to 38 F. for 9 and 14 hours. 2 Control chicks left at 75 F. Feed removed from both control and cold stressed chicks for duration of experiment. 3 Mean of 10 chicks per treatment. 4 Ten-fold normal vitamin supplementation plus 0.1% ascorbic acid. vitamin supplementation throughout the experiment, and was, in fact, significantly higher in the cold chicks at nine and fourteen hours, the increase in citric acid concentration was not significantly different in the two groups of chicks. High vitamin supplementation, therefore, did not significantly affect the citric acid response to cold stress. The results of the third study, designed to determine the effect of vitamin supplementation on the citric acid response to heat, are presented in Table 4. Under the influence of heat the blood citric acid level of the group of chicks fed the high level of vitamins increased. In that group of chicks fed the normal vitamin supplement there was no significant increase within the five hours of the experiment. These results indicate that supplementation of TABLE 4. Vitamin level and citric acid response to heat 1 Vitamin No. Houraon^erim^t level chicks n - citric acid jug./ml. + S.E. Normal High * 1 Chicks subjected to 110 F. for 5 hours. 2 Ten-fold normal vitamin supplementation plus 0.1% ascorbic acid. * Significantly (P<.05) higher than the high vitamin group at 0 time. the diet with high levels of vitamins stimulates the citric acid response to heat. The difference between these results and those with cold stress may be due to the fact that the heat stress is much more severe than is the cold. That this is so is indicated by the fact that four of the chicks receiving the normal levels of vitamins and three of the chicks receiving the high vitamin supplement died during the five hours of heat experiment, whereas there was no mortality in the cold experiment. The results of the study on the effect of vitamin supplementation on the citric acid response to starvation are presented in Table 5. As the experiment progressed both the normal and the high vitamin groups increased in blood citric acid concentration. There was no significant difference between the two groups until after ten hours of starvation. At fifteen, twenty, and twenty-four hours of starvation the group receiving the high vitamin supplement had a significantly higher blood citric acid concentration than did that group receiving the normal level. This is an indication that high vitamin supplementation stimulates the citric acid response to starvation as this stress becomes more severe. The results of this series of experiments indicate that the blood citric acid response to stressors can be increased by increasing the vitamin supplementation of the diet.

4 1314 C. H. HILL AND V. C. BAKER TABLE 5. Vitamin level and citric acid response to starvation Vitamin level Hours of starvation 0 10 IS citric acid ^g./ml. + S.E. 1 Normal 29.1 ± High ** ** ** 1 10 chicks per treatment. 2 Ten-fold normal vitamin supplementation plus 0.1% ascorbic acid. ** Significantly greater (P<.01) than normal vitamin group. The increase in response, however, was not apparent unless the stress had become severe. There was, for instance, no significant increase in response when the relatively mild stress of cold was applied, nor was there any in the early stages of starvation stress. This series of experiments resembles those previously reported for protein supplementation (Hill, 1960) in that higher supplementation of essential nutrients, in this instance vitamins, resulted in a greater citric acid response to the stressors used. Whether this has any meaning in relation to resistance to Salmonella infection, however, is doubtful. While high levels of protein increase susceptibility and increase the citric acid response to the stressors, high levels of vitamins increase resistance and increase the citric acid response to the stressors tested. The mechanism whereby increased vitamin levels increase the citric acid response to stressors is not revealed by these studies, nor can it be until the mechanism of the citric acid response to stressors has itself been elucidated. However, one possibility emerges from a study of the data obtained in these experiments. In every instance the initial blood citric acid concentration was higher in those groups receiving the high vitamin supplementation. This may indicate that this level of vitamin supplementation is itself a stressor. If this is so, the effect of the vitamin supplementation combined with the other stressors on the blood citric acid concentration may have been due to an additive effect of stressors. Further work to investigate this possibility is underway. SUMMARY Chicks were fed a corn-soybean oil meal diet supplemented with vitamins at two levels. The lowest level of supplement met the National Research Council's recommendation for the minimum requirements, while the highest level was tenfold the lowest and contained in addition 0.1% ascorbic acid. The chicks were subjected to cold, heat, and starvation stressors, as well as being injected with ACTH, and the blood citric acid response to these stressors measured. The high level of vitamin supplementation resulted in an increased citric acid response to the stressors of heat, and prolonged, but not short time, starvation. The citric acid response to ACTH was also accelerated with the high vitamin supplementation. These findings are discussed in relation to the effect of citrate on the resistance of chicks to Salmonella gallinarum infection. REFERENCES Berry, L. J., and R. B. Mitchell, The relation of the tricarboxylic acid cycle to bacterial infection. II. The effect of fluoroacetate, arsenite, citrate and succinate on Salmonella typhimurium infections in mice. J. Infectious Diseases, 93: Berry L. J., K. H. EWers and R. B. Mitchell, The relation of the tricarboxylic acid cycle to

5 BLOOD CITRIC ACID 1315 bacterial infection. J. Infectious Diseases, 94: Ettinger, R. H., L. R. Goldbaum and L. H. Smith, Jr., A simplified photometric method for the determination of citric acid in biological fluids. J. Biol. Chem. 199: Gilfillan, R. F., D. F. Holtman and R. T. Ross, Influence of Salmonella pullorum on various liver tricarboxylic acid enzymes and citrate levels in the chick. J. Bact. 72: Hill, C. H., and H. W. Garren, The effect of high levels of vitamins on the resistance of chicks to fowl typhoid. Ann. New York Acad. Sci. 63: SEVERAL reports have suggested beneficial effects of the use of tranquilizers n poultry nutrition and management practices. Burger, et al. (1959) and Fritz et al. (1959) obtained significant increases in chick growth with reserpine. Carlson (1959a) noted a similar growth response with a rapid growing strain of female broiler chicks. Meprobamate, fed at varying levels, produced a growth depression in the chick (Garren and Hill, 1957; Babcock and Taylor, 1957). Weiss (1959) reported that reserpine, injected at a level of 0.02 milligram per kilogram of body weight or fed at 2.0 milligrams per kilogram of diet, afforded laying birds protection against severe heat stress. Burger and Lorenz (1960) observed that reserpine and chlorpromazine in the diet prolonged the survival of chickens under defined conditions of experimental heat shock. Reserpine has been shown to support egg production and shell quality during and following periods of thermal stress (Van Hill, C. H., H. W. Garren, and J. W. Kelly, Studies on blood and liver citric acid levels in chicks. Poultry Sci. 39: Hill, C. H., M. K. Warren, and H. W. Garren, Blood citric acid concentration as affected by heat and cold stress and adrenocorticotrophic hormone. Poultry Sci. 40: 422^24. Hill, C. H., Dietary protein levels as they affect blood citric acid levels of chicks subjected to certain stresses. Poultry Sci. (In Press). National Research Council, Nutrient requirements for domestic animals. No. 1. Nutrient reauirements for poultry. The Effect of Tranquilizers on Egg Production and Egg Shell Quality in Caged Layers H. J. EOFF, R. E. DAVIES, T. M. FERGUSON AND J. R. COUCH Department of Poultry Science, Texas A &* M College, College Station, Texas (Received for publication November 28, 1960) Matre et al, 1957; Gilbreath et al, 1959). Carlson (1959b) reported thatreserpine fed to laying hens produced a slight increase in egg weight. Anderson and Smyth (1959) found significantly lower serum cholesterol and egg yolk cholesterol and egg yolk cholesterol levels in S. C. White Leghorn hens fed a diet containing 0.5 milligram of reserpine per kilogram of diet. The present experiment was designed to study the effect on continuous feeding of reserpine and meprobamate on performance factors, egg shell quality and certain physiological characteristics in caged layers. EXPERIMENTAL PROCEDURE One hundred ninety-two range-reared, strain-cross White Leghorn pullets were placed at random into individual cages (10"X18") on September 1, All birds were fed a practical type basal diet (Table 1) during the pre-experimental period. The duration of the experiment was 36