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Decreased Reproductive Potential and Reduced Feed Consumption in Mature White Leghorn Males Fed Aflatoxin
Decreased Reproductive Potential and Reduced Feed Consumption in Mature White Leghorn Males Fed Aflatoxin J. S. SHARLIN, B. HOWARTH, JR., F. N. THOMPSON,1 and R. D. WYATT Department of Poultry Science and Department of Physiology and Pharmacology University of Georgia Athens, Georgia 30602 (Received for publication March 4, 1981)
1981 Poultry Science 60:2701-2708 INTRODUCTION
The reproductive system of the mature White Leghorn male appears to be more susceptible to dietary aflatoxin than that of the mature broiler breeder male. After 4 to 5 weeks of dietary aflatoxin (20 Mg/g diet), a significant decline in semen volume and testes weights along with a disruption of testicular histology was found in White Leghorns (Sharlin et al, 1980), whereas the same variables observed in broiler breeder males were unaffected (Briggs et al, 1974). However, when compared to controls, both the White Leghorn and broiler breeder males showed approximately a 65% increase in liver weight and an 11% reduction in body weight in response to dietary aflatoxin. Among White Leghorn males fed aflatoxin a significant decrease in feed consumption occurred after 1 week of dietary treatment; during subsequent weeks feed consumption decreased steadily, reaching a minimum after 4 weeks of dietary aflatoxin at which time treated males consumed about half as much
Department of Physiology and Pharmacology.
feed as control males (Sharlin et al, 1980). Thus, while the 11% decline in body weight was most likely attributable to a reduction in feed consumption, the causative factor(s) for the impaired reproductive potential could be due to dietary aflatoxin and/or reduced feed intake. Mulinos and Pomerantz (1940) demonstrated that adult rats restricted to one-half their normal quantity of feed had severely atrophied testes and diminished or absent spermatozoa production. The testicular response was almost identical to rats which had been hypophysectomized. Decreased semen volume and fertility were observed in Rhode Island Reds (Parker and McSpadden, 1943) and White Leghorns (Parker and Arscott, 1964) after a body weight loss. Since the mature broiler breeder male has 2.5 times more crude fat than the mature White Leghorn male (Mitchell et al, 1926; 1931), the broiler breeder should be more tolerant than the White Leghorn male to a reduction in feed intake and body weight loss induced by dietary aflatoxin. Therefore, breed differences in reproductive response to aflatoxin can be explained by a difference in tolerance to undernutrition (Sharlin et al, 1980). The objective of this pair-feeding study was to determine the relative importance of ingestion
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ABSTRACT A pair-feeding study was conducted to investigate the relative importance of ingestion of aflatoxin (20 Mg/g diet) versus decreased feed consumption in explaining the effects of dietary aflatoxin on reproduction. Fifty-eight mature White Leghorn males were divided among three groups - control, 0 Mg/g diet ad lib; aflatoxin, 20 Mg/g diet ad lib; and pair-fed, 0 Mg/g diet pair fed to 20 Mg/g group. Aflatoxin and pair-fed males consumed significantly less feed than controls during weeks 1 through 5 of the 8-week aflatoxin feeding period. Measures of reproductive potential (semen volume, testes weights, spermatocrit, and plasma testosterone) for pair-fed males were not significantly different from males fed aflatoxin, although both groups were significantly lower than control males. Measures of aflatoxicosis (liver weight, liver fat, and plasma albumin) for pair-fed males were not significantly different from control males, although both groups were significantly different from males fed aflatoxin. In conclusion, decreased feed consumption did not produce symptoms of aflatoxicosis but accounted for 60% of the effects of aflatoxin on reproduction. Therefore, aflatoxin has nutritional and toxicological effects on reproduction. (Key words.- aflatoxin, reproduction, testosterone, feed consumption, White Leghorn)
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of toxin as compared to decreased food consumption in explaining the effects of dietary aflatoxin on reproduction. MATERIALS AND METHODS
Reproductive Measurements. Semen was collected weekly by abdominal massage. Individual semen volume was recorded to the nearest .01 ml using a 1 ml tuberculin syringe. Individual semen samples were drawn into capillary tubes, and spermatocrit readings, a measure of spermatozoa concentration, were determined using the method of Taneja and Go we (1961). Organ Weights. At the conclusion of the experiment, all birds were killed by cervical
The antisera (MSU #74) was diluted 1: 25,000 using .01M phosphate buffered saline (PBS). One-tenth milliliter of a labeled testosterone solution, .2 ml of antisera, and .2 ml PBS were added to each tube and vortexed. The tubes were held at 40 C for 10 min and then incubated at 4 C for 15 to 18 hr. The crossreactivity of the antisera (MSU #74) has been reported (Kiser et al, 1978). When testosterone equalled 100%, dihydrotestosterone was found to crossreact with this antisera at 60%. Thus, the assay measured both androgens in plasma samples, although the data are expressed in
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Animals and Feed. Fifty-eight 14-month-old mature White Leghorn males were housed individually in an unheated room with a constant photoperiod of 16 hr light and 8 hr dark. Aflatoxin was produced by growing Aspergillus parasiticus NRRL 2999 on sterile rice using the method of Shotwell et al. (1966) with the temperature modification of West et al. (1973). The rice was steamed to kill the fungus, ovendried at 80 C for 18 hr, and ground to a fine powder. The concentration of aflatoxin was determined by spectrophotometric analysis of the rice powder using the method of Nabney and Nesbitt (1965). Individual toxins were separated by high pressure liquid chromatography. The mixture contained 83.6% Bi, .2% B 2 ,and 16.2% G j . Experimental Design. The 10-week experiment (2 weeks pretreatment, 8 weeks aflatoxin feeding) consisted of three treatments in a completely randomized design. Sixteen birds served as controls and consumed the University of Georgia layer-breeder ration ad lib, 21 birds were given 20 /ig aflatoxin/g diet ad lib, and 21 birds consumed the control ration but were pair-fed to the aflatoxin group. During the 2-week pretreatment period, all birds consumed a layer breeder ration ad lib. Using data collected during the pretreatment period, birds were matched by closeness in body weight and feed consumption for pairfeeding during the aflatoxin feeding period. For pair-feeding, individual body weight and food consumption were measured daily at 1300 hr, and then each pair-fed bird was given a quantity of layer breeder ration equal to die amount of food consumed by its aflatoxin-fed partner during the previous 24 hr.
dislocation. Testes and liver were removed and weighed to the nearest .01 g. Collection of Plasma Samples. All birds were bled weekly from the brachial vein between 1000 and 1130 hr. A 23 gauge needle and 3 ml syringe were used to collect 3 ml blood from each bird. The blood was transferred from the syringe to a heparinized 12 X 75 mm tube for centrifugation. After centrifugation, plasma samples were frozen until analyzed. Laboratory Analysis. Immediately after collection, individual blood samples were drawn into heparinized capillary tubes, and the packed cell volume was determined by the microhematocrit method (Johnson, 1955). Plasma albumin was measured by the method of Doumas et al. (1971). Percent carcass fat and percent liver fat were measured by the method of VcAchetal. (1957). Within treatment variation for plasma albumin and liver fat was low enough that samples from nine randomly selected birds were analyzed per treatment group. Testosterone Analysis. Plasma testosterone was measured by radioimmunoassay. Extraction in duplicate of 100 fJ.1 plasma generally followed the procedure of Culbert (1976). Two milliliters of ligroine (boiling range 65 to 90 C) were added to 100 /txl plasma in 16 x 100 mm culture tubes. The tubes were stoppered and placed in a 50 C water bath and agitated with a mechanical shaker for 1 hr. The aqueous phase was frozen and the ether phase was poured into 12 X 75 mm culture tubes and dried under nitrogen. The plasma was similarly extracted a second time for 30 min. The ether phase from both extractions were combined. Recovery of 1, 2, 6, 7- 3 H testosterone during extraction of blood plasma averaged 90% (range 86 to 92%). Unknowns were not corrected for procedural loss.
AFLATOXIN ON REPRODUCTION AND FEED CONSUMPTION
The sensitivity of the assay was 100 pg/ml plasma. The addition of testosterone to a pooled sample either at 20, 40, 100, 200, or 1000 pg was assayed as 17, 46, 120, 280, and 1046 pg, respectively, after correcting for endogenous testosterone. Interassay variation (CV = 6%, n = 10) was calculated using a pooled plasma sample included in each assay. Testosterone levels were determined for 14 aflatoxinfed birds, 14 pair-fed birds, and 10 controls. Statistical Analysis. For variables measured every week, a one-way analysis of variance (ANOVA) was conducted separately for each week using the ANOVA procedure in the Statistical Analysis System (Helwig and Council, 1979). Variables measured at the conclusion of the study were analyzed using a one-way ANOVA. Mean separation was by Duncan's multiple range test at the .05 level. Significance is based on P<.05. Assuming no synergism between decreased feed consumption and ingestion of aflatoxin, the portion of the effect of dietary aflatoxin accounted for by decreased feed consumption was called the feed consumption factor (FCF). The FCF values were determined by calculating a ratio of the difference between controls and the pair-fed and aflatoxin groups using the formula: [(C-P)/(C-A)] X 100 where C = control, P = pair-fed, and A = aflatoxin. For example, if values for a hypothetical 2 Fisher Scientific Company, Manufacturing Division, Fair Lawn, NJ 07410.
variable were control, 100; pair-fed, 300; and aflatoxin, 500; then the FCF value would be 50%, that is, 50% of the effect of aflatoxin was accounted for by a decrease in feed consumption. The FCF values were calculated for only those weeks with a significant treatment effect. An FCF value for a given variable represents an average of the weekly FCF values. RESULTS
Feed Consumption and Body Weight. Feed consumption and body weight did not differ significantly between the aflatoxin and pair-fed groups during the entire study. Both groups consumed significantly less feed compared to controls during the first 5 weeks of the 8-week treatment period (Fig. la). Feed consumption of the toxin and pair-fed groups increased during treatment weeks 5 and 6 such that during the last 3 weeks of the experiment there were no significant differences for feed consumption among the three experimental groups. Body weight of the aflatoxin and pair-fed groups declined significantly from controls after 4 weeks of feeding aflatoxin and
PAIR-FED- OJJG /G PAIR-FED TO 10pG /a - CONTROL-Opo/G AD 111.
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FIG. 1. The effect of dietary aflatoxin and pairfeeding on feed consumption and body weight of mature White Leghorn males. When the F-test for any of the 11 analyses of variance was significant (P<.05 as shown along the top of each graph), treatments significantly different for that week have different letters plotted next to their mean.
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terms of testosterone. For all other steroids, crossreactivity was less than 2%. Unbound testosterone was removed from solution using dextran coated charcoal (1.23 g Norit A plus .123 g Dextran T70 in 500 ml PBS). Two-tenths milliliter charcoal solution were added to each assay tube. The tubes were centrifuged at 750 x g for 10 min at 4 C and the supernatant portion decanted into 7 ml scintillation vials. Five milliliters of Scinti Verse 2 were added to each vial, and radioactivity was determined with a liquid scintillation spectrophotometer (Beckman Model LS-7000). Duplicate testosterone standards of 10, 20, 40, 100, 200, 400, and 1000 pg dissolved in ethanol were included in each assay. The standard curve, plotted as percent bound against logio of standard, resulted in a straight line with an average R square value of 99%.
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- FAIR-FID - 0 pO /G PAIR-KD TO 20/KS / G GROUP - CONTROL-O^G/G AD LIB. - AILATOXIN-IOLIG/G AD LII. •
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FIG. 2. The effect of dietary aflatoxin and pair-feeding on semen volume, spermatocrit, and plasma testosterone of mature White Leghorn males. When the F-test for any of the 11 analyses of variance was significant (P<.05 as shown along the top of each graph), treatments significantly different for that week have different letters plotted next to their mean.
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4 5 6 TIME (WEEKS)
AFLATOXIN ON REPRODUCTION AND FEED CONSUMPTION
(Table 1). Packed Cell Volume. There were no significant differences between any of the groups for packed cell volume. Weekly treatment means ranged from 38 to 44% during any weekly period. Mortality. One aflatoxin-fed male and his pair-fed partner died during treatment week 6 when 36% of their original body weight had been lost. During the 2 weeks prior to death, only 15 g of feed were consumed. DISCUSSION
The response of the pair-fed group clearly supports the original hypothesis that reduced feed intake was an important factor in explaining the effects of dietary aflatoxin on reproduction in White Leghorn males (Sharlin et al., 1980). A decline in feed consumption accounted for over half the effects of aflatoxin on testes weight (62%), semen volume (59%), plasma testosterone (57%), body weight (80%), and almost all the effect on spermatocrit. However, the decline in feed consumption was unrelated to increased liver fat, increased liver weight, and decreased plasma albumin, all three of these responses being sensitive indicators of aflatoxicosis in the fowl (Gumbmann et al., 1970). Pair-feeding allows separation of aflatoxin's primary action, which causes disruption of carbohydrate and lipid metabolism, and inhibition of protein synthesis (Hsieh, 1979),
PAIR-FED- 0 U G / G PAIR-FID TO 20/JG / G GROUP CONTROL-OUG/G AD IIB. AFLATOXIN-JO U G / G AD IIB. 85
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FIG. 3. The effect of dietary aflatoxin and pairfeeding on plasma albumin of mature White Leghorn males. When the F-test for any of the 11 analyses of variance was significant (P<.05 as shown along the top of each graph), treatments which were significantly different for that week have different letters plotted next to their mean.
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continued to decrease during treatment week 5; thereafter, no further decline in body weight occurred (Fig. lb). The FCF value for body weight was 80%. Measures of Reproductive Potential. Semen volume of the aflatoxin-fed and pair-fed males declined significantly from controls after treatment weeks 4 and 5, respectively (Fig. 2a). During the last 5 weeks of the experiment, semen volume of the pair-fed males was usually significantly less than controls and nonsignificantly different from males fed aflatoxin. During the first 5 weeks of feeding aflatoxin there were no significant differences in spermatocrit (Fig. 2b). During the last 3 weeks of the experiment, spermatocrit values of the aflatoxin and pair-fed males declined significantly from controls but were nonsignificantly different from each other. After 8 weeks of feeding aflatoxin, testes weights and percent carcass fat of the aflatoxin and pair-fed males, while nonsignificantly different from each other, were significantly less than control males (Table 1). Plasma testosterone levels of males fed aflatoxin declined significantly from controls during weeks 4, 5, and 6 of toxin feeding (Fig. 2c). Pair-fed males had testosterone levels significantly lower than controls only at treatment week 5. The testosterone levels of the pair-fed group were between those of the aflatoxin and control groups during the last 5 weeks of the experiment. The FCF values for reproductive variables were semen volume 59%, testes weights 62%, and plasma testosterone 57%. For spermatocrit and carcass fat, FCF values were approximately 100% since the pair-fed males and males fed aflatoxin had nearly identical decreases from the control group. Measures of Aflatoxicosis. There were no significant differences in plasma albumin between the control and pair-fed groups (Fig. 3). Plasma albumin levels in males fed aflatoxin were significantly less than control males from the second week of toxin feeding until the end of the experiment. For birds fed aflatoxin, plasma albumin decreased markedly during treatment week 4 but remained fairly constant for the remainder of the toxin feeding period. Analysis of livers removed at the end of the experiment for percent fat and weight showed that control and pair-fed groups while nonsignificantly different from each other were both significantly less than males fed aflatoxin
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SHARLIN ET AL. TABLE 1. Organ weights and percent liver and carcass fat of mature White Leghorn males fed aflatoxin for 8 weeks1 Organ weights (g/lOOgbody wt)
Treatment
Percent carcass fat
Testes
Liver
Percent liver fat
Control 0 iig/g ad lib
6.8 a ± 1.3 (16)
.74 a ±.066 (16)
1.3 a ±.058 (16)
4.7 a ± .64 (9)
Pair-fed 0 Mg/g pair-fed to 20 jug/g group
2.8 b ± .42 (20)
.54 b ± .063 (20)
1.4* ±.059 (20)
4 . 2 a ± . 3 5 (9)
Aflatoxin 20 Mg/g ad lib
3.3 b i
.42 b ± .057 (20)
2 . 1 b ± .124 (20)
7.7 b ± .81 (9)
' Means within the same column bearing different superscripts are significantly different (P<.05).
'Means ± SE (number of observations).
from the toxin's other effects arising from reduced feed consumption. The quantity of control feed given to the pair-fed group was matched on a daily basis to the feed consumption of the males fed aflatoxin. Therefore, the response of the aflatoxin group was due to ingestion of toxin and decreased feed consumption whereas the response of the pair-fed group was only a result of decreased feed consumption. The relative effect on any variable of decreased feed consumption vs. ingestion of aflatoxin was determined by comparing the response of the aflatoxin and pairfed groups to controls. The response of feed consumption and semen volume by males fed aflatoxin was similar to that observed in an earlier study (Sharlin et ah, 1980). In both studies, after aflatoxin was fed for 4 weeks, semen volume of aflatoxin-fed males declined significantly from controls and feed consumption decreased to its lowest value. However, during treatment week 5 in both experiments, males fed aflatoxin increased their feed consumption. In this study feed consumption of the three experimental groups did not differ significantly during the last 3 weeks of the experiment. A similar response was observed when graded levels of ochratoxin were fed to male chicks from hatch to 8 weeks of age (Prior et ah, 1980). Feed consumption declined during the first 5 weeks, but then increased steadily; by week 8, treated chicks were consuming more feed than controls. Thus, it appears that chicks and adults can begin to overcome the appetite depressing
effects of dietary mycotoxins after 4 or 5 weeks of ingesting contaminated feed. Decreased feed consumption can partially explain the results of Ottinger and Doerr (1980) and Doerr and Ottinger (1980) who fed aflatoxin to immature Japanese quail and observed decreased body weight and testicular weight, reduced androgen levels, and delayed sexual maturity. Other workers have shown that these effects can be caused by a diet deficient in the quantity or quality of feedstuffs. Breneman (1940) restricted feed consumption of chicks and noted reduced body weight, testes weights, and comb weights. Low protein diets, when fed to cockerels, delayed sexual maturity (Wilson et ah, 1965), and when consumed by mature roosters for 2 weeks, they caused a 12% loss in body weight and a 59% decrease in plasma testosterone (Wilson et ah (1979). Leatham's (1970) statement that testes of immature animals are more sensitive to dietary changes than mature animals is consistent widi the conclusion of Ottinger and Doerr (1980) that young males are more sensitive to aflatoxin than mature birds. The significant decrease in plasma testosterone observed among the pair-fed and aflatoxin-fed males was probably not due to failure of the gonads but a result of diminished LH levels and the subsequent reduced stimulation of the Leydig cells. Howland (1975) demonstrated that underfed rats had reduced serum LH levels and increased pituitary concentrations of LH but not FSH. He concluded that underfeeding primarily impairs LH release
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a
.41 (20)
AFLATOXIN ON REPRODUCTION AND FEED CONSUMPTION
albumin, but reduced feed consumption can account for most of the effects of dietary aflatoxin on reproductive parameters. For reproductive measurements, the response of the pair-fed group was between the control group and the males fed aflatoxin. Therefore, at least half the effect of aflatoxin on reproduction can be explained by a decrease in feed consumption and the remainder by direct and other indirect actions of the toxin. In relation to reproduction, aflatoxicosis has nutritional and toxicological consequences.
REFERENCES Breneman, W. R., 1940. Limitation of food consumption as a factor influencing endocrine reactions in the chick. Endocrinology 26:1091— 1098. Briggs, D. M., R. D. Wyatt, and P. B. Hamilton, 1974. The effect of dietary aflatoxin on semen characteristics of mature broiler breeder males. Poultry Sci. 53:2115-2119. Culbert, J., 1976. Improved extraction of steroid hormones from the blood plasma of the domestic fowl (Gallus domesticus) using light petroleum at elevated temperatures. Analyst 101:391 — 395. Doerr, J. H., and M. A. Ottinger, 1980. Delayed reproductive development resulting from aflatoxicosis in juvenile Japanese quail. Poultry Sci. 59:1995-2001. Doumas, B. T., W. A. Watson, and H. G. Biggs, 1971. Albumin standards and the measurement of serum albumin with bromcresol green. Clin. Chim. Acta 31:87-96. Engster, H. M., L. B. Carew, Jr., and E. J. Cunningham, 1978. Effects of an essential fatty acid deficiency, pair-feeding and level of dietary corn on the hypothalmic - pituitary - gonadal axis and other physiological parameters in the male chicken. J. Nutr. 108:889-900. Folch, J., M. Lees and G. H. Sloan-Stanley, 1957. A simple method for the isolation and purification of total lipids from animal tissues. J. Biol. Chem. 226:497-509. Gumbmann, M. R., S. N. Williams, A. N. Booth, P. Vohra, R. A. Ernst and M. Bethard, 1970. Aflatoxin susceptibility in various breeds of poultry. Proc. Soc. Exp. Biol. Med. 134:683688. Helwig, J. T., and K. A. Council, ed., 1979. SAS User's guide. SAS Inst., Raleigh, NC. Howland, B. E., 1975. The influence of feed restriction and subsequent re-feeding on gonadotropin secretion and serum testosterone levels in male rats. J. Reprod. Fert. 44:429-436. Howland, B. E., 1979. Effect of underfeeding on the inhibition of gonadotropin secretion by testosterone propionate in rats. J. Reprod. Fert. 55:335-338. Hsieh, D.P.H., 1979. Basic metabolic effects of raycotoxins. Pages 43—55 in Interaction of mycotoxins in animal production. Nat. Acad. Sci., Washington, DC.
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and secondarily impairs LH synthesis. Engster et al. (1978) concluded that reduced energy intake by cockerels tended to decrease secretion of pituitary gonadotropins. In extending these conclusions, we hypothesize that the significant decline in semen volume and spermatocrit among aflatoxin and pair-fed males was due to impaired spermatogenesis resulting from decreased feed consumption which caused reduced LH and therefore reduced testosterone levels. Among aflatoxin-fed males, the significant decline in plasma testosterone occurred 1 week before the significant decline in semen volume and 3 weeks before the significant decline in sperm concentration, i.e., spermatocrit. Since spermatogenesis and sperm transport require 16 days in the fowl (Sturkie, 1976) and because the later stages of spermatogenesis are testosterone dependent (Murton and Westwood, 1977), a 3-week delay between a decline in testosterone levels and a decline in sperm concentration was expected. Howland (1979) showed that underfed male rats had decreased gonadotropin levels because their hypothalamic-pituitary axis had increased sensitivity to the inhibitory effects of androgens. Pituitaries of underfed rats could secrete adequate amounts of gonadotropins after the inhibitory influence of androgens was removed. Thus, current theory implicates the hypothalamus and pituitary as the active sites in balancing adequacy of nutrition and readiness for reproduction. Since reproduction increases energy requirements, a pituitary sensitive to poor nutrition would have survival value. How aflatoxin causes a temporary decrease in feed consumption remains speculative. Wyatt (1981, personal communication ) determined that broiler chicks showed no preference when given a choice between control feed and feed containing graded levels of aflatoxin grown on rice; therefore, poor palatability was probably not a factor in this experiment. Polin and Wolford (1973) hypothesized that appetite is controlled by receptors in the crop interacting with the hypothalamus and the whole system can be modified by metabolic alterations arising elsewhere in the body. Aflatoxin could, therefore, affect appetite as a result of its metabolic alterations to the liver. In summary, the response of the pair-fed group demonstrates that reduced feed consumption cannot explain the effects of aflatoxin on liver weight, liver fat, or plasma
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SHARLIN ET AL. feed restriction on fertility in male domestic fowls. Poultry Sci. 22:170-177. Polin, D., and J. H. Wolford, 1973. Factors influencing food intake and caloric balance in chickens. Fed. Proc. 32:1720-1726. Prior, M. G., J. B. O'Neil, and C. S. Sisodia, 1980. Effects of ochratoxin A on growth response and residues in broilers. Poultry Sci. 59:1254-1257. Sharlin, J. S., B. Howarth, Jr., and R. D. Wyatt, 1980. Effect of dietary aflatoxin on reproductive performance of mature White Leghorn males. Poultry Sci. 59:1311-1315. Shotwell, O. L., G. W. Hesseltine, R. D. Subblefield, and W. G. Sorenson, 1966. Production of aflatoxin on rice. Appl. Microbiol. 14:425—428. Sturkie, P. D., 1976. Reproduction in the male, fertilization and early embryonic development. Ch. 17 in Avian physiology. 3rd ed. P. D. Sturkie, ed. Springer-Verlag, New York, NY. Taneja, G. C , and R. S. Gowe, 1961. Spermatozoa concentration in the semen of two breeds of fowl estimated by three different methods. Poultry Sci. 40:608-615. West, S., R. D. Wyatt, and P. B. Hamilton. 1973. Improved yield of aflatoxin by incremental increases of temperature. Appl. Microbiol. 25:1018-1019. Wilson, E. R., J. C. Rogler, and R. E. Erb, 1979. Effect of sexual experience, location, malnutrition, and repeated sampling on concentrations of testosterone in blood plasma of Gallus domesticus roosters. Poultry Sci. 58:178— 186. Wilson, H. R., P. W. Waldroup, J. E. Jones, D. J. Duerre, and R. H. Harms, 1965. Protein levels in growing diets and reproductive performance of cockerels. J. Nutr. 85:29-37.
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Johnson, P. M., 1955. Hematocrit values for the chick embryo at various ages. Amer. J. Physiol. 180: 361-363. Riser, T. E., R. H. Milvae, H. D. Hafs, W. D. Oxender, and T. M. Lous, 1978. Comparison of testosterone and androstenedione secretion in bulls given prostaglandin F 2 a or luteinizing hormone. J. Anim. Sci. 46:436-442. Leathern, J. H., 1970. Nutrition. Pages 169-205 in The Testis. Vol. III. Influencing factors. A. D. Johnson, W. R. Gomes, and N. L. Vandemark, ed. Academic Press, New York, NY. Mitchell, H. H., L. E. Card, and T. S. Hamilton, 1926. The growth of White Plymouth Rock chickens. Illinois Agr. Exp. Sta. Bull. 278:69-132. Mitchell, H. H., L. E. Card, and T. S. Hamilton, 1931. A technical study of the growth of White Leghorn chickens. Illinois Agr. Exp. Sta. Bull. 367:81-139. Mulinos, M. G., and L. Pomerantz, 1940. Pseudohypophysectomy, a condition resembling hypophysectomy produced by malnutrition. J. Nutr. 19:493-504. Murton, R. K., and N. J. Westwood, 1977. Reproductive apparatus in the male. Ch. 3. In. Avian breeding cycles. Clarendon Press, Oxford. Nabney, J., and B. F. Nesbitt, 1965. A spectrophotometric method of determining the aflatoxins. Analyst 90:155-160. Ottinger, M. A., and J. A. Doerr, 1980. The early influence of aflatoxin upon sexual maturation in the male Japanese quail. Poultry Sci. 59:1750— 1754. Parker, J. E., and G. H. Arscott, 1964. Energy intake and fertility of male chickens. J. Nutr. 82: 183-187. Parker, J. E., and B. J. McSpadden, 1943. Influence of
1981 Poultry Science 60:2701-2708 INTRODUCTION
The reproductive system of the mature White Leghorn male appears to be more susceptible to dietary aflatoxin than that of the mature broiler breeder male. After 4 to 5 weeks of dietary aflatoxin (20 Mg/g diet), a significant decline in semen volume and testes weights along with a disruption of testicular histology was found in White Leghorns (Sharlin et al, 1980), whereas the same variables observed in broiler breeder males were unaffected (Briggs et al, 1974). However, when compared to controls, both the White Leghorn and broiler breeder males showed approximately a 65% increase in liver weight and an 11% reduction in body weight in response to dietary aflatoxin. Among White Leghorn males fed aflatoxin a significant decrease in feed consumption occurred after 1 week of dietary treatment; during subsequent weeks feed consumption decreased steadily, reaching a minimum after 4 weeks of dietary aflatoxin at which time treated males consumed about half as much
Department of Physiology and Pharmacology.
feed as control males (Sharlin et al, 1980). Thus, while the 11% decline in body weight was most likely attributable to a reduction in feed consumption, the causative factor(s) for the impaired reproductive potential could be due to dietary aflatoxin and/or reduced feed intake. Mulinos and Pomerantz (1940) demonstrated that adult rats restricted to one-half their normal quantity of feed had severely atrophied testes and diminished or absent spermatozoa production. The testicular response was almost identical to rats which had been hypophysectomized. Decreased semen volume and fertility were observed in Rhode Island Reds (Parker and McSpadden, 1943) and White Leghorns (Parker and Arscott, 1964) after a body weight loss. Since the mature broiler breeder male has 2.5 times more crude fat than the mature White Leghorn male (Mitchell et al, 1926; 1931), the broiler breeder should be more tolerant than the White Leghorn male to a reduction in feed intake and body weight loss induced by dietary aflatoxin. Therefore, breed differences in reproductive response to aflatoxin can be explained by a difference in tolerance to undernutrition (Sharlin et al, 1980). The objective of this pair-feeding study was to determine the relative importance of ingestion
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ABSTRACT A pair-feeding study was conducted to investigate the relative importance of ingestion of aflatoxin (20 Mg/g diet) versus decreased feed consumption in explaining the effects of dietary aflatoxin on reproduction. Fifty-eight mature White Leghorn males were divided among three groups - control, 0 Mg/g diet ad lib; aflatoxin, 20 Mg/g diet ad lib; and pair-fed, 0 Mg/g diet pair fed to 20 Mg/g group. Aflatoxin and pair-fed males consumed significantly less feed than controls during weeks 1 through 5 of the 8-week aflatoxin feeding period. Measures of reproductive potential (semen volume, testes weights, spermatocrit, and plasma testosterone) for pair-fed males were not significantly different from males fed aflatoxin, although both groups were significantly lower than control males. Measures of aflatoxicosis (liver weight, liver fat, and plasma albumin) for pair-fed males were not significantly different from control males, although both groups were significantly different from males fed aflatoxin. In conclusion, decreased feed consumption did not produce symptoms of aflatoxicosis but accounted for 60% of the effects of aflatoxin on reproduction. Therefore, aflatoxin has nutritional and toxicological effects on reproduction. (Key words.- aflatoxin, reproduction, testosterone, feed consumption, White Leghorn)
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of toxin as compared to decreased food consumption in explaining the effects of dietary aflatoxin on reproduction. MATERIALS AND METHODS
Reproductive Measurements. Semen was collected weekly by abdominal massage. Individual semen volume was recorded to the nearest .01 ml using a 1 ml tuberculin syringe. Individual semen samples were drawn into capillary tubes, and spermatocrit readings, a measure of spermatozoa concentration, were determined using the method of Taneja and Go we (1961). Organ Weights. At the conclusion of the experiment, all birds were killed by cervical
The antisera (MSU #74) was diluted 1: 25,000 using .01M phosphate buffered saline (PBS). One-tenth milliliter of a labeled testosterone solution, .2 ml of antisera, and .2 ml PBS were added to each tube and vortexed. The tubes were held at 40 C for 10 min and then incubated at 4 C for 15 to 18 hr. The crossreactivity of the antisera (MSU #74) has been reported (Kiser et al, 1978). When testosterone equalled 100%, dihydrotestosterone was found to crossreact with this antisera at 60%. Thus, the assay measured both androgens in plasma samples, although the data are expressed in
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Animals and Feed. Fifty-eight 14-month-old mature White Leghorn males were housed individually in an unheated room with a constant photoperiod of 16 hr light and 8 hr dark. Aflatoxin was produced by growing Aspergillus parasiticus NRRL 2999 on sterile rice using the method of Shotwell et al. (1966) with the temperature modification of West et al. (1973). The rice was steamed to kill the fungus, ovendried at 80 C for 18 hr, and ground to a fine powder. The concentration of aflatoxin was determined by spectrophotometric analysis of the rice powder using the method of Nabney and Nesbitt (1965). Individual toxins were separated by high pressure liquid chromatography. The mixture contained 83.6% Bi, .2% B 2 ,and 16.2% G j . Experimental Design. The 10-week experiment (2 weeks pretreatment, 8 weeks aflatoxin feeding) consisted of three treatments in a completely randomized design. Sixteen birds served as controls and consumed the University of Georgia layer-breeder ration ad lib, 21 birds were given 20 /ig aflatoxin/g diet ad lib, and 21 birds consumed the control ration but were pair-fed to the aflatoxin group. During the 2-week pretreatment period, all birds consumed a layer breeder ration ad lib. Using data collected during the pretreatment period, birds were matched by closeness in body weight and feed consumption for pairfeeding during the aflatoxin feeding period. For pair-feeding, individual body weight and food consumption were measured daily at 1300 hr, and then each pair-fed bird was given a quantity of layer breeder ration equal to die amount of food consumed by its aflatoxin-fed partner during the previous 24 hr.
dislocation. Testes and liver were removed and weighed to the nearest .01 g. Collection of Plasma Samples. All birds were bled weekly from the brachial vein between 1000 and 1130 hr. A 23 gauge needle and 3 ml syringe were used to collect 3 ml blood from each bird. The blood was transferred from the syringe to a heparinized 12 X 75 mm tube for centrifugation. After centrifugation, plasma samples were frozen until analyzed. Laboratory Analysis. Immediately after collection, individual blood samples were drawn into heparinized capillary tubes, and the packed cell volume was determined by the microhematocrit method (Johnson, 1955). Plasma albumin was measured by the method of Doumas et al. (1971). Percent carcass fat and percent liver fat were measured by the method of VcAchetal. (1957). Within treatment variation for plasma albumin and liver fat was low enough that samples from nine randomly selected birds were analyzed per treatment group. Testosterone Analysis. Plasma testosterone was measured by radioimmunoassay. Extraction in duplicate of 100 fJ.1 plasma generally followed the procedure of Culbert (1976). Two milliliters of ligroine (boiling range 65 to 90 C) were added to 100 /txl plasma in 16 x 100 mm culture tubes. The tubes were stoppered and placed in a 50 C water bath and agitated with a mechanical shaker for 1 hr. The aqueous phase was frozen and the ether phase was poured into 12 X 75 mm culture tubes and dried under nitrogen. The plasma was similarly extracted a second time for 30 min. The ether phase from both extractions were combined. Recovery of 1, 2, 6, 7- 3 H testosterone during extraction of blood plasma averaged 90% (range 86 to 92%). Unknowns were not corrected for procedural loss.
AFLATOXIN ON REPRODUCTION AND FEED CONSUMPTION
The sensitivity of the assay was 100 pg/ml plasma. The addition of testosterone to a pooled sample either at 20, 40, 100, 200, or 1000 pg was assayed as 17, 46, 120, 280, and 1046 pg, respectively, after correcting for endogenous testosterone. Interassay variation (CV = 6%, n = 10) was calculated using a pooled plasma sample included in each assay. Testosterone levels were determined for 14 aflatoxinfed birds, 14 pair-fed birds, and 10 controls. Statistical Analysis. For variables measured every week, a one-way analysis of variance (ANOVA) was conducted separately for each week using the ANOVA procedure in the Statistical Analysis System (Helwig and Council, 1979). Variables measured at the conclusion of the study were analyzed using a one-way ANOVA. Mean separation was by Duncan's multiple range test at the .05 level. Significance is based on P<.05. Assuming no synergism between decreased feed consumption and ingestion of aflatoxin, the portion of the effect of dietary aflatoxin accounted for by decreased feed consumption was called the feed consumption factor (FCF). The FCF values were determined by calculating a ratio of the difference between controls and the pair-fed and aflatoxin groups using the formula: [(C-P)/(C-A)] X 100 where C = control, P = pair-fed, and A = aflatoxin. For example, if values for a hypothetical 2 Fisher Scientific Company, Manufacturing Division, Fair Lawn, NJ 07410.
variable were control, 100; pair-fed, 300; and aflatoxin, 500; then the FCF value would be 50%, that is, 50% of the effect of aflatoxin was accounted for by a decrease in feed consumption. The FCF values were calculated for only those weeks with a significant treatment effect. An FCF value for a given variable represents an average of the weekly FCF values. RESULTS
Feed Consumption and Body Weight. Feed consumption and body weight did not differ significantly between the aflatoxin and pair-fed groups during the entire study. Both groups consumed significantly less feed compared to controls during the first 5 weeks of the 8-week treatment period (Fig. la). Feed consumption of the toxin and pair-fed groups increased during treatment weeks 5 and 6 such that during the last 3 weeks of the experiment there were no significant differences for feed consumption among the three experimental groups. Body weight of the aflatoxin and pair-fed groups declined significantly from controls after 4 weeks of feeding aflatoxin and
PAIR-FED- OJJG /G PAIR-FED TO 10pG /a - CONTROL-Opo/G AD 111.
GROUP
- AFLATOXIN-?O L I G / G AD LIB.
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7
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FIG. 1. The effect of dietary aflatoxin and pairfeeding on feed consumption and body weight of mature White Leghorn males. When the F-test for any of the 11 analyses of variance was significant (P<.05 as shown along the top of each graph), treatments significantly different for that week have different letters plotted next to their mean.
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terms of testosterone. For all other steroids, crossreactivity was less than 2%. Unbound testosterone was removed from solution using dextran coated charcoal (1.23 g Norit A plus .123 g Dextran T70 in 500 ml PBS). Two-tenths milliliter charcoal solution were added to each assay tube. The tubes were centrifuged at 750 x g for 10 min at 4 C and the supernatant portion decanted into 7 ml scintillation vials. Five milliliters of Scinti Verse 2 were added to each vial, and radioactivity was determined with a liquid scintillation spectrophotometer (Beckman Model LS-7000). Duplicate testosterone standards of 10, 20, 40, 100, 200, 400, and 1000 pg dissolved in ethanol were included in each assay. The standard curve, plotted as percent bound against logio of standard, resulted in a straight line with an average R square value of 99%.
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- FAIR-FID - 0 pO /G PAIR-KD TO 20/KS / G GROUP - CONTROL-O^G/G AD LIB. - AILATOXIN-IOLIG/G AD LII. •
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FIG. 2. The effect of dietary aflatoxin and pair-feeding on semen volume, spermatocrit, and plasma testosterone of mature White Leghorn males. When the F-test for any of the 11 analyses of variance was significant (P<.05 as shown along the top of each graph), treatments significantly different for that week have different letters plotted next to their mean.
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4 5 6 TIME (WEEKS)
AFLATOXIN ON REPRODUCTION AND FEED CONSUMPTION
(Table 1). Packed Cell Volume. There were no significant differences between any of the groups for packed cell volume. Weekly treatment means ranged from 38 to 44% during any weekly period. Mortality. One aflatoxin-fed male and his pair-fed partner died during treatment week 6 when 36% of their original body weight had been lost. During the 2 weeks prior to death, only 15 g of feed were consumed. DISCUSSION
The response of the pair-fed group clearly supports the original hypothesis that reduced feed intake was an important factor in explaining the effects of dietary aflatoxin on reproduction in White Leghorn males (Sharlin et al., 1980). A decline in feed consumption accounted for over half the effects of aflatoxin on testes weight (62%), semen volume (59%), plasma testosterone (57%), body weight (80%), and almost all the effect on spermatocrit. However, the decline in feed consumption was unrelated to increased liver fat, increased liver weight, and decreased plasma albumin, all three of these responses being sensitive indicators of aflatoxicosis in the fowl (Gumbmann et al., 1970). Pair-feeding allows separation of aflatoxin's primary action, which causes disruption of carbohydrate and lipid metabolism, and inhibition of protein synthesis (Hsieh, 1979),
PAIR-FED- 0 U G / G PAIR-FID TO 20/JG / G GROUP CONTROL-OUG/G AD IIB. AFLATOXIN-JO U G / G AD IIB. 85
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FIG. 3. The effect of dietary aflatoxin and pairfeeding on plasma albumin of mature White Leghorn males. When the F-test for any of the 11 analyses of variance was significant (P<.05 as shown along the top of each graph), treatments which were significantly different for that week have different letters plotted next to their mean.
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continued to decrease during treatment week 5; thereafter, no further decline in body weight occurred (Fig. lb). The FCF value for body weight was 80%. Measures of Reproductive Potential. Semen volume of the aflatoxin-fed and pair-fed males declined significantly from controls after treatment weeks 4 and 5, respectively (Fig. 2a). During the last 5 weeks of the experiment, semen volume of the pair-fed males was usually significantly less than controls and nonsignificantly different from males fed aflatoxin. During the first 5 weeks of feeding aflatoxin there were no significant differences in spermatocrit (Fig. 2b). During the last 3 weeks of the experiment, spermatocrit values of the aflatoxin and pair-fed males declined significantly from controls but were nonsignificantly different from each other. After 8 weeks of feeding aflatoxin, testes weights and percent carcass fat of the aflatoxin and pair-fed males, while nonsignificantly different from each other, were significantly less than control males (Table 1). Plasma testosterone levels of males fed aflatoxin declined significantly from controls during weeks 4, 5, and 6 of toxin feeding (Fig. 2c). Pair-fed males had testosterone levels significantly lower than controls only at treatment week 5. The testosterone levels of the pair-fed group were between those of the aflatoxin and control groups during the last 5 weeks of the experiment. The FCF values for reproductive variables were semen volume 59%, testes weights 62%, and plasma testosterone 57%. For spermatocrit and carcass fat, FCF values were approximately 100% since the pair-fed males and males fed aflatoxin had nearly identical decreases from the control group. Measures of Aflatoxicosis. There were no significant differences in plasma albumin between the control and pair-fed groups (Fig. 3). Plasma albumin levels in males fed aflatoxin were significantly less than control males from the second week of toxin feeding until the end of the experiment. For birds fed aflatoxin, plasma albumin decreased markedly during treatment week 4 but remained fairly constant for the remainder of the toxin feeding period. Analysis of livers removed at the end of the experiment for percent fat and weight showed that control and pair-fed groups while nonsignificantly different from each other were both significantly less than males fed aflatoxin
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SHARLIN ET AL. TABLE 1. Organ weights and percent liver and carcass fat of mature White Leghorn males fed aflatoxin for 8 weeks1 Organ weights (g/lOOgbody wt)
Treatment
Percent carcass fat
Testes
Liver
Percent liver fat
Control 0 iig/g ad lib
6.8 a ± 1.3 (16)
.74 a ±.066 (16)
1.3 a ±.058 (16)
4.7 a ± .64 (9)
Pair-fed 0 Mg/g pair-fed to 20 jug/g group
2.8 b ± .42 (20)
.54 b ± .063 (20)
1.4* ±.059 (20)
4 . 2 a ± . 3 5 (9)
Aflatoxin 20 Mg/g ad lib
3.3 b i
.42 b ± .057 (20)
2 . 1 b ± .124 (20)
7.7 b ± .81 (9)
' Means within the same column bearing different superscripts are significantly different (P<.05).
'Means ± SE (number of observations).
from the toxin's other effects arising from reduced feed consumption. The quantity of control feed given to the pair-fed group was matched on a daily basis to the feed consumption of the males fed aflatoxin. Therefore, the response of the aflatoxin group was due to ingestion of toxin and decreased feed consumption whereas the response of the pair-fed group was only a result of decreased feed consumption. The relative effect on any variable of decreased feed consumption vs. ingestion of aflatoxin was determined by comparing the response of the aflatoxin and pairfed groups to controls. The response of feed consumption and semen volume by males fed aflatoxin was similar to that observed in an earlier study (Sharlin et ah, 1980). In both studies, after aflatoxin was fed for 4 weeks, semen volume of aflatoxin-fed males declined significantly from controls and feed consumption decreased to its lowest value. However, during treatment week 5 in both experiments, males fed aflatoxin increased their feed consumption. In this study feed consumption of the three experimental groups did not differ significantly during the last 3 weeks of the experiment. A similar response was observed when graded levels of ochratoxin were fed to male chicks from hatch to 8 weeks of age (Prior et ah, 1980). Feed consumption declined during the first 5 weeks, but then increased steadily; by week 8, treated chicks were consuming more feed than controls. Thus, it appears that chicks and adults can begin to overcome the appetite depressing
effects of dietary mycotoxins after 4 or 5 weeks of ingesting contaminated feed. Decreased feed consumption can partially explain the results of Ottinger and Doerr (1980) and Doerr and Ottinger (1980) who fed aflatoxin to immature Japanese quail and observed decreased body weight and testicular weight, reduced androgen levels, and delayed sexual maturity. Other workers have shown that these effects can be caused by a diet deficient in the quantity or quality of feedstuffs. Breneman (1940) restricted feed consumption of chicks and noted reduced body weight, testes weights, and comb weights. Low protein diets, when fed to cockerels, delayed sexual maturity (Wilson et ah, 1965), and when consumed by mature roosters for 2 weeks, they caused a 12% loss in body weight and a 59% decrease in plasma testosterone (Wilson et ah (1979). Leatham's (1970) statement that testes of immature animals are more sensitive to dietary changes than mature animals is consistent widi the conclusion of Ottinger and Doerr (1980) that young males are more sensitive to aflatoxin than mature birds. The significant decrease in plasma testosterone observed among the pair-fed and aflatoxin-fed males was probably not due to failure of the gonads but a result of diminished LH levels and the subsequent reduced stimulation of the Leydig cells. Howland (1975) demonstrated that underfed rats had reduced serum LH levels and increased pituitary concentrations of LH but not FSH. He concluded that underfeeding primarily impairs LH release
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a
.41 (20)
AFLATOXIN ON REPRODUCTION AND FEED CONSUMPTION
albumin, but reduced feed consumption can account for most of the effects of dietary aflatoxin on reproductive parameters. For reproductive measurements, the response of the pair-fed group was between the control group and the males fed aflatoxin. Therefore, at least half the effect of aflatoxin on reproduction can be explained by a decrease in feed consumption and the remainder by direct and other indirect actions of the toxin. In relation to reproduction, aflatoxicosis has nutritional and toxicological consequences.
REFERENCES Breneman, W. R., 1940. Limitation of food consumption as a factor influencing endocrine reactions in the chick. Endocrinology 26:1091— 1098. Briggs, D. M., R. D. Wyatt, and P. B. Hamilton, 1974. The effect of dietary aflatoxin on semen characteristics of mature broiler breeder males. Poultry Sci. 53:2115-2119. Culbert, J., 1976. Improved extraction of steroid hormones from the blood plasma of the domestic fowl (Gallus domesticus) using light petroleum at elevated temperatures. Analyst 101:391 — 395. Doerr, J. H., and M. A. Ottinger, 1980. Delayed reproductive development resulting from aflatoxicosis in juvenile Japanese quail. Poultry Sci. 59:1995-2001. Doumas, B. T., W. A. Watson, and H. G. Biggs, 1971. Albumin standards and the measurement of serum albumin with bromcresol green. Clin. Chim. Acta 31:87-96. Engster, H. M., L. B. Carew, Jr., and E. J. Cunningham, 1978. Effects of an essential fatty acid deficiency, pair-feeding and level of dietary corn on the hypothalmic - pituitary - gonadal axis and other physiological parameters in the male chicken. J. Nutr. 108:889-900. Folch, J., M. Lees and G. H. Sloan-Stanley, 1957. A simple method for the isolation and purification of total lipids from animal tissues. J. Biol. Chem. 226:497-509. Gumbmann, M. R., S. N. Williams, A. N. Booth, P. Vohra, R. A. Ernst and M. Bethard, 1970. Aflatoxin susceptibility in various breeds of poultry. Proc. Soc. Exp. Biol. Med. 134:683688. Helwig, J. T., and K. A. Council, ed., 1979. SAS User's guide. SAS Inst., Raleigh, NC. Howland, B. E., 1975. The influence of feed restriction and subsequent re-feeding on gonadotropin secretion and serum testosterone levels in male rats. J. Reprod. Fert. 44:429-436. Howland, B. E., 1979. Effect of underfeeding on the inhibition of gonadotropin secretion by testosterone propionate in rats. J. Reprod. Fert. 55:335-338. Hsieh, D.P.H., 1979. Basic metabolic effects of raycotoxins. Pages 43—55 in Interaction of mycotoxins in animal production. Nat. Acad. Sci., Washington, DC.
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and secondarily impairs LH synthesis. Engster et al. (1978) concluded that reduced energy intake by cockerels tended to decrease secretion of pituitary gonadotropins. In extending these conclusions, we hypothesize that the significant decline in semen volume and spermatocrit among aflatoxin and pair-fed males was due to impaired spermatogenesis resulting from decreased feed consumption which caused reduced LH and therefore reduced testosterone levels. Among aflatoxin-fed males, the significant decline in plasma testosterone occurred 1 week before the significant decline in semen volume and 3 weeks before the significant decline in sperm concentration, i.e., spermatocrit. Since spermatogenesis and sperm transport require 16 days in the fowl (Sturkie, 1976) and because the later stages of spermatogenesis are testosterone dependent (Murton and Westwood, 1977), a 3-week delay between a decline in testosterone levels and a decline in sperm concentration was expected. Howland (1979) showed that underfed male rats had decreased gonadotropin levels because their hypothalamic-pituitary axis had increased sensitivity to the inhibitory effects of androgens. Pituitaries of underfed rats could secrete adequate amounts of gonadotropins after the inhibitory influence of androgens was removed. Thus, current theory implicates the hypothalamus and pituitary as the active sites in balancing adequacy of nutrition and readiness for reproduction. Since reproduction increases energy requirements, a pituitary sensitive to poor nutrition would have survival value. How aflatoxin causes a temporary decrease in feed consumption remains speculative. Wyatt (1981, personal communication ) determined that broiler chicks showed no preference when given a choice between control feed and feed containing graded levels of aflatoxin grown on rice; therefore, poor palatability was probably not a factor in this experiment. Polin and Wolford (1973) hypothesized that appetite is controlled by receptors in the crop interacting with the hypothalamus and the whole system can be modified by metabolic alterations arising elsewhere in the body. Aflatoxin could, therefore, affect appetite as a result of its metabolic alterations to the liver. In summary, the response of the pair-fed group demonstrates that reduced feed consumption cannot explain the effects of aflatoxin on liver weight, liver fat, or plasma
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SHARLIN ET AL. feed restriction on fertility in male domestic fowls. Poultry Sci. 22:170-177. Polin, D., and J. H. Wolford, 1973. Factors influencing food intake and caloric balance in chickens. Fed. Proc. 32:1720-1726. Prior, M. G., J. B. O'Neil, and C. S. Sisodia, 1980. Effects of ochratoxin A on growth response and residues in broilers. Poultry Sci. 59:1254-1257. Sharlin, J. S., B. Howarth, Jr., and R. D. Wyatt, 1980. Effect of dietary aflatoxin on reproductive performance of mature White Leghorn males. Poultry Sci. 59:1311-1315. Shotwell, O. L., G. W. Hesseltine, R. D. Subblefield, and W. G. Sorenson, 1966. Production of aflatoxin on rice. Appl. Microbiol. 14:425—428. Sturkie, P. D., 1976. Reproduction in the male, fertilization and early embryonic development. Ch. 17 in Avian physiology. 3rd ed. P. D. Sturkie, ed. Springer-Verlag, New York, NY. Taneja, G. C , and R. S. Gowe, 1961. Spermatozoa concentration in the semen of two breeds of fowl estimated by three different methods. Poultry Sci. 40:608-615. West, S., R. D. Wyatt, and P. B. Hamilton. 1973. Improved yield of aflatoxin by incremental increases of temperature. Appl. Microbiol. 25:1018-1019. Wilson, E. R., J. C. Rogler, and R. E. Erb, 1979. Effect of sexual experience, location, malnutrition, and repeated sampling on concentrations of testosterone in blood plasma of Gallus domesticus roosters. Poultry Sci. 58:178— 186. Wilson, H. R., P. W. Waldroup, J. E. Jones, D. J. Duerre, and R. H. Harms, 1965. Protein levels in growing diets and reproductive performance of cockerels. J. Nutr. 85:29-37.
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Johnson, P. M., 1955. Hematocrit values for the chick embryo at various ages. Amer. J. Physiol. 180: 361-363. Riser, T. E., R. H. Milvae, H. D. Hafs, W. D. Oxender, and T. M. Lous, 1978. Comparison of testosterone and androstenedione secretion in bulls given prostaglandin F 2 a or luteinizing hormone. J. Anim. Sci. 46:436-442. Leathern, J. H., 1970. Nutrition. Pages 169-205 in The Testis. Vol. III. Influencing factors. A. D. Johnson, W. R. Gomes, and N. L. Vandemark, ed. Academic Press, New York, NY. Mitchell, H. H., L. E. Card, and T. S. Hamilton, 1926. The growth of White Plymouth Rock chickens. Illinois Agr. Exp. Sta. Bull. 278:69-132. Mitchell, H. H., L. E. Card, and T. S. Hamilton, 1931. A technical study of the growth of White Leghorn chickens. Illinois Agr. Exp. Sta. Bull. 367:81-139. Mulinos, M. G., and L. Pomerantz, 1940. Pseudohypophysectomy, a condition resembling hypophysectomy produced by malnutrition. J. Nutr. 19:493-504. Murton, R. K., and N. J. Westwood, 1977. Reproductive apparatus in the male. Ch. 3. In. Avian breeding cycles. Clarendon Press, Oxford. Nabney, J., and B. F. Nesbitt, 1965. A spectrophotometric method of determining the aflatoxins. Analyst 90:155-160. Ottinger, M. A., and J. A. Doerr, 1980. The early influence of aflatoxin upon sexual maturation in the male Japanese quail. Poultry Sci. 59:1750— 1754. Parker, J. E., and G. H. Arscott, 1964. Energy intake and fertility of male chickens. J. Nutr. 82: 183-187. Parker, J. E., and B. J. McSpadden, 1943. Influence of