APPLIED AND ENVIRONMENTAL MICROBIOLOGY, May 1976, p. 68-684 Copyright X) 1976 American Society for Microbiology Vol. 31, No. 5 Printed in U.SA. Effect of Dietary Afla on Fertility, Hatchability, and Progeny Performance of Broiler Breeder Hens B. HOWARTH, JR.* AND R. D. WYATT Department of Poultry Science, University of Georgia, Athens, Georgia 362 Received for publication 12 January 1976 The effects of afla on egg production, fertility, hatchability, and progeny performance were investigated by feeding dietary afla at dose levels of, 5, and 1,ug/g to mature broiler breeder hens for 4 weeks. Sixteen hens were used for each dietary dose level. Egg production decreased significantly during weeks 3 and 4 after initiation of feeding for hens fed 1 and 5,ug of afla per g of diet, respectively. Whereas fertility was not affected by dietary afla, hatchability of fertile eggs decreased significantly within week 1 of feeding. Hatchability of fertile eggs collected during week 1 of the treatment period was 95.1, 68.9, and 48.5% for the control, 5- and 1-,ug/g groups, respectively. At the dose levels used in this study, no latent effects of the afla or its metabolites were observed on the performance of surviving chicks. Six hens from each experimental group were necropsied at the end of the 4-week treatment period. These birds exhibited typical symptoms of aflatoxicosis, including enlarged, fatty and friable livers, and enlarged spleens. Afla, produced by the fungus Aspergillus flavus, when present as a contaminant in feedstuffs, has caused severe economic losses to the poultry and animal industries. First discovered in 196 as a result of turkey "X" disease (3), afla is now considered as one of the most prevalent mycos in nature (8). Natural outbreaks of aflatoxicosis have been reported in chickens (1, 2), ducks (5), turkeys (1, 19), cattle (1), and swine (9, 1). In poultry, aflatoxicosis results in increased mortality, increased condemnations, decreased growth rates, and poor feed conversion (2). In laying hens aflatoxicosis causes poor egg production and decreases egg weight (12, 13). Recently, the influence of aflatoxicosis on reproductive performance of poultry breeding stock has become a question of economic concern. Briggs et al. (4) observed no alterations in semen characteristics of mature broiler breeder males experiencing aflatoxicosis. Sawhney et al. (17) observed decreased egg production and hatchability in Japanese quail with aflatoxicosis. Dietary ochra has been shown to adversely affect egg production, hatchability, and performance of chicks from laying hens (7). Little work has been done to determine the effect of aflatoxicosis on reproductive performance of broiler breeder females. This study was designed to investigate the effect of dietary afla on fertility, hatchability, and progeny performance in broiler breeder hens. MATERIALS AND METHODS Animals. Commercial broiler breeder hens (Cobb) were housed in a commercial type poultry house. All birds were placed in individual laying cages and fed a standard broiler breeder ration free of all medications. Throughout the experiment feed and water were available ad libitum. All hens were inseminated once weekly with.1 ml of fresh undiluted semen from broiler type White Rock males, which were housed in the same fashion as the females. Afla production. Afla was produced by growing Aspergillus parasiticus NRRL 2999 on sterile polished rice according to the method of Shotwell et al. (18) with the temperature modification of West et al. (21). The moldy rice was steamed to kill the fungus, dried at 8 C in a forced-draft oven for 18 h, and then ground to a fine powder. The rice powder was then analyzed for total afla content by the spectrophotometric method of Nabney and Nesbitt (16) with the modification of Wiseman et al. (22). The term "afla" as used in this report refers to a mixture of aflas composed of 87.5% B,, 1.9% B2, 9.6% G,, and 1.% G2. Aflatoxicosis was induced by incorporating known amounts of afla into the diet. Experimental design. For experimental purposes, the hens were randomly divided into three groups of 16 birds each. The experiment consisted of a 2-week pretreatment period during which all birds received the control ration. After the pretreatment period, one group remained on the basal ration and served as controls. The other two groups received rations containing either 5 or 1 A±g of afla/g of diet for 4 weeks. At the end of the 4-week treatment period, six birds randomly selected from each group were 68 Downloaded from http://aem.asm.org/ on May 15, 218 by guest
VOL. 31, 1976 necropsied to insure that typical symptoms of aflatoxicosis were present and that no othe disease was present. All remaining birds were placed on the basal ration and maintained for 4 additional weeks. The experiment was terminated after this recovery phase. Measurements. During all three experimental periods, daily egg production, egg weights, and bird weights were collected. Weekly collections of eggs of each treatment were stored and incubated to evaluate fertility and hatchability. After 5 days of incubation, all eggs were candled to determine fertility. Those eggs which appeared to be "infertile" were broken and examined macroscopically for evidence of embryonic development. The chicks that hatched were reared in battery brooders with commercial starter feed and water supplied ad libitum. Chick mortality was recorded daily for a period of 2 weeks posthatching. Weekly body weights were obtained on all surviving chicks. Statistical analyses. All data were statistically analyzed by an analysis of variance (6). Throughout.&'L. - &'L +& t_ -. e. - 9 -a a stat Ic this paper, the termn -sigmnicant- hn meaning based on P <.5. I the hens re- There were no significant differences in fer- tility of eggs from hens fed the afla-con- taining diets with the exception of eggs laid during week 2 of the posttreatment period RESULTS Throughout the first 6 weeks of the expri ment there was no significant ch age body weight of the hens on tl tal rations (Fig. 1). However, during the final week of the posttreatment period ceiving 1,ug of afla/g of di( et exhibited a significant decline in body weigiit (11%) from the pretreatment value. There was a significant deci weight for the 1-,ug/g afla I during a 3-week period beginning treatment period (Fig. 2). Howe ver, observa- B y 4.1 w E 3.8 G HT (Kg.) 3.5 w----@ I I--S--W o AFLATOXICOSIS IN BREEDER HENS 681 2 4 6 8 m 1 mental groups during the pretreatment period (2 weeks) or for the first 2 weeks of the afla feeding period. Beginning with week 3 of the TIME (WKS.] treatment period (week 5 of experiment) there FIG. 1. Effect of dietary afla on body weight of broiler breeder hens. E G w E G HT (9) 66 t 62 1 58 1 54 2-4 6-8 1 TIME (WKS.) FIG. 2. Effect ofdietary afla on egg weights. tions on egg weight during this period were based on small numbers of eggs due to a corresponding decline in egg production. Egg weight of the 1-pg/g group returned to essentially the tge eaperi- control value by week 3 of the recovery period. ange in aver- There was no significant difference in egg.ie experimen- weight between the 5-,ug/g afla group and the control group. rease in egg (week 8 of experiment) by hens which had rerwoeupk the ceived 1tg of afla per g of diet (Table 1). week4oftera Fertility of eggs from hens in this group de- clined to 64.3%. However, due to the effects of afla on egg production, these hens had virtually ceased laying during week 1 of the posttreatment period which accounts for the missing fertility value in Table 1. Only four out i g <uu of ten hens had returned to egg production by the week 2 posttreatment. It is possible that the condition of the hens returning to egg production at this time could have been responsible for the lowered fertility. By week 3 posttreatment, fertility of eggs from hens that had recieved 1 \ ug of afla per g had returned to the control value. The effect of afla on egg production and i 11Ogg hatchability by broiler breeder hens is shown in Fig. 3. There were no significant differences in egg production between the control and experi- was a significant reduction in egg production by hens fed 1,ug of afla per g. Egg produc- Downloaded from http://aem.asm.org/ on May 15, 218 by guest
682 HOWARTH AND WYArfT TABLE 1. Effects of dietary afta on fertility Fertility (%) Expt period (weeks) Oa 5 1 Pretreatmentb 1 84.4 ± 5.4a 92.6 ± 3.6a 92.5 ± 3.3a 2 89. t 4.8a 96.3 ± 2.44a 97.6 ± 1.88 Afla feeding period 3 88.7 t 5.1a 96.3 ± 1.9a 97.2 ± 1.7a 4 9.5 ± 3.2a 91.8 ± 4.5a 94.8 t 2.2a 5 92.6 ± 3.6a 93.3 t 4.4a 96.5 ± 3.1a 6 92.1 ± 3.9a 93.5 ± 6.98 84. ± 7.38 Posttreatment 7 82. t 9.38 94.6 ± 3.7a 8 88.1 ± 6.4a 91.4 ± 6.3a 64.3 ± 11. lb 9 85.7 ± 99.8 1. ±.8 87.5 t 2.a 1 85.2 ± 5.a 83.7 ± 8.6a 77.4 t 18.8a " Afla (micrograms per gram). b Values (x + standard error of the mean) followed by different superscripts differ significantly (P <.5) from the corresponding control value. " Only one egg was laid during this week. E 1 G G 75 p R 5 % 25 H 1 A T 75 c H A B 5 1 25 T y % O a % 'ki \I \%,, I N., -,% -"11, 2 4 6 TIME (WEEKS).,I APPL. ENVIRON. MICROBIOL. duction was dose related and required 3 to 4 weeks for its delayed expression after afla Z--. omg/g administration. 5 ug/g A significant dose-related decrease in hatchar..' O9g/g bility offertile eggs was observed within week 1 of administration (Fig. 3). Hatchability of eggs laid by the 5-,g/g group decreased 5 to 6 days after feeding and was significantly lower than the control value during all 4 weeks of the treatment period. There was no significant difference between the control and 5-pMg/g groups during any week of the recovery period. Hatchability of eggs laid by the 1-,mg/g group began to decline 48 h after feeding and decreased to minimal values of 22. and 21.% during weeks 2 and 3 of the treatment period, 8 1 respectively. Hatchability of the 1-,ug/g group did not return to the control value until week 4 posttreatment. The missing value for week 7 of FIG. 3. Effect of dietary afla Xon egg produc- the experiment was due to the corresponding tion and hatchability of fertile eggs. The missing low number of eggs laid during this week. No hatchability value for week 7 of the e. xperiment was consistent teratogenic effects of afla were due to the corresponding low numbeor of eggs laid observed in either hatched embryos or those during this week. failing to hatch. There was a significant decrease in day-old body weights of chicks hatched from hens re- a minimum ceiving 1,tg of afla per g for week 3 of the tion continued to decline reachingg value (2.8%) during week 1 of t;he posttreat- treatment period (Table 2). Progeny perform- and did not ance as evaluated by mortality, feed conver- ment period (week 7 of experiment) return to control values until week 4 posttreat- sion, and weight gain during the 2-week period ment (week 1 of experiment). For hens fed 5 posthatching indicated no significant differ- decrease in ences between control and experimental. ug of afla per g, a significanit egg production occurred during vveek 4 of the Six birds were chosen at random from each treatment period and week 1 of tthe posttreat- treatment at the end of the feeding period ment period (weeks 6 and 7 of the experiment). necropsied. The -fed birds exhibited en- By week 8 egg production for the 5-,ug/g group larged, pale, fatty livers, enlarged spleens, and had returned to the control value!. The magni- slight kidney enlargement. All control birds tude and duration of the decrease in egg pro- appeared clinically normal. Downloaded from http://aem.asm.org/ on May 15, 218 by guest
VOL. 31, 1976 Dietary afla AFLATOXICOSIS IN BREEDER HENS 683 TABLE 2. Day-old body weight of chicks hatched from hens receiving dietary afla Body wt (g) Afla feeding period (weeks) (,gig) Pretreatmenta b Posttreatment 1 2 3 4 41.6.2a 42.5.3a 43.8 ±.3a 41.5 ±.3a 42.1.3a 42.8 ±.4a 5 42.5 ±.3a 42.2 ±.3a 45.6 ±. 5a 42.4 ±. 3a 42.3 ±. 3a 42.9 ±.2a 1 42.5 ±.3a 42.2 ±.4a 42.4 ±.2a 39.8 ±.5b 41.3 ±.4a _C a Values (x ± standard error of the mean) followed by different superscripts differ significantly (P c.5) from the corresponding control value. b Values given for pretreatment and posttreatment are averages for 1 week before afla feeding and 1 week after afla feeding, respectively. r Only one chick hatched from eggs laid during the posttreatment period. DISCUSSION Aflatoxicosis was induced in mature broiler breeder hens by feeding dietary levels of afla of 5 and 1 ug/g for a 4-week period. During the course of the experiment, broiler breeder hens receiving afla exhibited a dose-related reduction in reproductive performance as measured by both egg production and hatchability of fertile eggs (Fig. 3). Egg production decreased significantly during weeks 3 and 4 after initiation of feeding for hens fed 1 to 5,ug of afla per g, respectively. This delayed onset in the decline of egg production of 2 to 3 weeks after the addition of to the diet was observed by Garlich et al. (11) in White Leghorn laying hens fed 1,ug of dietary afla per g. Hamilton et al. (Abstr. Poultry Sci., p. 1,932, 1974) have hypothesized that afla affects egg production primarily by inhibiting the commitment of ova to maturation and not the maturation process itself, thus leading to a delayed onset in the decline of egg production. Although fertility was not affected by dietary afla, hatchability of fertile eggs decreased significantly within the first week after initiation of feeding. Hatchability of fertile eggs collected during week 1 of the treatment period was 95.1, 68.9, and 48.5% for the control, 5- and 1-,ug/g groups, respectively. The importance of this observation is that afla can have an immediate effect on hatching eggs being produced by a flock in terms of their subsequent potential hatchability before any obvious indication of a problem, such as a decline in egg production would indicate. By the time a decrease in egg production becomes obvious, an equally economically important decrease in hatchability can simultaneously occur at the hatchery. This is a unique effect of dietary afla and one that apparently has not been reported previously. Kratzer et al. (15) observed a slight reduction in hatchability for eggs laid by White Leghorn hens fed a small dose of afla (2.7 ug/g). However, no decline in egg production was observed possibly due to the small dose of afla utilized. Aflatoxicosis in laying Japanese quail induced a significant reduction in hatchability 3 weeks after initiation of dietary at levels of 4 and 6,.g/g (17). In the present study, the rapid effect of afla on hatchability suggests an extremely rapid transfer of ingested afla or potent breakdown product to the egg. Observations by Jacobson and Wiseman (14) indicate that dietary afla B, (.1 to.4,ug/g) fed to broiler breeder hens can be transmitted into their eggs in measurable amounts and was found in both the whites and yolks. In addition to a rapid, direct effect of afla on hatchability, another attractive hypothesis might be an inadequate or altered chemical composition of the egg itself. Changes in the hen's protein, carbohydrate, and lipid metabolism occur rapidly after feeding dietary afla. Such alterations in metabolism could alter the chemical composition of the egg and its subsequent hatchability. Afla had virtually no effect on day-old body weights of chicks that hatched, with one exception. Chicks hatched from eggs laid during week 3 of the treatment period by hens receiving 1 Mg of afla per g had significantly reduced day-old body weights. Subsequent growth rates of these chicks, however, were comparable to control values. Thus, at the dose levels used in this study, no significantly latent effects of the afla or its metabolites on progeny preformance were observed. These data emphasize the need for additional work in the area of mycotoxicoses before the economical problems they present to the poultry and animal industries can be fully understood and controlled. LITERATURE CITED 1. Alicroft, R., and R. B. A. Carnaghan. 1963. Toxic prod- Downloaded from http://aem.asm.org/ on May 15, 218 by guest
684 HOWARTH AND WYATT ucts in groundnuts' biological effects. Chem. Industry 2:5-53. 2. Asplin, F. D., and R. B. A. Carnaghan. 1961. The toxicity of certain groundnut meals for poultry, with special reference to their effect on ducklings and chickens. Vet. Rec. 73:1215-1219. 3. Blount, W. P. 1961. Turkey "X" disease. J. Br. Turkey Fed. 9:52-61. 4. Briggs, D. M., R. D. Wyatt, and P. B. Hamilton. 1974. The effect of dietary afla on semen characteristics of mature broiler breeder males. Poult. Sci. 53:2115-2119. 5. Brown, J. M. M., and L. Abrams. 1965. Biochemical studies on aflatoxicosis. Onderstepoort J. Vet. Res. 32:119-146. 6. Bruning, J. L., and B. L. Kintz. 1968. Computational handbook of statistics. Scott Foresman Co., Glenview, Ill. 7. Choudhury, H., C. W. Carlson, and G. Semenink. 1971. A study of ochra toxicity in hens. Poult. Sci. 5:1855-1859. 8. Detroy, R. W., E. B. Lillehoj, and A. Ciegler. 1971. Afla and related compounds, p. 3-178. In A. Ciegler, S. Kadis, and S. J. Ajl (ed.), Microbial s: a comprehensive treatise, Fungal s, vol. 6. Academic Press Inc., New York. 9. Forgacs, J. 1965. Stachybotryotoxicosis and moldy corn toxicosis, p. 87-14. In G. N. Wogan (ed.), Mycos in foodstuffs. Massachusetts Institute of Technology Press, Cambridge, Mass. 1. Forgacs, J., and W. T. Carll. 1962. Mycotoxicoses. Adv. Vet. Sci. 7:273-282. 11. Garlich, J. D., Hsi-Tang Tung, and P. B. Hamilton. 1973. The effects of short-term feeding of afla on egg production and some plasma constituents of the APPL. ENVIRON. MICROBIOL. laying hen. Poult. Sci. 52:226-2211. 12. Hamilton, P. B. 1971. A natural and extremely severe occurrence of aflatoxicosis in laying hens. Poult. Sci. 5:188-1882. 13. Hamilton, P. B., and J. D. Garlich. 1971. Afla as a possible cause of fatty liver syndrome in laying hens. Poult. Sci. 5:8-84. 14. Jacobson, W. C., and H. G. Wiseman. 1974. The transmission of afla B, into eggs. Poult. Sci. 53:1743-1745. 15. Kratzer, F. H., D. Bandy, M. Wiley, and A. N. Booth. 1969. Afla effects in poultry. Proc. Soc. Exp. Biol. Med. 131:1281-1284. 16. Nabney, J., and B. F. Nesbitt. 1965. A spectrophotometric method of determining the aflas. Analyst 9:155-16. 17. Sawhney, D. S., D. V. Vadehra, and R. C. Baker. 1973. Aflatoxicosis in the laying Japanese quail (Coturnix coturnix japonica). Poult. Sci. 52:465473. 18. Shotwell,. L., C. W. Hesseltine, R. D. Stubblefield, and W. G. Sorrenson. 1966. Production ofafla on rice. Appl. Microbiol. 14:425428. 19. Siller, W. G., and D. L. Ostler. 1961. The histopathology of an enterohepatic syndrome of turkey poults. Vet. Rec. 73:134-138. 2. Smith, J. W., and P. B. Hamilton. 197. Aflatoxicosis in the broiler chicken. Poult. Sci. 49:27-215. 21. West, S., R. D. Wyatt, and P. B. Hamilton. 1973. Improved yield of afla by incremental increases of temperature. Appl. Microbiol. 25:118-119. 22. Wiseman, H. G., W. C. Jacobson, and W. C. Harmeyer. 1967. Note on removal of pigments from chloroform extracts of afla cultures with copper carbonate. J. Assoc. Off. Agric. Chem. 5:982-983. Downloaded from http://aem.asm.org/ on May 15, 218 by guest