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The Effect of Dietary Aflatoxin on Semen Characteristics of Mature Broiler Breeder Males1,2,3
2115
O R O O I L FOR BROILERS
Vogtmann, H., 1971. Der Einfluss von Art und Meng des Futterfettes auf die Verwertung von Canthaxanthin sowie der Vitamine A und E durch die Legehenne. Thesis no. 4549, ETHZ, Switzerland. Vogtmann, H., D. R. Clandinin and R. T. Hardin, 1973a. Utilization of rapeseed oils of high and low erucic acid contents. 1. Digestibility and energy utilization. Nutr. Metabol. 15: 252-266. Vogtmann, H., D. R. Clandinin and R. T. Hardin,
1973b. Utilization of rapeseed oils of high and low erucic acid contents. 2. Influence on tissues. Nutr. Metabol., submitted for publication. Vuilleumier, J. P., H. P. Probst und G. Brubacher, 1967. In Handbuch der Lebensmittelchemie Bd. II, 2. Teil, Vitamine, Provitamine und Carotinoide, p. 669-876. Winer, B. G., 1971. Statistical Principles in Experimental Design. McGraw-Hill, New York.
D . M . BRIGGS, R. D . W Y A T T AND P . B . HAMILTON
Department of Poultry Science and Department of Microbiology, North Carolina State Raleigh, North Carolina 27607
University,
(Received for-publication March 1, 1974)
ABSTRACT Aflatoxin (20 p.p.m.) incorporated into the feed of mature broiler breeder males for four weeks caused clinical aflatoxicosis as evidence by a significant (P < 0.05) decrease in body weight. Neither spermatozoa counts nor semen volume were altered significantly by the dietary aflatoxin. Chemical analyses of the semen for deoxyribonucleic acidfDNA), ribonucleic acid (RNA), and protein content revealed no significant alterations in these parameters associated with the treatment. Product moment correlations showed significant (P < 0.01) correlations between sperm count, DNA, RNA, and protein but not between these parameters and semen volume. It seems reasonable to conclude that aflatoxin does not affect significantly the semen characteristics of mature broiler breeder males. The refractoriness of sperm and associated nucleic acid and protein synthesis to aflatoxin would appear to be a divergence from the generally accepted mode of action based on the general inhibition of RNA polymerase. POULTRY SCIENCE 53: 2115-2119, 1974
INTRODUCTION
A
FLATOXIN is a mycotoxin produced in feedstuffs by the flavus-parasiticus group of the genus Aspergillus. It has been demonstrated to cause increased condemnations, poor feed conversion, decreased growth rate, and increased mortality in broiler 'Paper Number 4273 of the Journal Series of North Carolina State University Agriculture Experiment Station, Raleigh, North Carolina. 2 A preliminary report of part of this paper was given at the 52nd Annual Meeting of the Poultry Science Association, Brookings, South Dakota, August, 1973. 3 The use of trade names in this publication does not imply endorsement by the North Carolina Experiment Station of the product named, nor criticism of similar ones not mentioned.
chickens (Smith and Hamilton, 1970). An increased susceptibility to bruising (Tung et al., 1971), interference with lipid metabolism (Tung et al, 1972a; Donaldson et al., 1972) and interactions with infectious agents and other stresses (Hamilton and Harris, 1971) are further examples of how aflatoxin causes severe economic losses to the broiler industry. The primary manifestation of aflatoxicosis in the adult female chicken is a condition apparently identical to fatty liver syndrome (Hamilton and Garlich, 1971, 1972) in which aflatoxin causes a dose-related decrease in egg production. The effect of aflatoxin in the mature male has received little attention, except for a general survey of the physiolo-
Downloaded from http://ps.oxfordjournals.org/ at University of Georgia on May 25, 2015
The Effect of Dietary Aflatoxin on Semen Characteristics of Mature Broiler Breeder Males1-23
2116
D. M. BRIGGS, R. D. WYATT AND P. B. HAMILTON
gical effects of aflatoxinon the mature broiler breeder male by Wyatt et al. (1973). The purpose of this study was to characterize the influence of aflatoxin on the reproductive function of males by conducting a physiological survey of semen traits of mature broiler breeder males fed aflatoxin. MATERIALS AND METHODS
RESULTS The effect on body weight of feeding aflatoxin (20 p.p.m.) to mature broiler breeder males is shown in Fig. 1. There was a statistically significant (P < 0.01) reduction in body
•105r
"t
^ 95-
O CONTROL
D 20 p.p.m. flflatoxln
^ ^ ~ ^ \
e
*
^V
I 90LU
^ ^
>S 85'
'
'
'
'
SO
1
2
3
f
TIME (weeks) FIG. 1. The effect of feeding aflatoxin (20 p.p.m.) on the body weight of mature broiler breeder males.
Downloaded from http://ps.oxfordjournals.org/ at University of Georgia on May 25, 2015
Adult broiler roosters (Vantress) were obtained from a local breeder flock that was being phased out of production. Twenty roosters were housed individually in 1.2 x 1.8 m. floor pens three weeks prior to the experiment. Feed and water were available ad libitum at all times. No auxiliary light was used. At the start of the four week experimental period, ten roosters received commercial broiler ration containing 20 (jug. of aflatoxin per gram of diet. The remaining ten roosters received the commercial broiler ration without added aflatoxin. The aflatoxin was produced by growing A. parasiticus NRRL 2999 on rice (Shotwell et al., 1966) in flasks as described by Smith and Hamilton (1969). The moldy rice was steamed to kill the fungus, dried, ground to a fine powder, and analyzed by the spectrophotometric method of Nabney and Nesbitt (1965) as modified by Wiseman et al. (1967). Roosters were weighed at the start of the experiment and once a week during the four week study. At the end of the experiment, each male was killed and the testes removed for weighing. After weighing, the testes were fixed in 10% neutral buffered formalin and processed for histological examination. Semen was collected twice per week by a single operator using a massage technique similar to that described by Burrows and Quinn (1937). Semen was collected directly into graduated 15 ml. centrifuge tubes. A 5 JJUI. sample was removed for dilutions and the centrifuge tube was put in an ice bath for five minutes before determining the ejac-
ulate volume. The semen was diluted (1:1000) immediately in 0.85% NaCl at the farm for spermatozoa counts which were done later in the laboratory using a hemocytometer. The whole semen samples were carried in the ice bath to the laboratory where the total semen deoxyribonucleic acid (DNA), ribonucleic acid (RNA), and protein were measured. The nucleic acid determinations were done by using the procedures of Dische (1955). Semen total protein were determined according to Layne (1957). The data for each week were subjected to a nested analysis of variance in which significance was based on an F-ratio. In addition, an interclass correlation over the entire experiment was done (Snedecor and Cochran, 1967).
2117
AFLATOXTN AND SEMEN CHARACTERISTICS
TABLE 2.—Effect of dietary aflatoxin on the semen volume of mature broiler breeder males
TABLE 1.—Effect of dietary aflatoxin on spermatozoa counts of mature broiler breeder males Time (weeks) 0 1 2 3 4
Time (weeks)
Aflatoxin (p.p.m.) 0
20
5.3" 5.0 4.2 4.1 4.2
5.3 4.6 4.1 3.8 3.8
0 1 2 3 4
Aflatoxin (p.p.m.) 0
20
0.45" 0.50 0.48 0.51 0.48
0.80 0.78 0.94 0.81 0.82
"Each value represents the mean semen volume (ml.) of 10 birds. Values within a column do not differ significantly but an analysis of variance showed a significant (P < 0.05) difference between columns.
"Each value is the mean spermatozoa count (x 106/mm.3)for ten birds. An analysis of variance revealed no significant (P = 0.16) treatment effect.
Time (weeks) 0 1 2 3 4
DNA (jig. M O Control Toxin 9.17" 9.92 9.05 9.04 7.25 6.69 10.15 9.04 12.18 9.39
RNA (fig./Ml.) Toxin
Control 1.31 1.62 1.77 0.64
1.16 1.54 1.59 0.67
—
—
Protein (g. /100 ml.) Control
Toxin
4.30 3.55 3.60 3.26 2.70
3.98 3.78 2.72 2.25 2.06
"Each value in the table represents the mean of 10 birds. The toxin treated birds received 20 p.p.m. of dietary aflatoxin.
weights of treated males after one week. The effect of aflatoxin on the sperm count of mature broiler breeder males is shown in Table 1. There was no significant (P = 0.16) difference between males receiving the toxin and controls at any time during the experiment. Table 2 shows the effect of dietary aflatoxin on the semen volume of males. The semen volume was significantly (P < 0.01) greater in the treated males than in the control males at all time intervals. However, this effect was not treatment related since the volumes were unaltered from the pretreatment volume. This difference was the result of deliberate selection of high volume males for the treatment group so that if the volume were decreased by aflatoxin, enough semen would still be available for the other measurements. This selection was justified because there is a low correlation between volume and fertility (Lake, 1962).
TABLE 4.—Product moment correlations of the physiological parameters measured Sperm/mm.3 DNA RNA Protein DNA !62* RNA .50* .33* Protein .53* .45* .50* Volume -.09 -.10 -.01 -.13 *These correlations were significant at P < 0.01.
The DNA, RNA, and protein contents of semen are presented in Table 3. There were no statistically significant differences between the treatments for any of the measures at any time throughout the experiment. Table 4 gives the product moment correlations between all semen parameters surveyed in this experiment. Volume was not correlated with any other measure and thus offers internal evidence that the selection of high-volume males for the aflatoxin treatment was valid. The largest correlation (significant at P <
Downloaded from http://ps.oxfordjournals.org/ at University of Georgia on May 25, 2015
TABLE 3.—The effect of dietary aflatoxin on the DNA, RNA and total protein content of the semen of mature broiler breeder males
2118
D. M. BRIGGS, R. D. WYATT AND P. B. HAMILTON
0.01) was between the DNA content of semen and sperm count. The correlation of DNA and RNA with each other was not as great as these measures with the other traits. Figure 2 shows histological sections of testes from control males and from those receiving toxin. There were no discernible differences in the structure or spermatogenic development. Likewise, testicle weights revealed no difference. At the termination of the experiment the relative testes weight was 0.782 gm./lOO gm. body weight for both control and toxin fed males. DISCUSSION Broiler breeder males receiving 20 p.p.m. of dietary aflatoxin did not exhibit reduced reproductive preformance as measured by
sperm count, DNA, RNA, protein content or testes size. This level of aflatoxin is sufficient to produce clinical aflatoxicosis in broiler breeder males in the laboratory (Wyatt et al., 1973). Since the time interval required for a primary spermatocyte to mature and appear in ejaculated semen has been reported (Lake, 1971) to be around 15 to 16 days, any effect of aflatoxin on semen characteristics should have been apparent by the third and fourth week of the study. It seems reasonable to conclude that aflatoxin does not appreciably affect biochemical characteristics of semen of mature broiler breeder males as judged by the parameters measured in this experiment. It has been generally accepted that aflatoxin exerts its primary effect by a general
Downloaded from http://ps.oxfordjournals.org/ at University of Georgia on May 25, 2015
FIG. 2. Photomicrographs of histological sections of testis of control bird (A) and of testis from bird receiving 20 p.p.m. of dietary aflatoxin (B).
AFLATOXTN AND SEMEN CHARACTERISTICS
REFERENCES Burrows, W. H., and J. P. Quinn, 1937. The collection of spermatozoa from the domestic fowl and turkey. Poultry Sci. 16: 19-24. Clifford, J. I., and K. R. Rees, 1967. The action of aflatoxin B, on the rat liver. Biochem. J. 102: 65-75. Dische, Z., 1955. Color reactions of nucleic acid components. In: The Nucleic Acids, E. Chargaff and J. M. Davidson, eds., Academic Press, New York, Vol. 1, pp. 270-284. Donaldson, W. E., H. T. Tung and P. B. Hamilton, 1972. Depression of fatty acid synthesis in chicken liver (Gallus domesticus) by aflatoxin. Comp. Biochem. Physiol. 41B: 843-847. Hamilton, P. B., and J. R. Harris, 1971. Interaction of aflatoxicosis with Candida albicans infections and other stresses in chickens. Poultry Sci. 50: 906-912. Hamilton, P. B., and J. D. Garlich, 1971. Aflatoxin as a possible cause of fatty liver syndrome in laying hens. Poultry Sci. 50: 800-804.
Hamilton, P. B., and J. D. Garlich, 1972. Failure of vitamin supplementation to alter fatty liver syndrome caused by aflatoxin. Poultry Sci. 51:688-692. Lafarge, C , and C. Frayssinet, 1970. The reversibility of RNA and DNA synthesis induced by aflatoxin in rat liver. A tentative explanation for carcinogenic mechanism. Int. J. Cancer, 6: 74-83. Lake, P. E., 1962. Artificial insemination in poultry. In: The Semen of Animals and Artificial Insemination (J. P. Maule, ed.), Commonwealth Agricultural Bureaux, Farnham Royal, Bucks, England, Chapt. 19. Lake, P. E., 1971. The male in reproduction. In: Physiology and Biochemistry of the Domestic Fowl (D. J. Bell and B. M. Freeman, eds.), Academic Press, New York, Vol. 3., pp. 1411-1447. Layne, E., 1957. Spectrophotometric and turbidimetric methods for measuring proteins. In: Methods in Enzymology (S. P. Colowick and N. D. Kaplan, eds.), Academic 'Press, New York, Vol. 3., pp. 447-454. Nabney, J., and B. F. Nesbitt, 1965. A spectrophotometric method of determining the aflatoxins. Analyst, 90: 155-160. Pong, R. S., and G. N. Wogan, 1969. Time course alterations of rat liver polysome-profiles induced by aflatoxin B,. Biochem. Pharmacol. 18: 23572361. Shotwell, O. L., C. W. Hesseltine, R. D. Stubblefield and W. G. Sorenson, 1966. Production of aflatoxin on rice. Appl. Microbiol. 14: 425-428. Smith, J. W., and P. B. Hamilton, 1969. Technique for the aseptic addition of liquid to flask cultures. Appl. Microbiol. 17: 317. Smith, J. W., and P. B. Hamilton, 1970. Aflatoxicosis in the broiler chicken. Poultry Sci. 49: 207-215. Snedecor, G. W., and W. G. Cochran, 1967. Statistical Methods. Iowa State University Press, Ames, Iowa. Tung, H. T., J. W. Smith and P. B. Hamilton, 1971. Aflatoxicosis and bruising in the chicken. Poultry Sci. 50: 795-800. Tung, H. T., W. E. Donaldson and P. B. Hamilton, 1972a. Altered lipid transport during aflatoxicosis. Toxicol. Appl. Pharmacol. 22: 97-104. Tung, H. T., R. D. Wyatt, F. W. Cook and P. B. Hamilton, 1972b. Changes in the hemotopoietic system during aflatoxicosis. Abst. Am. Meet. Amer. Soc. Microbiol. 72: 27. Wiseman, H. G., W. C. Jacobson and W. C. Harmeyer, 1967. Note on removal of pigments from chloroform extracts of aflatoxin cultures with copper carbonate. J. Assoc. Off. Agr. Chem. 50: 982-983. Wyatt, R. D., D. M. Briggs and P. B. Hamilton, 1973. The effect of dietary aflatoxin on mature broiler breeder males. Poultry Sci. 52: 1119-1123.
Downloaded from http://ps.oxfordjournals.org/ at University of Georgia on May 25, 2015
inhibition of DNA dependent RNA polymerase (Clifford and Rees, 1967; Pong and Wogan, 1969; Lafarge and Frayssinet, 1970). If this mechanism were operating in the chicken, decreased production of sperm, DNA, RNA and protein would be expected. Nevertheless, significant decreases in these parameters were not observed even though the males experienced a clinical aflatoxicosis severe enough to cause a significant body weight loss. This represents yet another physiological system in chickens which does not appear to function as expected based on the contention that aflatoxin causes a general inhibition of RNA and consequentiy protein synthesis. Tung et al. (1972a) found that lipid transport from the liver of chickens was more sensitive to aflatoxin than was the RNA content of liver. Tung et al. (1972b) reported that both DNA and RNA syntheses were stimulated in the bone marrow of chickens with aflatoxicosis severe enough to cause retarded growth. It would appear from these results that the generally accepted mechanism for the action of aflatoxin does not apply in chickens for needs modification to allow for the observed exceptions and specificities.
2119
O R O O I L FOR BROILERS
Vogtmann, H., 1971. Der Einfluss von Art und Meng des Futterfettes auf die Verwertung von Canthaxanthin sowie der Vitamine A und E durch die Legehenne. Thesis no. 4549, ETHZ, Switzerland. Vogtmann, H., D. R. Clandinin and R. T. Hardin, 1973a. Utilization of rapeseed oils of high and low erucic acid contents. 1. Digestibility and energy utilization. Nutr. Metabol. 15: 252-266. Vogtmann, H., D. R. Clandinin and R. T. Hardin,
1973b. Utilization of rapeseed oils of high and low erucic acid contents. 2. Influence on tissues. Nutr. Metabol., submitted for publication. Vuilleumier, J. P., H. P. Probst und G. Brubacher, 1967. In Handbuch der Lebensmittelchemie Bd. II, 2. Teil, Vitamine, Provitamine und Carotinoide, p. 669-876. Winer, B. G., 1971. Statistical Principles in Experimental Design. McGraw-Hill, New York.
D . M . BRIGGS, R. D . W Y A T T AND P . B . HAMILTON
Department of Poultry Science and Department of Microbiology, North Carolina State Raleigh, North Carolina 27607
University,
(Received for-publication March 1, 1974)
ABSTRACT Aflatoxin (20 p.p.m.) incorporated into the feed of mature broiler breeder males for four weeks caused clinical aflatoxicosis as evidence by a significant (P < 0.05) decrease in body weight. Neither spermatozoa counts nor semen volume were altered significantly by the dietary aflatoxin. Chemical analyses of the semen for deoxyribonucleic acidfDNA), ribonucleic acid (RNA), and protein content revealed no significant alterations in these parameters associated with the treatment. Product moment correlations showed significant (P < 0.01) correlations between sperm count, DNA, RNA, and protein but not between these parameters and semen volume. It seems reasonable to conclude that aflatoxin does not affect significantly the semen characteristics of mature broiler breeder males. The refractoriness of sperm and associated nucleic acid and protein synthesis to aflatoxin would appear to be a divergence from the generally accepted mode of action based on the general inhibition of RNA polymerase. POULTRY SCIENCE 53: 2115-2119, 1974
INTRODUCTION
A
FLATOXIN is a mycotoxin produced in feedstuffs by the flavus-parasiticus group of the genus Aspergillus. It has been demonstrated to cause increased condemnations, poor feed conversion, decreased growth rate, and increased mortality in broiler 'Paper Number 4273 of the Journal Series of North Carolina State University Agriculture Experiment Station, Raleigh, North Carolina. 2 A preliminary report of part of this paper was given at the 52nd Annual Meeting of the Poultry Science Association, Brookings, South Dakota, August, 1973. 3 The use of trade names in this publication does not imply endorsement by the North Carolina Experiment Station of the product named, nor criticism of similar ones not mentioned.
chickens (Smith and Hamilton, 1970). An increased susceptibility to bruising (Tung et al., 1971), interference with lipid metabolism (Tung et al, 1972a; Donaldson et al., 1972) and interactions with infectious agents and other stresses (Hamilton and Harris, 1971) are further examples of how aflatoxin causes severe economic losses to the broiler industry. The primary manifestation of aflatoxicosis in the adult female chicken is a condition apparently identical to fatty liver syndrome (Hamilton and Garlich, 1971, 1972) in which aflatoxin causes a dose-related decrease in egg production. The effect of aflatoxin in the mature male has received little attention, except for a general survey of the physiolo-
Downloaded from http://ps.oxfordjournals.org/ at University of Georgia on May 25, 2015
The Effect of Dietary Aflatoxin on Semen Characteristics of Mature Broiler Breeder Males1-23
2116
D. M. BRIGGS, R. D. WYATT AND P. B. HAMILTON
gical effects of aflatoxinon the mature broiler breeder male by Wyatt et al. (1973). The purpose of this study was to characterize the influence of aflatoxin on the reproductive function of males by conducting a physiological survey of semen traits of mature broiler breeder males fed aflatoxin. MATERIALS AND METHODS
RESULTS The effect on body weight of feeding aflatoxin (20 p.p.m.) to mature broiler breeder males is shown in Fig. 1. There was a statistically significant (P < 0.01) reduction in body
•105r
"t
^ 95-
O CONTROL
D 20 p.p.m. flflatoxln
^ ^ ~ ^ \
e
*
^V
I 90LU
^ ^
>S 85'
'
'
'
'
SO
1
2
3
f
TIME (weeks) FIG. 1. The effect of feeding aflatoxin (20 p.p.m.) on the body weight of mature broiler breeder males.
Downloaded from http://ps.oxfordjournals.org/ at University of Georgia on May 25, 2015
Adult broiler roosters (Vantress) were obtained from a local breeder flock that was being phased out of production. Twenty roosters were housed individually in 1.2 x 1.8 m. floor pens three weeks prior to the experiment. Feed and water were available ad libitum at all times. No auxiliary light was used. At the start of the four week experimental period, ten roosters received commercial broiler ration containing 20 (jug. of aflatoxin per gram of diet. The remaining ten roosters received the commercial broiler ration without added aflatoxin. The aflatoxin was produced by growing A. parasiticus NRRL 2999 on rice (Shotwell et al., 1966) in flasks as described by Smith and Hamilton (1969). The moldy rice was steamed to kill the fungus, dried, ground to a fine powder, and analyzed by the spectrophotometric method of Nabney and Nesbitt (1965) as modified by Wiseman et al. (1967). Roosters were weighed at the start of the experiment and once a week during the four week study. At the end of the experiment, each male was killed and the testes removed for weighing. After weighing, the testes were fixed in 10% neutral buffered formalin and processed for histological examination. Semen was collected twice per week by a single operator using a massage technique similar to that described by Burrows and Quinn (1937). Semen was collected directly into graduated 15 ml. centrifuge tubes. A 5 JJUI. sample was removed for dilutions and the centrifuge tube was put in an ice bath for five minutes before determining the ejac-
ulate volume. The semen was diluted (1:1000) immediately in 0.85% NaCl at the farm for spermatozoa counts which were done later in the laboratory using a hemocytometer. The whole semen samples were carried in the ice bath to the laboratory where the total semen deoxyribonucleic acid (DNA), ribonucleic acid (RNA), and protein were measured. The nucleic acid determinations were done by using the procedures of Dische (1955). Semen total protein were determined according to Layne (1957). The data for each week were subjected to a nested analysis of variance in which significance was based on an F-ratio. In addition, an interclass correlation over the entire experiment was done (Snedecor and Cochran, 1967).
2117
AFLATOXTN AND SEMEN CHARACTERISTICS
TABLE 2.—Effect of dietary aflatoxin on the semen volume of mature broiler breeder males
TABLE 1.—Effect of dietary aflatoxin on spermatozoa counts of mature broiler breeder males Time (weeks) 0 1 2 3 4
Time (weeks)
Aflatoxin (p.p.m.) 0
20
5.3" 5.0 4.2 4.1 4.2
5.3 4.6 4.1 3.8 3.8
0 1 2 3 4
Aflatoxin (p.p.m.) 0
20
0.45" 0.50 0.48 0.51 0.48
0.80 0.78 0.94 0.81 0.82
"Each value represents the mean semen volume (ml.) of 10 birds. Values within a column do not differ significantly but an analysis of variance showed a significant (P < 0.05) difference between columns.
"Each value is the mean spermatozoa count (x 106/mm.3)for ten birds. An analysis of variance revealed no significant (P = 0.16) treatment effect.
Time (weeks) 0 1 2 3 4
DNA (jig. M O Control Toxin 9.17" 9.92 9.05 9.04 7.25 6.69 10.15 9.04 12.18 9.39
RNA (fig./Ml.) Toxin
Control 1.31 1.62 1.77 0.64
1.16 1.54 1.59 0.67
—
—
Protein (g. /100 ml.) Control
Toxin
4.30 3.55 3.60 3.26 2.70
3.98 3.78 2.72 2.25 2.06
"Each value in the table represents the mean of 10 birds. The toxin treated birds received 20 p.p.m. of dietary aflatoxin.
weights of treated males after one week. The effect of aflatoxin on the sperm count of mature broiler breeder males is shown in Table 1. There was no significant (P = 0.16) difference between males receiving the toxin and controls at any time during the experiment. Table 2 shows the effect of dietary aflatoxin on the semen volume of males. The semen volume was significantly (P < 0.01) greater in the treated males than in the control males at all time intervals. However, this effect was not treatment related since the volumes were unaltered from the pretreatment volume. This difference was the result of deliberate selection of high volume males for the treatment group so that if the volume were decreased by aflatoxin, enough semen would still be available for the other measurements. This selection was justified because there is a low correlation between volume and fertility (Lake, 1962).
TABLE 4.—Product moment correlations of the physiological parameters measured Sperm/mm.3 DNA RNA Protein DNA !62* RNA .50* .33* Protein .53* .45* .50* Volume -.09 -.10 -.01 -.13 *These correlations were significant at P < 0.01.
The DNA, RNA, and protein contents of semen are presented in Table 3. There were no statistically significant differences between the treatments for any of the measures at any time throughout the experiment. Table 4 gives the product moment correlations between all semen parameters surveyed in this experiment. Volume was not correlated with any other measure and thus offers internal evidence that the selection of high-volume males for the aflatoxin treatment was valid. The largest correlation (significant at P <
Downloaded from http://ps.oxfordjournals.org/ at University of Georgia on May 25, 2015
TABLE 3.—The effect of dietary aflatoxin on the DNA, RNA and total protein content of the semen of mature broiler breeder males
2118
D. M. BRIGGS, R. D. WYATT AND P. B. HAMILTON
0.01) was between the DNA content of semen and sperm count. The correlation of DNA and RNA with each other was not as great as these measures with the other traits. Figure 2 shows histological sections of testes from control males and from those receiving toxin. There were no discernible differences in the structure or spermatogenic development. Likewise, testicle weights revealed no difference. At the termination of the experiment the relative testes weight was 0.782 gm./lOO gm. body weight for both control and toxin fed males. DISCUSSION Broiler breeder males receiving 20 p.p.m. of dietary aflatoxin did not exhibit reduced reproductive preformance as measured by
sperm count, DNA, RNA, protein content or testes size. This level of aflatoxin is sufficient to produce clinical aflatoxicosis in broiler breeder males in the laboratory (Wyatt et al., 1973). Since the time interval required for a primary spermatocyte to mature and appear in ejaculated semen has been reported (Lake, 1971) to be around 15 to 16 days, any effect of aflatoxin on semen characteristics should have been apparent by the third and fourth week of the study. It seems reasonable to conclude that aflatoxin does not appreciably affect biochemical characteristics of semen of mature broiler breeder males as judged by the parameters measured in this experiment. It has been generally accepted that aflatoxin exerts its primary effect by a general
Downloaded from http://ps.oxfordjournals.org/ at University of Georgia on May 25, 2015
FIG. 2. Photomicrographs of histological sections of testis of control bird (A) and of testis from bird receiving 20 p.p.m. of dietary aflatoxin (B).
AFLATOXTN AND SEMEN CHARACTERISTICS
REFERENCES Burrows, W. H., and J. P. Quinn, 1937. The collection of spermatozoa from the domestic fowl and turkey. Poultry Sci. 16: 19-24. Clifford, J. I., and K. R. Rees, 1967. The action of aflatoxin B, on the rat liver. Biochem. J. 102: 65-75. Dische, Z., 1955. Color reactions of nucleic acid components. In: The Nucleic Acids, E. Chargaff and J. M. Davidson, eds., Academic Press, New York, Vol. 1, pp. 270-284. Donaldson, W. E., H. T. Tung and P. B. Hamilton, 1972. Depression of fatty acid synthesis in chicken liver (Gallus domesticus) by aflatoxin. Comp. Biochem. Physiol. 41B: 843-847. Hamilton, P. B., and J. R. Harris, 1971. Interaction of aflatoxicosis with Candida albicans infections and other stresses in chickens. Poultry Sci. 50: 906-912. Hamilton, P. B., and J. D. Garlich, 1971. Aflatoxin as a possible cause of fatty liver syndrome in laying hens. Poultry Sci. 50: 800-804.
Hamilton, P. B., and J. D. Garlich, 1972. Failure of vitamin supplementation to alter fatty liver syndrome caused by aflatoxin. Poultry Sci. 51:688-692. Lafarge, C , and C. Frayssinet, 1970. The reversibility of RNA and DNA synthesis induced by aflatoxin in rat liver. A tentative explanation for carcinogenic mechanism. Int. J. Cancer, 6: 74-83. Lake, P. E., 1962. Artificial insemination in poultry. In: The Semen of Animals and Artificial Insemination (J. P. Maule, ed.), Commonwealth Agricultural Bureaux, Farnham Royal, Bucks, England, Chapt. 19. Lake, P. E., 1971. The male in reproduction. In: Physiology and Biochemistry of the Domestic Fowl (D. J. Bell and B. M. Freeman, eds.), Academic Press, New York, Vol. 3., pp. 1411-1447. Layne, E., 1957. Spectrophotometric and turbidimetric methods for measuring proteins. In: Methods in Enzymology (S. P. Colowick and N. D. Kaplan, eds.), Academic 'Press, New York, Vol. 3., pp. 447-454. Nabney, J., and B. F. Nesbitt, 1965. A spectrophotometric method of determining the aflatoxins. Analyst, 90: 155-160. Pong, R. S., and G. N. Wogan, 1969. Time course alterations of rat liver polysome-profiles induced by aflatoxin B,. Biochem. Pharmacol. 18: 23572361. Shotwell, O. L., C. W. Hesseltine, R. D. Stubblefield and W. G. Sorenson, 1966. Production of aflatoxin on rice. Appl. Microbiol. 14: 425-428. Smith, J. W., and P. B. Hamilton, 1969. Technique for the aseptic addition of liquid to flask cultures. Appl. Microbiol. 17: 317. Smith, J. W., and P. B. Hamilton, 1970. Aflatoxicosis in the broiler chicken. Poultry Sci. 49: 207-215. Snedecor, G. W., and W. G. Cochran, 1967. Statistical Methods. Iowa State University Press, Ames, Iowa. Tung, H. T., J. W. Smith and P. B. Hamilton, 1971. Aflatoxicosis and bruising in the chicken. Poultry Sci. 50: 795-800. Tung, H. T., W. E. Donaldson and P. B. Hamilton, 1972a. Altered lipid transport during aflatoxicosis. Toxicol. Appl. Pharmacol. 22: 97-104. Tung, H. T., R. D. Wyatt, F. W. Cook and P. B. Hamilton, 1972b. Changes in the hemotopoietic system during aflatoxicosis. Abst. Am. Meet. Amer. Soc. Microbiol. 72: 27. Wiseman, H. G., W. C. Jacobson and W. C. Harmeyer, 1967. Note on removal of pigments from chloroform extracts of aflatoxin cultures with copper carbonate. J. Assoc. Off. Agr. Chem. 50: 982-983. Wyatt, R. D., D. M. Briggs and P. B. Hamilton, 1973. The effect of dietary aflatoxin on mature broiler breeder males. Poultry Sci. 52: 1119-1123.
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inhibition of DNA dependent RNA polymerase (Clifford and Rees, 1967; Pong and Wogan, 1969; Lafarge and Frayssinet, 1970). If this mechanism were operating in the chicken, decreased production of sperm, DNA, RNA and protein would be expected. Nevertheless, significant decreases in these parameters were not observed even though the males experienced a clinical aflatoxicosis severe enough to cause a significant body weight loss. This represents yet another physiological system in chickens which does not appear to function as expected based on the contention that aflatoxin causes a general inhibition of RNA and consequentiy protein synthesis. Tung et al. (1972a) found that lipid transport from the liver of chickens was more sensitive to aflatoxin than was the RNA content of liver. Tung et al. (1972b) reported that both DNA and RNA syntheses were stimulated in the bone marrow of chickens with aflatoxicosis severe enough to cause retarded growth. It would appear from these results that the generally accepted mechanism for the action of aflatoxin does not apply in chickens for needs modification to allow for the observed exceptions and specificities.
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