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The Effect of Dietary Ethylene Dibromide (EDB) on the Testes of Bulls
The Effect of Dietary Ethylene Dibromide (ED B) on the Testes of Bulls A Preliminary Report D. AMIR, PH.D., and R. VOLCANI, PH.D.
in Israel of large quantities of concentrates which may have been subjected to fumigation treatment with ethylene dibromide (EDB) has raised the question of whether residual quantities of this fumigant in the feed 'might adversely influence reproduction in the animal, as has been found in the case of laying hens. 2 , 3 When EDB, 4 mg.jkg. body weight, was administered orally to bulls on alternate days from calfhood or from the age of 14-16 months, it was found to affect semen properties adversely. The semen, when tested upon weekly collections, was found to have a low density and the motility of the spermatozoa was poor; the latter were abnormal, exhibiting various degrees of degeneration. This effect was shown to be reversible.! In order to extend the observations on the action of EDB on sperm production we considered it necessary to examine histologically the testes of bulls after EDB administration and after recovery from this treatment.
THE CONSUMPTION BY CATTLE
METHODS AND MATERIAL
Three bulls which had received a daily oral dose of 2 mg. EDB jkg. body weight from the age of 4 days (as described elsewhere!) were subjected to monolateral castration when they were 17}~22~ months old. The detrimental effect of the EDB upon their semen was ascertained by regular semen collections via an artificial vagina during a period of 4-6 months before operation. The right testis of 2 bulls and the left one of the third From the Division of Animal Science, The Volcani Institute of Agricultural Research, Rehovot, Israel. Supported by Grant FG-IS-1l7 from the U. S. Department of Agriculture, under P.L. MIO. The authors are indebted to A. Tadmor, D.V.M., for carrying out the surgical procedures, and to A. Shahar, D.V.M., Ph.D., for histological consultations.
144
VOL. 18, No.1, 1961
EbB
145
AND BULL TESTES
were weighed and dissected after removal. For the histologic examinations, samples from the testis, and the caput, corpus, and cauda epididymidis were fixed in Bouin's fluid. The preparations were stained with hematoxylin and eosin. EDB administration was discontinued after the operation and the recovery of normal semen properties was ascertained by examining semen collected regularly (every 7-10 days). In 2 bulls, recovery was complete after 3-4 months. In the third animal the density was lower than normal and the motility of the spermatozoa remained poor for several months, although the spermatozoa themselves, as in the other 2 bulls, showed no abnormalities about 1 month after the discontinuation of the treatment. Seven months after the operation, the bulls were slaughtered and the remaining testis in each animal was examined histologically as described above. Data on the bulls and their testes after castration and slaughter are given in Table 1. RESULTS AND COMMENTS
The histologic picture of the removed testis at castration showed a depopulation in the majority of the seminiferous tubules, the lumens of which were either empty or filled with cell debris (Fig. 1). According to the classification of Welch and Murphree, the predominating degree of denudation of the germinal epithelium was of Type II, III, and IV for the 3 bulls, No. 664, 667, and 692, respectively. The histologic picture of the TABLE 1. Age and Body Weight of Experimental Animals and Weight of the Testes (Including Epididymides) Bull
Age (months)
Body weight (kg.)
Testis weight (gm.)
AT CASTRATION TIME
667 664 692
17.5 22.5 22.2
413 578 450*
2001" 265, 245+
535 620 615
27 It 395+ 209,
AT SLAUGHTER
667 664
692 *Poor body condition.
tRight testis. :::Left testis.
25 30 29
146
AMIR & VOLCANI
FERTILI1'Y
& STERlLI1'Y
second testis, removed when the animals were slaughtered, showed normal and active hlbules in 2 of the bulls (Fig. 2). In the third animal (No. 692), however, the majority of the tubules remained inactive, showing hyalinization of the epithelium. In this bull a marked hyperplasia of the interstitial tissue was also observed in the second testis. The caput and corpus epididymidis at castration were empty of spermatozoa. The connective-tissue cells showed thickening, giving rise to a thick investment around the ductus (Fig. 3). A high pseudostratified epithelium, which protruded into the empty lumen, was observed both in the caput and in the corpus (Fig. 3). After discontinuation of treatment, the lumens of these parts were filled with spermatozoa (Fig. 4) and the epithelial and
2
4
Fig. 1. Cross section of the right testis of Bull 667 after castration. (X 81) Fig. 2. Cross section of the left testis of Bull 667 after slaughter. (X 81) Fig. 3. Cross section of caput epididymis of Bull 664 after castration. (X 81) Fig. 4. Cross section of the caput epididymis of Bull 664 after slaughter. (X 81)
VOL. 18, No.1, 1967
EDB AND BULL TESTES
147
connective tissue appeared normal in 2 bulls. In the third bull (No. 692), no such pronounced changes were observed on discontinuation of treatment. Spermatozoa were always found in the cauda epididymidis, but their density was higher in the samples taken after slaughter. The connective tissue surrounding the ductus was thicker in this part, as in the other parts of the epididymis, after castration than after slaughter. These differences were less apparent in Bull 692. At castration this bull was in rather poor condition, as manifested by low body weight in comparison with that of the other animals. Whereas in the other 2 animals the increase in weight of the testis remaining after castration was relatively greater than the total increase in body weight, the right testis of Bull 692 (which remained after castration) decreased in weight, although the total gain in body weight of this animal was the largest (Table 1). It is not clear if the EDB alone contributed to the pronounced and irreversible phenomena observed in this case, or whether some other, unknown factor was involved. The seminal changes described elsewhere! and the present histologic changes noted in the testes of the bulls after EDB administration since calfhood, can be compared with the changes occurring in human testes after starvation.4 These changes included obligospermia and degeneration of spermatozoa, "the remains of which, together with epithelial and Sertoli cells, filled the tubules with debris, whereas the walls of the tubules were thickened with connective tissue."4 The effect of EDB on the testes may be directly due to the higher concentration of bromine in this organ after treatment, 6 either by blocking normal nourishment of the reproductive tissue, or by its direct contact with the reproductive cells. The effect may be indirect-i.e., due to interference in the output of gonadotropins by the hypophysis; such an effect has been observed in rats made sterile by underfeeding, "which can both mate and successfully impregnate normal females, when injected with chorionic gonadotropin."5 Further work is to be carried out with a greater number of animals to study the effect of EDB on the testes of bulls, and its reversibility. The Volcani Institute of Agricultural Research Reho1)oth, P.D.B. 15 Israel
REFERENCES 1. AMIR, D., and VOLCANI, R. The effect of dietary ethylene dibromide (EDB) on bull semen. Nature 206:99, 1965. 2. BONDI, A., OLOMUCKI, E., and CALDERON, M. Problems connected with ethylene dibromide fumigation of cereals. II. Feeding experiments with laying hens. 1 Sci Food Agric 6:600, 1955.
148
AMIR & VOLCANI
FERTILITY & STERILITY
3. FULLER, H. L., and MORRIS, G. K. A study of the effect of ethylene dibromide fumigant components on egg production. Poultry Sci 41 :645, 1962. 4. KEYS, A., BROZEK, J., HENCHEL, A., MICHELSON, 0., and TAYLOR, H. L. "Sexual Function." In The Biology of Human Starvation. Univ. Minnesota Press, Minneapolis, 1950, p. 749. 5. MULINOS, M. G., and POMERANTZ, L. The reproductive organs in malnutrition: effect of chorionic gonadotropin upon atrophic genitalia of underfed male rats. Endocrinology 29:267, 1941. 6. VOLCANI, R, AMIR, D., and TAMIR, M. Effect of ethylene dibromide fumigated feed on ruminants. Annual Report to USDA, Research Grant FG-IS-1l7 (mimeo.), 1964, p. 2l. 7. WELCH, P. R, and MURPHREE, R L. Sperm production by chronically irradiated bulls. I Animal Sci 4: 1045, 1965.
in Israel of large quantities of concentrates which may have been subjected to fumigation treatment with ethylene dibromide (EDB) has raised the question of whether residual quantities of this fumigant in the feed 'might adversely influence reproduction in the animal, as has been found in the case of laying hens. 2 , 3 When EDB, 4 mg.jkg. body weight, was administered orally to bulls on alternate days from calfhood or from the age of 14-16 months, it was found to affect semen properties adversely. The semen, when tested upon weekly collections, was found to have a low density and the motility of the spermatozoa was poor; the latter were abnormal, exhibiting various degrees of degeneration. This effect was shown to be reversible.! In order to extend the observations on the action of EDB on sperm production we considered it necessary to examine histologically the testes of bulls after EDB administration and after recovery from this treatment.
THE CONSUMPTION BY CATTLE
METHODS AND MATERIAL
Three bulls which had received a daily oral dose of 2 mg. EDB jkg. body weight from the age of 4 days (as described elsewhere!) were subjected to monolateral castration when they were 17}~22~ months old. The detrimental effect of the EDB upon their semen was ascertained by regular semen collections via an artificial vagina during a period of 4-6 months before operation. The right testis of 2 bulls and the left one of the third From the Division of Animal Science, The Volcani Institute of Agricultural Research, Rehovot, Israel. Supported by Grant FG-IS-1l7 from the U. S. Department of Agriculture, under P.L. MIO. The authors are indebted to A. Tadmor, D.V.M., for carrying out the surgical procedures, and to A. Shahar, D.V.M., Ph.D., for histological consultations.
144
VOL. 18, No.1, 1961
EbB
145
AND BULL TESTES
were weighed and dissected after removal. For the histologic examinations, samples from the testis, and the caput, corpus, and cauda epididymidis were fixed in Bouin's fluid. The preparations were stained with hematoxylin and eosin. EDB administration was discontinued after the operation and the recovery of normal semen properties was ascertained by examining semen collected regularly (every 7-10 days). In 2 bulls, recovery was complete after 3-4 months. In the third animal the density was lower than normal and the motility of the spermatozoa remained poor for several months, although the spermatozoa themselves, as in the other 2 bulls, showed no abnormalities about 1 month after the discontinuation of the treatment. Seven months after the operation, the bulls were slaughtered and the remaining testis in each animal was examined histologically as described above. Data on the bulls and their testes after castration and slaughter are given in Table 1. RESULTS AND COMMENTS
The histologic picture of the removed testis at castration showed a depopulation in the majority of the seminiferous tubules, the lumens of which were either empty or filled with cell debris (Fig. 1). According to the classification of Welch and Murphree, the predominating degree of denudation of the germinal epithelium was of Type II, III, and IV for the 3 bulls, No. 664, 667, and 692, respectively. The histologic picture of the TABLE 1. Age and Body Weight of Experimental Animals and Weight of the Testes (Including Epididymides) Bull
Age (months)
Body weight (kg.)
Testis weight (gm.)
AT CASTRATION TIME
667 664 692
17.5 22.5 22.2
413 578 450*
2001" 265, 245+
535 620 615
27 It 395+ 209,
AT SLAUGHTER
667 664
692 *Poor body condition.
tRight testis. :::Left testis.
25 30 29
146
AMIR & VOLCANI
FERTILI1'Y
& STERlLI1'Y
second testis, removed when the animals were slaughtered, showed normal and active hlbules in 2 of the bulls (Fig. 2). In the third animal (No. 692), however, the majority of the tubules remained inactive, showing hyalinization of the epithelium. In this bull a marked hyperplasia of the interstitial tissue was also observed in the second testis. The caput and corpus epididymidis at castration were empty of spermatozoa. The connective-tissue cells showed thickening, giving rise to a thick investment around the ductus (Fig. 3). A high pseudostratified epithelium, which protruded into the empty lumen, was observed both in the caput and in the corpus (Fig. 3). After discontinuation of treatment, the lumens of these parts were filled with spermatozoa (Fig. 4) and the epithelial and
2
4
Fig. 1. Cross section of the right testis of Bull 667 after castration. (X 81) Fig. 2. Cross section of the left testis of Bull 667 after slaughter. (X 81) Fig. 3. Cross section of caput epididymis of Bull 664 after castration. (X 81) Fig. 4. Cross section of the caput epididymis of Bull 664 after slaughter. (X 81)
VOL. 18, No.1, 1967
EDB AND BULL TESTES
147
connective tissue appeared normal in 2 bulls. In the third bull (No. 692), no such pronounced changes were observed on discontinuation of treatment. Spermatozoa were always found in the cauda epididymidis, but their density was higher in the samples taken after slaughter. The connective tissue surrounding the ductus was thicker in this part, as in the other parts of the epididymis, after castration than after slaughter. These differences were less apparent in Bull 692. At castration this bull was in rather poor condition, as manifested by low body weight in comparison with that of the other animals. Whereas in the other 2 animals the increase in weight of the testis remaining after castration was relatively greater than the total increase in body weight, the right testis of Bull 692 (which remained after castration) decreased in weight, although the total gain in body weight of this animal was the largest (Table 1). It is not clear if the EDB alone contributed to the pronounced and irreversible phenomena observed in this case, or whether some other, unknown factor was involved. The seminal changes described elsewhere! and the present histologic changes noted in the testes of the bulls after EDB administration since calfhood, can be compared with the changes occurring in human testes after starvation.4 These changes included obligospermia and degeneration of spermatozoa, "the remains of which, together with epithelial and Sertoli cells, filled the tubules with debris, whereas the walls of the tubules were thickened with connective tissue."4 The effect of EDB on the testes may be directly due to the higher concentration of bromine in this organ after treatment, 6 either by blocking normal nourishment of the reproductive tissue, or by its direct contact with the reproductive cells. The effect may be indirect-i.e., due to interference in the output of gonadotropins by the hypophysis; such an effect has been observed in rats made sterile by underfeeding, "which can both mate and successfully impregnate normal females, when injected with chorionic gonadotropin."5 Further work is to be carried out with a greater number of animals to study the effect of EDB on the testes of bulls, and its reversibility. The Volcani Institute of Agricultural Research Reho1)oth, P.D.B. 15 Israel
REFERENCES 1. AMIR, D., and VOLCANI, R. The effect of dietary ethylene dibromide (EDB) on bull semen. Nature 206:99, 1965. 2. BONDI, A., OLOMUCKI, E., and CALDERON, M. Problems connected with ethylene dibromide fumigation of cereals. II. Feeding experiments with laying hens. 1 Sci Food Agric 6:600, 1955.
148
AMIR & VOLCANI
FERTILITY & STERILITY
3. FULLER, H. L., and MORRIS, G. K. A study of the effect of ethylene dibromide fumigant components on egg production. Poultry Sci 41 :645, 1962. 4. KEYS, A., BROZEK, J., HENCHEL, A., MICHELSON, 0., and TAYLOR, H. L. "Sexual Function." In The Biology of Human Starvation. Univ. Minnesota Press, Minneapolis, 1950, p. 749. 5. MULINOS, M. G., and POMERANTZ, L. The reproductive organs in malnutrition: effect of chorionic gonadotropin upon atrophic genitalia of underfed male rats. Endocrinology 29:267, 1941. 6. VOLCANI, R, AMIR, D., and TAMIR, M. Effect of ethylene dibromide fumigated feed on ruminants. Annual Report to USDA, Research Grant FG-IS-1l7 (mimeo.), 1964, p. 2l. 7. WELCH, P. R, and MURPHREE, R L. Sperm production by chronically irradiated bulls. I Animal Sci 4: 1045, 1965.