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Toxicological effects of cadmium chloride on the canine testis following various routes of administration
53
Toxicology Letters, 1 (1977) 53-58 o Elsevier/North-Holland Biomedical Press
TOXICOLOGICAL EFFECTS OF CADMIUM CHLORIDE ON THE CANINE TESTIS FOLLOWING VARIOUS ROUTES OF ADMINISTRATION P.A. DONNELLY and D.E. MONTY Jr. Division of Agriculture,
Arizona State University, Tempe, Arizona 85281
(U.S.A.)
(Received May lOth, 1977) (Accepted May 13th, 1977)
SUMMARY
The toxic effects of cadmium chloride on the canine testis were investigated. Various dosages of cadmium chloride were administered subcutaneously, intramuscularly, intravenously and intratesticularly. Single intravenous doses equivalent to 4.0 or 8.0 mg CdClJkg body weight were lethal. The study showed that the canine testis is sensitive to cadmium administered intratesticularly, but relatively insensitive to the subcutaneous, intramuscular and intravenous administration of cadmium. Single intratesticular infusions of 0.2 mg CdClJkg body weight caused testicular inflammation, hemorrhage, massive necrosis of central seminiferous tubules, and disorganization and degeneration of the peripheral seminiferous tubules. Leydig cell degeneration was also prominent. Atrophy of the seminiferous tubules was followed by interstitial revascularization and Leydig cell regeneration. The persistence of disorganized and degenerative seminiferous tubules was observed throughout the post-injection period (24 weeks), while signs of limited seminiferous tubule regeneration and spermatogenesis were present at 24 weeks. Epididymal damage occurred following cadmium infusion into the testes, however, it subsided by the 24th week after administration. In all cadmium-treated dogs evaluated, aspermic ejaculates were collected close to the time of sacrifice.
INTRODUCTION
A single, sublethal dose of cadmium chloride has been shown to selectively damage the testes of a variety of mammalian species. Testicular necrosis has been demonstrated in rats [1,2], mice [ 1,3], rabbits [4], guinea pigs [3], hamsters [ 31, opossums [ 11, goats [4] , dogs [ 51, and monkeys [ 4,6] . A single dose of cadmium chloride evokes testicular atrophy and sterility within 3-7 days after injection [ 5,6] . Cadmium chloride affects testicular blood vessels causing increased vascular permeability, with disruption of the bloodtestis barrier [7]. Destruction of the seminiferous tubules results in sterility.
54
Transitory disorganization and atrophy of the interstitium is followed by revascularization, proliferation of Leydig cells and restoration of androgenic function of the testes [5,6]. Secondary sexual characteristics and behavior are preserved by Leydig cell regeneration. In male rats, sterilized by a single subcutaneous injection of cadmium chloride, sexual behavior, including normal mating and ejaculatory activity, returned 2 months after injection [ES]. Cadmium may prove to be a useful tool in animal population control programs, as it may provide a way to produce sterile breeding males which could be added to a breeding population [5]. The objectives of the present study were two-fold. Preliminary studies were aimed at determining the testicular effects of cadmium chloride following various routes of administration in the dog. Subsequent studies were concerned with long-term ttesticular damage and regeneration. MATERIALS
AND METHODS
The male dogs used in this study were adult animals of mixed breeding between the ages of l-3 years and weighing 20-40 pounds. Preliminary studies involved subcutaneous, intramuscular, intravenous and intratesticular injections of 2 dosage levels of cadmium chloride. The dosages selected were based on previous studies on dogs and other mammals [1,5]. 4 and 8 mg CdClJkg body weight were administered subcutaneously, intramuscularly and intravenously to 6 dogs, and 0.1 and 0.2 mg CdCl,/kg body weight were infused into the testes of 2 dogs. A 0.5% solution of cadmium chloride in normal physiological saline was used for intratesticular infusions, while a 4% solution of cadmium chloride in 96% propylene glycol was injected subcutaneously and intramuscularly. A 4% solution of cadmium chloride in normal physiological saline was injected intravenously into the cephalic vein. At all sites of administration, the volumes injected were adjusted according to body weight and the desired dosage of cadmium chloride. Three control dogs received injections of the cadmium chloride solvents (subcutaneous, intramuscular and intratesticular solvents). Dogs receiving intratesticular injections were anesthetized with sodium thiamylal*. An aseptic technique was employed and each testis received half of the total dosage administered. The solution was uniformly infused throughout the testicular tissue by carefully adjusting the depth and direction of the syringe needle. All experimental and control dogs received 900 000 units of Bicillin** intramuscularly after the injection procedure. The dogs were sacrificed 7 days after the injection. The long-term study group was composed of five dogs injected intratesticularly with 0.2 mg CdClJkg body weight. One dog was sacrificed 3 weeks after injection, another 6 weeks after injection, and another at 9 weeks. Two * Surital, Parke, Davis and Co., Detroit, Michigan. ** Bicillin, Wyeth Laboratories Inc., Philadelphia,
Pa.
55
dogs were euthanized 24 weeks after injection. Two control dogs were injected intratesticularly with normal saline and were sacrificed 17 weeks after injection. The testes of all dogs were removed, fixed in Bouin’s fluid, and tissue sections were stained with hematoxylin-eosin. Testicular changes were evaluated by comparison with normal testicular microstructure. Semen was collected from the long-term study dogs prior to injection of cadmium chloride. Samples were obtained by manipulative ejaculation [9]. Following treatment, attempts to collect semen were made at lo-day intervals. Semen was evaluated for gross motility [lo] , individual sperm motility [lo] , and sperm morphology [ll]. The dogs were observed for symptoms of cadmium toxicity (anemia, bleaching of the incisor teeth, orchitis, diarrhea, and vomiting [ 5,121). The injection sites were examined for signs of local irritation and inflammation, and biopsies were taken. RESULTS
Subcutaneous injections of cadmium chloride did not produce histological changes in the testes, but did cause severe subcutaneous inflammation, edema, hemorrhage and necrosis at the site of injection. Similarly, intramuscular injections of cadmium chloride produced severe local inflammation, with minimal testicular effect. The intramuscular injection of 8.0 mg CdClJkg body weight caused particularly severe local inflammation and necrosis, and several hemorrhagic, necrotic foci were observed in the testes. Intravenous injections of 4.0 and 8.0 mg CdClJkg body weight proved fatal within 48 and 24 h after injection, respectively. Convulsions with uncontrollable vomiting and defecation were observed. At both intravenous dosage levels, some hemorrhagic and necrotic foci were observed within the testes. Intratesticular infusion of 0.1 mg CdClJkg body weight produced necrosis and hemorrhage within the central portion of the testes, however, peripheral tubules appeared normal and healthy. Massive testicular necrosis resulted from the infusion of 0.2 mg CdClJkg body weight, which was particularly extensive in the central portions of the testes. There were large areas of necrosis, hemorrhage, fibrin deposition, and -tissue debris. The peripheral areas of the testes contained a few intact seminiferous tubules, however, the majority were shrunken and disorganized. Multi-nucleated cells and eosinophilic, necrotic debris were commonly noted within the seminiferous tubules. A few tubules in the extreme periphery showed evidence of spermatogenesis, but degenerative changes, including giant cell formation, were also present. Sertoli cells were prominent in many peripheral tubules. Interstitial edema and hemorrhage were prominent. Few Leydig cells were seen, and they were confined to the extreme periphery. Intraepithelial cysts and mononuclear infiltration were observed in the epididymis. The lumen of epididymal tubules was almost completely devoid of sperm. Intratesticular infusion, at both dosage levels, caused scrotal inflammation, however, the scrotum returned to normal size and
56
appearance within 7 days. The testes of the control dogs showed normal testicular structure 7 days after injection. 3 weeks after testicular infusion of 0.2 mg Cd&/kg body weight extensive necrosis and atrophy of the seminiferous tubules was evident. Peritubular fibrosis was noted. In the central areas of the testes many seminiferous tubules were devoid of cellular elements, while others lacked intact membranes. Peripheral tubules showed internal disorganization, multi-nucleated elements, and arrested spermatogenesis. A number of peripheral seminiferous tubules contained only Sertoli cells. Some local areas of Leydig cell proliferation were present in the peripheral regions. Revascularization of the tissue bed was apparent. In the epididymis, there were focal areas of mononuclear infiltration, and intraepithelial cysts were common. There were no spermatozoa evident in the epididymis. Semen was not obtained for evaluation. 6 weeks after testicular infusion of CdCl, extensive fibrosis and atrophy of the seminiferous tubules was observed. Necrotic tubules were also present. Degenerative and disorganized seminiferous tubules were observed in peripheral areas of the testes, and spermatogenesis was arrested. Revascularization was evident throughout the necrotic tissue. Prominent nests of Leydig cells were present throughout the testes. There were no spermatozoa evident in the epididymal lumen, and some intraepithelial cysts were observed. Semen obtained 40 days after injection was devoid of sperm. The testes of the cadmium-treated dog which was sacrificed 9 weeks after treatment contained areas of mature fibrous tissue, with remnants of collapsed seminiferous tubules. Surrounding seminiferous tubules were degenerated, with vacuolization of the epithelial cells. Spermatogenesis was present in a few peripheral tubules. Prominent nests of Leydig cells were observed throughout the testes. Intraepithelial cysts and mononuclear infiltrates were distributed throughout the epididymis. Limited numbers of spermatozoa were present in the lumen of the epididymis. An ejaculate collected 40 days after injection was devoid of sperm. 24 weeks after testicular infusion, areas of vacuolated and atrophied seminiferous tubules were observed in the testes. In some areas, tubules were completely devoid of germinal cells. They were dilated and contained homogeneous material. In other areas, tubules contained only Sertoli cells. Degenerative seminiferous tubules containing giant cells and numerous vacuoles were also observed, and spermatogenesis was arrested. In some areas, seminiferous tubules showed evidence of germinal epithelial repair, but spermatogenesis was not present. A few peripheral seminiferous tubules appeared normal and spermatogenesis was present. Revascularization had progressed to normal distribution. Areas of mature fibrosis were observed. The interstitium contained many healthy Leydig cells. In the epididymis, there were a few spermatozoa, and a few mononuclear cells were noted in the stroma. Aspermic ejaculates were collected from both dogs 166 days after injection. The testes of the 2 long-term control dogs, injected intratesticularly with normal saline, revealed normal testicular microstructure and active spermato-
genesis 17 weeks after injection. Semen was collected from both control dogs 115 days after injection, and the sperm concentration, motility and morphology were similar to the pre-injection semen samples. DISCUSSION
Preliminary studies showed that, with the exception of intratesticular administration, the canine testes is rather insensitive to parenterally administered cadmium. These findings are consistent with previous studies on dogs which reported permanent sterility in male dogs following single intratesticular injections of 0.1-5.0 mg CdClz/kg body weight [5]. Histological examinations were performed 14 days after injection. It was also reported that subcutaneous administration of 1.0 and 2.0 mg Cd&/kg body weight in dogs produced no testicular damage [5], and the present study supports this observation at higher subcutaneous doses of cadmium chloride. In rats, mice, guinea pigs and rabbits, testicular necrosis is induced by subcutaneous or intraperitoneal injections of cadmium chloride at dosages of 0.02-0.04 mM/kg body weight [ 11. Testicular necrosis following subcutaneous injection of cadmium chloride appears to be species- and strain-specific, while intratesticular injection causes necrosis in all species tested, and at lower dose levels than is necessary with subcutaneous injection [ 1,6] . Many of the changes in the testes following the intratesticular injection of 0.2 mg CdClJkg body weight were consistent with previous reports on cadmium-induced sterility [ 3,5,6]. Initial inflammation, hemorrhage and necrosis was followed by seminiferous tubule atrophy, spermatogenic arrest, and Leydig cell regeneration. Severe impairment of spermatogenic activity was confirmed by histopathological evaluation of the testes and epididymis. Though epididymal damage subsided by 24 weeks after treatment, the presence of sperm in the epididymis was very limited. Semen evaluations confirmed histological observations of cadmium-induced, testicular damage. In all cadmium-treated dogs evaluated, aspermic ejaculates were collected close to the time of sacrifice. The testes of 2 dogs examined 24 weeks after injection showed areas of germinal tissue repair. Several active tubules were noted. Though some tubular regeneration appeared likely, spermatogenic activity was severely limited. Allanson and Deanesly [13] found evidence of seminiferous tubule regeneration in rats 20 weeks after subcutaneous injection of cadmium chloride. Full spermatogenesis returned to 2 out of 18 rats. This evidence, coupled with the findings of this study, suggest that canine testes may eventually regain some spermatogenic activity following cadmium-induced testicular injury, at the dosages used in this study. However, the extent of regeneration after 24 weeks was minimal. Also, reactions of individual dogs to intratesticularly administered cadmium may vary, and the 2 dogs sacrificed 24 weeks after injection may not have reacted as severely as the others, although this seems unlikely. The degree of testicular damage could also be influenced by the thoroughness of
58
infusion. The use of a higher dose of cadmium chloride may have produced more extensive testicular destruction, although intratesticular doses of 0.5 mg CdCl,/kg body weight have been reported to produce toxemia in dogs [ 51. ACKNOWLEDGEMENTS
The authors wish to thank Dr. B.I. Osburn, Department of Veterinary Pathology, School of Veterinary Medicine, University of California, Davis, California, 95616, for his aid in the histopathological evaluation of the testes. Additional thanks are due to Dr. Thomas E. Kelley, Rabies Animal Control Department, Health Services, Maricopa County, Arizona. REFERENCES 1 A.D. Chiquoine and V. Suntzeff, Sensitivity of mammals to cadmium necrosis of the testis, J. Reprod. Fert., 10 (1965) 455. 2 K.E. Mason, J.A. Brown, J.O. Young and R.R. Nesbitt, Cadmium-induced injury of the rat testes, Anat. Rec., 149 (1964) 135. 3 J. Parizek, Sterilization of the male by cadmium salts, J. Reprod. Fert., 1 (1960) 294. 4 A.B. Kar and R.P. Das, Sterilization of males by intratesticular administration of cadmium chloride, Acta Endocrinol., 40 (1962) 321. 5 S.N. Chatterjee and A.B. Kar, Chemical sterilization of stray dogs, Indian Vet. J., 45 (1968) 649. 6 A.B. Kar, Chemical sterilization of male Rhesus monkeys, Endocrinology, 69 (1961) 1116. 7 B.P. Setchell and G.M.H. Waites, Changes in the permeability of the testicular capillaries and of the blood-testis barrier after injection of cadmium chloride in the rat, J. Endocrinol., 47 (1970) 81. 8 J. Madlafousek, Z. Jlinak and J. Parizek, Sexual behavior of male rats sterilized by cadmium, J. Reprod. Fert., 26 (1971) 189. 9 J.H. Boucher, R.H. Foote and R.W. Kirk, The evaluation of semen quality in the dog and the effects of frequency of ejaculation upon semen quality, libido, and depletion of sperm reserves, Cornell Vet., 48 (1958) 67. 10 B. Zemjanis, Diagnostic and Therapeutic Techniques in Animal Reproduction, Williams and Wilkins, Baltimore, Md., 1970. 11 H.A. Herman and F.W. Madden, The Artificial Insemination of Dairy and Beef Cattle, Lucas Brothers, Columbia, Missouri, 1963. 12 D.F. Flick, H.F. Kraybill and J.M. Dimitroff, Toxic effects of cadmium, Environ. Res., 4 (1971) 71. 13 M. Allanson and R. Deanesly, Observations on cadmium damage and repair in rat testes and the effects on the pituitary gonadotrophins, J. Endocrinol., 24 (1962) 453.
Toxicology Letters, 1 (1977) 53-58 o Elsevier/North-Holland Biomedical Press
TOXICOLOGICAL EFFECTS OF CADMIUM CHLORIDE ON THE CANINE TESTIS FOLLOWING VARIOUS ROUTES OF ADMINISTRATION P.A. DONNELLY and D.E. MONTY Jr. Division of Agriculture,
Arizona State University, Tempe, Arizona 85281
(U.S.A.)
(Received May lOth, 1977) (Accepted May 13th, 1977)
SUMMARY
The toxic effects of cadmium chloride on the canine testis were investigated. Various dosages of cadmium chloride were administered subcutaneously, intramuscularly, intravenously and intratesticularly. Single intravenous doses equivalent to 4.0 or 8.0 mg CdClJkg body weight were lethal. The study showed that the canine testis is sensitive to cadmium administered intratesticularly, but relatively insensitive to the subcutaneous, intramuscular and intravenous administration of cadmium. Single intratesticular infusions of 0.2 mg CdClJkg body weight caused testicular inflammation, hemorrhage, massive necrosis of central seminiferous tubules, and disorganization and degeneration of the peripheral seminiferous tubules. Leydig cell degeneration was also prominent. Atrophy of the seminiferous tubules was followed by interstitial revascularization and Leydig cell regeneration. The persistence of disorganized and degenerative seminiferous tubules was observed throughout the post-injection period (24 weeks), while signs of limited seminiferous tubule regeneration and spermatogenesis were present at 24 weeks. Epididymal damage occurred following cadmium infusion into the testes, however, it subsided by the 24th week after administration. In all cadmium-treated dogs evaluated, aspermic ejaculates were collected close to the time of sacrifice.
INTRODUCTION
A single, sublethal dose of cadmium chloride has been shown to selectively damage the testes of a variety of mammalian species. Testicular necrosis has been demonstrated in rats [1,2], mice [ 1,3], rabbits [4], guinea pigs [3], hamsters [ 31, opossums [ 11, goats [4] , dogs [ 51, and monkeys [ 4,6] . A single dose of cadmium chloride evokes testicular atrophy and sterility within 3-7 days after injection [ 5,6] . Cadmium chloride affects testicular blood vessels causing increased vascular permeability, with disruption of the bloodtestis barrier [7]. Destruction of the seminiferous tubules results in sterility.
54
Transitory disorganization and atrophy of the interstitium is followed by revascularization, proliferation of Leydig cells and restoration of androgenic function of the testes [5,6]. Secondary sexual characteristics and behavior are preserved by Leydig cell regeneration. In male rats, sterilized by a single subcutaneous injection of cadmium chloride, sexual behavior, including normal mating and ejaculatory activity, returned 2 months after injection [ES]. Cadmium may prove to be a useful tool in animal population control programs, as it may provide a way to produce sterile breeding males which could be added to a breeding population [5]. The objectives of the present study were two-fold. Preliminary studies were aimed at determining the testicular effects of cadmium chloride following various routes of administration in the dog. Subsequent studies were concerned with long-term ttesticular damage and regeneration. MATERIALS
AND METHODS
The male dogs used in this study were adult animals of mixed breeding between the ages of l-3 years and weighing 20-40 pounds. Preliminary studies involved subcutaneous, intramuscular, intravenous and intratesticular injections of 2 dosage levels of cadmium chloride. The dosages selected were based on previous studies on dogs and other mammals [1,5]. 4 and 8 mg CdClJkg body weight were administered subcutaneously, intramuscularly and intravenously to 6 dogs, and 0.1 and 0.2 mg CdCl,/kg body weight were infused into the testes of 2 dogs. A 0.5% solution of cadmium chloride in normal physiological saline was used for intratesticular infusions, while a 4% solution of cadmium chloride in 96% propylene glycol was injected subcutaneously and intramuscularly. A 4% solution of cadmium chloride in normal physiological saline was injected intravenously into the cephalic vein. At all sites of administration, the volumes injected were adjusted according to body weight and the desired dosage of cadmium chloride. Three control dogs received injections of the cadmium chloride solvents (subcutaneous, intramuscular and intratesticular solvents). Dogs receiving intratesticular injections were anesthetized with sodium thiamylal*. An aseptic technique was employed and each testis received half of the total dosage administered. The solution was uniformly infused throughout the testicular tissue by carefully adjusting the depth and direction of the syringe needle. All experimental and control dogs received 900 000 units of Bicillin** intramuscularly after the injection procedure. The dogs were sacrificed 7 days after the injection. The long-term study group was composed of five dogs injected intratesticularly with 0.2 mg CdClJkg body weight. One dog was sacrificed 3 weeks after injection, another 6 weeks after injection, and another at 9 weeks. Two * Surital, Parke, Davis and Co., Detroit, Michigan. ** Bicillin, Wyeth Laboratories Inc., Philadelphia,
Pa.
55
dogs were euthanized 24 weeks after injection. Two control dogs were injected intratesticularly with normal saline and were sacrificed 17 weeks after injection. The testes of all dogs were removed, fixed in Bouin’s fluid, and tissue sections were stained with hematoxylin-eosin. Testicular changes were evaluated by comparison with normal testicular microstructure. Semen was collected from the long-term study dogs prior to injection of cadmium chloride. Samples were obtained by manipulative ejaculation [9]. Following treatment, attempts to collect semen were made at lo-day intervals. Semen was evaluated for gross motility [lo] , individual sperm motility [lo] , and sperm morphology [ll]. The dogs were observed for symptoms of cadmium toxicity (anemia, bleaching of the incisor teeth, orchitis, diarrhea, and vomiting [ 5,121). The injection sites were examined for signs of local irritation and inflammation, and biopsies were taken. RESULTS
Subcutaneous injections of cadmium chloride did not produce histological changes in the testes, but did cause severe subcutaneous inflammation, edema, hemorrhage and necrosis at the site of injection. Similarly, intramuscular injections of cadmium chloride produced severe local inflammation, with minimal testicular effect. The intramuscular injection of 8.0 mg CdClJkg body weight caused particularly severe local inflammation and necrosis, and several hemorrhagic, necrotic foci were observed in the testes. Intravenous injections of 4.0 and 8.0 mg CdClJkg body weight proved fatal within 48 and 24 h after injection, respectively. Convulsions with uncontrollable vomiting and defecation were observed. At both intravenous dosage levels, some hemorrhagic and necrotic foci were observed within the testes. Intratesticular infusion of 0.1 mg CdClJkg body weight produced necrosis and hemorrhage within the central portion of the testes, however, peripheral tubules appeared normal and healthy. Massive testicular necrosis resulted from the infusion of 0.2 mg CdClJkg body weight, which was particularly extensive in the central portions of the testes. There were large areas of necrosis, hemorrhage, fibrin deposition, and -tissue debris. The peripheral areas of the testes contained a few intact seminiferous tubules, however, the majority were shrunken and disorganized. Multi-nucleated cells and eosinophilic, necrotic debris were commonly noted within the seminiferous tubules. A few tubules in the extreme periphery showed evidence of spermatogenesis, but degenerative changes, including giant cell formation, were also present. Sertoli cells were prominent in many peripheral tubules. Interstitial edema and hemorrhage were prominent. Few Leydig cells were seen, and they were confined to the extreme periphery. Intraepithelial cysts and mononuclear infiltration were observed in the epididymis. The lumen of epididymal tubules was almost completely devoid of sperm. Intratesticular infusion, at both dosage levels, caused scrotal inflammation, however, the scrotum returned to normal size and
56
appearance within 7 days. The testes of the control dogs showed normal testicular structure 7 days after injection. 3 weeks after testicular infusion of 0.2 mg Cd&/kg body weight extensive necrosis and atrophy of the seminiferous tubules was evident. Peritubular fibrosis was noted. In the central areas of the testes many seminiferous tubules were devoid of cellular elements, while others lacked intact membranes. Peripheral tubules showed internal disorganization, multi-nucleated elements, and arrested spermatogenesis. A number of peripheral seminiferous tubules contained only Sertoli cells. Some local areas of Leydig cell proliferation were present in the peripheral regions. Revascularization of the tissue bed was apparent. In the epididymis, there were focal areas of mononuclear infiltration, and intraepithelial cysts were common. There were no spermatozoa evident in the epididymis. Semen was not obtained for evaluation. 6 weeks after testicular infusion of CdCl, extensive fibrosis and atrophy of the seminiferous tubules was observed. Necrotic tubules were also present. Degenerative and disorganized seminiferous tubules were observed in peripheral areas of the testes, and spermatogenesis was arrested. Revascularization was evident throughout the necrotic tissue. Prominent nests of Leydig cells were present throughout the testes. There were no spermatozoa evident in the epididymal lumen, and some intraepithelial cysts were observed. Semen obtained 40 days after injection was devoid of sperm. The testes of the cadmium-treated dog which was sacrificed 9 weeks after treatment contained areas of mature fibrous tissue, with remnants of collapsed seminiferous tubules. Surrounding seminiferous tubules were degenerated, with vacuolization of the epithelial cells. Spermatogenesis was present in a few peripheral tubules. Prominent nests of Leydig cells were observed throughout the testes. Intraepithelial cysts and mononuclear infiltrates were distributed throughout the epididymis. Limited numbers of spermatozoa were present in the lumen of the epididymis. An ejaculate collected 40 days after injection was devoid of sperm. 24 weeks after testicular infusion, areas of vacuolated and atrophied seminiferous tubules were observed in the testes. In some areas, tubules were completely devoid of germinal cells. They were dilated and contained homogeneous material. In other areas, tubules contained only Sertoli cells. Degenerative seminiferous tubules containing giant cells and numerous vacuoles were also observed, and spermatogenesis was arrested. In some areas, seminiferous tubules showed evidence of germinal epithelial repair, but spermatogenesis was not present. A few peripheral seminiferous tubules appeared normal and spermatogenesis was present. Revascularization had progressed to normal distribution. Areas of mature fibrosis were observed. The interstitium contained many healthy Leydig cells. In the epididymis, there were a few spermatozoa, and a few mononuclear cells were noted in the stroma. Aspermic ejaculates were collected from both dogs 166 days after injection. The testes of the 2 long-term control dogs, injected intratesticularly with normal saline, revealed normal testicular microstructure and active spermato-
genesis 17 weeks after injection. Semen was collected from both control dogs 115 days after injection, and the sperm concentration, motility and morphology were similar to the pre-injection semen samples. DISCUSSION
Preliminary studies showed that, with the exception of intratesticular administration, the canine testes is rather insensitive to parenterally administered cadmium. These findings are consistent with previous studies on dogs which reported permanent sterility in male dogs following single intratesticular injections of 0.1-5.0 mg CdClz/kg body weight [5]. Histological examinations were performed 14 days after injection. It was also reported that subcutaneous administration of 1.0 and 2.0 mg Cd&/kg body weight in dogs produced no testicular damage [5], and the present study supports this observation at higher subcutaneous doses of cadmium chloride. In rats, mice, guinea pigs and rabbits, testicular necrosis is induced by subcutaneous or intraperitoneal injections of cadmium chloride at dosages of 0.02-0.04 mM/kg body weight [ 11. Testicular necrosis following subcutaneous injection of cadmium chloride appears to be species- and strain-specific, while intratesticular injection causes necrosis in all species tested, and at lower dose levels than is necessary with subcutaneous injection [ 1,6] . Many of the changes in the testes following the intratesticular injection of 0.2 mg CdClJkg body weight were consistent with previous reports on cadmium-induced sterility [ 3,5,6]. Initial inflammation, hemorrhage and necrosis was followed by seminiferous tubule atrophy, spermatogenic arrest, and Leydig cell regeneration. Severe impairment of spermatogenic activity was confirmed by histopathological evaluation of the testes and epididymis. Though epididymal damage subsided by 24 weeks after treatment, the presence of sperm in the epididymis was very limited. Semen evaluations confirmed histological observations of cadmium-induced, testicular damage. In all cadmium-treated dogs evaluated, aspermic ejaculates were collected close to the time of sacrifice. The testes of 2 dogs examined 24 weeks after injection showed areas of germinal tissue repair. Several active tubules were noted. Though some tubular regeneration appeared likely, spermatogenic activity was severely limited. Allanson and Deanesly [13] found evidence of seminiferous tubule regeneration in rats 20 weeks after subcutaneous injection of cadmium chloride. Full spermatogenesis returned to 2 out of 18 rats. This evidence, coupled with the findings of this study, suggest that canine testes may eventually regain some spermatogenic activity following cadmium-induced testicular injury, at the dosages used in this study. However, the extent of regeneration after 24 weeks was minimal. Also, reactions of individual dogs to intratesticularly administered cadmium may vary, and the 2 dogs sacrificed 24 weeks after injection may not have reacted as severely as the others, although this seems unlikely. The degree of testicular damage could also be influenced by the thoroughness of
58
infusion. The use of a higher dose of cadmium chloride may have produced more extensive testicular destruction, although intratesticular doses of 0.5 mg CdCl,/kg body weight have been reported to produce toxemia in dogs [ 51. ACKNOWLEDGEMENTS
The authors wish to thank Dr. B.I. Osburn, Department of Veterinary Pathology, School of Veterinary Medicine, University of California, Davis, California, 95616, for his aid in the histopathological evaluation of the testes. Additional thanks are due to Dr. Thomas E. Kelley, Rabies Animal Control Department, Health Services, Maricopa County, Arizona. REFERENCES 1 A.D. Chiquoine and V. Suntzeff, Sensitivity of mammals to cadmium necrosis of the testis, J. Reprod. Fert., 10 (1965) 455. 2 K.E. Mason, J.A. Brown, J.O. Young and R.R. Nesbitt, Cadmium-induced injury of the rat testes, Anat. Rec., 149 (1964) 135. 3 J. Parizek, Sterilization of the male by cadmium salts, J. Reprod. Fert., 1 (1960) 294. 4 A.B. Kar and R.P. Das, Sterilization of males by intratesticular administration of cadmium chloride, Acta Endocrinol., 40 (1962) 321. 5 S.N. Chatterjee and A.B. Kar, Chemical sterilization of stray dogs, Indian Vet. J., 45 (1968) 649. 6 A.B. Kar, Chemical sterilization of male Rhesus monkeys, Endocrinology, 69 (1961) 1116. 7 B.P. Setchell and G.M.H. Waites, Changes in the permeability of the testicular capillaries and of the blood-testis barrier after injection of cadmium chloride in the rat, J. Endocrinol., 47 (1970) 81. 8 J. Madlafousek, Z. Jlinak and J. Parizek, Sexual behavior of male rats sterilized by cadmium, J. Reprod. Fert., 26 (1971) 189. 9 J.H. Boucher, R.H. Foote and R.W. Kirk, The evaluation of semen quality in the dog and the effects of frequency of ejaculation upon semen quality, libido, and depletion of sperm reserves, Cornell Vet., 48 (1958) 67. 10 B. Zemjanis, Diagnostic and Therapeutic Techniques in Animal Reproduction, Williams and Wilkins, Baltimore, Md., 1970. 11 H.A. Herman and F.W. Madden, The Artificial Insemination of Dairy and Beef Cattle, Lucas Brothers, Columbia, Missouri, 1963. 12 D.F. Flick, H.F. Kraybill and J.M. Dimitroff, Toxic effects of cadmium, Environ. Res., 4 (1971) 71. 13 M. Allanson and R. Deanesly, Observations on cadmium damage and repair in rat testes and the effects on the pituitary gonadotrophins, J. Endocrinol., 24 (1962) 453.