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Effects of intratesticular zinc gluconate treatment on testicular dimensions, echodensity, histology, sperm production, and testosterone secretion in American black bears (Ursus americanus)
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Theriogenology 75 (2011) 1444 –1452 www.theriojournal.com
Effects of intratesticular zinc gluconate treatment on testicular dimensions, echodensity, histology, sperm production, and testosterone secretion in American black bears (Ursus americanus) Leonardo F.C. Britoa, Patricia L. Serticha, William Rivesb, Marc Knobbea, Fabio Del Pieroa, Gordon B. Stullc,* a
Department of Clinical Sciences New Bolton Center, School of Veterinary Medicine, University of Pennsylvania, 382 West Street Road, Kennett Square, Pennsylvania, USA b Six Flags Great Adventure, Safari Department, P.O. Box 120, Jackson, New Jersey, USA c Millennium Wildlife Sciences Corporation, P.O. Box 2504, Vincentown, New Jersey, USA Received 30 May 2010; received in revised form 9 October 2010; accepted 28 October 2010
Abstract Eight adult American black bears were used to evaluate the effects of chemical castration by intratesticular zinc gluconate treatment on testicular dimensions, echodensity, histology, sperm production, and testosterone secretion. Treatment did not affect testicular dimensions and did not result in decreased resting or GnRH-stimulated testosterone secretion. Multifocal hyperchoic areas in the testicular parenchyma were observed on ultrasound examination, and white foci were observed on gross pathology examination after zinc gluconate treatment. Histologically, there were normal seminiferous tubules containing either round or elongated spermatids, along with abnormal tubules in all bears after treatment. Vacuolation of the seminiferous epithelium, sloughing of germ cells into the tubules’ lumen, presence of multinuclear giant cells, and reduced height of the seminiferous epithelium with missing generations of germ cells were commonly observed. The most severe testicular changes were multifocal and included fibrosis, complete degeneration of the seminiferous epithelium with shrinkage of the tubule, and sperm stasis. Epididymal sperm reserve was 982.74 ⫾ 654.16 ⫻ 106 sperm (mean ⫾ SEM) and motile sperm were observed in the epididymis of all but one of the bears. In conclusion, although intratesticular zinc gluconate treatment in black bears resulted in testicular degenerative changes detected by ultrasound and histology examinations, sperm production was not completely ablated. We inferred that normal fertility might have been compromised, but treatment unlikely resulted in sterility. © 2011 Published by Elsevier Inc. Keywords: Ursus americanus; Contraception; Testes; Sperm; Testosterone
1. Introduction American black bears (Ursus americanus) are the most common bear species in North America, with populations present in at least 11 provinces in Canada * Corresponding author. Tel.: ⫹ 1-609-268-2878; fax: ⫹ 1-609268-2878. E-mail address: [email protected] (G.B. Stull). 0093-691X/$ – see front matter © 2011 Published by Elsevier Inc. doi:10.1016/j.theriogenology.2010.10.036
and in 41 states in the USA. Conservation efforts after the great decline in black bear numbers in the early twentieth century have been successful and the estimated population growth rate during the period of 1987–1989 to 1999 –2001 in the USA was estimated to be 46% [1]. Increasing human-bear conflicts seems to be a direct consequence of the rapidly increasing black bear population, which is especially concerning due to the potential threat to personal safety. Therefore, con-
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trolling the black bear population, especially in areas of frequent conflicts, is an essential part of managing this species. Hunting has been the traditional method for controlling black bear populations in North America and the number of harvested bears, as well as the number of jurisdictions allowing bear hunt, has increased in recent years [1]. Other non-lethal methods of population control could perhaps be used in conjunction with traditional methods to improve population management efficiency, offering regulatory agencies alternatives with a more positive perception by the general public. Contraceptive methods involving antiprogestagens and zona pellucida vaccination have been tested in female black bears [2,3], but contraceptive methods for male black bears have not yet been evaluated. Intratesticular treatments with chemical compounds (chemical castration) that cause testicular degeneration and disruption of spermatogenesis have been used as alternative to orchiectomy. Glycerol [4,5] and calcium chloride [6,7] have been used for chemical castration of laboratory and domestic animals with relative success. Although the mechanism of action of these compounds is not completely understood, a direct toxic effect on testicular cells and/or disruption on the blood-testis barrier resulting in auto-immune testicular degeneration are possible effects. Intratesticular treatment with zinc gluconate neutralized with arginine is a method of sterilization approved by the Federal Drug Administration for use in dogs in the USA. A single treatment with this compound induces irreversible disruption of spermatogenesis, testicular degeneration, and aspermia/ azoospermia [8 –10]. The objective of the present study was to evaluate the effect of intratesticular zinc gluconate treatment on testicular dimensions, echotexture, histology, sperm production, and testosterone secretion in black bears.
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and microminerals, and vitamins). The ration was offered ad libitum and on average each bear consumed about 1.5 to 2.5 kg of the pelleted ration per day. In addition, bears were also fed various fruits, vegetables, and fish. Sterilization of male bears was chosen to control the growing number of this captive black bear population. Each bear was examined twice, with zinc gluconate intratesticular treatments performed during the first examination, and orchiectomy performed during the second. Examinations were conducted during the spring and fall, in order to avoid exposing anesthetized bears to excessive ambient temperatures. Variable time of treatment (spring or fall) and treatment-to-second examination intervals were used, as those might have affected the results. The first examination was performed in the spring (March-April), or in the fall (September-October), and all second examinations were performed in the spring (April-May). The intervals between the first and the second examination ranged from approximately 2 to 48 mo (Table 1). One bear died (from causes unrelated to the experiment) before the second examination, 17 mo after intratesticular zinc gluconate treatment. The testes from this bear were recovered for histophatology evaluation and the results of the examinations are included below; however, data on testicular dimensions and testosterone concentrations after treatment were excluded from the analysis. Before each examination, bears were immobilized in the field by an intramuscular injection via a dart blowgun with a combination of either 500 mg tiletamine hydrochloride and 500 mg zolazepam hydrochloride, or 10 mg medazalan, 6 mg metatomidine hydrochloride, and 500 mg ketamine hydrochloride. The bears were then transported (in less than 10 min) to the location
2. Materials and methods
Table 1 Details regarding administration of intratesticular zinc gluconate and subsequent orchiectomy in black bears.
2.1. Animals
Bear ID
Treatment season
Red #23 White #74 Red #2 Yellow #2 Yellow #12 White #21 Red #26 White#8
Spring Spring Spring Spring Spring Fall Fall Fall
This study was reviewed and approved by the State of New Jersey Department of Environmental Protection. Eight adult male bears (Ursus americanus), 6 to 15 y old, were selected for this study. The male black bears were maintained at Six Flags Great Adventure Park, a drive-through wildlife park located in Jackson, NJ, USA. The black bears in the park are confined in an area that mimics the natural environment. Bears were fed a manufactured omnivore pelleted ration (20% crude protein, 6% crude fat, 10% crude fiber, macro-
a b
Neutersol®/per testis; post-mortem.
Treatment-toorchiectomy interval (d)
Zinc gluconatea (mL)
1450 57 57 519b 729 591 592 567
1.5 2.0 2.25 2.0 2.0 2.5 2.5 2.5
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were all the procedures were performed. Anesthesia was maintained with isofluorine 2–3% supplemented with oxygen (12 L/min); additional ketamine was administered during the procedures when deemed necessary. Atipamezole hydrochloride (25–35 mg) was administered intramuscularly to reverse anesthesia, and the bears were also treated with 1.6 g danofloxacin and 600 mg ketoprofen. The bears were kept under observation in closed confinement for approximately 24 h after the procedures before being released into the exhibit area. 2.2. Zinc gluconate intratesticular treatment Zinc gluconate treatments were performed after blood samples were collected for testosterone evaluation, testicular dimensions and echodensity were evaluated, and a testicular biopsy was obtained (see below). Zinc gluconate (13.1 mg/mL) neutralized by arginine (Neutersol®; Technology Transfer Inc., Columbia, MO, USA) was injected into each testis using a 22G, 1 1/2 inches needle. The needle was fully introduced into the middle of the testicular parenchyma through the cranial pole of the testis parallel to the testis’ long axis and slowly withdrawn as the product was being injected. The volume injected into each testis ranged from 1.5 to 2.5 mL (Table 1); volume was based on the recommendations of the manufacturer for young dogs [9]. 2.3. Plasma testosterone concentration Blood samples were collected from the jugular vein as soon as the anesthesia equipment was set up and again 1 h after intravenous administration of 80 to 100 g gonadorelin (GnRH) diacetate tetrahydrate (Cystorelin®; Merial Limited, Duluth, GA, USA), according to the estimated bear weight (approximately 200 to 250 kg). Serum samples were extracted, stored at -20 °C, and submitted to a commercial laboratory for evaluation of testosterone concentrations by solid-phase RIA using a commercial kit (Coat-A-Count; Diagnostic Products Corporation, Los Angeles, CA, USA); the reported sensitivity of the assay was 0.04 ng/mL. Pre- and post-GnRH serum testosterone concentrations were determined for statistical analysis. GnRH challenge was not performed in one of the bears. 2.4. Testicular dimensions and echodensity During both examinations, the scrotum was clipped and the testes, epididymides, and spermatic cords were examined by palpation and ultrasonography. For these examinations, a B-mode ultrasound scanner equipped with a 7.5 MHz linear array transducer (MyLab 25;
Biosound Esaote, Inc., Indianapolis, IN, USA) was used to image the scrotal contents and measure testicular dimensions (length, width, and height). Testicular volume was calculated using the formula for an ellipsoid (volume ⫽ 4/3**length*width*height) and total testicular volume (left ⫹ right) was calculated for statistical analysis. 2.5. Sperm production Electroejaculation was attempted with bears in lateral recumbency using an electroejaculator equipped with a ram probe (Lane Manufacturing Inc., Denver, CO, USA). The probe was maintained approximately 5 cm past the anal sphincter, with the three electrodes facing the floor of the pelvis. Stimuli started at 1V and increased up to 7V with a 3 s stimulus, followed by 3 s of rest. Three stimuli for each voltage level were used with up to 10 stimuli for 7V. If the first attempt was not successful, the procedure was repeated up to three times with intervening 5 min rest periods. Ejaculates were recovered into a 15 mL plastic tube fitted with a plastic cone. The presence and motility of sperm were estimated under 400X magnification and phase-contrast with a microscope equipped with a warming stage. After orchiectomy (see below), a small incision was made on the tail of the epididymis and a small amount of fluid was collected with a Pasteur pipette. The fluid was examined for the presence and motility of sperm as described above. In addition, the epididymis was thoroughly homogenized and diluted with 0.05% Triton X-100 (w/v) saline solution, sperm were counted using hemocytometers, and epididymal sperm reserve was calculated based on dilutions. One bear had been vasectomized approximately 3 y prior to this study and no attempts were made to collect semen or to determine epididymal sperm reserves from this animal. 2.6. Testicular histopathology In the first examination, after attempted electroejaculation, the scrotum was scrubbed, a small stab incision was made on the scrotal skin, a testicular biopsy was obtained with a Quick-Core® biopsy needle (Cook Urological Inc., Spencer, IN, USA) from the cranio-ventral region of each testis, and the scrotal skin incisions were sutured with a single stitch using absorbable suture material. In the second examination, after scrotal examination, electroejaculation, and GnRH challenge, the scrotum was scrubbed and unilateral, closed orchiectomy was performed. The testis removed (left or right) was chosen randomly. The recovered testis was weighted, examined for gross pathological
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changes, and samples of the parenchyma were collected from the dorsal and ventral parts of the middle of the testis for examination. Testicular biopsy samples obtained before intratesticular zinc treatment and samples of the parenchyma obtained after orchiectomy were fixed in Bouin’s solution for 24 h, dehydrated and embedded in paraffin according to conventional techniques, sectioned 7-m thick, stained with haematoxylin and eosin-B, and examined for histopathological changes. 2.7. Statistical analysis Testosterone concentrations and testicular dimensions before and after zinc gluconate intratesticular treatment were compared using a paired Student’s t-test. 3. Results 3.1. Testicular dimensions and echodensity Palpation and ultrasonography of the scrotal contents prior to intratesticular zinc gluconate treatment revealed no abnormalities of the testes, epididymides, and spermatic cords, even in the bear subsequently diagnosed with unilateral testicular seminoma. On ultrasonography, the testicular parenchyma appeared homogenous and mildly echogenic with a prominent hyperchoic band in the middle of the testis corresponding to the mediastinum (Fig. 1A). There were evident changes in testicular echodensity after zinc treatment; however, the character and severity of these changes did not seem to be related to the time of treatment (Spring or Fall) or the treatment-to-examination interval. In some cases, hyperchoic areas were observed in few scattered areas (Fig. 1C and D), whereas in other cases hyperchoic areas were observed throughout the entire parenchyma (Fig. 1E and F). In some cases, hyperchoic areas blocked the ultrasound waves producing shadow artifacts. Despite changes in echodensity, testicular dimensions were not significantly different after intratesticular zinc gluconate treatment. Testicular length, width, and height (right and left testes average) before and after treatment were 6.2 ⫾ 0.7 vs 6.4 ⫾ 0.8, 3.2 ⫾ 0.4 vs 3.5 ⫾ 0.4, and 3.5 ⫾ 0.6 vs 3.6 ⫾ 0.6 cm, respectively (mean ⫾ SD). Combined testicular volume before and after treatment was 588.5 ⫾ 196.0 vs 700.2 ⫾ 250.5 cm3, respectively. 3.2. Testicular histopathology Testicular biopsy samples before intratesticular zinc gluconate treatment were not obtained from one bear,
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whereas examination of the sample obtained from another bear revealed that only connective tissue had been obtained. Testicular biopsy examination revealed unilateral testicular seminoma in one of the bears; bilateral orchiectomy was performed during the second examination on this bear, and the affected testis was not examined for this study. Biopsy samples were very small and only a few seminiferous tubules (⬍ 5) were observed. Crushing artifacts and mild hemorrhage were common. A distinct tubular lumen was not apparent in most of the tubules in all samples, but either round or elongated spermatids could be identified as the most developed germ cell in most of the tubules (Fig. 2A and B). Gross examination of the testes after intratesticular zinc gluconate treatment revealed multifocal, white areas in the testicular parenchyma (Fig. 1B). Testicular weight was 49.8 ⫾ 5.1 g (mean ⫾ SEM). Histology examination revealed normal seminiferous tubules containing either round or elongated spermatids along with abnormal tubules in all bears. Testicular histopathological changes did not seem to be related to the time of treatment or the treatment-to-examination interval. Vacuolation of the seminiferous epithelium and sloughing of germ cells into the tubules’ lumen was the most common abnormality observed (Fig. 2C), with multinuclear giant cells observed in some cases (Fig. 2D). The most severe changes were multifocal. In some tubules, the height of the seminiferous epithelium was reduced and complete generations of germ cells were missing (Fig. 3A and B). Other changes included fibrosis, complete degeneration of the seminiferous epithelium with shrinkage of the tubule, and sperm stasis (Fig. 3C–E). There were occasional intratubular mildly mineralized proteinaceus rings and vacuolated Sertoli cells. The cells inside the tubules were diffusely eosinophilic and hyaline with loss of cell details indicating necrosis in one bear (Fig. 3F). 3.3. Sperm production Electroejaculation produced erection in all bears, but semen was obtained from only three bears before intratesticular treatment (two in April and one in September) and three bears after treatment. Semen emission was observed after anesthesia in one bear during the second examination after introduction of the probe into the rectum, but before any stimulation. Contamination with urine was common, but motile sperm were observed in all samples. Similarly, motile sperm were observed in samples obtained from the epididymis immediately after orchiectomy in all but one bear. Epi-
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Fig. 1. (A) Normal, homogeneous, mildly echogenic testicular parenchyma before intratesticular zinc gluconate treatment; note the hyperechogenic band in the middle of the parenchyma corresponding to the mediastinum in a black bear. (B) Multifocal, white testicular lesions evident on gross pathology after zinc treatment. (C and D) Mild changes in testicular echotexture after zinc treatment characterized by few hyperechogenic areas throughout the parenchyma. (E and F) Severe changes in testicular echotexture after zinc treatment characterized by many hyper-echogenic areas throughout the entire parenchyma. Note that some hyper-echogenic areas blocked the ultrasound waves and produced shadow artifacts. (A): scale bar ⫽ 10 mm; (C–F): scale bar ⫽ 5 mm).
didymal sperm reserve was 982.74 ⫾ 654.16 ⫻ 106 sperm (mean ⫾ SEM), ranging from 3.8 to 4,860 ⫻ 106 sperm. 3.4. Plasma testosterone concentration Prior to intratesticular zinc gluconate treatment, resting serum testosterone concentration was extremely
elevated (29.7 ng/mL) in the bear subsequently diagnosed with unilateral testicular seminoma. Resting serum testosterone concentration decreased considerably after treatment (resting ⫽ 6.7 ng/mL and GnRH-stimulated ⫽ 8.6 ng/mL), but testosterone concentrations from this bear were excluded from the analysis. In addition, testosterone concentrations were not available
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Fig. 2. (A and B) Testicular biopsy sample before intratesticular zinc gluconate treatment in a black bear. Only a few seminiferous tubules were observed and a distinct tubular lumen was not apparent in most of the tubules, although either round or elongated spermatids could be identified as the most developed germ cell. (C) Vacuolation of the seminiferous epithelium and sloughing of germ cells into the tubules’ lumen was the most common abnormality observed after zinc gluconate treatment. (D) Multinuclear giant cells (*) were observed in some cases. (A): 10X, (B and D): 40X; (C): 20X.
prior to zinc gluconate intratesticular treatment in one bear. Testosterone concentrations increased (P ⬍ 0.05) after GnRH administration both before (0.8 ⫾ 0.6 vs 7.0 ⫾ 2.2 ng/mL, resting and GnRH-stimulated, respectively) and after zinc gluconate intratesticular treatment (8.8 ⫾ 1.9 vs 13.3 ⫾ 1.0 ng/mL, resting and GnRHstimulated, respectively). Overall, testosterone concentrations were greater (P ⬍ 0.05) after zinc gluconate treatment (11.0 ⫾ 1.2 ng/mL) than before treatment (3.9 ⫾ 1.4 ng/mL). 4. Discussion The safety of intratesticular zinc gluconate treatment in dogs has been demonstrated based on clinical parameters and behavior, but vomiting, anorexia, lethargy and neutrophilia are systemic adverse reactions sometimes observed after treatment. Local adverse reactions including scrotal necrotizing ulcers and development of draining tracts are also sometimes observed and believed to be associated with poor injection technique; these usually require medical care and surgical orchiectomy [9 –12]. In the present study, all bears recovered
uneventfully and no systemic adverse reactions were noted after treatment. In addition, no scrotal lesions were observed during the post-treatment examination. Although transient scrotal swelling and tenderness that usually resolves without treatment are common in the first few days following zinc gluconate treatment in dogs [9,11], no attempt was made to examine the scrotum and its contents in the weeks immediately after treatment in the present study. Since intratesticular zinc gluconate had not been previously used for chemical castration in black bears, the dose of the commercial product used in the present study was based on the recommendations of the manufacturer for young dogs, i.e., 1 mL (13.1 mg) for each testis 2.5–2.7 cm wide [9]. Zinc gluconate treatment in dogs resulted in a 23% reduction in testicular width in one study [10], but no changes were observed in another [12]. Considerable individual variation in testicular size has been reported after treatment in dogs, with some animals showing no change after treatment and others showing small and fibrotic testes [9]. In the present study, testicular dimensions were similar to that previously reported in black bears [13,14] and no sig-
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Fig. 3. The most severe changes in testicular histology after intratesticular zinc gluconate treatment in black bears were multifocal. (A) Complete generations of germ cells were missing in some tubules (spermatogonia only), while (B) some tubules were completely devoid of germ cells (Sertoli cells only). (C–E) Fibrosis accompanied by complete degeneration of the seminiferous epithelium with shrinkage of the tubules (*) and sperm stasis (s) were also observed. (F) Diffusely eosinophilic and hyaline intratubular cells indicating necrosis. (A): 40X; (B): 60X; (C–E): 20X.
nificant changes were observed after intratesticular zinc gluconate treatment. Although the histological abnormalities observed after intratesticular zinc gluconate treatment in the present experiment can also be observed in normal males [15,16], several factors indicate that these changes were likely the result of the treatment. All bears used in the present study were considered sexually mature, since active spermatogenesis is observed in all bears ⱖ3 y old [13] and testicular growth plateaus between 4 and 5 y of age [14]. The testicular samples for histology were all obtained in the spring, when testicular tissue
has already recrudesced after the non-breeding season and active spermatogenesis is observed [14,17]. While the small size of the biopsy samples obtained prior to the treatment limited the value of these as controls, the abnormalities observed after treatment were not present in the biopsy samples. Moreover, studies in llamas have demonstrated that testicular biopsy does not result in permanent, multifocal histological abnormalities [18]. Although the extent of testicular degeneration in the present study seemed less pronounced, the histological characterization was similar to that reported after intratesticular zinc gluconate in dogs [8,12]. Moreover, the
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changes in testicular echodensity were obvious and likely the result of the described histological abnormalities. Since the most severe testicular degenerative changes were multifocal instead of diffuse, it is likely that those resulted form direct contact with zinc gluconate. The areas with most severe degenerative changes also likely represented the hyperchoic areas on ultrasound examination and the white foci on gross pathology examination. Efficacy of intratesticular zinc gluconate treatment for chemical castration in dogs as determined by resulting aspermia or azoospermia has been reported to be ⬎98% [9,10] and mating of treated dogs with normal females has resulted in no pregnancies [9]. In another recent study, treatment of dogs with a more concentrated zinc gluconate product (26.2 mg/mL) containing 0.5% DMSO resulted in decreased sperm numbers in the ejaculate 15 d after treatment with minimal levels observed after approximately 4.5 mo, but aspermia or azoospermia were not observed [12]. Due to the difficulties associated with electroejaculation of black bears in this experiment, the efficacy of intratesticular zinc gluconate treatment could not be determined by quantifying motile sperm in the ejaculate. Although ultrasonographic and histopathological testicular changes were apparent in all bears after treatment, complete disruption of spermatogenesis was not observed, since motile sperm were observed in the epididymis of all but one of the bears. Seasonal changes in testicular function in black bears are likely regulated by changes in circulating testosterone. Testosterone concentrations start to increase during late winter and early spring while bears are still in their dens, peak during late spring and summer just prior or during the breeding season, and decrease to the lowest levels during the fall and early winter [14,19,20]. GnRH-stimulated testosterone production is also greater during the spring and summer than during the winter or fall [21]. Mean resting and GnRH-stimulated testosterone concentrations were greater after intratesticular zinc gluconate treatment in the present study because three bears were first examined (pre-treatment) during the fall when testosterone concentrations are normally low and all bears were re-examined during the spring when testosterone concentrations are normally high. In any case, these results demonstrate that treatment did not affect testosterone production. These observations are somewhat similar to those observed after zinc gluconate treatment in dogs, although more individual variation occurred in that species [9].
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In conclusion, although intratesticular zinc gluconate treatment in black bears resulted in testicular degenerative changes, sperm production was not completely ablated. These observations suggest that normal fertility might be compromised after treatment, but treatment would unlikely have resulted in sterility. Increasing the dose of zinc gluconate, modifying the method of intratesticular injection (e.g. multiple entry points), different vehicles (e.g. DMSO), or treating prepubescent bears might improve the efficacy of treatment and result in complete chemical castration. Acknowledgments This project was supported by the Millennium Wildlife Sciences Corporation, Six Flags Great Adventure, and In Defense of Animals. We thank Dr. Thomas Curro, Dr. Ken Keiffer, Dr. Robert Foglia, Kyle Covill, and the staff from Six Flags Great Adventure for their assistance during the study. References [1] Hristienko H, McDonald JE. Jr. Going into the 21st century: a perspective on trends and controversies in the management of the American black bear. Ursus 2007;18:72– 88. [2] Jewgenow K, Quest M, Elger W, Hildebrandt TB, Meyer HH, Strauss G, Goritz F. Administration of antiprogestin J956 for contraception in bears: a pharmacological study. Theriogenology 2001;56:601–11. [3] Lane VM, Liu IK, Casey K, vanLeeuwen EM, Flanagan DR, Murata K, Munro C. Inoculation of female American black bears (Ursus americanus) with partially purified porcine zona pellucidae limits cub production. Reprod Fertil Dev 2007;19: 617–25. [4] Wiebe JP, Barr KJ. The control of male fertility by 1,2,3trihydroxypropane (THP;glycerol): rapid arrest of spermatogenesis without altering libido, accessory organs, gonadal steroidogenesis, and serum testosterone, LH and FSH. Contraception 1984;29:291–302. [5] Wiebe JP, Barr KJ, Buckingham KD. Sustained azoospermia in squirrel monkey, Saimiri sciureus, resulting from a single intratesticular glycerol injection. Contraception 1989;39:447–57. [6] Jana K, Samanta PK, Ghosh D. Evaluation of single intratesticular injection of calcium chloride for nonsurgical sterilization of male Black Bengal goats (Capra hircus): a dose-dependent study. Anim Reprod Sci 2005;86:89 –108. [7] Jana K, Samanta PK. Sterilization of male stray dogs with a single intratesticular injection of calcium chloride: a dose-dependent study. Contraception 2007;75:390 – 400. [8] Oliveira EC, Moura MR, Silva VA, Jr., Peixoto CA, Saraiva KL, de Sa MJ, Douglas RH, de Pinho Marques A, Jr.Intratesticular injection of a zinc-based solution as a contraceptive for dogs. Theriogenology 2007;68:137– 45. [9] FDA. Neutersol® injectable solution for dogs (zinc gluconate neutralized by arginine), Freedom of Information Summary: Federal Drug Administration, 2003.
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[10] LaCroix CE. Evaluation of a single intratesticular injection of zinc gluconate neutralized by Arginine (Neutersol®) as a chemical sterilant in sexually mature, male dogs. International Symposium on Non-Surgical Contraceptive Methods for Pet Population Control. 2006, p.1–7. [11] Levy JK, Crawford PC, Appel LD, Clifford EL. Comparison of intratesticular injection of zinc gluconate versus surgical castration to sterilize male dogs. Am J Vet Res 2008;69:140 –3. [12] Soto FR, Viana WG, Mucciolo GC, Hosomi FY, Vannucchi CI, Mazzei CP, Eyherabide AR, de Fatima Lucio C, Dias RA, de Azevedo SS. Evaluation of efficacy and safety of zinc gluconate associated with dimethyl sulphoxide for sexually mature canine males chemical neutering. Reprod Domest Anim 2009;44:927–31. [13] Erickson A, Nellor J. The black bear in Michigan. In: Erickson A, Nellor J, Petrides G, editors. Breeding biology of the black bear. East Lansing: Michigan State University. 1964, p. 5– 45. [14] Garshelis DL, Hellgren EC. Variation in reproductive biology of male black bears. J Mammal 1994;75:175– 88. [15] Foley GL. Overview of male reproductive pathology. Toxicol Pathol 2001;29:49 – 63.
[16] Goedken MJ, Kerlin RL, Morton D. Spontaneous and agerelated testicular findings in beagle dogs. Toxicol Pathol 2008; 36:465–71. [17] Tsubota T, Howell-Skalla L, Nitta H, Osawa Y, Mason JI, Meiers PG, Nelson RA, Bahr JM. Seasonal changes in spermatogenesis and testicular steroidogenesis in the male black bear Ursus americanus. J Reprod Fertil 1997;109:21–7. [18] Heath AM, Pugh DG, Sartin EA, Navarre B, Purohit RC. Evaluation of the safety and efficacy of testicular biopsies in llamas. Theriogenology 2002;58:125–30. [19] McMillin JM, Seal US, Rogers L, Erickson AW. Annual testosterone rhythm in the black bear (Ursus americanus). Biol Reprod 1976;15:163–7. [20] Palmer SS, Nelson RA, Ramsay MA, Stirling I, Bahr JM. Annual changes in serum sex steroids in male and female black (Ursus americanus) and polar (Ursus maritimus) bears. Biol Reprod 1988;38:1044 –50. [21] Horan KT, Nelson RA, Palmer SS, Bahr JM. Seasonal response of the pituitary and testes to gonadotropin-releasing hormone in the black bear (Ursus americanus). Comp Biochem Physiol Comp Physiol 1993;106:175– 82.
Theriogenology 75 (2011) 1444 –1452 www.theriojournal.com
Effects of intratesticular zinc gluconate treatment on testicular dimensions, echodensity, histology, sperm production, and testosterone secretion in American black bears (Ursus americanus) Leonardo F.C. Britoa, Patricia L. Serticha, William Rivesb, Marc Knobbea, Fabio Del Pieroa, Gordon B. Stullc,* a
Department of Clinical Sciences New Bolton Center, School of Veterinary Medicine, University of Pennsylvania, 382 West Street Road, Kennett Square, Pennsylvania, USA b Six Flags Great Adventure, Safari Department, P.O. Box 120, Jackson, New Jersey, USA c Millennium Wildlife Sciences Corporation, P.O. Box 2504, Vincentown, New Jersey, USA Received 30 May 2010; received in revised form 9 October 2010; accepted 28 October 2010
Abstract Eight adult American black bears were used to evaluate the effects of chemical castration by intratesticular zinc gluconate treatment on testicular dimensions, echodensity, histology, sperm production, and testosterone secretion. Treatment did not affect testicular dimensions and did not result in decreased resting or GnRH-stimulated testosterone secretion. Multifocal hyperchoic areas in the testicular parenchyma were observed on ultrasound examination, and white foci were observed on gross pathology examination after zinc gluconate treatment. Histologically, there were normal seminiferous tubules containing either round or elongated spermatids, along with abnormal tubules in all bears after treatment. Vacuolation of the seminiferous epithelium, sloughing of germ cells into the tubules’ lumen, presence of multinuclear giant cells, and reduced height of the seminiferous epithelium with missing generations of germ cells were commonly observed. The most severe testicular changes were multifocal and included fibrosis, complete degeneration of the seminiferous epithelium with shrinkage of the tubule, and sperm stasis. Epididymal sperm reserve was 982.74 ⫾ 654.16 ⫻ 106 sperm (mean ⫾ SEM) and motile sperm were observed in the epididymis of all but one of the bears. In conclusion, although intratesticular zinc gluconate treatment in black bears resulted in testicular degenerative changes detected by ultrasound and histology examinations, sperm production was not completely ablated. We inferred that normal fertility might have been compromised, but treatment unlikely resulted in sterility. © 2011 Published by Elsevier Inc. Keywords: Ursus americanus; Contraception; Testes; Sperm; Testosterone
1. Introduction American black bears (Ursus americanus) are the most common bear species in North America, with populations present in at least 11 provinces in Canada * Corresponding author. Tel.: ⫹ 1-609-268-2878; fax: ⫹ 1-609268-2878. E-mail address: [email protected] (G.B. Stull). 0093-691X/$ – see front matter © 2011 Published by Elsevier Inc. doi:10.1016/j.theriogenology.2010.10.036
and in 41 states in the USA. Conservation efforts after the great decline in black bear numbers in the early twentieth century have been successful and the estimated population growth rate during the period of 1987–1989 to 1999 –2001 in the USA was estimated to be 46% [1]. Increasing human-bear conflicts seems to be a direct consequence of the rapidly increasing black bear population, which is especially concerning due to the potential threat to personal safety. Therefore, con-
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trolling the black bear population, especially in areas of frequent conflicts, is an essential part of managing this species. Hunting has been the traditional method for controlling black bear populations in North America and the number of harvested bears, as well as the number of jurisdictions allowing bear hunt, has increased in recent years [1]. Other non-lethal methods of population control could perhaps be used in conjunction with traditional methods to improve population management efficiency, offering regulatory agencies alternatives with a more positive perception by the general public. Contraceptive methods involving antiprogestagens and zona pellucida vaccination have been tested in female black bears [2,3], but contraceptive methods for male black bears have not yet been evaluated. Intratesticular treatments with chemical compounds (chemical castration) that cause testicular degeneration and disruption of spermatogenesis have been used as alternative to orchiectomy. Glycerol [4,5] and calcium chloride [6,7] have been used for chemical castration of laboratory and domestic animals with relative success. Although the mechanism of action of these compounds is not completely understood, a direct toxic effect on testicular cells and/or disruption on the blood-testis barrier resulting in auto-immune testicular degeneration are possible effects. Intratesticular treatment with zinc gluconate neutralized with arginine is a method of sterilization approved by the Federal Drug Administration for use in dogs in the USA. A single treatment with this compound induces irreversible disruption of spermatogenesis, testicular degeneration, and aspermia/ azoospermia [8 –10]. The objective of the present study was to evaluate the effect of intratesticular zinc gluconate treatment on testicular dimensions, echotexture, histology, sperm production, and testosterone secretion in black bears.
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and microminerals, and vitamins). The ration was offered ad libitum and on average each bear consumed about 1.5 to 2.5 kg of the pelleted ration per day. In addition, bears were also fed various fruits, vegetables, and fish. Sterilization of male bears was chosen to control the growing number of this captive black bear population. Each bear was examined twice, with zinc gluconate intratesticular treatments performed during the first examination, and orchiectomy performed during the second. Examinations were conducted during the spring and fall, in order to avoid exposing anesthetized bears to excessive ambient temperatures. Variable time of treatment (spring or fall) and treatment-to-second examination intervals were used, as those might have affected the results. The first examination was performed in the spring (March-April), or in the fall (September-October), and all second examinations were performed in the spring (April-May). The intervals between the first and the second examination ranged from approximately 2 to 48 mo (Table 1). One bear died (from causes unrelated to the experiment) before the second examination, 17 mo after intratesticular zinc gluconate treatment. The testes from this bear were recovered for histophatology evaluation and the results of the examinations are included below; however, data on testicular dimensions and testosterone concentrations after treatment were excluded from the analysis. Before each examination, bears were immobilized in the field by an intramuscular injection via a dart blowgun with a combination of either 500 mg tiletamine hydrochloride and 500 mg zolazepam hydrochloride, or 10 mg medazalan, 6 mg metatomidine hydrochloride, and 500 mg ketamine hydrochloride. The bears were then transported (in less than 10 min) to the location
2. Materials and methods
Table 1 Details regarding administration of intratesticular zinc gluconate and subsequent orchiectomy in black bears.
2.1. Animals
Bear ID
Treatment season
Red #23 White #74 Red #2 Yellow #2 Yellow #12 White #21 Red #26 White#8
Spring Spring Spring Spring Spring Fall Fall Fall
This study was reviewed and approved by the State of New Jersey Department of Environmental Protection. Eight adult male bears (Ursus americanus), 6 to 15 y old, were selected for this study. The male black bears were maintained at Six Flags Great Adventure Park, a drive-through wildlife park located in Jackson, NJ, USA. The black bears in the park are confined in an area that mimics the natural environment. Bears were fed a manufactured omnivore pelleted ration (20% crude protein, 6% crude fat, 10% crude fiber, macro-
a b
Neutersol®/per testis; post-mortem.
Treatment-toorchiectomy interval (d)
Zinc gluconatea (mL)
1450 57 57 519b 729 591 592 567
1.5 2.0 2.25 2.0 2.0 2.5 2.5 2.5
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were all the procedures were performed. Anesthesia was maintained with isofluorine 2–3% supplemented with oxygen (12 L/min); additional ketamine was administered during the procedures when deemed necessary. Atipamezole hydrochloride (25–35 mg) was administered intramuscularly to reverse anesthesia, and the bears were also treated with 1.6 g danofloxacin and 600 mg ketoprofen. The bears were kept under observation in closed confinement for approximately 24 h after the procedures before being released into the exhibit area. 2.2. Zinc gluconate intratesticular treatment Zinc gluconate treatments were performed after blood samples were collected for testosterone evaluation, testicular dimensions and echodensity were evaluated, and a testicular biopsy was obtained (see below). Zinc gluconate (13.1 mg/mL) neutralized by arginine (Neutersol®; Technology Transfer Inc., Columbia, MO, USA) was injected into each testis using a 22G, 1 1/2 inches needle. The needle was fully introduced into the middle of the testicular parenchyma through the cranial pole of the testis parallel to the testis’ long axis and slowly withdrawn as the product was being injected. The volume injected into each testis ranged from 1.5 to 2.5 mL (Table 1); volume was based on the recommendations of the manufacturer for young dogs [9]. 2.3. Plasma testosterone concentration Blood samples were collected from the jugular vein as soon as the anesthesia equipment was set up and again 1 h after intravenous administration of 80 to 100 g gonadorelin (GnRH) diacetate tetrahydrate (Cystorelin®; Merial Limited, Duluth, GA, USA), according to the estimated bear weight (approximately 200 to 250 kg). Serum samples were extracted, stored at -20 °C, and submitted to a commercial laboratory for evaluation of testosterone concentrations by solid-phase RIA using a commercial kit (Coat-A-Count; Diagnostic Products Corporation, Los Angeles, CA, USA); the reported sensitivity of the assay was 0.04 ng/mL. Pre- and post-GnRH serum testosterone concentrations were determined for statistical analysis. GnRH challenge was not performed in one of the bears. 2.4. Testicular dimensions and echodensity During both examinations, the scrotum was clipped and the testes, epididymides, and spermatic cords were examined by palpation and ultrasonography. For these examinations, a B-mode ultrasound scanner equipped with a 7.5 MHz linear array transducer (MyLab 25;
Biosound Esaote, Inc., Indianapolis, IN, USA) was used to image the scrotal contents and measure testicular dimensions (length, width, and height). Testicular volume was calculated using the formula for an ellipsoid (volume ⫽ 4/3**length*width*height) and total testicular volume (left ⫹ right) was calculated for statistical analysis. 2.5. Sperm production Electroejaculation was attempted with bears in lateral recumbency using an electroejaculator equipped with a ram probe (Lane Manufacturing Inc., Denver, CO, USA). The probe was maintained approximately 5 cm past the anal sphincter, with the three electrodes facing the floor of the pelvis. Stimuli started at 1V and increased up to 7V with a 3 s stimulus, followed by 3 s of rest. Three stimuli for each voltage level were used with up to 10 stimuli for 7V. If the first attempt was not successful, the procedure was repeated up to three times with intervening 5 min rest periods. Ejaculates were recovered into a 15 mL plastic tube fitted with a plastic cone. The presence and motility of sperm were estimated under 400X magnification and phase-contrast with a microscope equipped with a warming stage. After orchiectomy (see below), a small incision was made on the tail of the epididymis and a small amount of fluid was collected with a Pasteur pipette. The fluid was examined for the presence and motility of sperm as described above. In addition, the epididymis was thoroughly homogenized and diluted with 0.05% Triton X-100 (w/v) saline solution, sperm were counted using hemocytometers, and epididymal sperm reserve was calculated based on dilutions. One bear had been vasectomized approximately 3 y prior to this study and no attempts were made to collect semen or to determine epididymal sperm reserves from this animal. 2.6. Testicular histopathology In the first examination, after attempted electroejaculation, the scrotum was scrubbed, a small stab incision was made on the scrotal skin, a testicular biopsy was obtained with a Quick-Core® biopsy needle (Cook Urological Inc., Spencer, IN, USA) from the cranio-ventral region of each testis, and the scrotal skin incisions were sutured with a single stitch using absorbable suture material. In the second examination, after scrotal examination, electroejaculation, and GnRH challenge, the scrotum was scrubbed and unilateral, closed orchiectomy was performed. The testis removed (left or right) was chosen randomly. The recovered testis was weighted, examined for gross pathological
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changes, and samples of the parenchyma were collected from the dorsal and ventral parts of the middle of the testis for examination. Testicular biopsy samples obtained before intratesticular zinc treatment and samples of the parenchyma obtained after orchiectomy were fixed in Bouin’s solution for 24 h, dehydrated and embedded in paraffin according to conventional techniques, sectioned 7-m thick, stained with haematoxylin and eosin-B, and examined for histopathological changes. 2.7. Statistical analysis Testosterone concentrations and testicular dimensions before and after zinc gluconate intratesticular treatment were compared using a paired Student’s t-test. 3. Results 3.1. Testicular dimensions and echodensity Palpation and ultrasonography of the scrotal contents prior to intratesticular zinc gluconate treatment revealed no abnormalities of the testes, epididymides, and spermatic cords, even in the bear subsequently diagnosed with unilateral testicular seminoma. On ultrasonography, the testicular parenchyma appeared homogenous and mildly echogenic with a prominent hyperchoic band in the middle of the testis corresponding to the mediastinum (Fig. 1A). There were evident changes in testicular echodensity after zinc treatment; however, the character and severity of these changes did not seem to be related to the time of treatment (Spring or Fall) or the treatment-to-examination interval. In some cases, hyperchoic areas were observed in few scattered areas (Fig. 1C and D), whereas in other cases hyperchoic areas were observed throughout the entire parenchyma (Fig. 1E and F). In some cases, hyperchoic areas blocked the ultrasound waves producing shadow artifacts. Despite changes in echodensity, testicular dimensions were not significantly different after intratesticular zinc gluconate treatment. Testicular length, width, and height (right and left testes average) before and after treatment were 6.2 ⫾ 0.7 vs 6.4 ⫾ 0.8, 3.2 ⫾ 0.4 vs 3.5 ⫾ 0.4, and 3.5 ⫾ 0.6 vs 3.6 ⫾ 0.6 cm, respectively (mean ⫾ SD). Combined testicular volume before and after treatment was 588.5 ⫾ 196.0 vs 700.2 ⫾ 250.5 cm3, respectively. 3.2. Testicular histopathology Testicular biopsy samples before intratesticular zinc gluconate treatment were not obtained from one bear,
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whereas examination of the sample obtained from another bear revealed that only connective tissue had been obtained. Testicular biopsy examination revealed unilateral testicular seminoma in one of the bears; bilateral orchiectomy was performed during the second examination on this bear, and the affected testis was not examined for this study. Biopsy samples were very small and only a few seminiferous tubules (⬍ 5) were observed. Crushing artifacts and mild hemorrhage were common. A distinct tubular lumen was not apparent in most of the tubules in all samples, but either round or elongated spermatids could be identified as the most developed germ cell in most of the tubules (Fig. 2A and B). Gross examination of the testes after intratesticular zinc gluconate treatment revealed multifocal, white areas in the testicular parenchyma (Fig. 1B). Testicular weight was 49.8 ⫾ 5.1 g (mean ⫾ SEM). Histology examination revealed normal seminiferous tubules containing either round or elongated spermatids along with abnormal tubules in all bears. Testicular histopathological changes did not seem to be related to the time of treatment or the treatment-to-examination interval. Vacuolation of the seminiferous epithelium and sloughing of germ cells into the tubules’ lumen was the most common abnormality observed (Fig. 2C), with multinuclear giant cells observed in some cases (Fig. 2D). The most severe changes were multifocal. In some tubules, the height of the seminiferous epithelium was reduced and complete generations of germ cells were missing (Fig. 3A and B). Other changes included fibrosis, complete degeneration of the seminiferous epithelium with shrinkage of the tubule, and sperm stasis (Fig. 3C–E). There were occasional intratubular mildly mineralized proteinaceus rings and vacuolated Sertoli cells. The cells inside the tubules were diffusely eosinophilic and hyaline with loss of cell details indicating necrosis in one bear (Fig. 3F). 3.3. Sperm production Electroejaculation produced erection in all bears, but semen was obtained from only three bears before intratesticular treatment (two in April and one in September) and three bears after treatment. Semen emission was observed after anesthesia in one bear during the second examination after introduction of the probe into the rectum, but before any stimulation. Contamination with urine was common, but motile sperm were observed in all samples. Similarly, motile sperm were observed in samples obtained from the epididymis immediately after orchiectomy in all but one bear. Epi-
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Fig. 1. (A) Normal, homogeneous, mildly echogenic testicular parenchyma before intratesticular zinc gluconate treatment; note the hyperechogenic band in the middle of the parenchyma corresponding to the mediastinum in a black bear. (B) Multifocal, white testicular lesions evident on gross pathology after zinc treatment. (C and D) Mild changes in testicular echotexture after zinc treatment characterized by few hyperechogenic areas throughout the parenchyma. (E and F) Severe changes in testicular echotexture after zinc treatment characterized by many hyper-echogenic areas throughout the entire parenchyma. Note that some hyper-echogenic areas blocked the ultrasound waves and produced shadow artifacts. (A): scale bar ⫽ 10 mm; (C–F): scale bar ⫽ 5 mm).
didymal sperm reserve was 982.74 ⫾ 654.16 ⫻ 106 sperm (mean ⫾ SEM), ranging from 3.8 to 4,860 ⫻ 106 sperm. 3.4. Plasma testosterone concentration Prior to intratesticular zinc gluconate treatment, resting serum testosterone concentration was extremely
elevated (29.7 ng/mL) in the bear subsequently diagnosed with unilateral testicular seminoma. Resting serum testosterone concentration decreased considerably after treatment (resting ⫽ 6.7 ng/mL and GnRH-stimulated ⫽ 8.6 ng/mL), but testosterone concentrations from this bear were excluded from the analysis. In addition, testosterone concentrations were not available
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Fig. 2. (A and B) Testicular biopsy sample before intratesticular zinc gluconate treatment in a black bear. Only a few seminiferous tubules were observed and a distinct tubular lumen was not apparent in most of the tubules, although either round or elongated spermatids could be identified as the most developed germ cell. (C) Vacuolation of the seminiferous epithelium and sloughing of germ cells into the tubules’ lumen was the most common abnormality observed after zinc gluconate treatment. (D) Multinuclear giant cells (*) were observed in some cases. (A): 10X, (B and D): 40X; (C): 20X.
prior to zinc gluconate intratesticular treatment in one bear. Testosterone concentrations increased (P ⬍ 0.05) after GnRH administration both before (0.8 ⫾ 0.6 vs 7.0 ⫾ 2.2 ng/mL, resting and GnRH-stimulated, respectively) and after zinc gluconate intratesticular treatment (8.8 ⫾ 1.9 vs 13.3 ⫾ 1.0 ng/mL, resting and GnRHstimulated, respectively). Overall, testosterone concentrations were greater (P ⬍ 0.05) after zinc gluconate treatment (11.0 ⫾ 1.2 ng/mL) than before treatment (3.9 ⫾ 1.4 ng/mL). 4. Discussion The safety of intratesticular zinc gluconate treatment in dogs has been demonstrated based on clinical parameters and behavior, but vomiting, anorexia, lethargy and neutrophilia are systemic adverse reactions sometimes observed after treatment. Local adverse reactions including scrotal necrotizing ulcers and development of draining tracts are also sometimes observed and believed to be associated with poor injection technique; these usually require medical care and surgical orchiectomy [9 –12]. In the present study, all bears recovered
uneventfully and no systemic adverse reactions were noted after treatment. In addition, no scrotal lesions were observed during the post-treatment examination. Although transient scrotal swelling and tenderness that usually resolves without treatment are common in the first few days following zinc gluconate treatment in dogs [9,11], no attempt was made to examine the scrotum and its contents in the weeks immediately after treatment in the present study. Since intratesticular zinc gluconate had not been previously used for chemical castration in black bears, the dose of the commercial product used in the present study was based on the recommendations of the manufacturer for young dogs, i.e., 1 mL (13.1 mg) for each testis 2.5–2.7 cm wide [9]. Zinc gluconate treatment in dogs resulted in a 23% reduction in testicular width in one study [10], but no changes were observed in another [12]. Considerable individual variation in testicular size has been reported after treatment in dogs, with some animals showing no change after treatment and others showing small and fibrotic testes [9]. In the present study, testicular dimensions were similar to that previously reported in black bears [13,14] and no sig-
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Fig. 3. The most severe changes in testicular histology after intratesticular zinc gluconate treatment in black bears were multifocal. (A) Complete generations of germ cells were missing in some tubules (spermatogonia only), while (B) some tubules were completely devoid of germ cells (Sertoli cells only). (C–E) Fibrosis accompanied by complete degeneration of the seminiferous epithelium with shrinkage of the tubules (*) and sperm stasis (s) were also observed. (F) Diffusely eosinophilic and hyaline intratubular cells indicating necrosis. (A): 40X; (B): 60X; (C–E): 20X.
nificant changes were observed after intratesticular zinc gluconate treatment. Although the histological abnormalities observed after intratesticular zinc gluconate treatment in the present experiment can also be observed in normal males [15,16], several factors indicate that these changes were likely the result of the treatment. All bears used in the present study were considered sexually mature, since active spermatogenesis is observed in all bears ⱖ3 y old [13] and testicular growth plateaus between 4 and 5 y of age [14]. The testicular samples for histology were all obtained in the spring, when testicular tissue
has already recrudesced after the non-breeding season and active spermatogenesis is observed [14,17]. While the small size of the biopsy samples obtained prior to the treatment limited the value of these as controls, the abnormalities observed after treatment were not present in the biopsy samples. Moreover, studies in llamas have demonstrated that testicular biopsy does not result in permanent, multifocal histological abnormalities [18]. Although the extent of testicular degeneration in the present study seemed less pronounced, the histological characterization was similar to that reported after intratesticular zinc gluconate in dogs [8,12]. Moreover, the
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changes in testicular echodensity were obvious and likely the result of the described histological abnormalities. Since the most severe testicular degenerative changes were multifocal instead of diffuse, it is likely that those resulted form direct contact with zinc gluconate. The areas with most severe degenerative changes also likely represented the hyperchoic areas on ultrasound examination and the white foci on gross pathology examination. Efficacy of intratesticular zinc gluconate treatment for chemical castration in dogs as determined by resulting aspermia or azoospermia has been reported to be ⬎98% [9,10] and mating of treated dogs with normal females has resulted in no pregnancies [9]. In another recent study, treatment of dogs with a more concentrated zinc gluconate product (26.2 mg/mL) containing 0.5% DMSO resulted in decreased sperm numbers in the ejaculate 15 d after treatment with minimal levels observed after approximately 4.5 mo, but aspermia or azoospermia were not observed [12]. Due to the difficulties associated with electroejaculation of black bears in this experiment, the efficacy of intratesticular zinc gluconate treatment could not be determined by quantifying motile sperm in the ejaculate. Although ultrasonographic and histopathological testicular changes were apparent in all bears after treatment, complete disruption of spermatogenesis was not observed, since motile sperm were observed in the epididymis of all but one of the bears. Seasonal changes in testicular function in black bears are likely regulated by changes in circulating testosterone. Testosterone concentrations start to increase during late winter and early spring while bears are still in their dens, peak during late spring and summer just prior or during the breeding season, and decrease to the lowest levels during the fall and early winter [14,19,20]. GnRH-stimulated testosterone production is also greater during the spring and summer than during the winter or fall [21]. Mean resting and GnRH-stimulated testosterone concentrations were greater after intratesticular zinc gluconate treatment in the present study because three bears were first examined (pre-treatment) during the fall when testosterone concentrations are normally low and all bears were re-examined during the spring when testosterone concentrations are normally high. In any case, these results demonstrate that treatment did not affect testosterone production. These observations are somewhat similar to those observed after zinc gluconate treatment in dogs, although more individual variation occurred in that species [9].
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In conclusion, although intratesticular zinc gluconate treatment in black bears resulted in testicular degenerative changes, sperm production was not completely ablated. These observations suggest that normal fertility might be compromised after treatment, but treatment would unlikely have resulted in sterility. Increasing the dose of zinc gluconate, modifying the method of intratesticular injection (e.g. multiple entry points), different vehicles (e.g. DMSO), or treating prepubescent bears might improve the efficacy of treatment and result in complete chemical castration. Acknowledgments This project was supported by the Millennium Wildlife Sciences Corporation, Six Flags Great Adventure, and In Defense of Animals. We thank Dr. Thomas Curro, Dr. Ken Keiffer, Dr. Robert Foglia, Kyle Covill, and the staff from Six Flags Great Adventure for their assistance during the study. References [1] Hristienko H, McDonald JE. Jr. Going into the 21st century: a perspective on trends and controversies in the management of the American black bear. Ursus 2007;18:72– 88. [2] Jewgenow K, Quest M, Elger W, Hildebrandt TB, Meyer HH, Strauss G, Goritz F. Administration of antiprogestin J956 for contraception in bears: a pharmacological study. Theriogenology 2001;56:601–11. [3] Lane VM, Liu IK, Casey K, vanLeeuwen EM, Flanagan DR, Murata K, Munro C. Inoculation of female American black bears (Ursus americanus) with partially purified porcine zona pellucidae limits cub production. Reprod Fertil Dev 2007;19: 617–25. [4] Wiebe JP, Barr KJ. The control of male fertility by 1,2,3trihydroxypropane (THP;glycerol): rapid arrest of spermatogenesis without altering libido, accessory organs, gonadal steroidogenesis, and serum testosterone, LH and FSH. Contraception 1984;29:291–302. [5] Wiebe JP, Barr KJ, Buckingham KD. Sustained azoospermia in squirrel monkey, Saimiri sciureus, resulting from a single intratesticular glycerol injection. Contraception 1989;39:447–57. [6] Jana K, Samanta PK, Ghosh D. Evaluation of single intratesticular injection of calcium chloride for nonsurgical sterilization of male Black Bengal goats (Capra hircus): a dose-dependent study. Anim Reprod Sci 2005;86:89 –108. [7] Jana K, Samanta PK. Sterilization of male stray dogs with a single intratesticular injection of calcium chloride: a dose-dependent study. Contraception 2007;75:390 – 400. [8] Oliveira EC, Moura MR, Silva VA, Jr., Peixoto CA, Saraiva KL, de Sa MJ, Douglas RH, de Pinho Marques A, Jr.Intratesticular injection of a zinc-based solution as a contraceptive for dogs. Theriogenology 2007;68:137– 45. [9] FDA. Neutersol® injectable solution for dogs (zinc gluconate neutralized by arginine), Freedom of Information Summary: Federal Drug Administration, 2003.
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