Neutersol: Intratesticular Injection Induces Sterility in Dogs
Min Wang, MD
Center of Reproductive Science and Technology
111 Allton Building, School of Medicine
University of Missouri-Columbia
Columbia, MO 65212
Objective–To evaluate the clinical safety and efficacy of a single intratesticular injection of zinc gluconate neutralized by arginine (100 mg/mL) in causing sterility/infertility of male puppies and to document the incidence of sterility/infertility produced by such treatment as evidenced by semen analysis.
Animals–270 male puppies of various breeds ranging from 2½ to 10 months of age having testicular widths of 10 mm to 27 mm were injected intratesticularly with zinc gluconate neutralized by arginine (100 mg/mL) in a clinical trial conducted at 5 study sites.
Procedure–Six different doses were administered according to testicular width (Table 1). Blood for complete blood count was collected on day 0 and 3 days post-injection. Reaction to injection–biting and/or licking the testes–was assessed at 30 minutes, one hour and 2 hours post-injection. General attitude, appetite, ability to walk, scrotal pain when the testes were manipulated, rectal temperature, scrotal evaluation beyond swelling, and occurrence of irritation, dermatitis, or ulceration were assessed on days 1-3 post-injection. Physical examination and semen analysis were performed at 2 months and 6 months post-injection (and 10- or 12 months post-injection, if applicable).
Results–A total of 270 male puppies of various breeds, ranging in age from 2½ to 10 months of age, were injected intratesticularly with zinc gluconate neutralized by arginine at five different locations. Six different doses were administered according to testicular width which was determined by measuring the testes at their widest point using a caliper in metric scale (mm). Since the dose was based on the width of each testis, 23 of the 270 puppies had different doses of the test article injected into the right and left testes. For purposes of statistical analyses, these 23 animals were assigned to the dose group corresponding to the lower of the two doses. Sedation/tranquilizer, which was administered at the discretion of the veterinarian, was used in 66 of the 270 injected puppies (24%) and had no effect on the results.
Of the 270 puppies injected, 225 (83.3%) completed the study in accordance with the protocol and 45 failed to complete the study (dropped out). The most frequent cause of failure to complete the study was failure of the adoptive owner/owner to bring the animal for the follow-up visits. Table 2 indicates the number of animals injected per dose at each study site and total number injected per dose.
Safety–Post-injection scrotal reactions of biting and/or licking as the primary indicator of treatment safety–No biting or licking was recorded by the veterinarians at 30 minutes post-injection, one hour post-injection, and 2 hours post-injection for animals in which assessment could be made.
Scrotal pain–Scrotal pain when the testes were manipulated occurred in 5% of the animals on the 1st day post-injection, decreased to 2% on the 2nd day post-injection, and decreased to less than 1% on the 3rd day post-injection.
Post-injection scrotal irritation, dermatitis, and/or ulceration during the first 3 days post injection–Of the 270 animals followed for 3 days post-injection at the study sites, there were 6 animals (2%) in which scrotal irritation or dermatitis occurred; and no animals in which ulceration occurred. None of the 6 animals were treated with any medication.
Release to owner on 3rd day post-injection–Of the 270 animals, 268 animals (99.3%) were released to the owner on the 3rd day post-injection. Of the 2 animals kept for 7 days, only one was kept due to scrotal irritation and the other was kept due to pulmonic stenosis (a pre-existing condition) and scrotal bruising. No medication was administered, and both animals were released to the owner on 7th day post-injection.
Scrotal and general condition at 7 days post-injection–Abnormal scrotal condition at 7 days post-injection was recorded for 5 animals. When telephoned on the 7th day post-injection, 4 owners thought the scrotum of their animal was swollen or the testis was sore or there was minor irritation; however, no additional follow-up was necessary. The remaining animal had been returned to the study site, and the veterinarian recorded the scrotum as being “excessively dry” on the 7th day post-injection; no additional follow-up was necessary.
When telephoned on the 7th day post-injection, 10 owners reported abnormal general condition. Nine of the 10 owners reported various respiratory conditions, i.e., coughing, sneezing; and one owner reported that his/her animal was not eating well.
Rectal temperature–Biologically, there was no significant change in rectal temperature on the 1st, 2nd, and 3rd days post-injection as compared to day 0 for all 270 animals.
Complete blood count (CBC)–Biologically, there was no significant change in blood parameters at 3 days post-injection as compared to day 0 for 178 animals from which blood was collected for CBC.
. General attitude, appetite, ability to walk–Abnormal attitude was recorded for 6 animals (2%); abnormal appetite was recorded for 11 animals (4%); and no difficulty with walking after injection was recorded for any of the animals.
Post-injection follow-up at 2 months, 6 months, and, if applicable, 10 months or 12 months–A total of 496 physical examinations and scrotal examinations were performed during the follow-up period--249 at 2 months post-injection, 231 at 6 months post-injection, one at 10 months post-injection, and 15 at 12 months post-injection, and no abnormalities were found.
Unexpected reactions–Vomiting occurred as an unexpected reaction in 10 animals post-injection at the last two study sites which were private veterinary clinics. The vomiting was of short duration and not severe, and none of the animals received any anti-emetic medication. Withholding food and water for 12 hours prior to injection or use of some sedation by the veterinarian was recommended and no other cases of vomiting occurred.
Efficacy–Semen collection and analysis was the primary indicator of treatment efficacy, and semen collection was attempted in all dogs at the 2 month and 6 month post-injection follow-up visits and, if applicable, at the 10 month or 12 month post-injection follow-up visit. At 2 months post-injection, semen was not collected from 34 of the 249 animals available for semen collection because the animal was either too young or uncooperative. Of the 215 animals in which semen collection and analysis was completed, only 2 semen samples had live sperm in the ejaculate and both of these animals were found to be oligospermic (less than 20 million spermatozoa/mL). Of the remaining 213 animals, 127 had no semen ejaculated (59.1%), 52 had no sperm in the ejaculate (24.2%), and 34 had dead or motionless sperm in the ejaculate (15.8%).
At 6 months post-injection, semen was not collected from 7 of the 231 animals available for semen collection because the animals were uncooperative. Of the 224 animals in which semen collection and analysis was completed, only 2 semen samples had live sperm in the ejaculate; one of the animals was found to be oligospermic (less than 20 million spermatozoa/mL), and the other animal was found to have sperm greater than 20 million per mL. Of the remaining 222 animals, 171 were aspermic (76.3%), 50 were azoospermic (22.3%), and one was necrospermic (0.5%).
At 12 months post-injection, semen was not collected from 2 of the 15 animals available for semen collection because the animals were uncooperative. Of the remaining 13, 11 were aspermic and 2 had live sperm, one of which was oligospermic and one of which had live sperm greater than 20 million per mL.
Thus, semen analysis documenting the sterility/infertility produced by the drug at completion of the study (6 months or 10 months or 12 months post-injection) was obtained from 225 of the 270 animals injected. Table 3 indicates the number of animals in each semen analysis outcome. Ninety-nine percent of the 225 animals were aspermic, azoospermic, or necrospermic, and only 2 animals had motile sperm, one of which was oligospermic.
Failures to complete the trial–Of 270 animals, 45 failed to complete the trial. Three causes for failure to complete the trial were failure of the owner to bring the animal for the follow-up visits, death unrelated to injection, and surgical castration. Twenty-eight animals did not complete the trial because the owner failed to bring the animal for the follow-up visits. Seven animals did not complete the trial due to death unrelated to injection--3 animals were hit by a car and died; 2 animals were euthanized; one animal died due to a cage accident; and one animal was found dead in the owner’s yard prior to scheduling its 6 month follow-up visit. Ten animals did not complete the trial due to surgical castration which was requested by the owner in 9 of the 10 cases; the other animal was surgically castrated at the discretion of the veterinarian.
Conclusions and Clinical Relevance–Results indicate that intratesticular injection of zinc gluconate neutralized by arginine based on testicular width is an extremely effective method for non-surgical sterilization of male puppies and that it is safe regardless of age and breed of the animal.