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Author's address (for correspondence): C Donovan, Department of Animal and Rangeland Sciences, Oregon State University, 315 Withycombe Hall, Corvallis, OR 97331, USA. E-mail: email@example.com
We investigated the use of a commercial gonadotropin-releasing hormone (GnRH) vaccine as a method of temporary and reversible immunocastration in intact male dogs. Four privately owned dogs were vaccinated twice at 4-week intervals. Blood samples were collected at 0, 4, 12 and 20 weeks following the initial vaccination. These samples were analysed for GnRH antibody titres, luteinizing hormone (LH) and testosterone concentrations. Scrotal measurements were made at the time of sample collection, and testicular volume was calculated using the formula of an ellipsoid. As a result of vaccination, dogs displayed an elevated GnRH antibody titre, decreased LH and testosterone concentrations and decreased testicular volume, which reversed by the end of the study period. Therefore, these results suggest that immunizing against GnRH may be a possible choice for temporary and reversible immunocastration.
Non-surgical sterilization methods are highly desirable for domestic animals for population control purposes as well as for animals that are not candidates for surgery. The hypothalamic–pituitary–gonadal axis, which is the regulator of mammalian reproductive function, has specifically been a target of manipulation for immunocontraception development. Briefly, gonadotropin-releasing hormone (GnRH), a trophic decapeptide secreted from the hypothalamus, controls the release of follicle-stimulating hormone (FSH) and luteinizing hormone (LH) from the anterior pituitary. In turn, LH and FSH act upon the testes in the male to stimulate testosterone production, which then negatively feeds back on the hypothalamus and anterior pituitary to regulate GnRH secretion. By diminishing GnRH production, the production of LH, FSH and testosterone is diminished as well.
It has been shown previously in intact male dogs that vaccinating with a GnRH analogue effectively decreases testosterone concentration (Sabeur et al. 2003; Walker et al. 2007). A commercially manufactured GnRH vaccine in the United States (Canine Gonadotropin Releasing Factor Immunotherapeutic®; Pfizer Animal Health, Exton, PA, USA; referred to in this manuscript as a ‘commercial canine GnRH vaccine’), labelled for the twice-annual management of benign prostatic hyperplasia in intact male dogs, may also be an ideal option for a reversible means of immunocastration. The vaccine elicits antibodies that bind to GnRH and render it non-functional, ultimately decreasing testicular testosterone production (Pfizer Animal Health, Package Insert).
The objective of this study was to measure endocrine responses in intact male dogs after administering a commercial canine GnRH vaccine. Furthermore, because testosterone production is necessary for spermatogenesis and testicular volume largely reflects spermatogenesis (Gouletsou et al. 2008), testicular volume measurements were also recorded. It was expected that immunization against GnRH would temporarily elicit a GnRH antibody titre, decrease LH and testosterone concentrations and decrease testicular volume in intact males.
Materials and Methods
Animals, vaccination, sample collection and scrotal measurements
Four privately owned intact male dogs were used for this study. Dogs were vaccinated subcutaneously with 1 ml of commercial canine GnRH vaccine twice at 4-week intervals (at week 0 and 4). Animals were closely monitored for adverse reactions following each vaccination. Venous blood samples were collected prior to each vaccination (0 and 4 weeks) and at 12 and 20 weeks following initial treatment. Sera were separated, aliquoted and frozen at −20°C until analysis.
Scrotal length, width and height were measured using calipers at weeks 0, 4, 12 and 20. Testes were aligned side by side and forced down into the scrotum as far as possible for each measurement. This is the standard method for testis measurement in dogs and causes no observable pain to the animal. Measurements were used to calculate testicular volume using the formula of an ellipsoid, length × width × height × 0.5236, as described by Gouletsou et al. (2008).
Gonadotropin-releasing hormone antibody titres were determined by enzyme-linked immunosorbent assay (ELISA) using a technique modified from Elhay et al. (2007). Briefly, 96-well microtitre plates were coated with 100 μl of 5 μg/ml of LH-RH (71447-49-9; Sigma, St. Louis, MO, USA) in sodium bicarbonate buffer (pH 8.0) at 4°C overnight. After incubation, plates were washed with phosphate-buffered saline containing 0.05% Tween-20 (TPBS) (pH 8.0) and were then blocked with a serum dilution buffer (phosphate-buffered saline containing 0.5% bovine serum albumin, pH 8.0). After 1-h incubation at room temperature, plates were washed with TPBS and serum samples were diluted in the serum dilution buffer, yielding final serum dilutions ranging from 1 : 2 to 1 : 128. After washing with TPBS, antibodies were detected using horseradish peroxidase protein G conjugate (HRP) (10-1223; Invitrogen, Camarillo, CA, USA) diluted as 1 : 2000 in serum dilution buffer for 1 h at room temperature. After a final wash with TPBS, HRP was visualized with ABTS peroxidase substrate (50-66-01; KPL, Gaithersburg, MD, USA). Absorbances were read at 405 nm using a spectrophotometer (Bio-Tek Instruments Inc., Winooski, VT, USA). The antibody titre was expressed as an endpoint titre for each sample, which was calculated from a regression line of optical density against the sample dilution with a threshold of 0.200 optical density using a software program (KinetiCalc; Bio-Tek Instruments Inc.).
Serum samples were analysed for LH concentrations using an ELISA kit for canine serum (LH-Detect®; Repropharm, Nouzilly, France) and performed according to the manufacturer's instructions. The limit of detection was 0.12 ng/ml, and all assays were performed in duplicate. Serum samples were also analysed for testosterone at the Animal Health Diagnostic Center at Cornell University using Coat-A-Count® Total Testosterone radioimmunoassay (Diagnostics Products Corporation, Los Angeles, CA, USA), validated for dogs by Reimers et al. (1991) and performed according to the manufacturer's instructions. The detection limit of the assay was 0.04 ng/ml. The coefficients of variation for the intra-assay and inter-assay variability were 4–21% and 6–27%, respectively, depending on the testosterone concentration.
Gonadotropin-releasing hormone antibody titres, LH and testosterone concentrations and the per cent change in testicular volume were analysed as a repeated measure in time design using proc mixed in sas (version 9.2; SAS Institute Inc., Cary, NC, USA). Significance was defined as p ≤ 0.05. LH concentrations are expressed as mean ± SEM, and GnRH antibody titres, testosterone concentrations and per cent change in testicular volume are expressed as mean ± SD.
None of the dogs vaccinated experienced an adverse reaction to the vaccine and remained clinically healthy for the duration of the study. All dogs were seronegative for antibodies against GnRH prior to the first vaccination and were seropositive for antibodies against GnRH after receiving the first vaccination. Antibody titres peaked at 12 weeks post-vaccination and were significantly greater at week 12 compared to week 0 (p < 0.001), week 4 (p = 0.008) and week 20 (p = 0.01) (Fig. 1).
Luteinizing hormone concentrations remained at basal levels (<0.8 ng/ml) post-vaccination (Fig. 1). All dogs experienced a decrease in testosterone concentration following vaccination (Fig. 1). Concentrations of testosterone were below the detection limit of the assay at week 12, a significant decrease (p = 0.02) compared to pre-vaccination testosterone levels. At week 20, there was a slight but insignificant rise in testosterone concentrations.
All dogs experienced a decrease in testicular volume following GnRH vaccination (Fig. 2). Testicular volume was significantly decreased at week 12 compared to week 0 (p = 0.05), week 4 (p = 0.02) and week 20 (p = 0.01). At week 20, there was a slight but insignificant rise in testicular volume, consistent with the increase in testosterone concentrations.
An alternative to surgical castration is needed for population control in dogs that are not surgical candidates because of health limitations and other reasons. A vaccine that elicits antibodies against GnRH to effectively decrease steroid hormone production is a potential option for a temporary, reversible means of immunosterilization. Although this commercial canine GnRH vaccine has been studied to treat benign prostatic hyperplasia in intact male dogs, it has not been thoroughly studied as an immunocontraceptive, and therefore, the objective of this study was to measure endocrine responses and testicular volume changes in intact male dogs as a result of vaccination.
As approximately 70–80% of testicular mass consists of seminiferous tubules (Gouletsou et al. 2008), measurement of testicular volume is largely reflective of the capacity for spermatogenesis. The dogs used in this study all responded to the vaccine by developing a GnRH antibody titre, and this resulted in a significant decrease in testosterone production and also a significant decrease in testicular volume.
Luteinizing hormone concentrations remained at basal levels (<0.8 ng/ml) in all dogs post-vaccination, but this was not significant compared to week 0, perhaps because of the cyclical secretion pattern of LH. The time interval in between LH peaks in the intact male dog is approximately 103 min (Olson et al. 1992). Therefore, collecting multiple samples over a 2-h time interval is needed to determine whether LH secretion was suppressed as a result of vaccination. This frequency of sampling was not feasible in the current study because the dogs used were privately owned.
In conclusion, vaccinating intact male dogs with a commercial canine GnRH vaccine induces a temporary and reversible humoral response that results in decreased testosterone concentration and decreased testicular volume. Further study using a double-blind, placebo-controlled format that also evaluates sexual behaviour, semen quality and repeated immunization is a necessary next step.
We thank Pfizer Animal Health for product donation, Katherine Peed for research initiation, Steve Lamb for testosterone RIA, Tim Hazzard for GnRH assay assistance, Gerd Bobe for statistical assistance and the staff at the Veterinary Village LLC, International Canine Semen Bank-Wisconsin for technical assistance.
Conflicts of interest
None of the authors have any conflicts of interest to declare.
Donovan performed GnRH and LH ELISAs, analysed data, wrote manuscript; Greer administered vaccinations, collected samples (measurements and blood); Kutzler conceptualized, funded and provided oversight for the study.
This research was funded by the Department of Animal and Rangeland Sciences at Oregon State University and the Veterinary Village LLC, International Canine Semen Bank-Wisconsin.