SURGICAL VASECTOMY OR CHEMICAL EPIDIDECTOMY FOR STERILIZING FREE-ROAMING HORSES

• Sponsoring Institution: National Institute of Food and Agriculture
• Project Status: COMPLETE
• Funding Source: ANIMAL HEALTH
• Reporting Frequency: Annual
• Accession No.: 1004151
• Project Start Date: Sep 9, 2014
• Project End Date: Sep 30, 2014
• Program Code: [(N/A)]- (N/A)

Recipient Organization
OREGON STATE UNIVERSITY
(N/A)
CORVALLIS,OR 97331

Performing Department
Animal and Rangeland Sciences

Non Technical Summary
Between 2008 and 2011, free-roaming horse stallions were either surgically vasectomized or chemical epididectomized and then released onto the range to manage the horse population on the Sheldon National Wildlife Refuge. Sheldon Refuge is currently in the process of removing all free-roaming horses by 2015, including those animals previously sterilized and released back onto the range. In fall 2013, the U.S. Fish and Wildife Service gathered and castrated 228 stallions on the Refuge as routine in preparation for shipment to adoption agents. During the castration procedure, blood, seminal fluid, and testicular tissue samples were collected from the stallions and stored for later analysis. While ovariectomy and immunization against reproductive targets (such as the zona pellucida) have been used for mares, castration has been the only method used for stallions. Because dominant stallions are removed with castration, this technique upsets the herd hierarchy, which is detrimental for animal health and welfare. Surgical vasectomy and chemical epididectomy have been used in other species for male sterilization, but the side effects of these procedures have not been evaluated in horses. Widespread implementation of these methods to control free-roaming horse populations depends upon demonstration of their safety and efficacy. The objective of the proposed research is to determine the hormonal and gonadal effects 2-6 years after surgical vasectomy or chemical epididectomy. The hypothesis is that both methods will be equally effective as castration in causing sterility (by preventing sperm movement through the vas deferens) but will not lower testosterone concentrations, thereby preserving normal behavior.

Animal Health Component

100%

Research Effort Categories

Basic

0%

Applied

100%

Developmental

0%

Classification

Knowledge Area (KA)	Subject of Investigation (SOI)	Field of Science (FOS)	Percent
121	3810	1020	100%

Knowledge Area
121 - Management of Range Resources;

Subject Of Investigation
3810 - Horses, ponies, and mules;

Field Of Science
1020 - Physiology;

Keywords

surgical vasectomy

chemical epidididectomy

free-roaming horses

sterilization

overpopulation

Goals / Objectives
The major goals of the proposed research are to determine the hormonal and gonadal effects 2-6 years after surgical vasectomy or chemicalepididectomy. The hypothesis is that both methods will be equally effective as castration in causing sterility (by preventing sperm movement through the vas deferens) but will not lower testosterone concentrations, thereby preserving normal stallion-like behavior within the herd.The specific aims are:1. To compare sperm morphology from archived slides made from seminal fluid samples previously obtained after castration in stallionsthat had been surgically vasectomized, chemically epididectomized, or untreated.2. To compare testicular and epididymal histology from archived formalin-fixed tissue samples previously obtained after castration in stallions that had been surgically vasectomized, chemically epididectomized, or untreated.3. To compare circulating testosterone concentrations from archived serum samples previously obtained at the time of castration of stallions that had been surgically vasectomized, chemically epididectomized, or untreated.

Project Methods
Animals: Of the 228 stallions castrated, nearly half of the stallions were identified by microchip as being previously surgically vasectomized or chemically epididectomized. Of these stallions, blood, seminal fluid and tissue samples were collected from the first 29 surgically vasectomized and the first 21 chemically epididectomized stallions castrated. In addition, samples were collected from the first 44 previously untreated stallions castrated to serve as controls.Sperm Morphology :As mentioned previously, at the time of castration, a thin, semen smear was made by spreading a tiny drop of the seminal fluid across the slide with a spreader slide. The semen smear was allowed to air dry. Each slide was labeled with the stallion number and testis side (right or left) and stored in a slide box so that it could be archived for future determination of sperm morphology. For the proposed research, the slides will be stained using a commercial Romanowsky-type staining kit (Hema 3™ Manual Staining System, Fisher Scientific). After being plunged into the fixative for 10 seconds, the slides will be incubated with each stain for 10 minutes each. This longer staining incubation is necessary for staining sperm cells to determine sperm morphology. After staining, the slides will be rinsed in deionized water and air dried. The slides will be evaluated blindly by a single observer under oil immersion (1000X) without a coverslip using bright field microscopy. For each slide, the evaluator will count a minimum of 100 sperm and the percentage of sperm with abnormal morphology was determined.Serum testosterone concentration: Jugular venous blood samples were collected from each stallion prior to castration and allowed to sit overnight at 4°C in a glass tube without a clot activator or a serum separating gel. The blood samples were then centrifuged at 1,000Xg for 10 minutes to separate serum. Sera were stored at -20°C. The samples will be shipped overnight on ice to the Animal Health Diagnostic Center (AHDC) at Cornell University. Total serum testosterone concentrations will be analyzed using a Coat-A-Count® Total Testosterone radioimmunoassay (Diagnostics Products Corporation, Los Angeles, CA) and performed according to the manufacturer's instructions. The detection limit of the assay is 0.04 ng/ml.Histology: Following castration, a portion of tissue was collected from each testis and epididymis and placed in labelled jar containing 10% neutral buffered formalin. The fixed tissues were stored atroom temperature until processing for histologic examination. The formalin-fixed tissues will beparaffin embedded by the Oregon State University Veterinary Diagnostic Laboratory (Histology Department) and sectioned (5 μm) onto glass slides. Slides will be stained with hematoxylin and eosin on an automatic stainer and evaluated blindly at 100X and 400X using a bright field microscope (DMI 6000B Leica Microsystems, Buffalo Grove, IL). Represented areas will be digitally captured with a camera attached to the microscope. The Johnsen's Scoring System will be used for assessing the status of spermatogenesis.11 Johnsen's score for assessing the spermatogenesis depends on scoring each seminiferous tubule cross-section. From each testis, a group of five seminiferous tubules will be randomly selected. The criteria are as follows: 10, complete spermatogenesis; 9, many spermatozoa present but disorganized spermatogenesis; 8, only a few spermatozoa present; 7, no spermatozoa but many spermatids present; 6, only a few spermatids present; 5, no spermatozoa or spermatids present but many spermatocytes present; 4, only a few spermatocytes present; 3, only spermatogonia present; 2, no germ cell present; and 1,no germ cell or Sertoli cell present. A mean score count will be given to each stallion.Data Analysis: Data will be descriptive (presence of gross adhesions or other pathology) and numerical (sperm morphology, testosterone concentrations, spermatogenesis score). Averages and standard deviations will be determined for each group. For numerical data, an analysis of variance (ANOVA) will be used to analyze the data and make comparisons between groups. Post hoc tests will be used to interpret the data as appropriate. Significance will be defined as P<0.05. The results from these experiments will be analyzed using GraphPad Prism® (GraphPadSoftware, La Jolla, CA, USA) and summarized in a manuscript for publication in the Journal of Wildlife Management and Wildlife Monographs.

Progress 09/09/14 to 09/30/14

Outputs
Target Audience: Nothing Reported Changes/Problems: Nothing Reported What opportunities for training and professional development has the project provided?Nothing yet. How have the results been disseminated to communities of interest?Nothing yet What do you plan to do during the next reporting period to accomplish the goals?All of the stated goals.

Impacts
What was accomplished under these goals? Because of the timing of funding activation, the only activity to report has been the cutting in and paraffin embedding of the tissues for histology. Also, the assays were purchased for the testosterone concentrations and the sperm slides were organized for evaluation.

Publications

Progress 09/09/14 to 09/30/14

Outputs
Target Audience:The target audience reached by this research is quite wide. Three presentations (one posters and one podium) were given at the Research Advances in Wildlife, Fisheries and Ecology Symposium held at Oregon State University. Also, a poster was presented at the North West Reproduction Sciences Symposium in Cle Elum, Washington and two podium presentations were given at the Society for Theriogenology Conference (2014: Portland, Oregon; 2015: San Antonio, Texas). In addition to appearing as published abstracts in the proceedings of these four scientific meetings, the research efforts were accepted for full article publication in the Journal of Zoo and Wildlife Medicine. TheJournal of Zoo and Wildlife Medicine(JZWM) is the official publication of the American Association of Zoo Veterinarians and the European Association of Zoo and Wildlife Veterinarians, and it is supported by the American College of Zoological Medicine, World Association of Zoos and Aquariums, Association of Zoos & Aquariums, and the European Association of Zoos and Aquariums.isThe Journal of Zoo and Wildlife Medicine is considered one of the major sources of information on the biology and veterinary aspects in the field. This journal is published quarterly, is peer reviewed, is indexed by the major abstracting services, and is international in scope and distribution. Areas of interest include clinical medicine, surgery, anatomy, radiology, physiology, reproduction, nutrition, parasitology, microbiology, immunology, pathology (including infectious diseases and clinical pathology), toxicology, pharmacology, and epidemiology. This journal has a large and diverse circulation, with abstracts available Arabic, Czech, German, French, Italian, Japanese, Portuguese, Russian and Spanish. Changes/Problems: Nothing Reported What opportunities for training and professional development has the project provided?Dr. Clare Scully, veterinarian and Master's graduate student, and Rebecca Lee, senior undergraduate student, both received training and professional development as a result of opportunities provided within this project. Specifically, Dr. Clare Scully gave a poster presentation at the Northwest Reprodutive Sciences Symposium in Cle Elum, Washington and two podium presentations at the Society for Theriogenology conference in Portland, Oregon (2014) and San Antonio, Texas (2015). Rebecca Lee also gave two poster presentations at the Recent Advances in Wildlife, Fisheries, and Ecology Symposium at Oregon State University (2014, 2015). In addition, both students learned how to perform endocrine assays, read sperm morphologies and testicular histologies, and write a scientific manuscript. How have the results been disseminated to communities of interest?Dr. Clare Scully gave a poster presentation at the Northwest Reprodutive Sciences Symposium in Cle Elum, Washington and two podium presentations at the Society for Theriogenology conference in Portland, Oregon (2014) and San Antonio, Texas (2015). Rebecca Lee also gave two poster presentations at the Recent Advances in Wildlife, Fisheries, and Ecology Symposium at Oregon State University (2014, 2015). In addition, the results were compiled into a full-length scientific manuscript that has been accepted for publication in the next issue of the Journal of Zoo and Wildlife Medicine. What do you plan to do during the next reporting period to accomplish the goals? Nothing Reported

Impacts
What was accomplished under these goals? All of these major goals were accomplished. Specific Aim 1: Surgivally vasectomizd stallions had no sperm present in the remaining section of the proximal vas deferens, whereas all of the untreated and chemically vasectomized stallions had sperm present within the vas deferens seminal fluid samples. The difference in presence of sperm between the untreated and chemically vasectomized stallions compared to the surgically vasectomized stallions was highly significant (P < 0.001). For surgically vasectomized stallions that had no sperm present in the remaining section of the proximal vas, a sample of fluid was collected from the tail of the epididymis. There were more morphologically normal sperm in the untreated stallions compared to surgically and chemically vasectomized stallions (P < 0.01). In addition, chemically vasectomized stallions had more morphologically normal sperm than did surgically vasectomized stallions (P < 0.001). The majority of the defects recorded in all groups were detached heads, bent tails, and severely coiled tails. There were more detached heads in the surgically vasectomized stallions compared to chemically vasectomized (P < 0.01) and untreated stallions (P <0.001). However, there were more severely coiled tails in the chemically vasectomized stallions compared to surgically vasectomized stallions (P < 0.05). Specific Aim 2: The histomorphometry results showed no significant difference in seminiferous tubule diameter (P = 0.51) or basement membrane thickness (P = 0.07) mediated by changes in intratesticular pressure following treatment. In addition, spermatogenesis as scored using the Yoshida method did not differ between treatment groups (surgically vasectomized: 11.8 ± 0.3; chemically vasectomized: 12.0 ± 0.0; untreated: 12.0 ± 0.0). Specific Aim 3: The hormone analysis showed no significant differences in testosterone or estrone sulfate concentrations when comparing either sterilization method (surgically vasectomized or chemically vasectomized) to untreated stallions. However, surgically vasectomized stallions had significantly lower testosterone compared to chemically vasectomized stallions (P < 0.05). This difference was not evident when comparing estrone sulfate concentrations.

Publications

• Type: Journal Articles Status: Awaiting Publication Year Published: 2015 Citation: "Comparison of Chemical and Surgical Vasectomy on Testicular Activity in Free-Roaming Horses" by Scully, Clare; Lee, Rebecca; Pielstick, Leon; Medlock, Jan; Patton, Kristin; Collins, Gail; Kutzler, Michelle