Abstract
Semen collection and cryopreservation are becoming increasingly important for conserving the genetic diversity of declining wild felid populations. Traditionally, semen is collected from anesthetized cats using electroejaculation and then processed through multiple steps prior to straw freezing over liquid nitrogen vapor, requiring specialized equipment and technical skills. Recently, an alternative simplified approach to semen banking has been developed, combining semen collection via urethral catheterization and semen freezing via vitrification.1,2 Although effective in domestic cats, this cath-vit approach has not been tested in wild felids. In this study, objectives were to 1) compare the effectiveness of urethral catheterization/semen vitrification versus electroejaculation/straw freezing in ocelots (Leopardus pardalis), and 2) assess the capacity of zoo veterinarians to opportunistically apply this cath-vit approach for do-it-yourself (DIY) semen banking of their resident felids. For objective 1, urethral catheterization was performed on seven ocelots anesthetized with a combination of ketamine-medetomidine. An aliquot of recovered raw semen was diluted in vitrification medium, held for 5 min and pelleted by direct pipetting into liquid nitrogen. Remaining semen was combined with electroejaculated samples and processed via standard straw freezing methods. Post-thaw analysis included assessment of sperm motility and acrosome status over time and in vitro fertilization (IVF) success following insemination of in vitro matured domestic cat oocytes. In ocelots, four of seven catheterization attempts yielded high quality semen (mean±SE; 63.3±47.1x106 sperm, 80±4% motility) with additional semen (90.5±29.9x106 sperm) recovered via electroejaculation. Post-thaw sperm motility and acrosome status did not differ (p>0.05) between banking methods but fertilization success following IVF of domestic cat oocytes was lower (p<0.01) for cath-vit (35%, 53/150) than straw-frozen (54%, 39/72) samples. For objective 2, veterinarians at three zoos participated in initial field testing of DIY semen banking. Urethral catheterization was attempted with 12 males of five species (tiger, Panthera tigris; lion, Panthera leo; cheetah, Acinonyx jubatus; puma, Puma concolor; bobcat, Lynx rufus). Recovered urethral fluid was vitrified as described above and shipped to the Cincinnati Zoo for post-thaw assessment. Catheterization allowed recovery of urethral fluid (without obvious urine) from nine males; however, only six samples contained any spermatozoa (mean; 0.12±0.08x106/pellet), and just one vitrified sample (from a cheetah) contained sufficient sperm numbers (0.5x106/pellet) and post-thaw motility (10%) for fertilization (13%, 3/24) of domestic cat oocytes. Results suggest that the cath-vit approach may be used as a quick and simple method for semen recovery and cryopreservation in ocelots; however, the occurrence of urine contamination and poor sperm recovery in other felid species indicate that further refinement may be required for broader applicability of this cath-vit method for DIY semen banking by zoo veterinarians.
Acknowledgments
The authors thank the Institute of Museum and Library Services for financial support of this Collection Stewardship grant. The assistance of animal care staff at all participating institutions (Boise Zoo, Caldwell Zoo, Dallas Zoo, Denver Zoo, Houston Zoo, Oklahoma City Zoological Park, San Antonio Zoological Gardens & Aquarium, Texas Zoo, White Oak Conservation) and research staff (Dr. Raquel Gonzalez, Amy Miller) at CREW is greatly appreciated.
Literature Cited
1. Johnson JD, Bateman HL, Newsom J, Vansandt LM, Swanson WF. Semen vitrification in felids—a simplified cryopreservation method for field use. Proc Am Assoc Zoo Vet. 2015:124–125.
2. Swanson WF, Bateman HL, Vansandt LM. Urethral catheterization and sperm vitrification for simplified semen banking in felids. Reprod Dom Anim. 2016;51(Suppl. 3):1–6.