Semen Vitrification in Felids: A Simplified Cryopreservation Method for Field Use
Assisted reproductive technologies such as semen cryopreservation and artificial insemination (AI) are becoming increasingly important for genetic management and conservation of endangered felid species. However, current semen freezing methods for felids are not ‘field-friendly’, requiring advanced reproductive expertise and access to specialized equipment. One alternative to conventional semen freezing may be vitrification, using ultra-rapid cooling in liquid nitrogen to form a glass-like state without ice crystal formation. With this approach, wildlife veterinarians and biologists could easily cryopreserve felid semen in the wild for subsequent AI and genetic augmentation of captive populations. In this study, our objectives were to 1) compare post-thaw sperm parameters for domestic cat and nondomestic cat semen preserved with vitrification vs. conventional freezing; and 2) investigate production of viable kittens using vitrified domestic cat sperm for in vitro fertilization (IVF)/embryo transfer and AI. Semen was collected from domestic cats (n=2) and non-domestic felids (fishing cat, Prionailurus viverrinus, n=1; ocelot, Leopardus pardalis, n=1). Control samples were extended in a chemically defined soy lecithin-based medium with 4% glycerol, slow cooled and frozen in straws over liquid nitrogen vapor.4 For vitrification, raw semen was diluted in soy lecithin medium (without glycerol) containing sucrose (0.1 M, Trt 1; 0.2 M, Trt 2; 0.3 M, Trt 3), held for 5 min and vitrified by pipetting (30 µl) directly into liquid nitrogen. After thawing, control and vitrified samples were evaluated for acrosome status, progressive motility, and fertility in vitro. For IVF, oocytes collected laparoscopically from gonadotropin-treated domestic cats were inseminated with control or vitrified (Trt 2 only) semen and assessed for embryo cleavage. To evaluate in vivo viability, domestic cat embryos produced with vitrified sperm were transferred into three synchronized recipients, and three additional females were inseminated with vitrified sperm.3 In domestic cats, post-thaw sperm motility and acrosome status did not differ (p>0.05) for control, Trt 1 or Trt 2, but values were reduced (p<0.01) for Trt 3. IVF percentages were similar (p>0.05) for control (16/53, 30.2%) vs. vitrified sperm (13/53, 24.5%). In the fishing cat and ocelot, post-thaw motility was slightly decreased for vitrified (20–25%) compared to control (∼40%) sperm but acrosome status (43–48% intact, fishing cat; 26–27% intact, ocelot) was similar. IVF of domestic cat oocytes with vitrified sperm resulted in fertilization in both species (5/9, 55%, fishing cat; 2/9, 22%, ocelot). Domestic cat embryo transfer failed to produce any pregnancies; however, AI resulted in conception in all three females. Two pregnancies progressed to term, culminating in the birth of two healthy kittens—the first non-human offspring of any species ever produced from vitrified sperm AI.2 These findings suggest that vitrification may be a suitable option for cryopreservation of felid semen in a field situation, especially if combined with newer semen collection methods that do not require electroejaculation.1
The authors thank Procter & Gamble Pet Care for their generous funding of this study through the P&G Wildlife Conservation Scholarship program. We also thank the keeper staff at CREW for domestic cat care, and the veterinary staff at the Cincinnati Zoo & Botanical Garden for assistance with nondomestic cat procedures.
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