114 GETTING THE YOLK OUT: THE USE OF A SOY LECITHIN-BASED CRYOMEDIUM FOR SEMEN BANKING IN THE PALLAS’ CAT AND FISHING CAT

Abstract

Classically, semen from nondomestic felids has been frozen in an egg yolk-based cryomedium (TEY). However, the inclusion of egg yolk is problematic, resulting in possible batch to batch variation, microbial contamination, and regulatory concerns with international semen transport. Our previous research revealed that a soy lecithin-based cryopreservation medium (SOY) was superior to TEY for freezing domestic cat sperm. However, there is limited information on the efficacy of SOY in nondomestic felids. In the present study, our aim was to assess the effect of SOY v. TEY on motility, acrosome status, and fertilizing capacity of frozen-thawed sperm in 2 wild cat species: the Pallas’ cat (Otocolobus manul) and fishing cat (Prionailurus viverrinus). Semen was collected via electroejaculation from male cats (n = 3/species), split into 2 aliquots, extended in either SOY or TEY (containing 4% glycerol), slow-cooled, and frozen in straws over liquid nitrogen vapor. Sperm motility (percent progressively motile, PM; rate of progressive motility on 1 to 5 scale, RPM) was evaluated at 0, 1, 3, 6, and 24 h post-thaw and acrosome status (AS) was assessed via fluorescein isothiocyanate-peanut agglutinin staining at 0 and 6 h post-thaw. Heterologous IVF was performed using oocytes (n = 10–15/experimental unit, 2 replicates) collected laparoscopically from gonadotropin-treated domestic cats. At 48 h post-insemination, Hoechst33342 staining was used to determine oocyte stage, number of blastomeres, and number of accessory sperm bound to the zona pellucidae of embryos and mature oocytes. The PM, RPM, and AS were analysed with repeated-measures ANCOVA, using pre-freeze values as a covariate. Embryo cleavage % and accessory sperm number were analysed with ANOVA. All data are reported as mean ± s.e. In the Pallas’ cat, PM, RPM, and AS of SOY-treated sperm (35.0 ± 7.6% motile, 2.8 ± 0.4 RPM, 41.2 ± 5.4% intact; 0 h) did not differ (P > 0.05) from TEY-treated sperm (36.7 ± 4.4% motile, 2.8 ± 0.4 RPM, 45.3 ± 9.4% intact; 0 h) at any post-thaw time point. Similarly in the fishing cat, post-thaw PM, RPM, and AS of SOY-treated sperm (38.3 ± 7.3% motile, 2.6 ± 0.2 progression, 20.0 ± 2.9% intact; 0 h) did not differ (P > 0.05) from TEY-treated sperm (31.7 ± 8.7% motile, 2.4 ± 0.3 RPM, 18.3 ± 6.4% intact; 0 h) at any time point. In the Pallas’ cat, neither embryo cleavage % (42.6 ± 7.0% SOY; 61.4 ± 14.8% TEY) nor accessory sperm number (12.2 ± 2.8 SOY; 18.1 ± 4.0 TEY) differed (P > 0.05) between treatments. Fishing cat results were similar, with no difference (P > 0.05) between SOY and TEY for cleavage % (60.4 ± 9.4% SOY; 47.9 ± 5.0% TEY) or accessory sperm number (4.6 ± 1.0 SOY; 5.5 ± 1.0 TEY). Collectively, these findings demonstrate that our SOY medium is an effective alternative to TEY for sperm cryopreservation in 2 nondomestic felid species. The replacement of an egg yolk-based cryomedium with a chemically defined, animal protein-free option represents a significant advance in quality control and biosecurity for cat semen banking and may enhance the use of assisted reproductive technologies for population management of imperiled felids.

Research was funded by the Institute of Museum and Library Services.