102 Sperm cryopreservation with a soy lecithin-based medium in black-footed cats (Felis nigripes) and sand cats (Felis margarita)

Felid semen has historically been frozen using an egg yolk-based cryopreservation medium (TEY). However, the use of egg introduces several potential concerns, such as variability in composition, microbial contamination, and regulatory issues. Our recent research has focused on developing an animal protein-free medium containing soy lecithin (SOY). Our studies revealed that SOY was superior to TEY for freezing domestic cat sperm and provided similar results for freezing ocelot, Pallas’ cat, and fishing cat sperm. The objective of this study was to compare SOY to the standard TEY for sperm cryopreservation in 2 wild cat species: the black-footed cat and sand cat. Semen was collected from adult male cats (n=6/species) via electroejaculation, split into 2 aliquots, centrifuged, resuspended in either SOY or TEY, slow-cooled, and frozen in straws over nitrogen vapor. Sperm motility [percent progressively motile (PPM); rate of progressive motility on 0-5 scale (RPM)] was evaluated at 0, 1, 3, 6, and 24h post-thaw and acrosome status (AC) was assessed at 0 and 6h post-thaw. Heterologous IVF was performed using oocytes collected laparoscopically from gonadotropin-treated domestic cats. At 48h post-insemination, Hoechst33342 staining was used to determine oocyte stage, number of blastomeres, and number of accessory sperm (AS) bound to the zona pellucida of embryos and mature oocytes. Percent progressively motile, RPM, and AC were analysed with repeated-measures ANOVA; embryo cleavage, blastomere number, and AS number were analysed with one-way ANOVA. All data are reported as least squares means±average standard error. In the black-footed cat, PPM, RPM, and AC of SOY-treated sperm (32.5±4.0% motile, 2.8±0.2 RPM, 41.8±4.1% intact; 0h) did not differ from TEY-treated sperm (44.2±4.0% motile, 2.8±0.2 RPM, 46.8±4.1% intact; 0h) at any post-thaw time point (P > 0.05). Similarly, in the sand cat, post-thaw PPM, RPM, and AC of SOY-treated sperm (36.7±5.2% motile, 2.6±0.2 progression, 53.3±5.8% intact; 0h) did not differ from TEY-treated sperm (45.8±5.2% motile, 2.8±0.2 RPM, 51.0±5.8% intact; 0h) at any time point (P > 0.05). In black-footed cats, neither embryo cleavage (34.1±10.9% SOY; 58.5±10.9% TEY), blastomere number (7.8±0.8 SOY; 6.3±0.8 TEY), nor AS (3.5±0.8 SOY; 1.7±0.8 TEY) differed between treatments (P > 0.05). Sand cat results were similar, with no difference between SOY and TEY for cleavage (44.7±10.8% SOY; 40.6±10.8% TEY) or blastomere number (7.4±2.0 SOY; 6.7±2.0 TEY) (P > 0.05), but AS was higher in SOY-treated sperm (4.3±0.2 SOY; 3.5±0.2 TEY, P=0.0183). These data collectively demonstrate that our SOY medium was an effective substitute to TEY for sperm cryopreservation in the black-footed cat and sand cat. The replacement of an egg yolk-based cryomedium with a chemically defined, animal protein-free alternative represents a significant advance in quality control and biosecurity for felid semen banking and should augment the use of assisted reproduction for population management of imperiled cats. Funded by the Institute of Museum and Library Services.