37 Investigation of semen collection and cryopreservation techniques in the caracal (Caracal caracal)

Caracals are small savannah cats with populations in Africa, the Arabian Peninsula, and Northern India. Understanding their reproduction is crucial to maintaining zoo and wild populations, but virtually no data exist. This study aimed to (1) evaluate the ability of α-2 agonists to facilitate semen collection via urethral catheterization (UC), (2) compare soy lecithin and egg yolk–based cryomedia for semen cryopreservation, and (3) compare ultra-rapid freezing (URF) to traditional straw-freezing. Adult male caracals (n = 11) in North American zoos were anesthetized with a cocktail of drugs including an α-2 agonist. UC was performed 20–30 min after injection and electroejaculation (EEJ) was then immediately performed. Sperm were recovered from UC in 10 of 11 attempts, with only five attempts yielding samples satisfactory to cryopreserve. UC sperm were extended 1:5 in soy lecithin medium with 0.2 M sucrose (no glycerol), and ~30-µL droplets were pipetted directly into liquid nitrogen. All 11 EEJ attempts produced sperm. On average, 6.2 ± 4.0 × 10⁶ total sperm were recovered via UC, with 51.2 ± 25.6 × 10⁶ additional sperm recovered during successive EEJ. EEJ semen was split into two aliquots and centrifuged, and the resulting sperm pellets were resuspended in soy lecithin with 4% glycerol (SOY) or TEST-egg yolk with 4% glycerol (TEY), sealed in straws, cooled for 2 h, and frozen in two steps over liquid nitrogen vapor. Thawed samples were assessed for motility (% progressive motility, PM; rate of progressive motility on 1–5 scale, RPM) at 0, 1, 3, 6, and 24 h post-thaw; and acrosome status (AS) via FITC-peanut agglutinin staining at 0 and 6 h post-thaw. Heterologous IVF was performed via IVM with domestic cat oocytes. At 36–48 h after insemination, Hoechst33342 staining was used to determine cleavage rate and blastomere number. PM, RPM, and AS were analyzed with repeated measures ANOVA, using prefreeze values as a covariate. Embryo cleavage rate and blastomere number were analyzed with ANOVA. All data are reported as mean ± SE. There was a significant decrease for PM, RPM, and AS from prefreeze to 0 h for all treatments (P < 0.0001). PM and RPM of TEY-treated sperm at time 0 h (20.3 ± 2.6, 1.7 ± 0.1) were greater than SOY (7.2 ± 2.4, P = 0.01; 1.4 ± 0.2, P < 0.0001) or URF (7.6 ± 3.8, P = 0.05; 1.5 ± 0.2, P < 0.0001). There was no difference in AS (P = 0.321) by treatment. TEY had a significantly higher cleavage rate (34.0 ± 3.7) and blastomere number (9.1 ± 1.2) than SOY (19.2 ± 3.7, P = 0.025; 4.3 ± 1.2, P = 0.0132) and URF (18.2 ± 5.0, P = 0.0466; 4.0 ± 1.7, P = 0.045). In conclusion, EEJ was a superior method of semen collection. All cryomedia were effective in preserving fertilization-competent caracal sperm. SOY and URF both have the advantage of being chemically defined and animal-protein free. However, TEY yielded the highest post-thaw PM, RPM, and fertilization rates, suggesting that further refinement of the SOY and URF cryomedia are required.