221 BASAL SEMINAL TRAITS AND IN VITRO FERTILIZATION IN THE SAND CAT (FELIS MARGARITA)

Abstract

The sand cat (Felis margarita) is one of five small-sized cat species given priority for conservation in North American zoos. An improved understanding of sand cat reproductive biology would benefit captive breeding and facilitate use of assisted reproduction for genetic management. In this study, our objectives were to: (1) characterize basal seminal traits, (2) assess ovarian responses to exogenous gonadotropins, and (3) compare Ham's (HF10) F-10 with 5% fetal bovine serum (FBS) and feline optimized culture medium (FOCM) with 0.4% BSA for supporting gamete function and embryonic development in vitro. Semen was collected by electroejaculation from seven males (n = 10 ejaculates), washed, and resuspended (10 × 10⁶ motile sperm/mL) in HF10 or FOCM for culture (6% CO₂ in air at 38.7°C). Sperm motility (% motile and rate of forward progress, 0-5 scale) was evaluated at 0, 1, 3, and 6 h of culture and used to calculate a sperm motility index (SMI; [% + (5 * rate)]/2). Acrosomal integrity was evaluated by staining (fast green FCF-rose bengal) at 0 and 6 h. For IVF, ovarian follicles were aspirated laparoscopically from female sand cats (n = 4) treated at random times of the estrous cycle with 150 IU eCG and 100 IU hCG (84 h post-eCG) prior to oocyte recovery (25 h post-hCG). Grade 1 oocytes were co-incubated with 2 × 10⁵ motile sperm/mL in HF10 (n = 32) or FOCM (n = 33) for 20 h before transfer to fresh medium. Resulting embryos were either cryopreserved (n = 42) at 30 h post-insemination (hpi) or cultured until Day 7 pi after being moved to fresh medium (FOCM with 5% FBS (n = 10) or HF10 (n = 6)) on Day 3 pi. Ejaculates contained (mean ± SEM) 43.5 ± 11.0 × 10⁶ total spermatozoa, with 77.0 ± 2.3% motility, 43.8 ± 3.9% normal morphology, and 93.1 ± 1.3% intact acrosomes. During 6 h of culture, SMI and % intact acrosomes declined (P < 0.05) slightly (SMI, 73.8-74.8 at 0 h and 68.5-68.8 at 6 h; % intact acrosomes, 87.1-87.6% at 0 h and 69.0-74.2% at 6 h), but similarly (P > 0.05) in both media. Females produced 24.3 ± 5.6 follicles, with 19.3 ± 5.1 total oocytes and 16.5 ± 4.6 Grade 1 oocytes recovered per female. The proportions of oocytes cleaving at 20 and 30 hpi and the quality of the resulting embryos at 30 hpi were higher (P < 0.05) in FOCM (20 hpi, 76.5 ± 8.7%; 30 hpi, 92.9 ± 7.1%; 89.2 ± 7.9% Grade 1) than in HF10 (20 hpi, 29.8 ± 11.7%; 30 hpi, 55.9 ± 20.6%; 62.9 ± 7.2% Grade 1). Two blastocysts developed in FOCM (69.0 ± 19.0 cells), but the final cell numbers of all cultured embryos were not different (P > 0.05) between FOCM (26.9 ± 8.0 cells) and HF10 (19.3 ± 6.4 cells). Compared to other small felid species, sand cats exhibited excellent seminal traits, gonadotropin-induced ovarian responses, and fertilization success in vitro. Although sperm motility and acrosomal integrity were similar in FOCM and HF10, the medium developed specifically for domestic cat embryos (FOCM) better supported IVF and early embryonic development. These results indicate that IVF with fresh spermatozoa could be a valuable tool for genetic management of captive sand cat populations.

This work was supported by MAF D04ZO-72.