121 fixed-time laparoscopic oviductal artificial insemination (lo-ai) with frozen-thawed semen in pallas’ cats (otocolobus manul)

The Pallas’ cat, a small-sized felid native to Central Asia, is becoming increasingly threatened in the wild. Their ex situ conservation would benefit from effective assisted reproduction methods, especially AI using frozen semen. In our previous research, we showed that laparoscopic oviductal AI (LO-AI) with low numbers of freshly collected spermatozoa could be used to produce viable offspring in Pallas’ cats. In the current study, our goal was to assess the feasibility of LO-AI with frozen semen, using a fixed time AI approach that has proven highly successful in domestic cats. Our specific objectives were to 1) compare two doses of oral progestin for suppressing ovarian function in Pallas’ cats and 2) assess ovarian responsiveness and fertility following gonadotropin treatment and fixed time AI with frozen-thawed sperm. Pallas’ cats (n=5; 6 AI) housed at 4 zoos were fed altrenogest at 1 of 2 doses (0.088 or 0.176mgkg-1 of body weight; 3 cats/dose) for 14–30 consecutive days. Six days after altrenogest cessation, females were injected (IM) with 200IU of eCG followed 85h later with 1000IU of pLH, and inseminated (1–2.5×106 motile sperm/oviduct) using either frozen-thawed (5 AI) or freshly-collected (1 AI) sperm from 1 or 2 males. Endocrine profiles and pregnancy status were assessed with fecal estrogen/progestin EIA, and offspring paternity was determined with short tandem repeat molecular marker analysis. Following altrenogest treatment and gonadotropin injection, all females had pronounced estrogen surges and subsequently ovulated with no difference (P>0.05) between altrenogest doses in CL (9.0±1.5; mean±standard error of the mean) or follicle (1.5±1.1) number at time of AI. Following AI, five females had prolonged luteal phases (>50 days) consistent with early pregnancy. Two of these females had detectable urine relaxin at 45–55 days post-AI, indicative of functional placentae, but did not produce any kittens. A third female gave birth to a full-term (80g of BW) stillborn kitten after a 76-day gestation. Paternity analysis revealed that the kitten was conceived from semen frozen 22 years earlier. These results indicate that oral progestin treatment permits consistent induction of follicular growth and ovulation in Pallas’ cats for fixed-time LO-AI. Although at least 3 females had probable conception, their failure to produce viable kittens suggests possible aberrations in luteal function or pregnancy maintenance. The birth of the first Pallas’ cat kitten following LO-AI with frozen semen (stored for more than 2 decades) provides evidence that this approach may have conservation merit with further refinement. This study was funded by a Collection Stewardship Grant from the Institute of Museum and Library Services, with assistance of veterinary and animal care staff at the Cincinnati Zoo, Columbus Zoo, Pueblo Zoo and Red River Zoo.