The clouded leopard, a spotted, mid-sized cat native to southeast Asia, is classified as Endangered by the U.S. Fish and Wildlife Service. The Species Survival Plan (SSP) program has designated that clouded leopards in North American zoological institutions are a research population with the focus of enhancing management and developing assisted reproductive techniques. In this study, we examined (1) ovarian response to exogenous gonadotropin stimulation, (2) in vitro production of embryos by IVF and intracytoplasmic sperm injection (ICSI), and (3) in vivo developmental ability of in vitro-derived embryos. Eight females at Audubon Center for Research of Endangered Species (ACRES) in New Orleans, LA, were used as oocyte donors and embryo recipients. During non-estrual, but otherwise unknown, stages of the estrous cycle, females 4 to 10 years old at our first treatment were administered a total of 15 (n = 8) or 20 (n = 7) IU of porcine FSH (i.m; Sioux Biochemical Co., Sioux City, IA) daily for 4 days. On Day 5, 10 (n = 8) or 15 (n = 7) IU of porcine LH (i.m; Sioux Biochemical Co.) was given and laparoscopic oocyte retrieval (LOR) was done 24 h later. One, 2, or 3 LOR were performed on 3, 3, and 2 females, respectively (total n LOR = 15), 10 (67%) of which were done on females that were 8 to 12 years of age. A total of 176 pre-ovulatory oocytes (mean = 14.7; range = 3-31) were recovered from 12 LOR performed on 6 females. Two females, a 5-year-old and an 11-year-old, did not respond to gonadotropin treatment. Of 5 females with ≥2 LOR, an average of 16.2 and 14.6 oocytes were recovered at the first and second LOR, respectively. Luteal tissue was present on the ovaries at 3 of 4 LOR performed during January-April as compared with 0 of 11 LOR performed from June-December. Semen was obtained by electroejaculation of 3 males (6, 9, and 11 years of age) and used after cryopreservation [TEST-yolk (media prepared with egg yolk, TES, and Tris) + 6% glycerol] or after storage at 4°C for 24 h (cooled). In vitro production, cryopreservation, and transfer of embryos were done as described by Pope et al. (2006 Theriogenology 66, 1518-1524). Cleavage frequency was 43% overall, 55% (64/117) after IVF, and 20% after ICSI with frozen (10/43) or cooled (2/16) sperm. After IVF with frozen sperm, cleavage rate was 63% (48/76) v. 39% (16/41) with cooled sperm. On Days 5, 57, 26, and 17% of embryos were morula (> 16 cells), early morula (≈16 cells) and pre-morula (< 16 cells), respectively. Forty-four Day 5 embryos were cryopreserved at a slow, controlled rate, 24 of which were thawed and transferred by laparotomy to the uteri of 3 Day 5 gonadotropin-treated recipients (7-9 embryos/female). Similarly, 28 Day 5 fresh embryos were auto-transferred to 3 Day 5 recipients (5-15 embryos/female). Most embryos transferred were morulae (83%) produced by IVF using frozen sperm (71%). None of the recipients established pregnancies, a failure that, undoubtedly, was multifaceted. Age may have been a factor because most recipients were 8 to 11 years old. Nonetheless, we have (1) shown that clouded leopard females are responsive to exogenous gonadotropins, (2) produced embryos in vitro, both by IVF and ICSI, using both cryopreserved and cooled sperm, and (3) provided further evidence of seasonality in ovarian activity.
Trans-vaginal oocyte retrieval and subsequent in vitro production of embryos from a cow involuntarily culled : case report