122 BIRTH OF BLACK-FOOTED CAT KITTENS AFTER TRANSFER OF CRYOPRESERVED EMBRYOS PRODUCED BY IN VITRO FERTILIZATION OF OOCYTES WITH CRYOPRESERVED SPERM

Abstract

The black-footed cat (Felis nigripes), a diminutive spotted cat whose native habitat is arid grasslands in South Africa, Namibia and Botswana, is classified as vulnerable by the International Union for Conservation of Nature and is listed as CITES Appendix I. They are perhaps the rarest of the African cats and their status is threatened by habitat deterioration and poisoning from ingestion of baited carcasses intended for other species of cats. Here, we examined (1) ovarian response of black-footed cat females to exogenous gonadotropin stimulation, (2) in vitro production of embryos by IVF with cooled vs cryopreserved sperm and (3) in vivo developmental ability of in vitro–derived embryos. Six females, 1.5 to 2.5 years of age at first treatment, were administered a total of 3.0 to 3.6 IU of porcine FSH (IM; Sioux Biochemical Co., Sioux City, IA) daily over 4 days. On Day 5, 3.0 (n = 12) or 5.0 (n = 2) IU of porcine LH (IM; Sioux Biochemical Co.) was given and laparoscopic oocyte retrieval (LOR) was done 24 h later. One, two, three, or four LOR were done on 1, 3, 1 and 1 females, respectively (total = 14 LOR). The average age at LOR was 3.3 years (range = 1.5–7.5 years). Semen was obtained by electroejaculation of 3 males (1.5, 6.0, 7.5 years). Anaesthesia for LOR and electroejaculation was induced and maintained, after intubation, with 5 and 2.5% isoflurane, respectively. Sperm samples were used after storage at 4°C for 24 h (TEST yolk, TY) or after cryopreservation (TY + 6% glycerol). Luteal tissue was present on the ovaries at 4 of 5 LOR done during January to May as compared with none of 9 LOR done from June to December. Of 165 oocytes (mean = 11.8) recovered, 38/54 (70%) and 50/106 (47%) underwent cleavage after IVF with cooled or cryopreserved sperm, respectively (P < 0.01, chi-square). None of 5 oocytes cleaved after intracytoplasmic sperm injection with cryopreserved sperm. Procedures for in vitro embryo production were as described previously (Gómez et al. 2006 Theriogenology 66, 72–81; Pope et al. 2006 Theriogenology 66, 1518–1524). Four laparoscopic oviducal embryo transfer procedures were done on Day 1: 2 recipients received fresh Day 2 embryos (n = 5, 8) and 2 recipients received embryos that had been cryopreserved on Day 1 (n = 6) or 2 (n = 8) at a slow, controlled rate in 1.4 M of propylene glycol/0.125 M of sucrose/10% dextran 70. Each recipient (1.75 to 4.5 years) had undergone LOR on Day 0 (5–19 oocytes recovered). Upon ultrasonographic examination on Day 50, a 2.3-year-old recipient of cryopreserved embryos was determined to be pregnant. She delivered 2 live male kittens, without assistance, on Day 69. When first examined at 15 days of age, the kittens weighed 156 and 198 g. At 5 months, their weights were 1.62 and 1.81 kg. The sperm sample used to produce the embryos (in 2005) that resulted in the births of kittens (in 2011) was collected from a male at the Henry Doorly Zoo, Omaha, NE (in 2003), extended and transported overnight at 4°C to New Orleans, LA, before cryopreservation. In summary, we have further demonstrated that assisted reproductive technology can be used for conservation of rare and vulnerable small felids.