SUCCESSFUL LAPAROSCOPIC OVIDUCTAL ARTIFICIAL INSEMINATION IN THE CLOUDED LEOPARD (NEOFELIS NEBULOSA) IN THAILAND.

Captive breeding of clouded leopards (Neofelis nebulosa) is challenging because of mating incompatibility, high incidence of teratospermia in males, and inconsistent ovulation patterns in females. Assisted reproductive techniques, therefore, are necessary to overcome these issues and maintain the genetic diversity in the captive population. The objective was to use laparoscopic oviductal artificial insemination (AI) to breed genetically valuable females (n = 4; aged 4.5-5 yr) that were unsuccessfully paired. Fecal hormone metabolites (estrogen and progesterone) were extracted and measured by enzyme immunoassay for monitoring of ovarian activity 45 days before and 65 days after laparoscopic AI. For timed insemination, females were injected with 200 IU equine chorionic gonadotropin and 1,000 IU porcine luteinizing hormone (pLH) at the 82-hr interval. Ovarian assessment was performed by laparoscopy 44 hr after pLH administration. One nulliparous female out of four presented two ovulation sites on each ovary. The single female that had ovulated was inseminated with chilled semen collected from two males (8 × 106 and 2.7 × 106 motile spermatozoa, respectively, in each oviduct). A significant increase in fecal progesterone concentrations was observed after AI with a concentration peak (500 µg/g dry feces) detected on day 24 after pLH injection, which was then sustained for more than 45 days after the pLH injection. The delivery of two cubs occurred on day 92 after pLH. Microsatellite marker analysis determined that both cubs were sired by the same male. This is the first report of a successful oviductal AI in the clouded leopard.

The impact of ovarian stimulation protocol on oocyte quality, subsequent in vitro embryo development, and pregnancy after transfer to recipients in Eld's deer (Rucervus eldii thamin).

Propagating genetically valuable individuals through oocyte collection, in vitro fertilization (IVF) and embryo transfer is critical to maintain sustainable populations of the endangered Eld's deer. The objectives of this study were to assess the impact of exogenous FSH injections on (1) the number and in vitro competence of oocytes collected and (2) the developmental potential of resulting IVF embryos after transfer into recipients during the breeding season (February-April). In a pilot experiment, estrus synchronization was conducted in three surplus females (using intravaginal progesterone-releasing devices, CIDRG for 14 days and injections of buserelin (a GnRH agonist). Five days after CIDR removal, ovaries were excised, minced and a total of 133 oocytes were recovered. Following in vitro maturation (IVM) and IVF, 63% of the oocytes formed embryos but only 5% reached the blastocyst stage. In a subsequent study, follicle numbers and diameters were compared between synchronized does stimulated with 0 or 80 mg FSH (-FSH and +FSH; n = 8 does in each group) and oocytes collected either by laparoscopic ovum pick-up or ovariectomy. FSH stimulation increased the main follicular diameter from 2-3 mm to 4-5 mm (P < 0.05) but not the oocyte number (∼20/donor) or the percentage of good quality oocytes (57%) regardless of the treatment. FSH stimulation did not either affect the percentage of cleaved embryos after IVF (25-35%; P > 0.05). Lastly, embryos at the 2-to 8-cell stage (from either + FSH or -FSH groups) were transferred into the oviducts of 11 synchronized recipients. With the +FSH embryos, three pregnancies failed between 90 and 120 days of gestation and two fawns that were born preterm (Days 215 and 224 of gestation) died at birth. In the -FSH group one healthy female fawn was born on Day 234 of gestation. This is the first report of a successful in vitro embryo production and subsequent birth of a live Eld's deer fawn. Further investigations are required to improve IVM/IVF success and the developmental potential of the embryos.