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.

Genetic diversity of the captive Asian tapir population in Thailand, based on mitochondrial control region sequence data and the comparison of its nucleotide structure with Brazilian tapir.

The Asian tapir (Tapirus indicus) has been classified as Endangered on the IUCN Red List of Threatened Species (2008). Genetic diversity data provide important information for the management of captive breeding and conservation of this species. We analyzed mitochondrial control region (CR) sequences from 37 captive Asian tapirs in Thailand. Multiple alignments of the full-length CR sequences sized 1268 bp comprised three domains as described in other mammal species. Analysis of 16 parsimony-informative variable sites revealed 11 haplotypes. Furthermore, the phylogenetic analysis using median-joining network clearly showed three clades correlated with our earlier cytochrome b gene study in this endangered species. The repetitive motif is located between first and second conserved sequence blocks, similar to the Brazilian tapir. The highest polymorphic site was located in the extended termination associated sequences domain. The results could be applied for future genetic management based in captivity and wild that shows stable populations.

The complete mitochondrial genome of the Asian tapirs (Tapirus indicus): the only extant Tapiridae species in the old world.

Asian tapir (Tapirus indicus) is categorized as Endangered on the 2008 IUCN red list. The first full-length mitochondrial DNA (mtDNA) sequence of Asian tapir is 16,717 bp in length. Base composition shows 34.6% A, 27.2% T, 25.8% C and 12.3% G. Highest polymorphic site is on the control region as typical for many species.

PCR-based method for sex identification of Eastern sarus crane (Grus antigone sharpii): implications for reintroduction programs in Thailand.

Due to human activity and a reduction in the size and quality of wetland habitats, populations of the Eastern sarus crane (Grus antigone sharpii) have declined dramatically across their range in Southeast Asia. Conservation efforts in Thailand have focused on reintroduction of the founders harboring the highest genetic diversity. One of the most important requirements to ensure the persistence of the reintroduced populations is a balanced sex ratio. In this study we tested three simple PCR-based methods which may be used for reliable sex identification in G. a. sharpii. The first method employs two combined primer sets based on a 0.6 kb EcoRI fragment (EE0.6). The second method is based on the intronic length polymorphism of the chromo-helicase DNA binding protein (CHD). The last technique relies on PCR-RFLP technique. The sex of six known and 24 unknown cranes were successfully identified by all three methods. These PCR-based sex identification methods are also useful for captive breeding management of G. a. sharpii.

Environmental management procedures following fatal melioidosis in a captive chimpanzee (Pan troglodytes).

A 40-yr-old male captive chimpanzee (Pan troglodytes) presented with depression and anorexia for 7 days. The tentative diagnosis, following a physical examination under anesthesia, was pneumonia with sepsis. Despite antibiotic treatment and supportive care the chimpanzee died a week following presentation. Gross pathology confirmed severe purulent pneumonia and diffuse hepatosplenic abscesses. Detected in serum at the time of the initial examination, the melioidosis serum antibody titer was elevated (> 1:512). Soil samples were collected from three sites in the exhibit at three depths of 5, 15, and 30 cm. By direct and enrichment culture, positive cultures for Burkholderia pseudomallei were found at 5 and 15 cm in one site. The other two sites were positive by enrichment culture at the depth of 5 cm. To prevent disease in the remaining seven troop members, they were relocated to permit a soil treatment with calcium oxide. The exhibit remained empty for approximately 1 yr before the chimpanzees were returned. During that period, the soil in the exhibit area was again cultured as before and all samples were negative for B. pseudomallei. Following the soil treatment in the exhibit, all chimpanzees have remained free of clinical signs consistent with melioidosis.