Semen characteristics and second successful artificial insemination of Asian elephant (Elephas maximus) in Thailand.

Background and Aim: As the number of wild Asian elephants (Elephas maximus) continues to decline, maintaining healthy populations under human care is vital. Male fertility assessment is essential for understanding the reproductive status, which can help to uncover underlying problems and improve the rate of pregnancy success. The objectives of this study in Asian elephants were as follows: (1) To investigate the semen characteristics; (2) to compare the relative seminal vesicle size and semen characteristics; (3) to compare the semen characteristics between good-motile (>60% progressive motility) and poor-motile (<60% progressive motility) ejaculates; and (4) to investigate the pregnancy success rate after artificial insemination (AI) with combined chilled and frozen semen. Materials and Methods: In total, 153 ejaculates were collected by manual rectal stimulation from 25 bulls. The volume, pH, sperm concentration, progressive motility, viability, morphology, and membrane integrity were investigated in each ejaculate. Assessment of accessory sex glands was conducted using transrectal ultrasonography to compare the relative seminal vesicle size and semen characteristics, and the bulls were divided into two groups according to the size of the ampulla (<7 or ≥7 cm2). For the comparison of good and poor-motile ejaculates and semen characteristics, the samples were divided into two groups: Good-motile (>60% progressive motility) and poor-motile (<60% progressive motility) ejaculates. Semen ejaculates for AI were collected from three bulls. The estrous cycles of four females were monitored using an enzyme immunoassay. Seven AI attempts were conducted using frozen and/or chilled semen by endoscopic visualization. AI was repeated 1 day before the luteinizing hormone surge, on the day of the surge, and 1 day after the surge. Pregnancy was confirmed by monitoring the serum progesterone profile and the abdomen and mammary glands changes. Results: From 153 ejaculates, the mean±standard error values of progressive motility, semen volume, sperm concentration, pH, and viability were 40.18%±2.28%, 40.94±3.86 mL, 1,205.58±62.26×106 sperm/mL, 7.50±0.10, and 56.17%±1.96%, respectively. Comparing ampulla size and semen characteristics revealed that the bulls with ampullae of ≥7 cm2 yielded significantly larger volume ejaculates. However, there were no significant differences in sperm motility and concentration. The comparison of semen characteristics between good- and poor-motile ejaculates revealed that the former had significantly higher pH, viability, normal acrosomes, intact membranes, and normal head and tail morphology but often had a significantly lower volume and sperm concentration. From seven AI attempts in four females, one female had a confirmed pregnancy (14.3% pregnancy rate), and delivered a healthy live female baby weighing 128 kg at 21 months and 12 days of gestation. The baby is now 3 years old and in a healthy condition, with normally developing growth and behavior. Conclusion: The semen characteristics of Asian elephants can be used as the baseline reference for further applications. The ampullae size indicates semen quantity but not quality. Our success in producing an elephant calf from AI using frozen and chilled semen demonstrated that AI can be used as an alternative approach for the breeding management of Asian elephants. However, the semen of Asian elephants is of poor quality, especially in terms of membrane integrity; thus, the improvement in semen quality through intensive and careful management of elephant health and fertility remains a challenge for the future. Furthermore, a sperm bank should be established to develop sperm cryopreservation, which will be invaluable for improving the genetic diversity of the Asian elephant.

Biological and environmental factors associated with the detection of elephant endotheliotropic herpesvirus in Asian elephants (Elephas maximus) in Thailand.

Elephant endotheliotropic herpesvirus (EEHV) infection is one of the most common diseases in young elephants, causing severe fatal hemorrhagic disease. Subclinical infection was previously described; however, information about the factors associated with virus shedding and reactivation were scarce. To identify the biological and environmental factors related with EEHV detection, blood and oral swab samples were collected from nine captive Asian elephants in Thailand for one year and tested for EEHV presence using real-time PCR. Data including hematological values, management, environmental temperature, and serum cortisol levels were also recorded and analyzed. Results showed that the viral detection frequency ranged from 0-25%. The highest detection frequency was found in the two youngest elephants, aged less than 15 years. Three types of viruses, EEHV1, EEHV4, and EEHV5, were found in this study, which also detected mixed infection in five elephants. Additionally, the study found that sample type, changes in hematological values, management and health issues, and serum cortisol levels were not associated with herpesvirus detection in the elephants. However, EEHV detection percentage was significantly increased in the summer (mid-Feb to mid-May), possibly due to body fitness reduction from food source limitation and low nutrient content. To obtain a broad aspect of EEHV management, long-term EEHV monitoring is highly recommended in every captive elephant herd.

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.