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.

Keywords: artificial insemination, Asian elephant, hormone, semen quality, sex accessory glands.