Refining Asian elephant semen technologies: The need for improved quality analysis and preservation techniques

The ultimate aim of a breeding management programme for captive Asian elephants is to establish a self-sustaining population, while maintaining maximal genetic diversity. In this respect, artificial insemination (AI) is considered a crucial tool for facilitating the exchange of genetic material between animals housed in different areas and emanating from different founder populations. Moreover, an AI programme can be most effectively implemented if spermatozoa are cryopreserved, since this allows storage of unlimited duration and makes it easier to ensure semen availability when a female is at the appropriate stage of the cycle. At present, two of the major obstacles to more widespread implementation of AI within captive Asian elephant populations are the poor quality of semen collected from many adult bulls, and difficulties in maintaining adequate sperm fertilizing capacity during cooled storage or cryopreservation. In this thesis, the potential contribution of sperm DNA damage to the loss of fertility during the storage of elephant sperm was assessed by developing a dynamic test of sperm DNA stability based around the sperm chromatin dispersion (SCD) test. It transpired that Asian elephant spermatozoa are highly susceptible to iatrogenic DNA damage during storage or preservation, probably as a result of the low cysteine residue content of the P1 protamine and relatively high contribution of protamine P2 to the total protamine pool employed in sperm DNA packaging. The susceptibility to DNA damage was emphasized by the demonstration that a semen diluent designed to prevent DNA damage was better for preserving DNA integrity during cooled storage than the diluent used more commonly for storing elephant sperm, when examined using the dynamic test. This suggests that attention to the effects of diluent and storage technique on DNA integrity should be considered important components of selecting and refining semen preservation protocols for Asian elephants; inclusion of additional antioxidants in the diluents may further improve sperm DNA stability during preservation. It was also demonstrated that the quality of fresh semen significantly influences post-thaw quality of Asian elephant spermatozoa. For this reason, the high proportion of fresh ejaculates from Asian elephants that have very poor sperm quality is a major obstacle to attempts to develop semen preservation protocols that can be applied to a large proportion of potential breeding males, and thereby meet the goal of preserving a genetically diverse breeding population. Increasing age, sperm accumulation as a result of infrequent ejaculation, and small size of the ampullae were all associated with poor sperm quality in Asian elephants. In particular, we demonstrated that sperm accumulation may be a significant contributor to poor semen quality in some Asian elephant males, because semen quality could be improved by frequent (alternate day) semen collection. Since it is important to retain as many males from different genetic lines as possible within the breeding pool, further studies to investigate and ameliorate the origins of poor semen quality and techniques to maintain the fertilizing capacity of spermatozoa during cooled or frozen storage are considered critical to the viability of the captive Asian elephant population.