Background:
The reduced post-thaw motility and the fertility of cryopreserved ruminant spermatozoa impede the success of artificial insemination significantly. Further, the average post-thaw motility of frozen-thawed buffalo spermatozoa is substantially low as compared to that of cattle spermatozoa (30-40% vs. 50-60%). Consequently, the average conception rate in buffaloes inseminated with frozen-thawed semen is lower than that of cattle (approx. 26% vs. 35%). One of the reasons for low sperm motility/fertility in frozen-thawed buffalo semen maybe, over the decades, the semen extender that is used for cryopreservation of cattle semen is also used for buffalo semen even though it has been aptly established that the sperm structure and seminal plasma composition varies substantially between cattle and buffalo. Thus, currently, a suitable species-specific semen extender is not available for buffaloes. Technology Details:
This novel buffalo-specific semen extender contains the essential metabolites that are generally found deficient in buffalo seminal plasma as compared to that of the cattle. The performance of this extender has been validated at the semen cryopreservation center of BAIF, Pune, Maharashtra. This extender improves the post-thaw progressive motility of the cryopreserved buffalo spermatozoa by 5-47% (depending upon the bull) as compared to the extender used traditionally for buffalo. Approximately, 80% of the buffalo bulls responded to this new semen extender and exhibit improved post-thaw sperm qualities (motility, viability and acrosomal integrity) of the cryopreserved semen. Freshly ejaculated mammalian semen is composed of spermatozoa and seminal plasma. The composition of seminal plasma is highly complex and varies among species. Various vital components of seminal plasma are required for sperm survival, metabolism, motility and fertility. Thus, when semen is diluted for cryopreservation, the vital components of the seminal plasma are also diluted and thus this procedure affects the motility and fertility of the semen. Present-day cattle semen extender generally includes a buffering agent such as tris-citrate, an energy source such as fructose, a source of phospholipid for membrane protection such as egg-yolk, a cryoprotective agent such as glycerol and antibiotics to prevent microbial contamination. For the last 2-3 decades, various agents have been added empirically to the semen extender for improving the post-thaw viability, motility and fertility of cryopreserved spermatozoa. However, these approaches remained primarily unsuccessful and most of them do not work and hence could not find a place in the semen cryopreservation protocol of semen stations. Further, most of these agents have been added to the semen extender without knowing whether those are required for the spermatozoa. Because various constituents of the seminal plasma are of paramount importance for sperm health and function; providing a microenvironment similar to seminal plasma during semen dilution and cryopreservation is of utmost importance for successful cryopreservation. To have a comparative perspective, the seminal plasma of both cattle and buffaloes were subjected to mass spectrometry for analysis of selected metabolites. The analyzed data revealed that concentrations of several metabolites were significantly low in the seminal plasma of buffaloes as compared to cattle. Subsequently, a new buffalo-specific semen extender was developed using such deficient metabolites. The developed species-specific novel semen extender improved the average post-thaw motility of cryopreserved buffalo sperm significantly as compared to control (53.25±3.02 in a treated group vs. 31.62±3.18 in the control group).