Identification And Cryopreservation Of Type A Spermatogonia In Chinese Crossbred Buffalo(Swamp×River)

Buffalo is an important livestock species especially in Asia, where, it has been used for milk and meat production and for draught purposes. Importance of this species in this region necessitates improvement in reproduction using different reproductive techniques. Male germline stem cells have received much attention as these cells can be isolated and cultured in vitro. Spermatogonial stem cells (SSCs) can be changed genetically and have further application in producing transgenic buffaloes with desired traits following transplantation in recipient testis. Therefore, this study was designed to optimize isolation, identification, enrichment and cryopreservation procedures in Chinese crossbred buffaloes. Different isolation and enrichment strategies were applied and their effect on viability and yield of spermatogonia was evaluated. Later on, an optimized cryopreservation protocol was established to immortalize these germ cells for long time from proven sires. Below are some salient results for these experiments;1. Isolation, identification and enrichment of type A spermatogonia from the testis of Chinese crossbred buffaloes (Swamp×River)The proportion of type A spermatogonia in the isolated testis cells is a prerequisite for conducting experiments and the manipulation of these germ cells. Thus, the present study was designed to examine the wide range of strategies for the isolation, identification and enrichment of type A spermatogonia in prepubertal buffalo calves (3-6mo). Histological findings revealed the presence of maximum number of type A spermatogonia at5months, which was further confirmed by DBA immunohistochemistry. In a newly modified strategy for isolation of testis tissues, mincing followed by trituration and two rounds of digestion with collagenase, hyaluronidase and DNase yielded more than95%testis cell population. Differential plating with laminin, poly-L-lysine and gelatin significantly (P<0.05) affected the purity of type A spermatogonia. Among these extracellular matrix (ECMs) molecules, laminin and gelatin performed well and reached at a purity of39.38±1.21%and32.15±1.60%, respectively. In addition, combination of laminin and gelatin followed by percoll centrifugation performed the best and yielded>90%type A spermatogonial purity. Moreover, viability of the cells was not affected (P>0.05) irrespective of different enrichment methods. 2. Cryopreservation of Type A spermatogonia in Chinese crossbred buffaloes (Swamp x River)Type A spermatogonia are the continuous source of production for male gametes. Cryopreservation is the potential option to preserve spermatogonial cells. There is a dire need to develop cryopreservation procedure with improved viability and less harmed by reactive oxygen species (ROS). The aim of this study was to develop an optimized cryopreservation technique for the preservation of type A spermatogonia using taurine (2-aminoethanesulfonic acid) as an antioxidant. For this purpose, enriched type A spermatogonia were used and taurine was added at different concentrations (0,15,30,50mM) to freezing media. Viability of spermatogonia was monitored after isolation (enrichment), addition of freezing media and after thawing. After thawing, oxidative stress parameters (MDA, SOD, CAT, GSH, GSH-Px) of frozen-thawed type A spermatogonia were also measured. It was noted that after thawing viability rate was significantly (p<0.004) improved. The highest concentration of taurine (50mM) improved viability as compared to other concentrations and control group. Interestingly, SOD activity significantly (p<0.05) in taurine50mM compared to control, while, MDA and CAT activity decreased significantly (p<0.05) in the same group.3. Expression analysis of spermatogonia specific markers in Chinese crossbred buffaloes (Swamp×River)Spermatogonial stem cells (SSCs) represent an undifferentiated cell population among germ cells. Isolation and enrichment of these cells are critical steps for long-term culture/transplantation/cryopreservation. Identifying spermatogonia using intracellular and cell surface markers (species specific) is important for yielding enriched population and subsequent use. This study was conducted to evaluate four markers i.e. CD34, podocalyxin, CDX2and Rexl as spermatogonia-specific markers which are previously in use for enrichment of other type of cells. Testes from prepubertal (3-6months) and adult (>2-3years) buffalo bulls were collected following surgical castration. Immunohistochemistry, RT-PCR and qPCR and western blotting for these four markers were performed. Immunohistochemistry results revealed that CD34and Rexl were exclusively expressed on spermatogonia, while, PODXL and CDX2were not only expressed on spermatogonia but also by myoid cells. QPCR and western blotting results showed highest levels of expression for all these genes/proteins in prepubertal bulls except for CDX2where both stages showed the same expression level. CD34and CDX2 showed truncated fragments. These findings strongly suggest that spermatogonial cells isolated from prepubertal bulls can be used to establish germ cell line ultimately aiding in transgenic buffalo production.