Executive Summary : | Spermatogonial stem cells (SSCs) are unipotent adult stem cells of the testis, continually responsible for spermatogenesis and production of sperm. The various applications of SSCs technology in livestock production are a) propagation of superior germplasm through transplantation of SSCs from the elite animal b) excellent model for transgenic animal production as SSCs are the only adult stem cells transferring genetic information to the next generation, c) preservation of genetic material of elite animals and endangered species. Recently, the application of SSCs has been focused towards generation of designer bulls for production of either X or Y chromosome bearing sperm, as an alternative to sperm sexing. The applications of this technology will be more effective and feasible once the cryopreservation and transplantation protocol for SSCs are established. Cryopreservation of SSCs has been carried out using different methods of freezing and cryomedia, wherein the post-thaw survivability varied from 40-70%. Since these results are inconsistent and lower than the reported viability of more than 80% for other stem cells, the cryopreservation protocol for SSCs need to be optimized. In addition, cryopreservation studies need to be accompanied by a transplantation study, in order to demonstrate the functional ability. The transplantation of cryopreserved SSCs from long-term culture (P1-P10) of SSCs are very limited. The cryoinjury may be attributed to a) uncontrolled freezing method b) production of reactive oxygen species and c) apoptosis like changes. The damage of cells due to ice-crystallization and solution effect during freezing and recrystallization during thawing are attributed to variations in the susceptibility of cells to different freezing and cooling rates. The major cryoinjury occurs in the critical temperature range of -15⁰C to -60⁰C during cryopreservation. Controlling rate of temperature during cryopreservation can minimize cryoinjury. Very limited study followed the cryopreservation of SSCs using controlled freezing protocol with post-thaw viability of 70%. The use of apoptosis inhibitors and antioxidant in the cryopreservation and thawing media could improve the cryotolerance, post thaw recovery and transplantation efficiency of SSCs. However, no such finding has been reported in livestock. Perusal of studies in livestock revealed that cryopreservation of SSCs based on uncontrolled conventional freezing method provided inconsistent results; freshly isolated SSCs were used for cryopreservation and transplantation studies. Hence there is a need to optimize freezing protocol for the long-term cultured SSCs with maximal viability and functional ability. Hence the present study is proposed with the following hypothesis and objectives: Hypothesis: Improvement in freezing protocol with the additives and controlled cooling rate enhance the cryosurvivability and transplantation efficiency of SSCs. |
Co-PI: | Dr. S. Selvaraju, ICAR- National Institute Of Animal Nutrition And Physiology, Bangalore, Karnataka-560030, Dr. Arangasamy Arunachalam, ICAR- National Institute Of Animal Nutrition And Physiology, Bangalore, Karnataka-560030, Dr. Krishnappa Kariyappa Balaganur, ICAR- National Institute Of Animal Nutrition And Physiology, Bangalore, Karnataka-560030, Dr. Arindam Dhali, ICAR- National Institute Of Animal Nutrition And Physiology, Bangalore, Karnataka-560030, Dr. Ashish Mishra, ICAR- National Institute Of Animal Nutrition And Physiology, Bangalore, Karnataka-560030, Dr. N Ramachandran, ICAR- National Institute Of Animal Nutrition And Physiology, Bangalore, Karnataka-560030 |