Virmani, MeenakshiVijayalakshmy.K2017-12-142017-12-142017http://krishikosh.egranth.ac.in/handle/1/5810037510Keeping in view the importance of sperm RNA and its correlation with fertility, the proposed study was carried out to determine the gene expression between high fertile and sub-fertile buffalo bull spermatozoa and identify the genes potentially associated with bull fertility through transcriptome analysis. These fertilityassociated genes were further validated with qRT-PCR and their possible role in fertilization through in-vitro fertilization. Ten bulls were classified on the basis of conception rates (CR), where bulls having CR <40% and 50% were considered as sub- and high-fertile bulls, respectively. Total motility, rapid motility, average path velocity, curvilinear velocity, amplitude of lateral head, beat cross frequency, straightness, linearity and intactness of plasma membrane of sperm for high-fertile bulls were significantly higher than sub-fertile bulls. Semen samples from these bulls were utilized for sperm enrichment using three different density gradient methods viz., Percoll, BoviPure and Iodixanol. Purified sperm pellets were subjected to RNA isolation using conventional and kit methods with and without TRIZOL. Maximum yield of RNA was achieved when the semen sample was purified using BoviPure as density gradient followed by isolation of RNA by RNAqueous + heated TRIZOL method. Further, AKAP4, CRISP2, Fertilin- â and PLCZ1 transcripts showed significantly higher expression in high- fertile than sub-fertile groups. Transcriptome analysis was done by Next generation sequencing and a total of 33715 and 21788 transcripts were expressed in sub- and high-fertile groups, respectively. When these transcripts were annotated with the available sequence of buffalo, 1248 and 802 transcripts were uniquely expressed in the sub- and high-fertile groups, respectively and 2527 transcripts were identified as common in both the groups. When in silico translated these transcripts, 258 proteins were observed to be commonly present in both the groups. Apart from these, 6 and 18 proteins were specifically present in the high and sub-fertile group, respectively. The expression of AKAP4, CRISP2, Fertilin- â and PLCZ1 was evaluated in IVF derived embryos. All the four transcripts differentially expressed only in sperm and embryo up to cleaved and blastocyst stage, but these genes didn‟t express in immature and mature oocytes. Thus, the present study provides the first evidence of differential expression of AKAP4, CRISP2, Fertilin â and PLCZ1 genes in high and sub-fertile bulls and their transmission to embryo indicating the role of sperm RNA in fertility and embryo development.enThesis