Cryostress induces fragmentation and alters the abundance of sperm transcripts associated with fertilizing competence and reproductive processes in buffalo

The study aimed to assess the influence of cryostress on RNA integrity and functional significance in sperm fertilizing ability. The fresh and post-thawed buffalo sperm ( n = 6 each) samples were evaluated for their functional attributes, and sperm total RNA was subjected to transcriptome sequencing followed by validation using real-time PCR and dot blot. Overall, 6911 genes had an expression of FPKM > 1, and among these 431 genes were abundantly expressed (FPKM > 20) in buffalo sperm. These abundantly expressed genes regulate reproductive functions such as sperm motility ( TEKT2 , SPEM1 , and PRM3 , FDR = 1.10E-08), fertilization ( EQTN , PLCZ1 , and SPESP1 , FDR = 7.25E-06) and the developmental process involved in reproduction ( SPACA1 , TNP1 , and YBX2 , FDR = 7.21E-06). Cryopreservation significantly ( p < 0.05) affected the structural and functional membrane integrities of sperm. The expression levels of transcripts that regulate the metabolic activities and fertility-related functions were compromised during cryopreservation. Interestingly, cryostress induces the expression of genes involved ( p < 0.05) in chemokine signaling ( CX3CL1 , CCL20 , and CXCR4 ), G-protein coupled receptor binding ( ADRB1 , EDN1, and BRS3 ), translation ( RPS28 , MRPL28 , and RPL18A ), oxidative phosphorylation ( ND1 , ND2 , and COX2 ), response to reactive oxygen species ( GLRX2 , HYAL2 , and EDN1 ), and immune responses ( CX3CL1 , CCL26 , and TBXA2R ). These precociously expressed genes during cryopreservation alter the signaling mechanisms that govern sperm functional competence and can impact fertilization and early embryonic development.