Differential proteomics highlights specific testicular proteins associated with chicken sperm motility and fertility potential

Sperm motility, the percentage of sperm with forward progressions, is one of the semen quality traits that is highly related with male fertility potential in farm animals. The proportion of roosters with low sperm motility impedes the reproduction efficiency. This study aimed to determine crucial testicular proteins linked to sperm motility defects as causes of subfertility in roosters. iTRAQ was performed with the testis from five low sperm motility roosters and three high ones. A total of 2345 proteins were identified, of which, 86 were differentially expressed (DEPs), including 5 up-regulated and 88 down-regulated proteins in the low group. These DEPs were mainly enriched in cytoskeleton and cilium cell component, and biological processes related to sperm motility, and protein transport, and cellular protein localization. A cluster of 13 down-regulated proteins such as IFT88, TEKT1, ACTN2, DNAH5, RSPH9, and SPAG6, were associated with axoneme assembly. Further western blot and immunohistochemical analysis confirmed the down-regulated SPAG6 expression in low group, and indicated its expression in other cell types in testis beyond round spermatids, and that its pattern was in accordance with testis development and recession pace. Silencing transcription factor SOX5 down-regulated SPAG6 transcripts and impaired the cell proliferation and migration. In summary, this study highlights that down-regulated sperm flagellar structure associated proteins were the potential cause of low sperm motility. More specifically, the highly conserved SPAG6 protein across species is a positive regulator for testis development, spermatogenesis, and sperm motility regulation in chickens.