Functional sperm quality markers to predict bull fertility have been actively investigated. Among them, proAKAP4, which is the precursor of AKAP4, the main structural protein in the fibrous sheath of spermatozoa; appears to be promising, especially since spermatozoa lacking AKAP4 expression were shown to be immotile, abnormal, and infertile. In this study, the objective was to evaluate proAKAP4 concentration values with the classic sperm motility descriptors and fertility outcomes (NRR at 90 days) in post-thawed conditions of 10 bulls’ semen. ProAKAP4 expression was confirmed by Western blotting and proAKAP4 concentrations were determined by ELISA. Variations in proAKAP4 concentrations were observed independently of the motility sperm descriptors measured using computer-assisted semen analysis (CASA). A ProAKAP4 concentration of 38.67 ± 8.55 ng/10 million spermatozoa was obtained as a statistical mean of all samples. Threshold values of proAKAP4 were then determined between 19.96 to 96.95 ng/10 million spermatozoa. ProAKAP4 concentrations were positively correlated with progressive motility and the linearity coefficient. The sperm showing the lowest progressive motility were the samples exhibiting proAKAP4 concentrations below 20 ng/10 million spermatozoa. Furthermore, proAKAP4 concentrations were significantly higher in bulls with a higher NRR in the field. Our results demonstrate a correlation between the semen concentration of proAKAP4 and NRR-90d (p = 0.05) in post-thawed bull semen, highlighting the potential of proAKAP4 as a predictive marker of bull fertility.

Recently, ProAKAP4 has been described as a pertinent indicator of sperm quality in humans, pigs, and stallions. In knockout mouse models lacking AKAP4 expression, the male mice were infertile. As high proAKAP4 levels were significantly correlated with a lower proportion of abortions in intrauterine insemination settings in human reproduction, proAKAP4 could be considered a pertinent new sperm parameter for assessing embryo quality. Our main goal was to assess the proAKAP4 concentrations in Holstein bull semen for comparison with the motility sperm parameters and fertility outcomes in post-thawed conditions. Straws issued from 52 ejaculates from 13 bulls, retrospectively identified with known nonreturn rates (NRR) as a fertility indicator, were provided by Evolution XY. Expression of ProAKAP4 and AKAP4 was assessed using enzyme-linked immunosorbent assay, western blotting, flow cytometry, and microscopy methods. Using the Bull 4MID kit (4BioDx), striking variations in proAKAP4 concentrations were observed independently of the classic sperm parameters that were measured using computer-assisted semen analysis. A mean proAKAP4 concentration of 44.42ng per 10 million spermatozoa was obtained through all our series. Interestingly, the variations in proAKAP4 concentrations were positively correlated with progressive motility and with the linearity coefficient parameter. Furthermore, the post-thawed concentrations of proAKAP4 were significantly higher in bulls with a higher NRR in a field study of more than 190 000 AI. We then demonstrated for the first time a correlation between the semen concentration of proAKAP4 and NRR (P=0.05) in bulls. Threshold values of proAKAP4 were then determined, with good values being between 25 and 60ngmL−1. Below 25ngmL−1, the sperm were of poor quality. The proportion of functional spermatozoa (i.e. spermatozoa expressing proAKAP4 in ejaculates) was assessed using flow cytometry. We observed that the cell debris and dead spermatozoa were never immunolabeled with proAKAP4 antibodies. On testis tissue sections, proAKAP4 was expressed only from the spermatids stages up to the ejaculated spermatozoa, being influenced by external factors and reflecting good spermatogenesis. Our preliminary study highlighted the pertinence of proAKAP4 in assessing sperm quality in bulls. It could be interesting to further analyse the effect of proAKAP4 level of expression on capacitation and IVF. As high levels of proAKAP4 were significantly correlated with fertility rates and with progressive motility, proAKAP4 could be proposed as a predictive marker of bull fertility and could be further investigated to evaluate the quality of invitro-produced embryos.