145 ProAKAP4 concentrations in semen as a predictive tool of bull fertility: A preliminary study

Abstract

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.

Influence of sperm cryopreservation on sperm motility and proAKAP4 concentration in mice

“…In stallions, 21 bulls, 22 and dogs, 23 the proAKAP4 concentration of sperm samples was positively correlated with sperm motility and other sperm kinematic parameters investigated, suggesting that proAKAP4 levels could be uniformly used as a marker for sperm quality and potentially even male fertility. 22 , 24 The roles of both proAKAP4 and AKAP4 in mice have been studied in the context of oxidative stress, 25 but to our knowledge, no study has investigated the relationship between proAKAP4 concentration and sperm quality in laboratory mice, the most commonly used model organism in biomedical research. Given the broad application of sperm cryopreservation and in vitro fertilization in mice, a reliable sperm quality marker would allow for selecting highly fertile males as sperm donors, thereby reducing the number of females needed and contributing to 3R.…”

“…Recently, several studies have directly investigated the link between proAKAP4 and sperm quality. In stallions, 21 bulls, 22 and dogs, 23 the proAKAP4 concentration of sperm samples was positively correlated with sperm motility and other sperm kinematic parameters investigated, suggesting that proAKAP4 levels could be uniformly used as a marker for sperm quality and potentially even male fertility 22,24 . The roles of both proAKAP4 and AKAP4 in mice have been studied in the context of oxidative stress, 25 but to our knowledge, no study has investigated the relationship between proAKAP4 concentration and sperm quality in laboratory mice, the most commonly used model organism in biomedical research.…”

ProAKAP4 as Novel Molecular Marker of Sperm Quality in Ram: An Integrative Study in Fresh, Cooled and Cryopreserved Sperm

“…AKAP4 and proAKAP4 are the most abundantly expressed proteins of the sperm fibrous sheath in all mammals, being part of the principle piece of the flagellum [ 51 , 52 , 53 ]. Moreover, the proAKAP4 precursor molecule has recently emerged as a fertility indicator in a number of species such as humans, boar, and bulls [ 47 , 48 , 50 , 54 , 55 , 56 , 57 , 58 ] providing it with an added value as a diagnostic biomarker of overall semen quality.…”

Cellular and Molecular Insights Into the Etiology of Subfertility/Infertility in Crossbred Bulls (Bos taurus × Bos indicus): A Review

“…In gist, several studies have been conducted on sperm proteomics and transcriptomics in relation to fertility in bull ( Moura et al, 2006 ; Peddinti et al, 2008 ; D’Amours et al, 2010 ; Card et al, 2013 , 2017 ), stallion ( Suliman et al, 2018 ; Griffin et al, 2020 ), and boar ( Kwon et al, 2015 ; Gòdia et al, 2018 ; Dai et al, 2019 ; Fraser et al, 2020 ) and, as a result, few molecular biomarkers for male fertility and semen quality were well established. In case of breeding bulls, proAKAP4 (4MID ® technology, SPQI, Lille, France) based kits were developed ( Sergeant et al, 2019 ; Ruelle et al, 2020 ) for fertility prediction. However, majority of these studies were conducted in purebred bulls.…”