ProAKAP4 Concentration Is Related to Sperm Motility and Motile Sperm Subpopulations in Frozen–Thawed Horse Semen

Abstract

ProAKAP4 is the precursor of AKAP4 (A-kinase Anchor protein 4), the main structural protein of the fibrous sheath of sperm. The amount of proAKAP4 reflects the ability of spermatozoa to maintain the flagellum activity and functionality up to the site of fertilization and is positively correlated with progressive motility in several mammalian species. The aim of this study was to investigate the relationship between proAKAP4 concentration with horse sperm motility descriptors and spermatic motile subpopulations. For this purpose, a total of 48 ejaculates from 13 different stallions were analyzed. Spermatic motility descriptors were obtained by the CASA system, and four motile subpopulations (SP) with specific motility patterns were statistically identified. ProAKAP4 concentrations were evaluated by ELISA. The relationship between motility descriptors of sperm subpopulations and proAKAP4 concentrations was evaluated. Following a hierarchical cluster statistical analysis, ejaculates were divided into two groups according to their proAKAP4 concentrations, either having low proAKAP4 concentrations (5.06–35.61 ng/10M spz; n = 23) or high (39.92–82.23 ng/10M spz; n = 25) proAKAP4 concentrations (p < 0.001). ProAKAP4 concentrations were positively correlated (p < 0.05) with total and progressive motility, as well as with parameters of velocity. ProAKAP4 amount also showed a negative correlation (p < 0.05) with sperm motile subpopulation number 3, which was the subpopulation with the lowest velocity parameters. In conclusion, proAKAP4 concentration in stallion semen positively reflects sperm progressive motility with the functional velocity kinematic descriptors. Concentrations of proAKAP4 higher than 37.77 ng/10M spz were correlated with a very good quality frozen/thawed stallion semen.

Evaluation of a commercial proAKAP4 kit for the assessment of fresh and frozen–thawed feline spermatozoa

“…ProAKAP4 as a semen quality marker has been gaining increasing attention, possibly due to the availability of an easy‐to‐use commercial ELISA kit and publications confirming a positive correlation between proAKAP4 concentration and sperm motility parameters in many species (Blommaert et al., 2019; Dordas‐Perpinyà, Sergeant, et al., 2022; Dordas‐Perpinyà, Yánez‐Ortiz, et al., 2022; Le Couazer et al., 2019; Malo et al., 2021; Riesco et al., 2020). We did not find such correlations for feline spermatozoa, and even found negative correlations with VAP and VSL for cryopreserved samples, similar to one study in mice (Boersma et al., 2022).…”

“…For example, in horses, the first reported values for frozen–thawed semen were between 3 and 12 ng/mL (isolated from around 7.5 × 10 6 spermatozoa), while 2 years later, the same team in another study obtained a mean concentration 60.9 ± 2.1 ng/10 million spermatozoa (Blommaert et al., 2019, 2021). Another study of frozen–thawed horse semen classified values of 5.06–35.61 ng/10 million spermatozoa as ‘low’ and values of 39.92–82.23 ng/10 million spermatozoa as ‘high’ (Dordas‐Perpinyà, Yánez‐Ortiz, et al., 2022). A proAKAP4 concentration of between 3.67 and 10.73 ng/10 million spermatozoa was reported for fresh camel semen (Malo et al., 2021), whereas it was around 60 ng/mL per million spermatozoa for fresh ram semen (Riesco et al., 2020), and around 300 ng/mL per million spermatozoa in rabbits (Gardela et al., 2023).…”

“…Therefore, proAKAP4 concentrations are considered to be a marker of the ability of spermatozoa to exhibit sustained motility (Delehedde et al., 2019). Furthermore, proAKAP4 concentrations have been correlated with total and progressive motility in multiple species, such as stallions (Blommaert et al., 2019; Dordas‐Perpinyà, Yánez‐Ortiz, et al., 2022), bulls (Dordas‐Perpinyà, Sergeant, et al., 2022), dogs (Le Couazer et al., 2019), rams (Riesco et al., 2020) and camels (Malo et al., 2021).…”

“…. Another study of frozen-thawed horse semen classified values of 5.06-35.61 ng/10 million spermatozoa as 'low' and values of 39.92-82.23 ng/10 million spermatozoa as 'high'(Dordas-Perpinyà,Yánez-Ortiz, et al, 2022). A proAKAP4 concentration of between 3.67 and 10.73 ng/10 million spermatozoa was reported for fresh camel semen(Malo et al, 2021), whereas it was around 60 ng/mL per million spermatozoa for fresh ram semenFrozen-…”

ProAKAP4 as Indicator of Long-Lasting Motility Marker in Post-Thaw Conditions in Stallions

“…ProAKAP4 is a marker of sperm quality and male fertility in different species [12], including in stallions [13][14][15][16][17]. ProAKAP4 is the precursor of A-kinase anchor protein 4, which is the main protein of the fibrous sheath of the principal piece of the flagellum [12,18].…”