ProAKAP4 as a protein marker of sperm quality: Insights from frozen-thawed ejaculated and epididymal sperm

The AKAP4 protein is the most abundant protein of the sperm fibrous sheath. This protein is considered instrumental in regulating sperm flagellum motion and its precursor, proAKAP4, is considered a biomarker for sperm quality in different species. Therefore, this study aimed to evaluate the relationship of proAKAP4 concentration on feline sperm kinematic, morphology, and membrane integrity of cryopreserved ejaculated and epididymal sperm. A total of fifteen adult mixed breed male cats between 1 and 3 years of age, with a mean weight of 4.21 ± 0.55 kg were included in this study. The first step of the study involved semen collection by urethral catheterization, using a sterile probe. In the second step, sperm were recovered from the epididymal tail after elective orchiectomy. The samples were cryopreserved in a TRIS-yolk extender. Thawed sperm straws were analyzed regarding their sperm kinematic, morphology and plasma membrane integrity. Sperm proAKAP4 concentration was determined using a commercial proAKAP4 ELISA kit. The relationship between proAKAP4 concentrations and sperm kinematics were evaluated by linear regressions, considering the proAKAP4 concentrations as independent variable and kinematic parameters as dependent variable. To compare concentration of proAKAP4 protein in both collection methods, paired t-test was used (P < 0.05). When comparing proAKAP4 concentrations in ejaculated and epididymal sperm, it was observed that the mean of proAKAP4 in ejaculated sperm were higher, 31.43 ± 3.36 ng/mL (15.26-59.18 ng/mL) than epididymal sperm 19.58 ± 1.67 ng/mL (12.15-32.14 ng/mL) (P = 0.004). It was possible to observe the relationship between proAKAP4 concentration and the parameters: TM (r2 = 0.574, P = 0.001) and dance (r2 = 0.267, P = 0.049) in ejaculated sperm. Also, a relationship between the proAKAP4 concentration and TM (r2 = 0.670, P < 0.001) and the percentage of static sperm cells (r2 = 0.477, P = 0.004) in epididymal sperm was seen. In conclusion, our findings indicated that proAKAP4 concentrations are correlated with kinematic parameters in both ejaculated and epididymal sperm post-thaw, suggesting its role in sustaining flagellar architecture and modulating capacitation-associated motility of felines.