ProAKAP4 is a sperm structural protein that regulates motility through the PKA-dependent cAMP signaling pathway, which is synthesized as an X chromosome-linked member of the gene family. This study aims to determine the optimal level of proAKAP4 for evaluating sexed semen through investigating its relationship with the longevity of sperm quality in sexed Holstein bull sperm. A total of 30 sexed sperm samples (bearing X chromosomes) from 30 distinct Holstein bulls (n = 30) were analyzed. The frozen bull sperm samples were assessed for their proAKAP4 levels, mitochondrial membrane potential, plasma membrane and acrosome integrity (PMAI), and spermatozoa movement parameters at hours 0 and 3 after thawing. The proAKAP4 levels in the sexed sperm samples ranged from 16.35 to 72.10 ng/10 M spz, with an average of 37.18 ± 15.1 ng/10 M spz. A strong positive correlation was observed between proAKAP4 levels and total motility, progressive motility, PMAI, high mitochondrial membrane potential, VAP, and VCL values after 3 h of incubation, when compared to post-thaw analyses. The results also reveal that spermatozoa with proAKAP4 levels of ≥40 ng/10 M spz exhibit higher quality. In conclusion, the level of proAKAP4 in sexed sperm aligns with previous studies and shows potential as a biomarker for assessing the longevity of sexed sperm quality.

In this project, ejaculates (n=34) were collected from three Brown Swiss bulls using the artificial vagina method, then divided into seven equal parts and diluted to 60×10⁶ spermatozoa/mL using the following extenders: Control, Control+1mM carnosic acid (CR1), Control+3mM carnosic acid (CR3), Control+2.5mM glutamine (G2.5), Control+7.5mM glutamine (G7.5), Control+CR1+G7.5, and Control+CR3+G2.5. All diluted aliquots were frozen after 3 hours of equilibration. Thawed samples were analyzed for motility, plasma membrane and acrosome integrity (PMAI), mitochondrial membrane potential (MMP), and DNA integrity. The Control, G7.5, and G2.5 groups showed the highest total and progressive motility. The highest PMAI value was observed in the Control group, while the lowest was in the CR3 group. The PMAI values of G2.5, G7.5, and G7.5+CR1 were significantly higher than those of CR1, CR3, and CR3+G2.5. There were no statistically significant differences in MMP among the groups. The lowest DNA damage was observed in the Control group and the highest in CR3+G2.5. Control, G2.5, G7.5, and CR1+G7.5 groups had statistically similar kinematic values (VAP, VSL, VCL) and outperformed the others. Although 2.5 mM and 7.5 mM glutamine showed a synergistic interaction, they did not significantly improve spermatological parameters. Carnosic acid appeared to act as an antagonistic antioxidant.

The main objective of sperm production centers is to produce as many straws as possible from the obtained ejaculates using the optimal dilution rate. To this end, this study is the first to evaluate the effect of different semen extender rates on Anatolian buffalo semen quality. Ejaculates were collected by artificial vagina from three Anatolian buffalo bulls (n=105). These ejaculates were divided into three aliquots and filled into 0.25 ml straws with soy-based extenders at the concentrations of 35, 25, and 15 million spermatozoa/straw. The straw samples of different spermatozoa concentrations were frozen. The quality of spermatozoa was evaluated after thawing (37 °C, 30 sec) and following the thermoresistance test (37 °C, 3 h). The post-thaw total motility and progressive motility values were similar between the groups. However, following the thermoresistance test, there was a significant decrease in total motility in the 35 million spermatozoa/straw group, and the progressive motility was significantly higher in the 25 million spermatozoa/straw group. There was no statistically significant difference between the groups in terms of sperm kinetic parameters, except VSL after thawing except the VAP and LIN values following the thermoresistance test. In conclusion, it is recommended to dilute the Anatolian buffalo semen at a concentration of 25 or 100 million spermatozoa/ml per straw when freezing it with a soy-based semen extender. In addition, it is considered that soy-based extenders compensate for cryo-damage to sperm motility for a short time, and the thermoresistance test should be applied for objective evaluation in dose optimization studies.