Transmembrane protein 95 as a promising molecular marker of ram sperm functionality

The optimization of preservation protocols (refrigeration and freezing) in ovine species is necessary for a wider diffusion of artificial insemination in this species. Besides the ram sperm quality assays, the characterization of novel proteins could be crucial for improving these protocols employing biomarkers. The protein transmembrane 95 (TMEM95) is a sperm membrane protein associated with oocyte-sperm fusion previously described in bull or mouse. However, this protein has not yet been characterized in the ram until now. In this work, different experimental groups based on sperm functionality: capacitated, refrigerated at different times (5 °C 24 h, 5 °C 48 h, and 5 °C 72 h), and frozen-thawed sperm samples were analyzed and compared to initial sperm quality samples (15 °C 3 h) to characterize the expression of this novel protein and its relationship with other sperm quality markers (motility, kinetic parameters, viability, apoptosis-like events, mitochondrial function, acrosome-reacted, zinc content as marker of capacitation). In addition, capacitation status was tested by Fluozin-3, a novel fluorescent probe measuring zinc content used for the first time in ram sperm. After capacitation induction, as expected, acrosome reactive spermatozoa and zinc signature 2 and 3 were significantly increased, while linearity was significantly (P < 0.05) decreased compared to non-capacitated samples. Concerning TMEM95, its profile was significantly (P < 0.05) increased after the capacitation process, confirming its relationship with this spermatozoa status. Attending to preservation processes, as expected, semen quality decreased progressively during liquid storage, and a significant (P < 0.05) decrease was observed at 24 h according to fast progressive motility and linearity. TMEM95 profile showed the same decrease tendency, showing a significant reduction (P < 0.05) at 48 h with respect to the control samples. Finally, after the cryopreservation process, the semen quality of the thawed samples suffered a detrimental effect compared to the initial control sample, concerting all studied parameters accomplished by a significant (P < 0.05) decrease in TMEM95 profile compared to initial control samples. When we analyzed the TMEM95 correlation with other sperm quality markers, the highest positive correlations observed were with low sperm quality parameters in capacitated samples, such as apoptosis-like changes and acrosome-reaction. On the other hand, the highest positive correlations observed between TMEM95 and sperm quality parameters in preservation process samples were observed with suitable sperm quality parameters (motility, viability, and mitochondrial functionality). According to our results, this novel protein could be considered a predictor of early damage in ram sperm preservation protocols (cooling and freezing), considering its relationship with capacitation and membrane integrity status.