First Detection of γH2AX in Equine Spermatozoa Through Flow Cytometry and Its Association with DNA Status and Apoptotic-like Changes

Histone H2A family member X phosphorylated at serine 139 (γH2AX) is a marker of the cellular response to DNA double-strand breaks. To our knowledge, this study reports the detection of γH2AX by flow cytometry in equine spermatozoa and provides preliminary evidence through its association with DNA status n and apoptotic-like changes. A total of 64 semen samples from 12 stallions were analyzed under routine field conditions. Sperm viability, mitochondrial membrane potential, Annexin V positivity, and γH2AX levels were assessed using multiparametric flow cytometry. γH2AX showed a wide distribution among samples and was correlated with Annexin V positivity and PI positivity. Linear mixed-effects models, including stallion as a random effect, identified significant associations between γH2AX levels and both Annexin V positivity and PI positivity. In contrast, γH2AX was not significantly related to sperm viability or mitochondrial membrane potential. These findings indicate that γH2AX-associated fluorescence can be detected in equine spermatozoa and is associated with indicators of apoptotic-like changes and DNA instability, which may be related to nuclear DNA damage rather than general sperm functional status. The observed associations suggest that γH2AX may have potential as an additional parameter in multiparametric flow cytometry approaches for the assessment of sperm nuclear status in stallions. However, further validation against established DNA damage assays and appropriate specificity controls is required to determine whether γH2AX fluorescence provides a specific measure of sperm nuclear DNA damage.