Abstract
Background:
The cryopreservation process can negatively affect the functional integrity of thawed ram spermatozoa. Evaluating post-thaw changes in sperm motility, kinematic characteristics, sperm subpopulations, and mitochondrial activity may improve sperm quality and functional stability assessment.

Aim:
This study aimed to evaluate the effects of post-thaw incubation time on sperm motility, kinematic parameters, sperm subpopulations, and high mitochondrial membrane potential (HMMP) in frozen-thawed ram sperm.

Methods:
This study used 48 frozen semen straws obtained from 6 breeding rams. Sperm samples were evaluated 0, 1, and 2 h after thawing. Motility and kinematic analyses were performed using a computer-aided sperm analysis (CASA) system, while HMMP ratios were determined by flow cytometry using the 5,5′,6,6′-tetrachloro-1,1′,3,3′-tetraethylbenzimidazolylcarbocyanine iodide (JC-1) staining method. Functional sperm subpopulations were created based on velocity classes and k-means clustering analysis using CASA data.

Results:
The total and progressive motility rates and percentage of spermatozoa with HMMP decreased significantly depending on post-thaw incubation time (P<0.001). In kinematic analyses, while curve velocity (VCL) values remained largely stable, incubation-related decreases were observed in mean path velocity (VAP), linear velocity (VSL), linearity of the curved path (LIN), linearity of the mean path (STR), and wobble/instability (WOB) values. The significant timeXram interaction found in progressive motility, VSL, LIN, and STR parameters revealed that the spermatozoa’s post-thaw functional stability can vary among individuals. In velocity-based sperm subpopulation analyses, a significant decrease occurred in the proportion of fast and moderately fast spermatozoa during incubation, whereas the proportion of slow and immobile spermatozoa increased. In functional subpopulation assessments based on K-means clustering analysis, a significant decrease in the proportion of spermatozoa exhibiting high velocity and high linearity was detected.

Conclusion:
The post-thaw incubation process in frozen ram semen caused not only a loss of motility but also significant changes in sperm motility dynamics, functional sperm subpopulations, and mitochondrial activity. In this context, the combined evaluation of CASA-based sperm subpopulation analyses and mitochondrial function parameters can contribute to a comprehensive interpretation of sperm functionality after thawing.

Key words: CASA kinematics; Frozen ram semen; Mitochondrial membrane potential; Post-thaw incubation; Sperm subpopulation.