Effects of post-thaw incubation time on sperm functional dynamics and mitochondrial activity in frozen-thawed ram semen
Open Veterinary Journal, cilt.16, sa.9, ss.6312-6321, 2026 (ESCI, Scopus)
- Yayın Türü: Makale / Tam Makale
- Cilt numarası: 16 Sayı: 9
- Basım Tarihi: 2026
- Doi Numarası: 10.5455/ovj.2026.v16.i9.37
- Dergi Adı: Open Veterinary Journal
- Derginin Tarandığı İndeksler: Emerging Sources Citation Index (ESCI), Scopus
- Sayfa Sayıları: ss.6312-6321
- Anahtar Kelimeler: CASA kinematics, Frozen ram semen, Mitochondrial membrane potential, Post-thaw incubation, Sperm subpopulation
- Hatay Mustafa Kemal Üniversitesi Adresli: Hayır
Özet
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 six breeding rams. Sperm samples were evaluated 0, 1, and 2 hours 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 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 values remained largely stable, incubation-related decreases were observed in mean path velocity, linear velocity (VSL), linearity of the curved path (LIN), linearity of the mean path (STR), and wobble/instability (WOB) values. The significant time × ram 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.