Optimization of centrifugation protocols for frozen–thawed ovine semen in IVF applications

The use of frozen–thawed semen is a key tool in ovine assisted reproduction, particularly for in vitro fertilization (IVF), which remains the most widely applied technique as intracytoplasmic sperm injection (ICSI) is not yet fully optimized in this species [1]. However, cryopreservation introduces structural and functional damage to spermatozoa and leaves residual cryoprotectants that may exert toxic effects on oocytes and early embryos [2]. Therefore, the identification of effective sperm processing protocols able to remove these compounds while preserving sperm quality is essential to improve IVF outcomes. This study evaluated different centrifugation protocols applied to frozen–thawed ovine semen to identify the most suitable method for IVF applications. After thawing, semen samples were diluted 1:1 with SOFM medium to facilitate the removal of cryoprotectants. Four experimental conditions were tested: (i) dilution only without centrifugation (control), (ii) centrifugation at 150 × g for 10 minutes, (iii) centrifugation at 100 × g for 10 minutes, and (iv) centrifugation at 100 × g for 15 minutes, all performed at room temperature. Sperm motility parameters were analyzed using a Computer-Assisted Sperm Analysis (CASA) system, including total motility, progressive motility, and the percentage of rapid cells. The mean motility values recorded immediately after thawing were 38% for total motility, 10% for progressive motility, and 12% for rapid cells. The dilution-only protocol maintained the initial levels of progressive motility and rapid cells, but resulted in a decrease of total motility of approximately 16%. Centrifugation at 150 × g for 10 minutes at room temperature yielded the best results, with an average increase in total motility of 7.7%, along with a 2% increase in both progressive motility and rapid cells percentage, indicating an overall improvement in sperm kinetic performance. Conversely, centrifugation at 100 × g for 10 minutes negatively affected sperm quality, leading to a reduction of total motility by 16%, progressive motility by 7%, and rapid cells by 8%. Increasing the centrifugation time at the same force (100 × g for 15 minutes) resulted in a smaller decrease in total motility (−10.8%) and no variation in progressive motility, although the percentage of rapid cells still declined by 8%. These findings demonstrate that centrifugation conditions markedly influence the quality of frozen–thawed ovine spermatozoa. Among the tested protocols, centrifugation at 150 × g for 10 minutes appears to represent the most effective compromise between removal of cryoprotectants and preservation, or even improvement, of sperm motility parameters. Ongoing studies are currently assessing the impact of these treatments on fertilization rates and embryo development in IVF systems to validate their practical application in ovine reproductive technologies. References: [1] Gómez MC et al.; Cleavage, development and competence of sheep embryos fertilized by intracytoplasmic sperm injection and in vitro fertilization. Theriogenology. 1998 Apr 15;49(6):1143-54. doi: 10.1016/s0093-691x(98)00062-4. PMID: 10732052. [2] Varisli O et al.; Relationship between toxicity of cryoprotectants, osmotic and oxidative stresses in awassi ram sperm. Cryo Letters. 2022 Mar-Apr;43(2):120-128. PMID: 36626153.