JBRA Assisted Reproduction 2025;29(Suppl.2 SBRA 2025):31
Poster Presentation
29^th Annual Congress of the SBRA. São Paulo/SP - Brazil, 2025
doi: 10.5935/1518-0557.20263555

P-019. Assessment of Seminal Viability and Sperm DNA Fragmentation Following Cryopreservation with Different Cooling Rates

Vera Lucia Langaro Amaral¹, Vittorya Fischer Schirmer¹, Rafaela Silva Maciel Borges¹, Jaiara Veber¹, Alfred Paul Senn²

¹Universidade do Vale do Itajaí - UNIVALI - Itajai - SC - Brasil
²University of Geneva, Switzerland

Objective: This study aimed to simultaneously evaluate the effects of different cooling curves on sperm viability and DNA fragmentation in semen samples subjected to cryopreservation.
Methods: Twenty semen samples from healthy individuals were analyzed. Samples were pre-diluted 1:1 in an egg yolk-free cryoprotectant medium containing glycerol (Ingámed), packaged in 0.5 mL straws, and distributed in triplicate. The straws were exposed to liquid nitrogen vapor for 10 minutes at distances of 2 cm, 5 cm, or 10 cm from the surface. The average cooling rates were −20 °C/min (2 cm), −16 °C/min (5 cm), and −8.5 °C/min (10 cm). Afterwards, the straws were immersed in liquid nitrogen and stored until thawing, which was performed at room temperature for 15 minutes. Progressive motility, total motility, and viability were assessed by comparing pre- and post-cryopreservation values. Additionally, sperm DNA fragmentation was evaluated using a commercial chromatin dispersion kit (Sperm Select^®, Ingámed, Brazil), with 400 spermatozoa analyzed per group. Sperm cells were classified into five halo categories, where categories 1 and 2 indicated intact chromatin and categories 3 to 5 represented different levels of DNA fragmentation.
Results: Progressive motility recovery rates were 45% (2 cm), 42% (5 cm), and 48% (10 cm); total motility recovery was 60%, 59%, and 64%, respectively. Viability recovery rates were 59% (2 cm), 63% (5 cm), and 63% (10 cm). Although all groups showed significant reductions compared to fresh samples, no statistically significant differences were observed among the different cooling curves. Mean DNA fragmentation rates were 18.6% (2 cm), 20.0% (5 cm), and 19.1% (10 cm), also with no significant differences among groups.
Conclusion: Despite numerical variations, no statistically significant differences were found among the tested cooling rates. These findings suggest that, under the evaluated conditions, different vapor-phase cooling rates do not significantly impact sperm motility, viability, or DNA integrity. This supports the feasibility of using various vapor exposure protocols for semen cryopreservation, even without strict standardization of vapor exposure distance.