Assessing in vitro fertilization ability of pre-freeze holding stage and post-thaw boar epididymal sperm on oocytes matured in two media

Résumé

The use of post-thawed sperm in pigs for in vivo/vitro fertilization (IVF) presents unique challenges due to its compromised molecular and structural alterations. Maturation of pig oocytes is a critical step in IVF success, influencing both fertilization rates and embryo development. This study aimed to investigate the fertilization potential of boar epididymal sperm during two stages (pre-freeze holding at 18 °C and post-thaw) and pig oocytes that have undergone maturation in North Carolinah State University 23 (NCSU-23) and modified NCSU-23 (mNCSU-23) media. The epididymal sperm were retrieved once from six cauda epididymides, pooled to prevent individual variability, and treated as a single biological sample. The pooled sample was split for in vitro fertilization and cryopreservation following 3 h of holding at 18 °C (pre-freeze). The same batch of frozen sperm samples was reanalyzed post-thaw after three months of storage, and in vitro fertilized with oocytes matured in two media. A total of 280 oocytes were equally allocated between the two media (NCSU-23 and mNCSU-23) and matured for 44 h prior to IVF. Following maturation, 100% cumulus cell expansion was recorded, and only 80 oocytes per maturation medium were vortexed and assessed for polar bodies. The remaining 200 oocytes per maturation medium were subjected to IVF, with a total of 100 oocytes being allocated to each sperm type. Data were evaluated with the aid of a general linear model. Post-thaw sperm total motility of 85.9% was noted. The use of mNCSU-23 media resulted in a higher rate of oocytes with polar body extrusion (81%) compared with NCSU-23 (46.2%). No significant difference in the prevalence of two pronuclei was observed between the pre-freeze holding stage and post-thaw sperm, with values remaining below 30% regardless of whether mNCSU-23 or NCSU-23 media were used. Additionally, oocytes matured in mNCSU-23 media exhibited higher overall IVF rates of 91.2% for pre-freeze holding stage sperm and 81.6% for post-thawed sperm, primarily due to the polyspermy. Therefore, both mNCSU-23 and NCSU-23 media show potential but remain substantially limited by high levels of polyspermy. It is recommended that future studies in pigs should address the challenge of polyspermy.

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