Optimizing Rat In Vitro Fertilization for Rat Model Cryo-Resuscitation from Frozen-Thawed Sperm

Optimizing cryo-resuscitation from frozen sperm would improve access to cryopreserved rat models. In this study, the possibility of replacing intracytoplasmic sperm injection (ICSI) with in vitro fertilization (IVF) for model cryo-resuscitation from frozen-thawed sperm was investigated. Rat IVF protocol was modified to allow the procedures to be performed during a 9 h workday. The possibility of genetic background-specific modification of the superovulation protocol for the improvement in IVF outcomes was explored. Wild-type and genetically modified Sprague Dawley (SD), Long Evans (LE) and Fischer 344 (F344) rats were used. Sperm freezing and IVF were conducted as previously described. Cleavage, blastocyst formation and hatching of the resulting embryos were used to assess their developmental potential in vitro. The results showed that, with limited repetitions, current sperm freezing and IVF protocols resulted in cleavage rates ranging from 58 ± 11% to 87 ± 7% and blastocyst rates ranging from 21 ± 25% to 54 ± 23%, which are acceptable for the cryo-resuscitation of rat models. With slight modifications, the procedure can be fit into a 9 h workday (SD: 48 ± 35%; F344: 36 ± 13%). Strain/stock-specific differences in oocyte maturation timing were observed: LE females had a two-hour delay compared to SD and F344 rats in response to the same superovulation protocol. However, modifying the protocol for LE rats did not significantly improve IVF outcomes (34 ± 6 vs. 32 ± 12%). Overall, while IVF with frozen-thawed sperm is a promising alternative to ICSI, significant variability remains across strains/stocks and protocols. Continued research is necessary to advance our understanding of factors affecting the efficiency and repeatability of rat sperm freezing and IVF.