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

## TL;DR

This study improves rat in vitro fertilization (IVF) to replace the costly ICSI method for recovering rat models from frozen sperm, making the process more accessible and efficient.

## Contribution

The study optimizes rat IVF protocols to enable reliable cryo-resuscitation of rat models using frozen sperm, reducing reliance on ICSI.

## Key findings

- Modified IVF procedures can be completed within a 9-hour workday with acceptable embryo development rates.
- Cleavage and blastocyst rates from frozen sperm IVF are sufficient for rat model cryo-resuscitation.
- Strain-specific differences in oocyte maturation timing exist but adjusting protocols for LE rats did not improve outcomes.

## Abstract

Rat is one of the most important models in biomedical research. Like mice, rat models are also maintained as frozen sperm, frozen embryos or both. However, the recovery of rat models from frozen sperm must rely on costly and difficult procedure-intracytoplasmic sperm injection (ICSI) in contrast to mouse model’s recovery through in vitro fertilization (IVF), which is relatively simple and cost-effective. Recent advances in rat IVF have made it possible in replacement of ICSI, though significant variation still exists among different strains/stocks. The present study aimed at optimizing the current IVF protocol so that it can be used as a reliable, cost-effective alternative to ICSI for rat model recovery. The results showed that satisfactory cleavage and blastocyst rates can be obtained with limited repetition of IVF. By adjusting the IVF procedures, IVF can be accomplished within a regular 9 h workday schedule. Modified superovulation protocol based on the timing of first polar body extrusion in LE strain did not result in significant improvement in IVF outcomes. Current results demonstrate that IVF can be routinely used for cryorecovery of rat lines with ICSI as a back-up procedure in situations where repeated IVF failures occur.

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.