Naturally sterile *Mus spretus* hybrids are suitable for the generation of pseudopregnant embryo transfer recipients

For the preparation of embryo transfer recipients, surgically vasectomized mice are commonly used, generated by procedures associated with pain and discomfort. Sterile transgenic strains provide a nonsurgical replacement, but their maintenance requires breeding and genotyping procedures. We have previously reported the use of naturally sterile STUSB6F1 hybrids for the production of embryo transfer recipients and found the behavior of these recipients to be indistinguishable from those generated by vasectomized males. The method provides two substantial 3R impacts: refinement (when compared with surgical vasectomy) and reduction in breeding procedures (compared with sterile transgenic lines). Despite initial promise, the 3Rs impact of this innovation was limited by difficulties in breeding the parental STUS/Fore strain, which precluded the wider distribution of the sterile hybrid. The value of a 3R initiative is only as good as the uptake in the community. Here we, thus, select a different naturally sterile hybrid, generated from strains that are widely available: the B6SPRTF1 hybrid between C57BL/6J and Mus spretus. We first confirmed its sterility by sperm counting and testes weight and then trialed the recovery of cryopreserved embryos and germplasm within three UK facilities. Distribution of sperm for the generation of these hybrids by in vitro fertilization was found to be the most robust distribution method and avoided the need to maintain a live M. spretus colony. We then tested the suitability of B6SPRTF1 sterile hybrids for the generation of embryo transfer recipients at these same three UK facilities and found the hybrids to be suitable when compared with surgical vasectomized mice and a sterile transgenic strain. In conclusion, the potential 3Rs impact of this method was confirmed by the ease of distribution and the utility of sterile B6SPRTF1 hybrids at independent production facilities.