C57BL/6J and B6129F1 Embryo Transfer: Unilateral and Bilateral Transfer, Embryo Number and Recipient Female Background Control for the Optimization of Embryo Survival and Litter Size

SIMPLE SUMMARY: Embryo transfer is a common procedure in rodent facilities related to rederivation protocols, recovery of cryopreserved embryos and production of genetically engineered animals. This procedure consists of the transfer of mouse embryos into the oviduct of a pseudopregnant recipient female in order to obtain live pups. The aim of this study is to further characterize the optimal conditions to perform embryo transfer using wild type strains and particularly the bilateral transfer. C57BL/6J and B6129F1 embryos were freshly collected and transferred to recipient females, after overnight culture to a 2-cell stage and tested for different conditions (unilateral and bilateral surgical procedures, variable number of embryos and reciprocity between recipient mother and embryo’s genetic background). The results achieved show that C57BL/6J transfers with a low number of embryos provide higher success rates when using unilateral transfers, but for bilateral transfers a minimum number of embryos seems to be necessary. B6129F1 presented similar results, but bilateral transfers were more effective with low number of embryos. These results allow a better planning of the embryo transfer procedure, considering low number of embryos and the choice of unilateral transfers as the ideal condition for an optimal outcome. This optimization has a positive impact on the 3R’s application: it can help to reduce the number of recipient and donor females and to improve recipient female’s welfare through the use of a less invasive technique. ABSTRACT: Embryo transfer (ET) is a common procedure in rodent facilities. Optimizing this technique may help to reduce the number of animals, but little information is available regarding wild type strains and the conditions that affect embryo transfer. To explore this theme, 2-cell C57BL/6J embryos were transferred after overnight culture of freshly collected zygotes using different conditions: unilateral transfers using a total of 6, 8, 12, 15, 20 and 25 embryos were performed initially; then, this strain was also used for bilateral transfers using a total of 6, 12 and 20 embryos equally divided by the two oviducts. Groups of 25 embryos were not tested for the bilateral technique, since this condition produced the lower success rate when using the unilateral technique and 20 embryos would still represent a large number of embryos. A group of 2-cell B6129F1 embryos was also transferred using unilateral and bilateral ET with 6, 12 and 20 embryos. Crl:CD1(ICR) were used as recipient females for non-reciprocal transfers and C57BL/6J were used to test reciprocal transfers (only tested for six C57BL/6J unilateral transfers). Unilateral transfers using C57BL/6J mice produced higher success rates using six embryos, compared to the other groups transferred unilaterally (p-values between 0.0001 and 0.0267), but the mean number of pups per litter was not different among groups. Bilateral transfer produced higher number of pups when 20 embryos were divided by the two oviducts compared to six (p = 0.0012) or 12 (p = 0.0148) embryos, but with no differences in success rates. No statistical differences were found between the groups of B6129F1, but better results were obtained on bilateral transfers using a total of six embryos. For the strain tested (C57BL/6J), the uterine environment (Crl:CD1(ICR) or C57BL/6J recipient) does not impact the outcome of the technique. These results complement previous work published using genetically engineered mice strains and show that unilateral transfers using low number of embryos (6), produce better outcomes when compared to bilateral or unilateral transfers using more embryos. It also highlights differences between the outcome of bilateral transfers in the two strains tested. A set of historical data of genetically engineered mice at a C57BL/6J background was also included, confirming that lower embryo numbers are related to higher success rates. Together, the outcome of these experiments can be important to reduce the number of recipient and donor females, optimize embryo transfers and improve animal welfare discouraging the use of a more invasive technique.