Absence of inter-horn migration validates bilateral embryo transfer as a within-dam experimental design in mice

Embryo transfer (ET) is a foundational technique in mouse reproductive biology. Bilateral ET, in which embryos of different genotypes are placed in opposite uterine horns of a single recipient, offers strict control over maternal variables but requires that embryos remain in the horn to which they were transferred. Here, we tested this assumption directly by transferring BTBR T⁺Itpr3^tf/J (BTBR) and C57BL/6J (B6) blastocysts into opposite horns and genotyping mid-gestation fetuses by strain-specific PCR. Across four experiments varying embryo load, recipient strain, and donor-recipient synchrony, no genotype-discordant fetus was detected contralaterally. These findings validate bilateral co-transfer of genotypically distinct embryos as a methodologically safe design that halves the number of recipient females while providing rigorous within-dam experimental control.