This will give rise to fertilized embryos that are 0.5 d.p.c. Mice that mate earlier may yield a much higher number of fertilized embryos that have already fused their pronuclei and lost any visible pronuclear membrane, or have advanced past the first cellular division to the 2-cell stage of development. Neither of these embryos can be successfully microinjected, as the placement of the injection needle to deliver the DNA construct cannot be determined. Any mice to be used as pseudopregnant embryo recipients for these injected eggs should be mated in synchrony with these mice also. The stud males' ability to plug female mice should be carefully monitored and tracked to ensure maximum mating efficiency and a high yield of fertilized eggs harvested per superovulated female.
Several factors control the reproductive performance of superovulated females and stud males.
Breeding conditions such as light cycle and timing must be carefully controlled and regulated. The time of administration of PMS and hCG and the light-dark cycle of the animal facility is critical to the synchronous development of the eggs and the number harvested from the superovulated female. If female mice are ordered from an outside supplier, they should be allowed approximately one week to adjust to the animal rooms light-dark cycle before administration of superovulatory hormones. This will also synchronize the endogenous release of LH stimulated by the PMS injection. Controlling the release of endogenous LH is important because the hCG must be administered prior to the natural LH surge in order to precisely control ovulation.