Pseudopregnant recipient females are used as surrogate mothers to nurture surviving microinjected eggs to birth. Females are made pseudopregnant by mating them with vasectomized males. To increase the chances of a successful mating, only females in estrous are coupled with the males. Female rats and mice maintained on the light-dark cycle described here will ovulate roughly every 4 d. The stage of the estrous cycle for mice can be determined by the appearance of the vagina as detailed in Table 1. For rats, a vaginal smear is required. On the afternoon before the day of implantation, estrous females are paired with a vasectomized male. On the following morning, female mice are examined for the presence of a copulatory plug, evidence that a successful mating has occurred (50-80% of females that are visually determined to be in estrous will be impregnated). In rats, the copulatory plug will often have fallen out by the morning, and there will not be sperm in the vagina to indicate that a successful mating has taken place. However, vaginal stimulation can induce pseudopregnancy, so as a backup procedure, female rats are gently prodded with a smooth blunt probe for 1 min before being placed with the vasectomized males.
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