Inducing Pseudopregnancy in Female Mice Without the Need for Vasectomized Males Prior to Non-Surgical Embryo Transfer or Artificial Insemination.

For successfully maintaining pregnancy with embryo transfer or artificial insemination, female recipient mice must be induced into a pseudopregnant state. Female mice are traditionally paired overnight with vasectomized males, and the following morning, the presence of a copulation plug is assessed. To increase the efficiency of producing pseudopregnant females, a cervical manipulation technique has been standardized to be used in combination with non-surgical embryo transfer or artificial insemination techniques in mice. The blunt end of a small plastic rod is inserted vaginally to contact the cervix and is vibrated for 30 s by contact with a trimmer. The procedure is quick and does not require anesthesia or analgesia. This technique increases the reliability and predictability of producing pseudopregnant females and entirely eliminates the requirement for vasectomized males. For CD1 mice, the efficiency of pseudopregnancy induction using cervical manipulation was 83% for females in estrus (N = 76) but only 38% of females in estrus were plugged by vasectomized males (N = 24). Artificial insemination in CD1 mice was performed by estrus synchronization with hormones, cervical manipulation, and the uterine transfer of sperm. Artificial insemination recipients receiving cervical manipulation (N = 76) had a pregnancy rate of 72% and an average litter size of 8.3 pups. This method can also be used to produce pseudopregnant females for non-surgical embryo transfer. Therefore, inducing pseudopregnancy by cervical manipulation is a convenient and efficient alternative to mating with a vasectomized male when performing non-surgical assisted reproduction techniques. Using cervical manipulation provides 3Rs (replacement, reduction, and refinement) benefits for assisted reproduction techniques by reducing the number of animals required and eliminating the necessity for surgically altered males.

Adrenal influence on the persistence of pseudopregnancy in rats.

The effect of adrenalectomy on the duration of pseudopregnancy was investigated in rats. When adrenalectomy was performed three days before cervical stimulation or when it was done on the first day (day 1, day of estrus) or day 2 of pseudopregnancy, the duration of the pseudopregnancy was significantly prolonged (3.0, 2.5 and 1.8 days respectively). This effect of adrenalectomy was not seen when operation was delayed until day 4 of pseudopregnancy. Adrenalectomy on day 2 of pseudopregnancy significantly increased prolactin (PRL) release at the time of the nocturnal PRL surge (5:00) on day 7. When rats were ovariectomized simultaneously with adrenalectomy on day 1, the stimulating effect of adrenalectomy on PRL release was more evident. The effect of active immunization against corticosterone on the continuation of pseudopregnancy was also examined. Neutralization of plasma corticosterone extended the duration of pseudopregnancy and the binding activity of the antiserum positively correlated with the length of continuing diestrus (P < 0.05). These results indicate the negative effect of the adrenal glands, which is probably due to corticosterone, on PRL release in pseudopregnant rats and that the early relief of this inhibition extends the duration of pseudopregnancy.

Hemodynamic changes in pseudopregnancy in chronically instrumented, conscious rats are preserved after hysterectomy.

Hemodynamic changes in early-pregnant and pseudopregnant rats are comparable, indicating that the trophoblast does not contribute to these changes. It is unclear whether the presence of the uterus is needed for the normal early-pregnancy hemodynamic adaptation. In this study we tested the hypothesis that uterine factors do not contribute to the systemic hemodynamic changes in early pseudopregnancy. To this end, we studied systemic hemodynamics in conscious pseudopregnant rats subjected to a hysterectomy, and compared these results with those obtained in a control group of pseudopregnant rats. The animals were studied on days 4, 8, 12 and 19 postmating. On day 8 of pseudopregnancy, cardiac output has increased by 23+/-7% in the hysterectomized group and 15+/-5% in the control group. In both groups this rise in cardiac output was entirely accomplished by a rise in stroke volume, by 28+/-8% and 19+/-5%, respectively. Mean arterial pressure did not change appreciably. Therefore, total peripheral resistance also decreased in both groups (17+/-6%) by day 8. After day 12 the hemodynamic parameters returned to baseline. We conclude that systemic hemodynamic changes in hysterectomized pseudopregnant rats closely resemble those in intact pseudopregnant rats. Therefore, the uterus does not seem to play a role in these changes. This supports the hypothesis that only hormones from ovarian origin trigger the initial hemodynamic adaptation to early pregnancy.