Using Vaginal Impedance Measurement to Identify Proestrus in Rats Given Luteinizing Hormone Releasing Hormone (LHRH) Agonist

Abstract

Vaginal cytology is the most common method of monitoring the estrous cycle in rats; however, this test requires specific technical training and can be subject to interpretation. Vaginal impedance offers a quicker and less technically challenging alternative and has been used successfully to identify estrus in normally cycling breeder rats. We hypothesize that vaginal impedance can also be used to stage the estrous cycle in rats that have been given luteinizing hormone releasing hormone (LHRH) for timed mating. Vaginal impedance measurements and vaginal cytology were performed in LHRH-primed female rats (n = 36) at the expected peak of proestrus and paired with proven stud males. Breeding success was determined by gross necropsy to detect embryo implantation sites in the female rats. We found that the predictive rates of vaginal cytology and impedance measurement for proestrus were similar; however, both methods resulted in high proportions of false positive and false negative determinations (28% and 31%, respectively). We further hypothesized that females respond to LHRH at variable rates, resulting in variable times of peak proestrus. To test this, vaginal impedance measurements were performed multiple times throughout the expected day of proestrus in LHRH-primed female rats (n = 36). Females were either paired with a male 24 h after reaching the proestrus threshold (n = 18) or paired according to our standard protocol at 1300 h on the day after the expected proestrus (n = 18). Sequential measurements reduced false positive and negative rates (14% and 8%, respectively). Pregnancy rates did not differ based on the time of pairing during expected estrus. Overall, we determined vaginal impedance can be more successful than vaginal cytology at identifying proestrus in the rat, but only if multiple measurements are taken.

Figure 1.

Protocol for evaluating the effectiveness of using vaginal impedance to stage estrous in female rats given LHRH by (A) comparing vaginal impedance and vaginal cytology, and (B) evaluating impedance values throughout the expected day of proestrus. (A) Three groups of 12 female SD rats between 10-12 wk of age were injected with 0.2 mL luteinizing hormone releasing hormone (LHRH) at 0900 h on day 0. At 1300 h on day 3, vaginal impedance measurements and vaginal cytology samples were taken (in that order). Females were paired with proven males 24 h later (day 4). At 0700 h on day 5, females were inspected for vaginal plugs. Ten days postmating, females were euthanized and evaluated for pregnancy status. (B) Three groups of 12 female Crl:SD rats between 10-12 wk of age were injected with 0.2 mL (40 µg) LHRH at 0900 h on day 0. On day 3, the expected day of proestrus, vaginal impedance measurements were taken at 0900, 1100, 1300, 1500 and 1700 h, or until the female reached an impedance value ≥ 3kΩ. The next day (day 4), half of the females were paired with a proven male exactly 24 h after their positive proestrus impedance reading and half were paired at 1300 h per standard protocols. The next morning at 0700 h, females were inspected for vaginal plugs and were euthanized 10 d after mating to evaluate pregnancy status.

Figure 2.

Representative images of Diff-Quik (modified Giemsa)-stained vaginal smears from rats showing the relative cellular composition of 3 out of the 4 stages of the estrous cycle at 200× magnification. (A) Proestrus; cells tend to appear in clumps with an abundance (more than 80%) of basal and nucleated intermediate and superficial epithelial cells. (B) Estrus; More than 75% nonnucleated (cornified) superficial squamous cells. (C) Diestrus; more than 60% polymorphonuclear cells with occasional nucleated epithelial cells.

Figure 3.

Ninety-five percent confidence intervals around estimates of sensitivity and specificity (A) Sensitivity for single (black circle) and sequential impedance readings (gray circle). 95% CI around estimate of sensitivity for sequential readings is narrower than for single readings. (B) Specificity for single reading (42%) and sequential readings (44%).

Figure 4.

Time at which females reached an impedance reading ≥ 3kΩ on day 3. Once females reached a reading of greater than or equal to 3kΩ, they were not measured again to reduce the chance of inducing pseudopregnancy.