Determination of Reproductive Competence by Confirming Pubertal Onset and Performing a Fertility Assay in Mice and Rats

This method can help answer key questions in the reproductive field, such as how to determine pubertal onset in mice and rats through preputial separation, vaginal opening, and establishing first estrus prior to performing a fertility study. The main advantage of this technique is that by establishing pubertal onset using the demonstrated non-invasive approach, it is possible to follow and conduct a fertility study in animals that are sexually mature. The implications of this technique extend towards diagnosis of deficits in reproduction, as caused by genetic mutations or treatments susceptible to impact reproductive competence, such as endocrine disrupting chemicals.

Though this method can provide insight into reproductive competence in mice, it can also be applied to other model systems, such as rats and hamsters. Visual demonstration of this method is critical, as it requires good and delicate animal handling skills. It is particularly important to hold the mice correctly for the following visual identification of preputial separation and vaginal opening.

To prepare the work area, place a pad on the table. Then place a clean mouse cage top with the grid facing up, on the pad. Next, set up a scale in the work area.

Place a clean 500 to 1000 milliliter beaker on the scale and tare it. Then place a sheet next to the work area to record the data. After this, place the mouse cage in the work area.

Open the cage and remove the lid, water bottle, and food holder to the table. While holding the mouse by the tail, place it on the clean mouse cage top. While maintaining hold of the tail, allow the mouse to explore the grid of the cage top.

Gently pull the tail backward and approach the other hand to the skin close to the neck and ears. Grasp the loose skin between the shoulders and neck with the thumb and forefinger. Then grab the skin on the back with the remaining fingers and hold the mouse by gently pressing it against the hand.

With the free hand, gently push the skin around the penis back. At preputial separation, the preputial skin slides backwards, exposing the glans penis. After examination, record the presence or absence of preputial separation.

After this, weigh the mouse and record its weight. Then reintroduce the mouse to the housing cage by holding the beaker over the cage floor and allowing the mouse to exit on its own. Place a cotton ball, a large beaker, and a bottle of sterile water in the work area.

Pour sterile water into the beaker. Then, humidify the cotton ball with the sterile water in the beaker, and place it next to the clean cage top. Place the mouse cage in the work area.

Then transfer the mouse to the cage top by holding it by the base of its tail. Gently pull on the mouse's tail to encourage a forward movement by the mouse. After this, lift the tail while supporting the hips with three fingers.

Gently clean the vulva with the humidified cotton ball, then examine the vulva to determine if the vagina is completely open. Record if vaginal opening has occurred. Then weigh the mouse and return it to the home cage.

Prepare a clean mouse cage with lid, water, food, bedding, and nesting material for each breeding pair of mice. Then fill out the cage cards with the requisite information. Place the prepared bedding cage on a clean table, then slowly introduce a gloved hand into the mouse cage and leave it in the cage for a short period of time.

Slowly approach the mice with the gloved hand. When the mice run over the hand, grab the tail of one mouse between the thumb and index finger, then lift the mouse by the tail. The mouse can be held by the tail without support of the body for up to two to three seconds.

Place the mouse in the prepared breeding cage, then close the cage after ensuring that the male and female mice have food, water, and nesting material. Using the presented method, two different transgenic mouse models were studied. Vaginal opening and preputial separation occurred at the same age and weight in both the heterozygous mice and the control mice.

Conversely, vaginal opening and preputial separation were significantly delayed in conditional knockout females and males, and associated with increased body weight. At 4.5 weeks of age, female controls and the conditional knockout mice had comparable body weight, while the conditional knockout males were slightly heavier than the controls. This indicates that the delay in vaginal opening and preputial separation of the conditional knockout mice was not associated with a delay in weight gain.

After completion of pubertal onset, the fertility study was started at age 10 to 16 weeks. Both male and female heterozygote mice were sub-fertile. Heterozygote females generated fewer litters in the three month study, and both male and female heterozygote mice generated smaller litters.

To illustrate the importance of confirming pubertal onset prior to setting up a fertility assay, reproductive competence was evaluated in conditional knockout mice. During the 70 day fertility study, neither conditional knockout males nor females generated any litters. While attempting this procedure, it is important to remember to handle the mice with care and remain calm while approaching and handling the animals.

Following this procedure, other methods like measuring circulating sex hormone levels can be performed to answer additional questions, like whether infertility or other subfertility was caused by abnormal testosterone, estrogen, follicle stimulating hormone, and luteinizing hormone levels. Don't forget that working with mice can be hazardous due to the risk of biting and development of allergies. To reduce exposure, precautions such as wearing gloves and a lab coat, and careful handling of the mice, should always be taken while performing this procedure.