Timed pregnancies in mice can be set up in two ways: 1) use vaginal cytology to determine the stage of a mouse's estrous cycle and the appropriate time for mating; and/or 2) check females for vaginal plugs to determine if mating has occurred.
Evaluating vaginal cytology
To obtain a vaginal specimen for determining the stage of the estrous cycle, insert a moistened cotton-tipped swab into the vaginal opening of a manually restrained mouse. Most commercially available swabs are too large to fit easily into the vaginal opening of an average-sized mouse, but a swab of the appropriate size can be made by wrapping a small amount of sterile cotton wool around the blunt end of a toothpick. Rotate the swab gently but firmly within the mouse's vagina, taking care to maintain contact with the vaginal wall. After removing the swab, wipe it on a clean glass microscope slide to transfer the vaginal specimen to the slide. Allow the slide to air dry, then apply a 0.1% aqueous solution of methylene blue to stain the vaginal material. After the stain dries, examine the specimen under a microscope. The stage of estrous can be determined by the types and relative numbers of cells present:
- If the mouse is in diestrus, you will see mostly polymorphonuclear leukocytes (PMNs) and some epithelial cells.
- If the mouse is in proestrus, you will see mostly nucleated and cornified epithelial cells. You will see some PMNs if the mouse is in early proestrus.
- If the mouse is in estrus, you will see mostly cornified epithelial cells. You may see a few nucleated cells if the mouse is in early estrus.
- If the mouse is in metestrus, you will see mostly cornified epithelial cells and PMNs and some nucleated epithelial cells.
Both mating and ovulation typically (but not invariably) occur during estrus, which generally occurs approximately six hours after the onset of the dark period and lasts 12-14 hours. For timed breeding, place the female with a male during the proestrus phase. To verify that mating has occurred, check the female for a vaginal plug the next morning (see below).
Checking for vaginal plugs
Although evaluating vaginal cytology is commonly used to determine the best time for breeding in some domestic species (such as dogs), it is not as practical for mice because they have a very short estrous cycle (4-5 days total). A more practical approach for mice is to simply place the male and female together, then check the female early the following morning for a vaginal plug. The plug is made of coagulated secretions from the coagulating and vesicular glands of the male. It generally fills the female's vagina and persists for 8-24 hours after breeding. To see the plug, lift the female by the base of her tail and examine her vaginal opening for a whitish mass. It may be difficult to see, especially if you are less experienced in this procedure. You may see it better if you spread the lips of the vulva slightly with a cotton-tipped swab or the blunt end of a flat toothpick. If there is no plug, leave the female with the male and check her each morning until you see a plug.
If you need large numbers of timed pregnant females, you can take advantage of the influence of pheromones and social factors on the estrous cycle. House the females in large groups with no males nearby. By doing this, diestrus is prolonged and estrus is suppressed, a phenomenon known as the Whitten effect. Because pheromones from male mice can interfere with this effect, olfactory stimulation of the group-housed females by male mice must be prevented. When the females are placed with males (typically 1-3 females per male), the estrous cycle of most females will restart within three days. To identify those that have mated, examine them daily for a vaginal plug.
The presence of a vaginal plug does not guarantee pregnancy: it only indicates that sexual activity occurred. The likelihood of pregnancy after mating varies with the mouse strain and with the phase of the estrous cycle when mating occurs. For most strains, the rate of pregnancy is highest among estrus-suppressed females with vaginal plugs found the third day after being placed with a male. As examples, whereas the rate of pregnancy is ~100% and 44% respectively for C3H/HeJ and BALB/cJ females with plugs found on the third day, it is only 62% and 31% respectively for those with plugs found on the fifth day. In contrast, whereas the rate of pregnancy for C57BL/6J females is only 39% for those with plugs found on the third day, it is 69% for those with plugs found on the fourth day.
If the plugged female is pregnant, the first day of gestation is considered to be the day after the plug is found.