Millions of mice and rats are bred for use in biomedical research every year, and many institutions choose to this in-house to reduce costs and increase research options. The advantages of in-house breeding are, 1) researchers are able to manipulate the genetics of the animals through selective breeding 2) they can time pregnancies to meet research needs, and 3) they can work with embryos and neonates as necessary. However, to setup a successful breeding scheme, one should understand the rodent estrous cycle. In addition, they should have knowledge about the different mating schemes, the factors affecting rodent breeding behavior and the weaning considerations…all of which will be discussed in this video presentation.

Let’s begin with review of the rodent estrous cycle. Both female rats and mice are polyestrous, meaning they have more than one estrus cycle in a year or a breeding season. Each cycle lasts for 4-5 days and can be divided into 4 stages: metestrus, diestrus, proestrus, and estrus. Estrus is the ovulatory phase, which means that if the female is in the proestrus or the estrus phase, she is ready to conceive.

One way to determine the estrous cycle stage is by visual examination. Manually restrain the animal by the tail and with the forepaws resting on a cage lid and carefully inspect the size of the vaginal opening and the surrounding tissue. During the proestrus phase, the vaginal opening is wide and is characterized by swelling of the surrounding tissue, which is pink in color and very moist. Often there are wrinkles or striations along the dorsal and ventral edges of the opening. During the estrus, the swelling surrounding the vaginal opening is reduced and the tissues are not as moist and pink. During the metestrus, the vaginal opening is minimal and there is negligible swelling. During diestrus, there is no swelling of the tissues around the vaginal area and the opening is small or closed.

Another, more accurate approach for determining the estrous cycle stage is vaginal cytology for which cell samples are collected by either lavage or swabbing. For lavage, place a latex bulb on the end of a sterile 200-microliter tip and draw up approximately 100 microliters of sterile double distilled water into the pipette. Next, lift the mouse out of the cage and place her on the wire bar cage top with her tail towards you. Firmly grasp the tail and elevate the hindquarters of the mouse such that only the front paws are grasping the lid. Place the end of the filled tip at the opening of the vaginal canal without penetrating the orifice. Gently depress the bulb to expel approximately 25-50 microliters of water at the vaginal opening. The liquid will spontaneously aspirate into the canal without tip insertion. Slowly release the pressure exerted on the bulb and the fluid will withdraw back into the tip. Repeat 4-5 times using the same tip, bulb, and fluid to obtain a sufficient number of cells in a single sample. Place the fluid on glass slide, and allow the smear to completely dry at room temperature. Once dry, these estrous smears can be stored for later use, or they can be stained immediately using the Wright-Giemsa stain, which is a one-step stain and does not requiring fixation. The slide is placed in the stain for 45 to 60 seconds.

On the other hand, for swabbing, wet a 2 mm cotton-tipped applicator with saline. Insert the tip of the applicator into the vagina of the restrained mouse and gently turn and roll the tip against the vaginal wall. Then remove the swab carefully and transfer the cells to a dry glass slide by rolling the swab across the slide. This procedure is considered stressful and can cause damage if not performed gently, with proper restraint, and with the correct sized cotton swabs. Like lavage, once the slide is dry, it can be stained with the Wright-Giemsa stain. Following staining, the slides can be examined under a microscope.

If the female is in proestrus, the cells are seen as clusters of round, well-formed, nucleated epithelial cells with a nucleus that stains darker than the cytoplasm. If she is in estrus phase, the majority of the cells are cornified squamous epithelial cells that lack a nucleus. They are angular in appearance and are in densely packed clusters. If the female is in metestrus, the cells are typically white blood cells, specifically neutrophils, with some cornified squamous epithelial cells present. During diestrus, the cells present are normally white blood cells with the occurrence of a few nucleated epithelial cells.

Now that you have an understanding of the rodent estrous cycle, let’s discuss how to set-up mating. First step is determination of the sex, which is done by comparing the anogenital distances. In females, the distance between the anus and the external genitalia is shorter than it is for the males. Based on research needs and the strain’s breeding efficiency, the mating scheme can be monogamous — where one female is bred to one male OR polygamous — where two or more females are bred to one male.

In terms of timing, given that the rodent estrous cycle is short-only 4-5 days long-one can set up mating randomly. Or they can set up “timed mating”, which involves introducing female to the mating cage when she is at the point of maximum receptivity and fertility-that is during the proestrus or the estrus phase. In either case, the mating should be set up at the end of a day, as rodents are nocturnal and tend to mate at night. The following morning, the female is examined for a copulatory plug – a whitish mass consisting of vaginal fluid and semen that persists for 12-24 hours post-copulation. If the copulatory plug is not visually obvious, gently insert the tip of a blunt probe into the vaginal opening. The presence of a copulatory plug will impede the advancement of the probe within 0.5 cm of the vaginal opening. In case of random mating, copulatory plug appearance commonly takes three days. The presence of a plug confirms mating does not guarantee that the female is pregnant.

Once a copulatory plug is observed, the female should be monitored for signs of pregnancy such as weight gain. If the plugged female is pregnant, then the day the plug was found is embryonic day zero or E0 and the next day is considered as the first day of gestation, or E1, and so on right up to parturition-that is giving birth-which is between 19 to 21 days. Approximately 20-24 hours post partum, both female rats and mice undergo an estrus and can conceive again.

In an intensive breeding scheme, which is commonly used for animals with a short breeding life span due to a genetic mutation, the male animals are left in the cage with the female and pups, so that the female can conceive another litter immediately. This scheme can be stressful to the female as she is continually lactating and gestating. On the contrary, the non-intensive breeding scheme, involves separating the female once she is visibly pregnant, and not returning her to the male’s cage until her litter has been weaned, making this a less demanding breeding scheme.

There are many factors that can influence the breeding performance of mice and rats. Let’s review some of them, which come up more frequently. The vigor of female breeders largely depends on the level of inbreeding. Animals that are outbred are much more hardy and vigorous, thus they produce larger and stronger litters. Another factor that can affect breeding performance is aggressive behavior. Some commonly used strains, such as C57BL/6 mice, have a tendency to display aggression, which can interfere with breeding. When breeding an aggressive strain, all litters should be closely watched. Animals from a litter containing aggressive pups should not be used for breeding. Temperature, humidity, and lighting fluctuations can cause decreases in breeding efficiency. Noise and vibrations within the breeding rooms have also been shown to cause deleterious effects. Animals with genetic modifications tend to be less hardy and fertile, and some of the mutations may result in lethality of the pups before or soon after birth.

Now let’s briefly review how to wean pups of these lab animals. The time to start weaning differs with the breeding scheme. In case of non-intensive breeding, the young may be weaned at 21-28 days of age. But in case of intensive breeding, the pups of each litter are weaned at day 20 to prevent the older pups from being present when the next litter in born. Since rodents can begin mating as young as 8 weeks, male and female pups are separated at weaning. Whenever possible, newly weaned pups should not be housed singly.

If a litter contains only one pup of a given sex, attempts should be made to house this pup with pups of the same gender from another litter. The possible housing options for weaning pups are listed in the manuscript below. Weanling mice and rats should be checked daily to assure that they are thriving. For food supply, a small amount of food, one pellet per mouse, should be placed on the cage floor for the first 7-10 days, with additional food in the cage top. This will encourage the animals to transition to having rodent chow as their sole food source. For water supply, a bottle should be added even if the animals are housed on racks with automatic watering system. This is to prevent dehydration.

Lastly, let’s see how scientists are using the in-house breeding approach to their advantage. One of the most common applications of setting up mating is to develop mice with altered genotype. To study a gene’s function, researchers often disrupt its genetic code. However, these animals like humans are diploid and thus have two copies of the same gene. Therefore, in order to disrupt the gene completely, the altered mice need to be bred to produce an animal with both copies dysfunctional, in other words a homozygous knockout. Mice with one copy dysfunctional are called heterozygous or hets.

The other advantage of setting up in-house mating is testing the effect of prenatal exposure to test compounds. For example, here researchers provided pregnant female a liquid diet containing alcohol from E7 to E13. Then on E13, they dissected the pregnant female, obtained fetal brains, and sliced them into thin sections. Lastly, staining of the slides revealed that prenatal alcohol exposure increased cell death in the neural tissue.

Finally, in-house breeding also allows for the study of post pregnancy disorders like postpartum depression. In this study, scientists first moved the litter away from the dam during the lactating period. Then, 60 minutes later, reintroduced the pups to the dam. And to induce stress in the female, added a novel male intruder to the cage. Then, the researchers observed the dam for maternal aggression, which includes attacking and biting of the intruder male as well as for different maternal care behaviors like pup grooming, and nursing. The data obtained revealed that stress has a significant effect on both postpartum maternal aggression and care.

You’ve just watched JoVE’s video on fundamentals of breeding and weaning mice and rats in the laboratory. You should now have a better understanding of the rodent estrus cycle and also know how to determine the cycle stage and how to use it to set-up successful mating schemes. We also reviewed the factors that can affect the breeding behavior, and explained how and when to wean mice and rat pups. As always, thanks for watching!