Breeding Information: Females
- When to Begin Breeding
- Estrous Cycle
- The Effect of Pheromones on the Estrous Cycle
- Hormonal Manipulation
- Mixing Breeding Pairs
- Plug Checking
- Pregnancy Determination
- Postpartum Estrous and Delayed Implantation
- Handling Pregnant Females
- Parturition Preparation
- Cesarean Section
- Foster Rearing
- Nutrition of the Pregnant Mouse
- Breeding Life Expectancy
When to Begin Breeding
In general, female mice reach sexual maturity, and estrous cycles commence around 4 weeks of age. However, inbred strains often reach sexual maturity a little later than outbred strains. Consequently, many labs begin breeding females at 5-6 weeks of age.
Mice have a 4-5 day estrous cycle that is divided into four phases-proestrus, estrus, metestrus and diestrus.
- Proestrus: Ovarian follicular development occurs. Estrus: Female is sexually receptive to male; ovulation occurs; duration 6-10 hours. Metestrus: Corpora lutea forms; mature eggs move through oviduct into uterus. Diestrus: Follicles begin to undergo rapid development for next ovulation; previous ova are eliminated.
Ovulation usually occurs 3-5 hours after onset of the dark cycle. Males will generally copulate with females in estrus at about the midpoint of the dark period. In addition, the estrous cycle is profoundly affected by the light cycle (hours of light and dark); interruption of the dark cycle can affect reproductive performance. In general, breeding mice should be maintained on a 12:12 cycle (12 hrs light: 12 hrs dark).
Each phase of estrus can be identified by the appearance of vaginal epithelium and vulva. It is possible to identify females in estrus by examining the color, moistness and degree of swelling of the vagina, see below.
- Proestrus: Vagina is gaping; tissues are reddish-pink and moist; numerous longitudinal folds or striations visible on the dorsal or ventral lips.
- Estrus: Vaginal signs similar to above, but tissues are lighter pink and less moist; striations are more pronounced
- Metestrus: Vaginal tissues are pale and dry; dorsal lip less edematous; whitish cellular debri may line the inner walls and/or partially fill the vagina.
- Diestrus: Vagina has small opening; tissues are blue and very moist.
The Effect of Pheromones on the Estrous Cycle
Pheromones are scents emitted by mice that cause behavioral and physiological changes in other mice, both same and opposite sex. Pheromones can produce significant influence on reproductive performance of mice and can be used to an investigator's advantage.
Below are several pheromone-induced reproductive effects:
- Suppression of estrus in grouped females
Group housed female mice tend to stop cycling and display either pseudopregnancy (Lee-Boot effect) or anestrus. This phenomenon can be exploited in some strains by grouping females together before exposure to a male (see below).
- Induction of estrus by a male (Whitten effect)
Whether a female mouse is housed alone or in an all female group, exposure to a male has an immediate effect upon her cycle. A new estrus cycle is initiated in group housed females, while individually housed females begin to cycle regularly. To utilize this effect, single or group housed females are exposed to a male, or male mouse urine (dirty bedding). A majority of females will then be in estrus the 3rd night after exposure to the male mouse.
- Male-induced pregnancy block (Bruce effect)
Pregnancy can be blocked during the preimplantation period by exposure to a strange male. Exposure to a male of a different strain than original male causes failure of implantation, followed by return to estrus in 4-5 days.
Superovulation regimens are commonly used to maximize the number of ova released at a particular ovulation, such as collection of ova for production of transgenic animals. A combination of Pregnant Mare's Serum Gonadotropin (PMSG) and Human Chorionic Gonadotropin (HCG) is often used. For more information on superovulation regimens, please contact the Transgenic Mouse Facility at 824-8579.
Mixing Breeding Pairs
Breeding animals should be mixed by placing a female mouse (or mice) into a male's cage. Placing a male into a female's cage often results in lack of mating and may result in the female mouse attacking the male.
The ejaculate from the male's accessory sex glands forms a short-lived, white to yellowish plug in the vagina of the female. Presence of a vaginal plug is often used to determine if copulation occurred between mice. By checking female breeding mice each morning, the presence of a vaginal plug allows one to estimate the approximate time of mating as the middle of the preceding night.
It is important to remember that the presence of a vaginal plug indicates that a mating took place, and does not mean that a pregnancy will result from the mating. It should also be remembered that lack of a vaginal plug does not always mean that mating did not take place, as the plug may have already dislodged. It is common to check for plugs early in the morning, as most matings take place during the dark cycle each evening.
- External visualization of the vulva and plug. However, deeper plugs may be missed by this method.
- Use of a small forceps to gently part the vulvar lips to identify the plug in the vagina.
- Use of a blunt probe (i.e.: capillary tube, glass rod) to examine the vulva and vagina for presence of a plug.
- Pregnant mice will fail to return to estrus and will not breed when placed with male mice.
- Abdominal distention is apparent in most mice by 12-14 days of gestation. Mice with large litters may show distention slightly earlier.
- Palpation of the uterus: By days 12-14 a small "string of pearls" may be palpated. Palpation takes practice, patience and a soft touch.
Gestation in mice varies slightly by strain and ranges from 19-21 days. Smaller litters are often carried longer than larger litters.
Postpartum Estrous and Delayed Implantation
Mice have a fertile estrus that occurs 12-24 hours after delivery (postpartum). If a mating takes place, the embryos resulting from the postpartum mating undergo developmental arrest and delayed implantation. Consequently, the delivery date of the pups from the postpartum mating may be delayed 4-5 days.
Handling Pregnant Females
Near term females with greatly distended abdomens should be disturbed as little as possible. Rather than lifting them the tail, it is recommended to gently lift them in the palm of the hand. Care should also be taken to insure the mouse will not jump out the handler's cage and injure itself.
- Cage Preparation: If possible, it is recommended to minimize disturbances to the female within 2 days of delivery and for 2-3 days after delivery. If the female is singly housed, or paired with a male, the cage should be changed 2-3 days before delivery and nesting material added to the cage. The cage should then be left undisturbed for 2-3 days after delivery. If the female is housed as part of a harem, it is recommended that the female be separated into a single cage several days prior to delivery. Delivery in a harem cage may result in an overcrowded cage and occasionally results in trampling and death of the pups.
- Nesting material: Nesting material is not required for successful rearing of pups, but it may aid in decreasing cannibalism in nervous dams and strains with a history of cannibalism. Nesting material also helps create a warm microenvironment for the pups during the early neonatal period. Popular nest material includes Kimwipes, cotton nestlets and shredded paper towels. The dams will usually shred the material and create a nest. Nests also allow easy transfer of young litters between cages at cleaning time. The whole nest can be gently grasped and transferred between cages with little disturbance to the mouse pups.
Occasionally, mice experience difficulties during delivery (dystocia) and a cesarean section (C-section) is required. C-sections can also be used as a method of rederiving mice that are contaminated with certain viral, bacterial and parasitic agents. If you have questions about performing C-sections in mice, please contact Veterinary Services.
Foster rearing involves transferring pups from one dam to another to be raised. It is performed for many reasons, including strain history of poor maternal behavior, maternal neglect, poor milk production by the dam, death of the dam, excessively large litters and experimental dam.
- Selection of foster dams: The best candidate for a foster mom is a strain with a history of good maternal behavior. Generally, outbred strains (e.g. CD-1) are usually better foster moms than inbred strains such as C57BL/6.
- Age of pups: Ideally, a foster mom should have a litter of her own that is within approximately 1 day of age as the pups to be fostered. In addition, it is also recommended to select a dam that has successfully raised a litter previously.
- Suggested procedure for cross fostering:
- Remove the foster dam from her cage, while leaving her pups in the original cage. If the size of the foster dam's original litter must be decreased to allow room for the incoming pups, this should be performed now. In general, the final litter size should be between 6-10 pups, depending on the strain.
- Transfer the pups to be fostered to the foster cage. Mix the foster pups with the original pups to pick up their scent, and the scent of the foster mom. Many find it successful to hold all the pups (foster and original) in their cupped hands to mix them up and thoroughly mix the scents. Also, if the fostering pups are cool, warming them in your hands may increase the likelihood of acceptance by the foster mom.
- Place all the pups in the foster mom's original cage. If nesting material is available, place all the pups in the nest.
- Return the foster mom to her cage. If she has not rejected the litter in 10-15 minutes, she will usually continue to care for them.
- Identification of fostered pups: If it is necessary to identify the fostered pups to allow subsequent separation from the foster dam's original litter, several options are available. The easiest method is to select a foster dam (and litter) with a different coat color. If the coat color is the same, procedures such as tail snips or ear notching can be performed to identify the foster animals.
Nutrition of the Pregnant Mouse
Most outbred, and many inbred strains perform well with standard rodent chow. However, a few inbred and mutant strains may require a higher fat (and protein) diet for breeding and lactation, and "breeder" diets are available for this purpose. The increased fat in such diets may increase body weight and rate of gain during pregnancy compared to regular rodent diets. However higher fat diets may cause male mice to become obese and lead to poor breeding performance. Please contact ULAR for information on ordering higher fat diets.
Breeding Life Expectancy
Reproductive performance of female mice tends to decrease with increasing age and number of prior pregnancies. Few females of inbred strains, with the exception of FVB/N, will produce more than five litters. And, irrespective of past reproductive history, most inbred females exhibit greatly reduced fecundity by the age of 8-10 months. Reproductive characteristics for specific strains of mice are usually available from commercial vendors. See section I below for reproductive characteristics of selected inbred strains.
Outbred mice and F1 hybrids will routinely surpass inbred strains when comparing reproductive performance. Productive matings (those resulting in live offspring) often approach 100% in outbred animals, the age of first mating can be as early as 5 weeks, and the females may remain fertile up to 18 months of age.