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4.4.1 Postnatal development

A mouse is born naked with closed ears and eyes, and if a female, with a closed vagina. Hair begins to appear at 2-4 days, ears open at 3-5 days, and eyes open at about 14 days. Typically, the vagina opens at 24-28 days of age, but it can be delayed in some mice until they are 35-40 days old. As soon as the eyes are fully functional, at about 16 days, pups will begin to eat solid food. However, nursing can continue to at least the end of the third week and sometimes a week or more longer. By the end of the third week of life, a young mouse resembles the adult in every aspect other than size and sexual differentiation.

4.4.2 Determination of sex

The sex of the newborn mouse can be determined from both the distance that separates the genital papilla and the anal opening, and from the general appearance of the urogenital-anal region. The genital-anal distance in newborn males is generally 50% greater than in newborn females. In addition, the male genitalia are often more prominent, and in the pre-scrotal region below, a dark pigmentation is often visible. If a litter is large enough, it is likely to have both males and females. The simplest way to become adept at distinguishing gender is through pairwise comparisons of the pups — in each hand, a newborn pup can be held gently, but firmly, between the index finger and the thumb in an upside-down position. As neonates age, gender determination becomes somewhat more difficult. It becomes easier again at 8-10 days with the appearance of nipples along the ventral side of the female, and at the age of weaning (18-28 days), when the penis has developed more fully in the male.

4.4.3 Lactation, culling, and supplementing litters

The most important factor in the growth of infant mice is the amount of milk available for suckling. Thriving newborns will begin to nurse immediately after birth, and within a matter of hours, it is possible to clearly see the milk in their stomachs through their translucent bodies. The amount of milk present is an excellent gauge of the likelihood with which a young pup will become a vibrant, healthy adult. When little or no milk is present by 6 hours after birth, it is almost certainly the case that something is wrong with either the pup or the mother.

For litters of four or more pups, the amount of milk produced by a lactating mother will increase with the number of young, but the increase will not be proportional (Grüneberg, 1943). However, if there are two or more mature females in a cage, all can be induced to lactate in response to a single litter. This phenomenon makes sense from an evolutionary point of view since females living together in a deme are likely to be related — often as sisters — and thus child care sharing serves to enhance the survival of the common gene pool. For the mouse geneticist, however, it means that when there are two or more females in a breeding cage, one can not use a state of lactation as a means to distinguish the birth mother.

When the number of pups is eight or more, a single inbred mother will not be able to provide the nourishment required for the optimal growth and development of all. Thus, when it is not detrimental to the experimental protocol, it makes sense to cull litters soon after birth. In some cases, one will wish to select pups according to sex (as described in the previous section), or other visible phenotypes such as eye or coat color (at day 2-4), or gross morphological characters. To ensure optimal growth of selected animals, litters should be culled to 5-6 pups during the first days after birth. A further reduction to 3-4 youngsters can be carried out between days 10 and 14.

With the common strains of mice, it is not often the case that only one or two pups will be born in a litter. However, this situation can arise more frequently with the breeding of animals that carry embryonic lethal mutations, and it is also problematic. Especially for first-time mothers, 1-2 pups may not provide the level of suckling stimulation required to effectively stimulate milk production. When this problem arises (as indicated by an insufficient level of milk in the stomach), the simplest solution is to supplement the litter with 2-3 age-matched pups that are clearly distinguishable from those born to the mother.

4.4.4 Foster mothers

When newborn animals are not receiving sufficient amounts of milk and it is likely that the mother is the problem, one can consider fostering as a last resort. The foster mother should be an experienced female with her own newborn litter. This entire litter should be removed from the foster mother's cage and placed onto a clean surface. The pups to be fostered can then be added to this group and an equivalent (or greater) number of the foster mother's pups can be removed; when there is a choice, the largest and best-fed of these pups should be eliminated. The new mixed litter can now be placed back into the foster mother's cage. Obviously, it is critical to be able to easily distinguish the pups of the foster mother from those that have been added. This is most readily accomplished by known coat color differences between the two litters that have been mixed together.

4.4.5 Age of weaning

Mice can be weaned from their mothers when they are as young as 18 days old. However, especially for inbred strains or those that carry deleterious mutations, it is best to wait until they are 4 weeks of age. When young are kept with their mothers for a longer period, they are more likely to thrive as adults.

4.4.6 Postpartum estrus

Amazingly, within 28 hours of giving birth, a nursing mother will normally go into a postpartum estrus that can allow her to become pregnant again immediately. There is a tendency to ovulate 12-18 hours after the time of birth, but this can be countered by the tendency to ovulate nocturnally (Bronson et al., 1966). The level of postpartum estrus fertility is reduced somewhat relative to that achieved during a normal estrus cycle. A postpartum pregnancy can have negative consequences for the litter already born as well as the one on the way. Since the mother is forced to split her resources between two sets of "progeny," her milk production will fall off more quickly than would otherwise be the case. In addition, the duration of the postpartum pregnancy can be extended for up to two weeks. Finally, when the second litter is born, there will be competition between the new pups and the older ones (if they are not yet weaned), and the new ones can suffer malnourishment or death.

4.4.7 Genetically controlled variation in the adult mouse

Although it is possible to make general statements about the gross characteristics of all laboratory mice — they reach adult weights of 22-40 g, they have life spans of 1-3 years, they have a gestation period of 18-22 days, and an average litter size of 5-10 pups — much more discrete numbers can be obtained for individual inbred strains. It is not surprising that the members of an inbred strain show much less variance in these numbers since they are, for all practical purposes, genetically identical. Statistical evaluations of the growth and reproductive characteristics of the older, established inbred lines have been compiled in two different handbooks. One is published by the Federation of American Societies for Experimental Biology, abbreviated FASEB (Altman and Katz, 1979). The second is published by the Jackson Laboratory in regularly updated editions (Green and Witham, 1991). The Jackson Laboratory handbook is available without charge and has detailed information on each of the inbred strains that are sold to investigators. Click here to order the current vesion of the JAX Mice Handbook.

A quick survey of the information provided in these books provides ample evidence of the wealth of genetic variation that exists among the classical inbred strains in terms of gross morphological and physiological characters. Although most of this variation shows a high degree of heritability, it is polygenic and, as a consequence, it was not readily accessible to the types of genetic analyses carried out in the past. However, with the new genetic markers described in Section 8.3, polygenic traits are no longer beyond reach, and it is only matter of time before many of the common forms of variation in mice — which often have human counterparts — will be linked to individual loci and, ultimately, to cloned genes (see Section 9.5).

One note of caution is the possibility that genetic drift within an inbred line can lead to a drift in gross phenotype. This is a concern because mutations occur constantly and inbred lines are continuously re-derived through two-member population bottlenecks, which can lead to the rapid fixation of new genetic variants. In general, gross phenotypic features are controlled by multiple genes, with changes in each having small additive effects. The extent of genetic drift should be roughly linear with time; thus, the longer the period that has elapsed since a study was performed, the more likely it is that a gross characteristic can change in a statistically significant manner. Genetic drift is much more likely to occur in outbred laboratory stocks, which are heterogeneous to begin with, but also pass through narrow population bottlenecks (Papaioannou and Festing, 1980).

The anatomy of the laboratory mouse is described with numerous illustrations by Cook (1983). The average body weight of a full-grown adult member of the standard inbred strain C57BL/6J is 30 g for males and 25 g for females (Altman and Katz, 1979). The largest of the commonly available inbred strains is AKR/J with males that reach 40 g in weight. The smallest is 129/J with full- grown males and females that weigh 27 g and 22 g respectively. Hybrids between inbred strains are usually larger than either parent.

The life span of a mouse is highly strain dependent. At one extreme, the AKR/J mouse has a mean life span of only 10 months due to its propensity to develop lymphatic leukemia; at the opposite extreme, the life span of the B6 mouse is among the longest of the common inbred lines. B6 animals have a median life span of 27-28 months — somewhat over two years (Zurcher et al., 1982; Green and Witham, 1991). The longevity of inter-strain hybrids tends to be greater than either inbred parent. The hybrid formed by a cross between B6 and DBA/2J (called B6D2F1) has a median life span of over 2.5 years and some animals survive as long as 3.5 years (Green and Witham, 1991).

The genetic factors responsible for longevity have been studied by a number of different investigators. In one study, the second generation F2 offspring derived from an outcross-intercross between the inbred strains B6 and DBA/2J were analyzed for correlations between longevity and genotype at three different autosomal loci — H-2, b (brown), and d (dilute) — as well as the two sex chromosomes. Statistically significant correlations were observed between longevity and particular genotypes for each of the loci analyzed. This does not mean that the tested loci, in and of themselves, have any bearing on longevity, but rather that genes in their vicinity do. The fact that an effect was observed with all of the loci tested points to a strong likelihood that the number of genes involved in this polygenic trait is many. This result should not be unexpected since one can imagine that many, many different phenotypic characteristics will have an indirect effect on life span.

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