Reproductive performance can be measured according to several different parameters including age at first mating, number of litters sired, number of pups per litter, and the frequency with which a strain has productive matings. Table 4.1 shows the values obtained for these different parameters with the most commonly used inbred strains of mice. As the table shows, inbred strains vary widely in their reproductive fitness.

The first important measure of reproductive fitness is the frequency with which a mating pair will produce any offspring at all. With some strains, such as C3H/HeOuJ, CBA/CaJ and FVB/N, over 90% of all matings that are set up will produce offspring. The C3H/HeOuJ strain is at the extreme end of this group with a 99% frequency of productive matings. At the opposite extreme, among the most well-characterized strains, is BALB/cJ with a frequency of non-productive matings that is over 50%. A second measure of fitness is the age at which females first become pregnant. This can vary from an early 5.9 weeks for C3H/HeOuJ to a late 8.0 weeks for BALB/cJ. The third measure of reproductive fitness is litter size. Once again, BALB/cJ performs the worst in this category with an average litter size of just 5.2. All but one of the remaining inbred strains have average litter sizes in the range of 5.4 to 7.0. The one strain that outperforms all others in this category is FVB/N with a much larger average litter size of 9.5. The final measure of reproductive fitness is the average number of litters that a single female can produce in a lifetime. This varies from a low of 2.2 litters with AKR/J females to a high of 4.8 litters with FVB/N females.

Three of the easily quantitated measures of reproductive performance — frequency of productive matings, litter size, and number of litters — have been multiplied together to give a sense of the overall fecundity associated with any one inbred strain in comparison to the others (Table 4.1). Far and away, the highest fecundity (41.0) is associated with the relatively new FVB/N strain. It is for this reason, as well as others, that FVB/N has become the strain of choice for use in the production of transgenic animals (see section 6.2). Among the traditional inbred strains, C57BL/6J (B6) and C3H/HeOuJ show a fecundity (23.5 and 23.4) that is significantly above all others. The lowest fecundity (9.3) is associated with the BALB/cJ strain.

The fecundity of female mice declines with both age and number of prior pregnancies. Few inbred females of any strain, with the exception of FVB/N, will produce more than five litters (Green and Witham, 1991). Irrespective of their past reproductive history, most inbred females exhibit greatly reduced fecundity by the age of 8 to 10 months. Male mice, like male humans, can remain fertile throughout their lives. However, older males that have become obese or sedentary are unlikely to breed.

Reproductive performance is among the characteristics most affected by inbreeding. Outbred animals and F1 hybrids of all types will routinely surpass the inbred strains in all of the categories listed in Table 4.1 as a consequence of "hybrid vigor". With non-inbred animals, the frequency of productive matings is close to 100%, the age of first mating can be as early as five weeks, and litters can have as many as 16 pups. Finally, non-inbred females can sometimes remain fertile up to 18 months of age, and bring as many as 10 litters successfully to weaning.

Male germ cell differentiation occurs continuously in the seminiferous tubules of the testes throughout the life of a normal animal. This process has been very well characterized in the mouse (Bellvé et al., 1977; Eddy et al., 1991), and only its salient features will be summarized here. Spermatogenic cells at different stages are classified into four broad categories — spermatogonia, spermatocytes, spermatids, and spermatozoa — with numerous substages defined within each category. All pre-meiotic cells are called spermatogonia; these include regenerating stem cells as well as those that have taken the path to terminal differentiation. With the commencement of meiosis, germ cells are called spermatocytes, and subsequent to meiosis, haploid cells are called spermatids. Finally, with the release of the morphologically mature product, the germ cells are called spermatozoa or, more simply, just sperm.

The timing of the stages of spermatogenesis in the mouse was described originally by Oakberg (1956a; 1956b). At birth, the testis contains only undifferentiated type A1 spermatogonia, which will serve as a self-renewing stem cell population throughout the life of a male mouse. By day three, differentiation has begun through a series of mitotic divisions into more advanced spermatogonial stages (A2, A3, A4, intermediate and type B spermatogonia). By 8 to 10 days, spermatocytes are observed for the first time in the leptotene phase of meiosis (Nebel et al., 1961). The meiotic phase is relatively long, extending over a 13 day period. When the male has reached 17 to 19 days of age, approximately 50% of the seminiferous tubules are found to contain cells in the late pachytene stage. The earliest postmeiotic cells — round spermatids — are not observed until after 20 days (Nebel et al., 1961). During the next 13 days, the round spermatids differentiate into elongating spermatids in which the sperm tail forms and the nucleus condenses. At the end of this process, morphologically-mature sperm are released into the fluid-filled lumen.

The entire process of differentiation from stem cell to released spermatozoa is called spermatogenesis. The term spermiogenesis refers specifically to the final morphological differentiation of haploid cells into sperm. At the time of release into the lumen of the seminiferous tubules, sperm cells are still not physiologically mature. After leaving the testes, they pass through the epididymis where they undergo further biochemical changes. From the epididymis, they go to the vas deferens, where they are stored until ejaculation. The final stage of sperm maturation — known as capacitation — is required for fertilizing activity and does not occur until after contact has been made with the milieu of the female reproductive tract.

Female germ cell differentiation operates under a two-phase time course dramatically different from that found in the male. By the twelfth or thirteenth day after fertilization, the primordial oocytes within the fetal ovary have undergone their last mitotic division and are referred to as oogonia. At this point, the young female, still not born, has produced all of the germ cells that she will ever have; the total number is somewhere between 30,000 and 75,000. All of these oogonia progress into meiosis, and by five days after birth, they reach the diplotene stage of prophase of the first meiotic division. At this point, also called the dictyate stage, the oogonia become arrested and remain quiescent until sexual maturation. As they move into the dictyate stage, all primordial oocytes acquire a coat of follicle cells; the complete coat surrounding each oocyte is called a follicle.

With the onset of puberty, the ovaries become activated by hormone stimulation, and every four days, a new group of oogonia are stimulated to proceed forward toward their ultimate differentiated state. This second phase of differentiation occurs over a period of twenty days. During this entire period — until a few hours prior to ovulation — the oocytes still remain fixed in the dictyate stage of meiosis, but they become highly active metabolically and increase greatly in size from 15 to 80 mm. The size of each follicle also increases through the addition of follicle cells up to a total of 50,000 per ovum. At 20 days after activation, oocytes have become competent for ovulation, which occurs in response to the correct hormonal cues during the estrus cycle described in the next section. During each natural cycle, only 6-to-16 oocytes are stimulated to undergo ovulation. The stimulated follicles swell with fluid and move to the periphery of the ovary where they burst out to begin their journey into the oviduct and further down the reproductive tract.

Stimulation to ovulate also releases the oocyte (now also called an egg) from its state of arrest and induces it to continue through meiosis. The first meiotic division is completed and the first polar body is formed prior to release from the ovary. The second meiotic division begins immediately but stops at metaphase, where the oocyte remains arrested until fertilization. Penetration by the sperm triggers completion of the final meiotic division and the formation of the second polar body.

Surprisingly, at least 50% of the oocytes present at birth degenerate before the mouse reaches three weeks of age. The vast majority of the remaining oocytes are never ovulated — many degenerate throughout the life of the animal, and all that remain are eliminated at the time of mouse menopause, which occurs at approximately 12-14 months of age.

The onset of puberty — when ovulation first occurs in a female, and when males have achieved full spermatogenic activity — is variable even among different animals within the same inbred strain. Although it is possible for some outbred females to reach puberty by the age of four weeks, the majority of females from most inbred strains first ovulate naturally between six and eight weeks after birth (Table 4.1). Numerous environmental factors appear to have an effect on the timing of this event (Whittingham and Wood, 1983). Exposure to adult males or their urine can bring it on sooner, whereas adult females or their urine may retard its onset. Furthermore, three-to-six week old females can be induced to ovulate with a specific regimen of hormone treatment as described in section 6.2.2.1. The onset of male puberty in most laboratory strains usually occurs between 34 and 38 days, however, it is sometimes possible for non-inbred males to reach sexual maturity by 30-32 days after birth. Thus, if one does not want littermates to mate with each other, they should be separated according to sex before the appropriate age is reached.

The normal estrus cycle of a laboratory mouse is four to six days in length. The cycle has been divided into four phases which are distinguished by changes in physiology, morphology, and behavior. (1) The proestrus portion of the cycle begins when a new batch of eggs reach maturity within ovarian follicles that are ripe and large. External examination of the female will usually show a bloated vulva with an open vagina. (2) Estrus begins with the ovulation of fully mature oocytes. The vulva remains in an extended state with an open vagina, and females are maximally receptive to male advances. When mice are maintained on a standard light-dark cycle, the estrus phase will usually begin soon after midnight and last for 6 to 8 hours. (3) The metestrus phase follows, when mature eggs move through the oviducts and into the uterus. The vulva is no longer bloated, and the vagina is now closed.

At the end of metestrus, a physiological branch point occurs with the direction to be taken dependent on whether a successful copulation has occurred. The act of successful copulation induces hormonal changes that prepare the uterus for a pregnancy which will ensue under normal circumstances. However, a sterile copulation — one that does not lead to fertilization — can induce a state of pseudopregnancy (see section 6.2.3.2). A pseudopregnancy can extend the metestrus phase by as long as 10-13 days.

(4) If pregnancy does not occur, the metestrus phase is ultimately followed by the last phase of the estrus cycle: diestrus. Unfertilized eggs are eliminated, the vagina and vulva are at a minimum size, and new follicles begin to undergo a rapid growth for the next ovulation. (The proestrus and estrus phases together constitute the follicular phase; the metestrus and diestrus phases together constitute the luteal phase.)

Once animals have been together for more than a few days, mating will be restricted to the late proestrus/early estrus portion of the female cycle. It is only during this period that a female will be receptive and that a male will normally be interested. (However, in some instances, when a new couple is first brought together in a cage, the male will rape his partner, irrespective of her estrus phase). Mating typically occurs over a period of 15 to 60 minutes with clear strain-specific differences: DBA males are quick (20 minutes) and BALB/c males are slow (one hour) according to Wimer and Fuller (1966). The male first examines the female genitalia and then mounts his mate and withdraws from one to one hundred times until ejaculation occurs during a final mounting. The male is quiet for a short period of time and then resumes normal activity. Although a full sperm count is not built up again for two days, it is possible for a male, especially an outbred one, to mate with up to three females in a single night, causing all to become pregnant. Different inbred strains have very different average times for recovery of libido, defined operationally as the time between attempted matings. DBA/2 mice can mate again within one hour, whereas B6 males usually wait for four days (Wimer and Fuller, 1966).

In some instances, one may want to maximize the rapid output of offspring from a single male. This situation could arise with rare genotypes such as new mutants or first generation transgenic founders. For this purpose, a single male can be rotated among sets of females (two or three per cage) in three or four cages. The factors that play a role in the length of each rotation have just been discussed: the length of the estrus cycle, the time it takes for a male to recover a full sperm count, and the libido recovery time. Together, these factors suggest an optimal rotation period of four days in each cage. For full optimization of offspring output, a male should receive two new, 8-week old, virgin females in his cage, every four days.

Fertilization takes place in the upper reaches of the oviduct (a region referred to as the ampulla). The egg remains viable for 10-15 hours after ovulation, although a gradual aging process slowly reduces the probability that fertilization will occur. Fertilization causes an immediate activation of the egg and induces the completion of the second meiotic division which leads to the formation of the second polar body within two hours.

The actual process of fertilization can be divided into a series of highly ordered steps that lead ultimately to the joining of a single sperm cell with an ovulated egg (Wassarman, 1993). The first step in this process occurs with the binding of multiple spermatozoa to the zona pellucida, a thick extracellular coat that surrounds the egg. The association between the zona and the sperm surface triggers the acrosome reaction which affects an elongated sperm-specific membrane-bound organelle just below the surface that contains a specialized protease called acrosin. The acrosome reaction is a form of exocytosis that results in the complete loss of the plasma membrane overlying the acrosome in hybrid vesicles along with the outer acrosomal membrane. The acrosomal contents are released, and these allow the resulting "acrosome-reacted" sperm to protease-digest its way through the zona pellucida to reach the perivitelline space between the zona and the egg plasma membrane. Finally, fusion occurs between the egg plasma membrane and the plasma membrane overlying the equatorial region of a single sperm cell. Fusion leads to the activation of the egg and the initiation of embryonic development.

The ultimate fusion reaction is not species-specific and can occur between heterologous gametes when the zona pellucida is first removed from the egg. Thus, in general, the main biochemical barrier to cross-species fertilization appears to lie within the initial interaction between the sperm plasma membrane and the egg zona pellucida. The specificity of this interaction implicates the existence of specific complementary molecules on egg and sperm, referred to respectively as the "sperm receptor" and the "egg binding protein" or EBP. The sperm receptor has been identified as a specific zona protein called ZP3 (Wassarman, 1990). The identity of the sperm surface EBP is still under investigation with multiple candidates described to date.

After a successful copulation has been completed, particular components of the male ejaculate will coagulate to form a hard plug that occludes the entrance to the vagina. The plug is a coagulum of fluids derived from both the vesicular and coagulating glands, and as such, it can be produced even by a vasectomized male. Usually the plug is visible through a simple visual examination of the vulva. In other cases, a probe will be required to detect a plug located further back in the vagina. The most common probe used for this purpose is a simple dental tool with a blunt end. "Plugging" should be performed as early as possible in the morning after a potential mating. By noon, some inbred strain plugs will begin to disappear, however, most will persist for 16 to 24 hours after copulation. Plugs formed by outbred mice can persist for several days.

Later in the pregnancy, from 10-to-12 days post-conception and beyond, it becomes possible to feel the maturing fetuses within the uterus by simple palpation. Pregnancy palpation is most readily carried out on older, multiparous females who have looser skin and are more accustomed to being handled. Right-handed workers should hold the female in the left hand (left-handers should hold the mouse in the right hand), with the thumb and forefinger grasping the skin behind the neck, and the smallest finger holding back the tail. The other fingers of this hand should be brought in behind the mouse to arch her forward. When the female is securely held by one hand and relatively calm, one should use the other hand to close down firmly on the abdomen close to the spine on one side at a time with the forefinger and thumb and then gently move the fingers out. Initially, a pregnant female will seem to have a string of beads on each side of her body. As development proceeds, these "beads" will mature into larger, more-defined shapes. With experience, this method can be used to determine the gestational stage of a pregnancy to within a single day.

It is possible to identify a state of pregnancy in young females by a simple visual inspection that does not even require one to handle the animal. The gestational day at which this becomes possible is greatly dependent on a number of factors including the age of the female, the number of fetuses inside, and whether she has given birth previously. For first-time pregnant females carrying large litters, tell-tale bulges from the center of her body can be detected by day 15. At the opposite extreme, older multiparous females with small litters never "show" in this way. Fortunately, these older animals are easier to palpate when a prenatal determination is required.

The gestation period for the mouse ranges from 18 to 22 days. Different strains have different averages within this range but even within a single strain, and even for a single female, there can be significant differences from one pregnancy to the next. Many different factors can have an effect on the length of pregnancy. For example, larger litters tend to be born earlier (Rugh, 1968), as is the case with humans as well. Non-inbred females tend to have shorter pregnancies then inbred ones, but this may be simply because they tend to produce larger litters as well as larger pups. Birth occurs most frequently between the hours of midnight and 4:00 A.M. when animals are maintained under a standard light-dark cycle; however, it can occur anytime of the day or night.

The gestation period can be greatly extended when the pregnant mother continues to nurse a previous litter. Prolongation up to seven days is not uncommon, and birth can sometimes be pushed back by as many as 16 days (Grüneberg, 1943; Bronson et al., 1966). This fact should be kept in mind when trying to count back from the day of birth to the day of conception in order to determine paternity for females in contact with sequential males.

The fertilized embryo is a free-floating entity in the female reproductive tract for the first 4.5-to-5 days of development. It is during this pre-implantation period that external events can play a role in determining whether a successful implantation will occur. Obvious disturbances to the mental health of the pregnant female — such as erratic lighting, extremes in temperature or humidity, high noise levels, or insufficient food and water — can cause a failure to implant. In addition, there is one other less obvious disturbance that is highly significant in the eyes of the female — the introduction into her cage of a male other than the one with whom she had mated. If the foreign male is not genetically identical to her partner, he can cause a premature termination of the pregnancy through a mechanism which is almost certainly a hormonally-induced block to implantation (Bruce, 1959; Bruce, 1968). This pregnancy block is also known as the Bruce Effect (after its discoverer) and it provides an obvious selective advantage by ensuring that females will use their resources only to raise offspring who carry the genes of the intruding male (who is presumably more fit since he has displaced the original mating male). With the previous pregnancy terminated, the female can quickly become pregnant again with her new partner.

It is interesting that females do not recognize males from the same inbred strain as foreign (Bruce, 1968). On the other hand, a pregnancy block is induced in nearly all other cases. These findings indicate that one or more genetic differences are responsible for the distinction between the original and the intruder male, but in addition, they clearly show that the genetic recognition system is highly polymorphic. Further studies with congenic and coisogenic strains have demonstrated conclusively that a major component of this recognition system is the highly polymorphic class I family of genes in the major histocompatibility complex (Yamazaki et al., 1986).

The Bruce effect has important implications for the management of a breeding mouse colony. Quite simply, if a mating event has occurred and one wants to recover live-born offspring from this mating, for the first five days that follow, the pregnant female should not be placed either into a cage with a foreign male or in contact with bedding that has been soiled by a foreign male. After this initial stage, there is no longer any problem. In fact, if one wishes to quickly set up a new mating pair, one should be sure to do it before the litter is born. If a foreign male is in the cage at the time of birth, he will normally accept the newborn pups. (Presumably, he "thinks" these pups are his own.) On the other hand, if a male is placed into a new cage that already has newborn pups, he is likely to kill them ("knowing" that he couldn’t possibly have been the father).

A mouse is born naked with closed ears and eyes, and if a female, with a closed vagina. Hair begins to appear at two to four days, ears open at three to five days, and eyes open at about 14 days. Typically, the vagina opens at 24 to 28 days of age, but it can be delayed in some mice until they are 35 to 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.

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 to 10 days with the appearance of nipples along the ventral side of the female, and at the age of weaning (18 to 28 days), when the penis has developed more fully in the male.

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 six 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 two to four), or gross morphological characters. To ensure optimal growth of selected animals, litters should be culled to five to six pups during the first days after birth. A further reduction to three to four 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. But, 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, one-to-two 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 two-to-three age-matched pups that are clearly distinguishable from those born to the mother.

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 biggest, 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.

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 four weeks of age. When young are kept with their mothers for a longer period, they are more likely to thrive as adults.

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 to 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.

Although it is possible to make general statements about the gross characteristics of all laboratory mice — they reach adult weights of 22 to 40 grams, they have life spans of one to three years, they have a gestation period of 18 to 22 days, and an average litter size of five to ten 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.

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.

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 grams for males and 25 grams for females (Altman and Katz, 1979). The largest of the commonly available inbred strains is AKR/J with males that reach 40 grams in weight. The smallest is 129/J with full-grown males and females that weigh 27 grams and 22 grams 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 to 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 two and half years and some animals survive as long as three and half 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.

Although artificial insemination is a critical tool for reproductive biologists working with other species (including humans), it is not often used by mouse geneticists. It’s major use in other species is to initiate a successful pregnancy when, for any of a number of reasons, the male cannot or should not, be directly involved in the process of mating. Artificial insemination has been a boon to the cattle industry because the semen from one good bull can be shipped around the world to impregnate unlimited numbers of females. Male mice are somewhat smaller than bulls and, as a consequence, the whole animal can be shipped for a cost that is likely to be the same (or less) than one would pay for frozen semen alone. Furthermore, obtaining semen from a mouse is a "one-shot" deal. Since assisted masturbation of the male mouse is not practical, sperm must be recovered from the epididymis after the animal has been sacrificed.

There are some special cases where artificial insemination can be used as an experimental tool for the study of the mouse. One example is in those cases where, for behavioral reasons, males of a particular strain refuse to mate with selected females of another strain. This scenario is most likely to occur when the males and females are members of different Mus species. West and colleagues (1977) used artificial insemination to overcome this problem in order to determine the viability of various hybrid embryos formed between distantly related members of the Mus genus.

Another use of artificial insemination is in those cases where one wants to alter the composition of the sperm pool. For example, Olds-Clarke and Peitz (1985) were able to analyze the relative fertilizing potentials associated with sperm obtained from two different males by mixing equal numbers together before insemination. Finally, there will always be the case where a one-of-a-kind male — such as a first generation transgenic or another new mutant — refuses to participate in the mating process. As a last resort, one can recover epididymal sperm from such an animal for a single chance at achieving a pregnancy. Detailed protocols for sperm recovery and artificial insemination have been described elsewhere (Rugh, 1968; West et al., 1977; Olds-Clarke and Peitz, 1985).

When a choice is possible, females to be inseminated should not be inbred; F1 hybrids and random-bred animals will always have higher levels of fertility. A successful fertilization can only occur when the inseminated female is in the late proestrus/early estrus stage of the estrus cycle. Appropriately staged females can be obtained either by visual inspection of naturally cycling animals (as described earlier in this chapter) or through superovulation (see section 6.2). The implantation of fertilized embryos will occur only in females that have been stimulated into a state of pseudopregnancy (section 6.2.3). If the investigator intends to use a sterile stud male for this purpose, the mating should be performed after the insemination (within 0.5-2 hours) so that the vaginal plug does not interfere with the protocol (Olds-Clarke and Peitz, 1985). If pseudopregnancy is to be induced manually, it should be accomplished in the fully alert female prior to the insemination protocol (see section 6.2.3 for details).

In a small number of instances, females that express certain mutations may be fertile in the sense that they are able to produce functional oocytes but infertile in the sense that they are physically unable to bring offspring to term. Such females may not be able to mate, they may not be fit enough to allow gestation to proceed properly or they may be unable to birth live offspring. In such cases, it is possible to transplant the ovaries from these incapacitated females into healthy females of another strain as means for obtaining germ line transmission (Russell and Hurst, 1945; Stevens, 1957). This protocol is commonly used at the Jackson Laboratory to maintain several mutant strains of mice, including those that carry the obese mutation or the dystrophia muscularis (musculuar dystrophy) mutation.

A third method of assisted reproduction entails fertilization outside the female reproductive tract. There are two general types of circumstances when in vitro fertilization becomes useful: when the female partner of a cross is unable to carry litters to term for one reason or another; and when an investigator wants to establish the timing of fertilization to a more precise degree and/or wants to synchronize the development of a batch of embryos for later recovery and analysis. A detailed discussion of this procedure and all other aspects of embryo manipulation are provided in the manual by Hogan and her colleagues (Hogan et al., 1994).