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4.5.1 Artificial insemination

Although artificial insemination is a critical tool for reproductive biologists working with other species (including humans), it is not often used by mouse geneticists. Its 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).

4.5.2 Transplantation of ovaries

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, the dwarf mutation, or the dystrophia muscularis (muscular dystrophy) mutation. (Green and Witham, 1991).

4.5.3 In vitro fertilization

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 (1994).

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