section, as opposed to 0.9 percent of calves produced by standard AI techniques. Because of this, it is becoming more common to deliver IVP offspring by elective caesarian section (Eyestone, 1999). Again, the number of times that this procedure should be performed on any individual animal during her lifetime is an issue of concern. The selection of older, higher parity cows as recipients is important to decrease the incidence of dystocia.

There also is a potential for IVF to have longer-term effects, although detailed data for livestock are lacking (Van Reenen et al., 2001). Even though they are heavier at birth, and might have enlarged organs, IVP-produced bulls seem to have normal semen quality and heifers show normal reproductive maturation (Van Wagtendonk-de Leeuw et al., 2000). IVP calves have normal growth rates and slaughter weights (Farin and Farin, 1995; McEvoy et al., 1998). Studies with mice, however, have shown that in vitro manipulation can result in long-term phenotypic changes (Reik et al., 1993), including retarded growth and abnormal DNA methylation patterns; these changes can be transmitted to the offspring (Römer et al., 1997). Intracytoplasmic sperm injection (ICSI) is under development for fertilizing livestock embryos (Chapter 1), and ICSI procedures have been combined with microinjection to produce transgenic animals (Perry et al., 1999). A concern is that, since the normal fertilization method of sperm and egg membrane fusion is bypassed—as is the sperm selection that normally would take place in the female reproductive tract (Galli and Lazarri, 1996)—embryos can be produced from abnormal sperm (Liu et al., 1995), possibly resulting in abnormal offspring.

EFFICIENCY OF PRODUCTION AND NUMBER OF ANIMALS NEEDED

Microinjection (Chapter 2) is an extremely inefficient method for producing transgenic offspring. Although the success of the method varies by species and gene construct, it has been estimated that less than one percent of microinjected livestock embryos result in transgenic offspring, and, of those, typically fewer than half actually express the transgene (Pursel et al., 1989; Rexroad, 1994). Ebert and Schindler (1993) reported efficiencies of between 0 to 4 percent for production of transgenic pigs, cattle, sheep, and goats. About 80 to 90 percent of the mortality occurs very early during development, before the eggs are even mature enough to be transferred to the recipient female (Eyestone, 1994), but postnatal mortality also occurs (Pursel et al., 1989).

Even if an individual does express the transgene, it might not be transmitted to subsequent generations. Approximately 30 percent of transgenic mice are mosaics, which means that they carry the transgene in only some of their cells (Wilkie et al., 1986). High rates of mosaicism are observed in other animals as well (e.g., fish, Hallerman et al., 1990; Gross et al., 1992). In one



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