Embryos can be bisected from the two-cell stage through the hatched blastocyst stage. However, because of logistical constraints, such as the need for surgical recovery, dividing pre-morula stage embryos will not be considered here (see Willadsen, 1982). There are two main reasons for splitting embryos. The first is to obtain identical twins, which are very useful for research as well as for certain commercial goals. The second is to increase productivity. Under above average commercial conditions, about 50 percent more calves result per two demi-embryos than per whole embryo.
Splitting bovine embryos is easy provided that the technician has patience, proper training, and appropriate equipment (Figure 30). Splitting need not involve complex technology, which means it is appropriate for less developed countries provided that there is a legitimate application.
Example of a micromanipulator system (A) and microtools for bisecting embryos (B)
Dozens of procedures for bisection of embryos have been published, from the very complex (Ozil, 1983) to the very simple (Williams and Moore, 1988). Most of these procedures consist of two stages: immobilizing the embryo and bisecting it. Immobilization can be done by applying suction to the zona pellucida, making a depression or cul-de-sac in the container, constructing a device that traps the embryo, or making the embryo stick to a surface, e.g. by roughening the surface of the container or using protein-free culture medium. The bisection is usually done with a broken fragment of razor blade or a fine glass needle (Figure 31). After the blastocyst stage, the embryo must be bisected so that both halves receive cells from the inner cell mass. After the late morula stage, there is no need to return demi-embryos to a zona pellucida (Warfield et al., 1987).
Pregnancy rates will be high provided that: (1) the embryo is immobilized without damaging it; (2) the bisection process does not damage too many cells, and (3) embryos are bisected reasonably symmetrically. Embryos also may be divided into thirds or quarters, but this lowers success rates considerably.
Immobilizing a blastocyst by means of suction using a micropipette and bisecting the embryo with a fragment of razor blade (A), and removing one demi-embryo from the zona pellucida. (These figures first appeared in Williams et al., Theriogenology, 22:524, 1984)
One or two demi-embryos may be transferred per recipient. It is probably best to place one demi-embryo in each uterine horn rather than placing both ipsilateral to the corpus luteum (Rowson et al., 1971). Twinning results in more calves per recipient. The main disadvantage is that there is increased morbidity and mortality when cows have two calves, and the calves will be slightly smaller than singles. Under most circumstances, twinning heifers is not recommended (see Chapter 2).