As an alternative to cesarian section, embryo transfer has been used over the last 20 years. The advantages to cesarian section are that the embryos may be kept for long periods in liquid nitrogen and used for further rederivation whenever needed. The technique is also a must for the production of transgenic
Donor females \ Recipient females
Donor females \ Recipient females
animals. Disadvantageous is that more specialized equipment is needed for this method than for cesarian section. Embryo transfer rederivation is also a multistep method (Figure 11.5)
Embryos are normally harvested from superovulated females, i.e., multiple ovulations have been induced by hormonal treatment. In mice and rats, this is done by injection of pregnant mare serum gonadotropin (PMSG) combined with human chorionic gonadotropin (HCG) and best results are achieved in premature animals. HCG is normally given 48 h later than PMSG and has to be correlated to the light cycle. Females are then mated with the appropriate male the night after giving HCG. Next morning, the females are checked for plugs. In premature rats, these more frequently remain in the vagina than is the case for adult rats as used for cesarian section (see above). Natural matings compared to superovulated matings lead to a significantly lower embryo recovery.
The donor female is euthanized for harvesting the embryos. For rederivation procedures, two-cell stages are mostly used, as these are the most appropriate for freezing. These are recovered from the salpinx, 36 h after mating. Having euthanized the donor female, the salpinx is excised and transferred into an appropriate medium, in which a needle is used to flush out the embryos. Dependent on the risk of collecting hazardous infections, such as Mycoplasma spp., along with the embryos, the embryos are submitted to a series of rinsing steps. The embryos may be cultivated in vitro in order to synchronize with mating of the recipient female. When placed in straws in some appropriate medium supplemented with sucrose and some freezing protectant, embryos may be frozen in liquid nitrogen. The straws may simply be dropped into the nitrogen; a procedure known as vitrification. However, survival may be increased by the use of a so-called cryostat, i.e., equipment that slowly reduces the temperature. For thawing, the embryos are placed in a 37° water bath and the straws are emptied into a petri dish containing a dilution medium. The embryos are then submitted to a number of elution steps. There are no known limits for how long the embryos can be stored in liquid nitrogen and in professional hands the viability is normally above 90%.
If the outcome needs to be germ-free, transfer of embryos and all related procedures should be performed in isolators. The recipient mother needs to be mated with a vasectomized male to induce pseudopregnancy. This is, in principle, done and checked as described for time mating of donors for cesarian section. The mating is synchronized with production and cultivation of embryos and the stage in which these are to be transferred. The pseudopregnant female is placed in a surgical isolator and anaesthetized and the abdomen is entered through the back skin and both sides of the flank muscles as described for ovariectomy. The ovary with the salpinx is carefully grasped from the abdominal cavity, and while the bursa is opened with great care, the embryos are collected with a transfer pipette. The embryos are placed in the salpinx through the infundibulum. Equal amounts of embryos are placed in both salpinces. The wound is closed with sutures, and the recipient female is returned to develop her pregnancy in the isolator. From this point, the steps are similar to those of cesarian section.
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