Embryo Freezing

The full potential of embryo transfer can only be reached if other techniques such as long-term preservation of the embryo (freezing), embryo sexing, and in vitro production of embryos are developed.

Preservation of embryos by deep freezing presents many obvious advantages such as long-term storage of genetic material from the female, ease of transportation and international exchange, and improvement of recipient management. Dromedary embryo freezing has been attempted by our laboratory as well as by others.^{(62, 63)} The method of freezing used in our laboratory is similar to those described for the bovine and equine species. The embryos are dehydrated by successive passage in a medium with increasing concentration of a cryoprotectant such as glycerol, then frozen in 0.25 ml straws.

Glycerolization

Embryos of good quality are selected for freezing. There are washed in fresh preservation medium without glycerol then transferred successively into 4 different media with increasing concentration of glycerol (0.5 mM, 0.75 mM, 1.0 mM, and 1.5 mM). The embryos are maintained in each solution for 10 minutes before being transferred to the next highest glycerol concentration. When all these steps are completed, the embryo is loaded into a 0.25 ml straw and allowed to equilibrate for another 10 minutes before freezing.

Freezing curve

A programmed embryo freezer designed for the standard freezing of equine and bovine embryos is used (Figure 11.12). The temperature in the straw is monitored continuously to follow a standard embryo freezing curve. The embryos are cooled from room temperature to seeding temperature (-7°C) at the rate of 0.3° to l°C/minute. The straws are held at -7°C for 10 minutes and water crystallization is induced (seeding) by touching the straws with a very cold rod. The seeding phase is following by further cooling of the straw from -7°C to -30°C at the rate of 0.3°C/minute. The straws are held at -30°C for 10 minutes then plunged directly into liquid nitrogen.
 

Thawing and deglycerolization

Embryos are thawed by placing the straws in a water bath at 37°C for 1 minute. The content of the straw is emptied into a small Petri dish. Glycerol is removed (deglycerolization) by successive passage in a medium containing sucrose and/or decreasing glycerol concentration. The embryo is then placed in a new straw and transferred into a recipient in the same manner described for fresh embryos.

Results and future developments

Initial experiments with frozen dromedary embryos in our laboratory yielded very poor pregnancy results (10%). However, research is in progress to determine the best conditions (stage of embryos, medium, nature and concentration of cryoprotectant, freezing curve) for freezing embryos from the species. The major limitation for embryo freezing in the dromedary is the relatively large size of the embryos collected from the uterus. Size of the embryo has been identified as the major factor in the success of embryo freezing in the equine. In this species, embryos that are larger than 250 µm do not survive the freezing process well. Unfortunately, collection of embryos before hatching is not easy in the dromedary.