Cryopreservation of Preimplantation Embryos of Cattle, Sheep, and Goats

Abstract

Preimplantation embryos from cattle, sheep, and goats may be cryopreserved for short-or long-term storage. Preimplantation embryos consist predominantly of water, and the avoidance of intracellular ice crystal formation during the cryopreservation process is of paramount importance to maintain embryo viability. Embryos are placed into a hypertonic solution (1.4 -1.5 M) of a cryoprotective agent (CPA) such as ethylene glycol (EG) or glycerol (GLYC) to create an osmotic gradient that facilitates cellular dehydration. After embryos reach osmotic equilibrium in the CPA solution, they are individually loaded in the hypertonic CPA solution into 0.25 ml plastic straws for freezing. Embryos are placed into a controlled rate freezer at a temperature of -6°C. Ice crystal formation is induced in the CPA solution surrounding the embryo, and crystallization causes an increase in the concentration of CPA outside of the embryo, causing further cellular dehydration. Embryos are cooled at a rate of 0.5°C/min, enabling further dehydration, to a temperature of -34°C before being plunged into liquid nitrogen (-196°C). Cryopreserved embryos must be thawed prior to transfer to a recipient (surrogate) female. Straws containing the embryos are removed from the liquid nitrogen dewar, held in room temperature air for 3 to 5 sec, and placed into a 37°C water bath for 25 to 30 sec. Embryos cryopreserved in GLYC are placed into a 1 M solution of sucrose for 10 min for removal of the CPA before transfer to a recipient (surrogate) female. Embryos cryopreserved in EG, however, may be directly transferred to the uterus of a recipient.

How Developments in Cryobiology, Reproductive Technologies and Conservation Genomics Could Shape Gene Banking Strategies for (Farm) Animals

“…This is certainly the case for cattle (Van Wagtendonk de Leeuw et al. 1997; Youngs 2011) and to a lesser extent also for sheep (Youngs 2011). Live offspring or pregnancies have also been reported for other farm animal species, including pigs (Li et al.…”

“…Yet, easy and cost-effective use of embryos in gene banks may be limited to these species for which collection and transfer techniques are available and operational. This is certainly the case for cattle (Van Wagtendonk de Leeuw et al 1997;Youngs 2011) and to a lesser extent also for sheep (Youngs 2011). Live offspring or pregnancies have also been reported for other farm animal species, including pigs (Li et al 2009), horse (Ulrich and Nowshari 2002;Galli et al 2007), goat (Rodrı´guez-Dorta et al 2007) and rabbit (Naik et al 2005), but in most of these species, the techniques are not routine.…”

Multiple Ovulation and Embryo Transfer in Cattle

“…Average pregnancy rates obtained after freezing-thawing the embryos from farm animals using the vitrification method are between 60-75%. In addition, the most suitable embryo development stage for the vitrification technique is the blastocyst stage (Youngs 2011).…”

Modern Reproductive Technologies to Improve Cattle Production

“…Methods for embryo cryopreservation have been reviewed recently (Youngs et al 2010;Youngs 2011a;Youngs 2011b). Historically, embryos had been frozen using glycerol as the cryoprotective agent to dehydrate the cells of the embryo prior to freezing.…”

“…However, pregnancy rates after transfer of frozen-thawed IVP embryos is typically only 40-50% (Youngs 2011a). Substantial research in underway on the cryopreservation of bovine IVP embryos using the ultra-rapid cooling method known as vitrification, and in the near future it is likely that pregnancy rates after transfer of vitrified/warmed embryos will approach those obtained with fresh IVP embryos.…”