Cryopreservation of equine embryos: ice or glass?

ABSTRACT

Background: Gamete cryopreservation has permitted widespread preservation of valuable genetics. However, cryopreservation of equine embryos is still not a routine procedure in the industry due to biological reasons [16] and inconsistent results in the field. Review The basic principle of cryopreservation is to avoid ice crystal formation. Small (< 250 mm) equine embryos can be successfully frozen by slow-cooling techniques to allow slow dehydration of the embryo, thus avoiding ice crystal formation and subsequent lysis of plasma membranes, cytoskeleton disruption, and organelle dysfunction [10,14-15]. Embryos are dehydrated with glycerol, loaded in 0.25 ml straws and frozen by a programmable freezer with liquid nitrogen. Embryos are thawed in a water bath followed by serial dilutions in glycerol and sucrose [14]. Recently, a variation of the slow freezing technique [1] in field conditions produced 16 day pregnancy rates (70%), not different from controls. Vitrification is an alternative to slow-freezing and is defined as a physical state similar to glass [13]. There is no ice crystal formation or concentration of solutes. The methodology requires a high concentration of cryoprotectants, small volumes, high viscosity and rapid cooling rates. Numerous studies with different formulas and containers have resulted in successful vitrification of small equine embryos [6,8,10-11]. An economical and simple vitrification technique was developed [6] that permits in-straw dilution of embryos for direct transfer in the field. Pregnancy rates on day 16 were 65% and not different from controls. In a subsequent study [9], embryos (n=40) < 250 mm in diameter were obtained from eFSH treated mares, half cooled for 16-20 h and vitrified and the other half vitrified only. The pregnancy rate on day 16 after direct transfer under field conditions was 65 and 70% respectively. However, personal communications from practitioners around the world have found poor pregnancy rates (250 mm) have poor survival rate after cryopreservation. One explanation is that the equine embryonic capsule composed of mucin like glycoproteins [12] that replaces the zona pellucida and surrounds the embryo until embryonic attachment (16- 21 d) may impede the proper penetration of cryoprotectants, leading to intracellular ice crystal formation. Another possibility is that the small surface area to volume ratio of embryos with large amounts of fluid in the blastocoele cavity may slow addition and removal of cryoprotectants [2]. A vitrification technique was developed with a modified three-step vitrification procedure for large embryos [3]. However, the pregnancy rate on day 16 (37 %) was unacceptable for commercial use. In a recent and promising study [5] after piercing the embryonic capsule with micromanipulation tools, pregnancy rates were 70% (5/7) at day 16. In vitro produced embryos have been cryopreserved successfully. Pregnancies (~50%) after slow freezing and thawing of equine blastocysts produced after ICSI have been consistently obtained [7]. In addition, pregnancies (65%, 5/8) have been obtained after vitrification in super open pulled straws , warming and transfer of 2- to 8-cell embryos produced by ICSI [4]. However, more studies are needed in this area. Conclusions: Even though vitrification offers theoretical advantages to minimize cell damage, the learning curve and practicality of the methods need to be standardized for commercial use. Nonetheless, depending on embryological stage, both methods (ice or glass) of cryopreservation can be used successfully to produce pregnancies.