114 EFFECT OF CYTOCHALASIN B TREATMENT ON VITRIFICATION OF CAPRINE PARTHENOGENIC BLASTOCYSTS

Abstract

Production of human recombinant proteins in the milk of transgenic animals has been shown to be a viable production system. Protection of the animal genetics involved is paramount. Vitrification of embryos is a simple, time-efficient way of preserving an animal’s genetics without the formation of damaging ice crystals during the freezing process. Cytochalasin B has been shown to increase the viability of porcine blastocysts by reducing damage to microfilaments and other cytoskeletal components. These experiments utilized caprine parthenogenic blastocysts as a model to compare the viability of parthenotes treated with or without cytochalasin B prior to and during vitrification. Abattoir oocytes were in vitro-matured in M199 with 10% goat serum containing FSH, LH and gentamycin for 18 to 21 h. Parthenogenic blastocysts were produced by treating in vitro matured abattoir oocytes with ionomycin for 5 min (5 μM) and with 6-dMAP (3 mM) for 3 h followed by culturing in SOF + 0.8% BSA for 7 to 8 days at 38°C with 6% O₂, 5% CO₂, and 89% N₂ in a modular incubator chamber. The experimental group was treated with cytochalasin B (5 μg/mL)in the culture media for 30 to 45 min prior to and thereafter throughout the vitrification process. All blastocysts (both the experimental group and the control group) were washed through two ovum culture media (OCM) droplets for 5 min each. The blastocysts were incubated in vitrification solutions 1 and 2 (10% glycerol in OCM and 10% glycerol + 20% ethylene glycol in OCM, respectively) for 5 min each, followed by vitrification solution 3 (25% glycerol + 25% ethylene glycol in OCM). They were then aspirated immediately into a 0.25 cc cryopreservation straw, followed by an air bubble, and then a 0.25 M sucrose solution in OCM. The straws were immediately plunged into liquid nitrogen and stored at −196°C. One to four days later, straws were thawed in air for 5 s at room temperature, then in 22°C water for 15 s. After thawing, the contents of the straw were expelled, mixed, held for 5 min, and finally placed in OCM for 5 min. Recovered embryos were placed in SOF + 20% FBS and incubated at 38°C with 5% CO₂ in air overnight. Viability was determined by re-expanding and subsequent hatching of the blastocyst. As shown in Table 1, there were no significant differences between re-expansion and hatching of blastocysts with cytochalasin B treatment compared to blastocysts not treated with cytochalasin B. These results suggest that, unlike porcine embryos (Dobrinsky et al., 2000 Biol Reprod 62, 564–570), cytochalasin B treatment does not improve the post-thaw viability of vitrified caprine parthenogenic blastocysts.

Table 1