85 ASSISTED HATCHING IMPROVES POST-WARMING IN VITRO VIABILITY OF VITRIFIED PORCINE BLASTOCYSTS

Abstract

The objective of this study was to improve the vitrification protocol that was used to obtain the first reported litter of pigs resulting from the transfer of vitrified zona pellucida-intact blastocysts (Beebe et al. 2000 Theriogenology 53: 249 abstr). We had previously noticed that vitrified and warmed early blastocysts rarely hatched after 48 h in vitro culture, starting to degenerate after 24 h. These experiments examined whether assisted hatching, initially done by removing the zona, then by zona thinning, would improve post-warming in vitro viability. Early blastocysts were surgically recovered from mature Large White x Landrace sows five days after mating. Embryos were washed 3 times in modified phosphate-buffered saline (mPBS; Quinn et al. 1982 J. Reprod. Fert. 66:161–168), and then washed twice and cultured in 40-μL droplets of NCSU23 + 10% fetal bovine serum under mineral oil at 39°C until ready for vitrification. Embryos were cultured for 25 min with 7.5 μg mL⁻¹ cytochalasin B, placed into a 0.5-mL eppendorf tube in 30 μL of the culture medium, centrifuged for 13 min at 13000g and then recovered back to the culture medium. All media involved in the vitrification protocol included cytochalasin B. Centrifuged embryos were equilibrated in 2 M ethylene glycol in mPBS at 25°C for 5 min, washed briefly in 8 M ethylene glycol + 7% polyvinylpyrrolidone in mPBS for approximately 20 sec, loaded into open pulled straws and plunged into liquid nitrogen. Warming and rehydration was performed by immersing the end of the straw containing the embryos into 1.2 mL 1 M sucrose in mPBS at 39°C, allowing the thawing media to enter the straw and recovering the embryos from the straw with a pipette, followed by 2 min in 1 M ethylene glycol in mPBS, and 2 min in 0.5 M ethylene glycol, both at 25°C. The zona-intact group (ZI) was placed in mPBS at 39°C for at least 5 min, then cultured in vitro for 24 h. The zona-free group (ZF) was washed briefly in mPBS, and the zona was removed with 0.5% pronase in PBS for 30 sec, then washed extensively in mPBS, all at 25°C, and then washed and cultured as described for 24 h. After 24 h embryos that had reformed the blastocoel and expanded were considered viable. Viable embryos were fixed with 100% ethanol and the nuclei stained with 10 ng mL⁻¹ bisbenzimide, visualized and counted using fluorescent microscopy. There were 3 experiments. Removing the zona did not affect survival rates (ZI 81% n = 21; ZF 91% n = 23) but improved the cell count by 56% (cell number ± SEM; ZI 39.1 ± 2.8 n = 17; ZF 60.8 ± 4.3 n = 20; P < 0.05 by ANOVA). A subsequent series of experiments found that zona-thinned embryos (0.25% pronase for 10 sec) had the same survival and cell count as zona-free embryos. These experiments show that vitrified porcine blastocysts benefit from assisted hatching, whether the zona pellucida is removed or just thinned.