Changes to porcine blastocyst vitrification methods and improved litter size after transfer

Abstract

The objective was to improve the protocol that was used to obtain the first reported piglets from transferred vitrified and warmed zona-intact blastocysts. Blastocysts were collected from superovulated sows and gilts, centrifuged to polarize lipid, vitrified, warmed and cultured for 24h or transferred immediately. Removing the zona pellucida after warming increased the number of cells in the surviving blastocysts (zona-free 60.8+/-4.3, zona-intact 39.1+/-2.8; P<0.05). Thinning the zona pellucida produced similar results to zona removal. Changing the basal medium of the vitrification and warming solutions from modified PBS to phosphate buffered NCSU-23 increased the number of cells (44.7+/-2.2 versus 56.0+/-3.9, respectively; P<0.05). Reducing the plunge temperature of the liquid nitrogen from -196 degrees C to less than -204 degrees C improved the embryo survival rate (61.9% versus 82.9%, respectively; P<0.05). These modifications were incorporated into the vitrification protocol that was used to vitrify and warm 105 blastocysts (that were subsequently transferred into four recipients). Three recipients became pregnant, farrowing three litters (average litter size, 5.3; 18.8% embryo survival in farrowing sows). Changing the warming protocol to using sucrose rather than ethylene glycol resulted in a trend towards improved embryo survival (73.5% versus 91.2%) but this was not statistically significant. Incorporating this modification, 203 blastocysts were vitrified, warmed and transferred into seven recipients. Five became pregnant and 36 fetuses were recovered (average litter size 7.2; 24.8% embryo survival in pregnant sows) at Day 40 of pregnancy. In conclusion, changes made to the vitrification protocol improved pregnancy rate and in vivo embryo survival compared to an earlier study using the original protocol.