Piglets born from centrifuged and vitrified early and peri-hatching blastocysts

Cryopreservation of zona-intact porcine embryos has been relatively unsuccessful to date, although some success has been obtained with lipid reduced morulae and early blastocysts. This study adapted some vitrification protocols used successfully with late blastocysts for use with early zona-intact blastocysts, using actin depolymerization, centrifugation, and open-pulled (OPS) straws. Initially, Day 6 peri-hatching blastocysts were collected, cultured for 40 min in 7.5 microg/ml cytochalasin B and vitrified in 6.5 M glycerol and 6% BSA (VS1) in either heat-sealed (HS) or open straws (OS). The post-thaw survival of those stored in HS was 15.4% after 24 and 48 h in vitro; storage in OS significantly improved survival (58.8% for both 24 and 48 h). When similar stage blastocysts were cultured in cytochalasin B and vitrified with 8 M ethylene glycol and 7% polyvinylpyrrolidone (PVP; VS2) in OS, survival was 44.4 and 33.3% for 24 and 48 h, respectively. Day 5 late morulae and early blastocysts were collected, cultured with cytochalasin B, and centrifuged or left intact (control), then vitrified with VS1 in HS or OS, or vitrified in VS2 in OS only. None of the intact control embryos survived thawing and 48 h culture in vitro. Centrifuged early blastocysts vitrified with VS1 showed good post-thaw survival in culture when stored in HS (62.8 and 60.5% for 24 and 48 h, respectively), or OS (75 and 63.6%). When vitrified with VS2 in OS, survival improved (80 and 76.7%). Peri-hatching blastocysts were vitrified in VS1, and early blastocysts were vitrified with VS1 and VS2. All blastocysts were stored in OS. The embryos were recovered and transferred to Day 4 and 5 pseudopregnant recipients (for Day 5 and 6 blastocysts, respectively). Of the five recipients receiving peri-hatching blastocysts, two became pregnant and delivered a total of eight piglets. All three recipients of early blastocysts vitrified in VS1 had a delayed return to estrus; while of the four receiving embryos vitrified with VS2, two were delayed in returning to estrus, and one was confirmed pregnant after 45 days. A litter of five piglets, one male and four female, was produced at 116 days of gestation. To our knowledge, this is the first litter of piglets produced from early blastocysts vitrified without micromanipulation to remove polarized lipid droplets.