High hydrostatic pressure treatment of porcine oocytes before handmade cloning improves developmental competence and cryosurvival

An innovative technique, called the high hydrostatic pressure (HHP) treatment, has been recently reported to improve the cryosurvival of gametes or embryos in certain mammalian species. The aim of the present study was to investigate the in vitro and in vivo developmental competence and cryotolerance of embryos produced by handmade cloning (HMC) after pressure treatment of recipient oocytes. In vitro-matured porcine oocytes were treated with a sublethal hydrostatic pressure of 20 MPa (200 times greater than atmospheric pressure) and recovered for either 1 or 2 h (HHP1 and HHP2 groups, respectively) before they were used for HMC. After 7 days of in vitro culture, blastocyst rates and mean cell numbers were determined. Randomly selected blastocysts were vitrified with the Cryotop method based on minimum volume cooling procedure. The blastocyst rate was higher in the HHP2 group than in the control group (68.2 +/- 4.1% vs. 46.4 +/- 4.2%; p < 0.01), while there was no difference between HHP1 and control group (52.1 +/- 1.2% vs. 49.0 +/- 2.7%; p > 0.05). Similar mean cell numbers of produced blastocysts were obtained in HHP2 and control groups (56 +/- 4 vs. 49 +/- 5; p > 0.05). Subsequent blastocyst vitrification with the Cryotop method resulted in significantly higher survival rate after thawing in the HHP2 group than in the control group (61.6 +/- 4.0% vs. 30.2 +/- 30.9%; p < 0.01). Fifty-six and 57 day 5 to day 7 fresh blastocysts in HHP1 group were transferred into two recipient sows on day 5 of the estrous cycle. One recipient was diagnosed pregnant and gave birth to two healthy piglets by naturally delivery on day 122 of gestation. This pilot study proved that the sublethal HHP treatment of porcine oocytes before HMC results in improved in vitro developmental competence and cryotolerance, and supports embryonic and fetal development as well as pregnancy establishment and maintenance up to the birth of healthy piglets.