title: High hydrostatic pressure treatment of porcine oocytes before handmade cloning improves developmental competence and cryosurvival.
abstract: 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.
authors.affiliation: Population Genetics and Embryology, Institute of Genetics and Biotechnology, University of Aarhus, Tjele, Denmark.
authors.forename: Yutao
authors.initials: Y
authors.lastname: Du
authors.forename: Lin
authors.initials: L
authors.lastname: Lin
authors.forename: Mette
authors.initials: M
authors.lastname: Schmidt
authors.forename: Ingrid B
authors.initials: IB
authors.lastname: Bøgh
authors.forename: Peter M
authors.initials: PM
authors.lastname: Kragh
authors.forename: Charlotte B
authors.initials: CB
authors.lastname: Sørensen
authors.forename: Juan
authors.initials: J
authors.lastname: Li
authors.forename: Stig
authors.initials: S
authors.lastname: Purup
authors.forename: Csaba
authors.initials: C
authors.lastname: Pribenszky
authors.forename: Molnar
authors.initials: M
authors.lastname: Molnár
authors.forename: Masaabc
authors.initials: M
authors.lastname: Kuwayama
authors.forename: Xiuqing
authors.initials: X
authors.lastname: Zhang
authors.forename: Huanming
authors.initials: H
authors.lastname: Yang
authors.forename: Lars
authors.initials: L
authors.lastname: Bolund
authors.forename: Gábor
authors.initials: G
authors.lastname: Vajta
mesh_terms: D000818:Animals; D001755:Blastocyst / Q000166:cytology / Q000502:physiology; D002470:Cell Survival; D019976:Cloning, Organism / Q000379:methods*; D015925:Cryopreservation / Q000379:methods / Q000662:veterinary; D046149:Embryo Culture Techniques / Q000379:methods / Q000662:veterinary; D005260:Female; D006801:Humans; D006874:Hydrostatic Pressure; D053652:Nuclear Transfer Techniques; D009865:Oocytes; D011247:Pregnancy; D011897:Random Allocation; D034421:Sus scrofa / Q000196:embryology