title: Calcium concentration in vitrification medium affects the developmental competence of in vitro matured ovine oocytes.
abstract: The present study was designed to determine whether different calcium concentrations in the vitrification solutions could improve the developmental competence of in vitro matured ovine oocytes after cryopreservation. In vitro matured oocytes were vitrified with 16.5% ethylene glycol (EG) + 16.5% dimethylsulfoxide (DMSO) vitrification media. The base media contain different calcium concentrations, so that five experimental groups were obtained: TCM/FCS (TCM 199 + 20% fetal calf serum (FCS), [Ca(2+)] 9.9 mg/dl); PBS/FCS (Dulbecco Phosphate Buffered Saline (PBS) + 20% FCS, [Ca(2+)] 4.4 mg/dl); PBS(CaMg free)/FCS (PBS without Ca(2+) and Mg(2+) + 20% FCS [Ca(2+)] 2.2 mg/dl); PBS/BSA (PBS + 0.4% bovine serum albumin (BSA), [Ca(2+)] 3.2 mg/dl) and PBS(CaMg free)/BSA (PBS without Ca(2+) and Mg(2+) +0.4% BSA, [Ca(2+)] 0.4 mg/dl). After warming, the oocytes from the five experimental groups were assessed for survival, spontaneous parthenogenetic activation and developmental capacity via in vitro fertilization. Oocyte survival after vitrification procedures was better preserved in group PBS(CaMg free)/FCS compared to the others (P < 0.05). In addition, a positive correlation was found between calcium concentration in vitrification solutions and spontaneous parthenogenetic activation (correlation index 0,82; P < 0.001). Development of vitrified oocytes was significantly affected by vitrification media composition (P < 0.01). In particular, oocytes from group PBS(CaMg free)/FCS led to higher cleavage rates and blastocyst rate compared to the others. Our data showed that lowering calcium concentration in the vitrification medium improves the blastocyst rate of vitrified ovine oocytes, probably reducing the effect of EG + DMSO during vitrification. On the contrary, the replacement of FCS with BSA dramatically reduces the developmental potential of these oocytes.
authors.affiliation: Department of Animal Biology, University of Sassari, Sassari, Italy. succus@uniss.it
authors.forename: Sara
authors.initials: S
authors.lastname: Succu
authors.forename: Fiammetta
authors.initials: F
authors.lastname: Berlinguer
authors.forename: Giovanni G
authors.initials: GG
authors.lastname: Leoni
authors.forename: Daniela
authors.initials: D
authors.lastname: Bebbere
authors.forename: Valentina
authors.initials: V
authors.lastname: Satta
authors.forename: Francisco
authors.initials: F
authors.lastname: Marco-Jimenez
authors.forename: Valeria
authors.initials: V
authors.lastname: Pasciu
authors.forename: Salvatore
authors.initials: S
authors.lastname: Naitana
mesh_terms: D000818:Animals; D001755:Blastocyst / Q000187:drug effects; D002118:Calcium / Q000494:pharmacology*; D015925:Cryopreservation / Q000662:veterinary*; D003470:Culture Media; D047108:Embryonic Development / Q000187:drug effects; D005307:Fertilization in Vitro / Q000662:veterinary; D009865:Oocytes / Q000187:drug effects* / Q000254:growth & development; D010312:Parthenogenesis; D012756:Sheep
chemical_list: D003470:Culture Media; D002118:Calcium