title: Effects of different cryopreservation methods on post-thaw culture conditions of in vitro produced bovine embryos.
abstract: The aim of this work was to evaluate the effect of cryopreservation protocols on subsequent development of in vitro produced bovine embryos under different culture conditions. Expanded in vitro produced blastocysts (n = 600) harvested on days 7-9 were submitted to controlled freezing [slow freezing group: 10% ethylene glycol (EG) for 10 min and 1.2°C/min cryopreservation]; quick-freezing [rapid freezing group: 10% EG for 10 min, 20% EG + 20% glycerol (Gly) for 30 s]; or vitrification [vitrification group: 10% EG for 10 min, 25% EG + 25% Gly for 30 s] protocols. Control group embryos were not exposed to cryoprotectant or cryopreservation protocols and the hatching rate was evaluated on day 12 post-insemination. In order to evaluate development, frozen-thawed embryos were subjected to granulosa cell co-culture in TCM199 or SOFaa for 4 days. Data were analyzed by PROC MIXED model using SAS Systems for Windows®. Values were significant at p < 0.05. The hatching rate of the control group was 46.09%. In embryos cultured in TCM199, slow freezing and vitrification group hatching rates were 44.65 ± 5.94% and 9.43 ± 6.77%, respectively. In embryos cultured in SOFaa, slow freezing and vitrification groups showed hatching rates of 11.65 ± 3.37 and 8.67 ± 4.47%, respectively. In contrast, the rapid freezing group embryos did not hatch, regardless of culture medium. The slow freezing group showed higher hatching rates than other cryopreservation groups. Under such conditions, controlled freezing (1.2°C/min) can be an alternative to cryopreservation of in vitro produced bovine embryos.
authors.affiliation: Department of Animal Reproduction, College of Veterinary Medicine and Animal Science, University of São Paulo, São Paulo, Brazil.
authors.forename: Alessandra Corallo
authors.initials: AC
authors.lastname: Nicacio
authors.forename: Renata
authors.initials: R
authors.lastname: Simões
authors.forename: Fabiola Freitas
authors.initials: FF
authors.lastname: de Paula-Lopes
authors.forename: Flavia Regina Oliveira
authors.initials: FR
authors.lastname: de Barros
authors.forename: Maria Angelica
authors.initials: MA
authors.lastname: Peres
authors.forename: Mayra Elena Ortiz D'Avila
authors.initials: ME
authors.lastname: Assumpção
authors.forename: Jose Antonio
authors.initials: JA
authors.lastname: Visintin
mesh_terms: D000818:Animals; D001755:Blastocyst / Q000166:cytology* / Q000187:drug effects; D002417:Cattle; D015925:Cryopreservation / Q000379:methods*; D003451:Cryoprotective Agents / Q000494:pharmacology; D003470:Culture Media; D046149:Embryo Culture Techniques / Q000379:methods*; D019855:Ethylene Glycol / Q000494:pharmacology; D005260:Female; D005307:Fertilization in Vitro; D005615:Freezing; D005990:Glycerol; D006107:Granulosa Cells / Q000166:cytology; D058989:Vitrification
chemical_list: D003451:Cryoprotective Agents; D003470:Culture Media; D019855:Ethylene Glycol; D005990:Glycerol