title: Preservation of bovine preantral follicle viability and ultra-structure after cooling and freezing of ovarian tissue.
abstract: Bovine preantral follicles within ovarian fragments were exposed and cryopreserved in absence or presence of 1.5M glycerol (GLY), ethylene glycol (EG), propanediol (PROH) or dimethyl sulfoxide (DMSO), undergoing a previous cooling at 20 degrees C for 1h (protocol 1) or at 4 degrees C for 24h (protocol 2) in 0.9% saline solution. At the end of each treatment, preantral follicles were classified as non-viable/viable when they were stained/not stained with trypan blue, respectively. To confirm viability staining, ultra-structure of the follicles was evaluated by transmission electronic microscopy (TEM). Data were compared by Chi-square test (P<0.05). The storage of the ovaries at 20 degrees C for 1h (78%) and 4 degrees C for 24h (80%) did not reduce significantly the percentage of viable preantral follicles when compared to the control (75%). Similar results were obtained when ovarian fragments, respectively, for protocols 1 and 2, were exposed to MEM (78 and 77%), 1.5M EG (78 and 71%), as well as frozen in 1.5M EG (74 and 77%). Percentages of viable follicles in control were similar to those observed after exposure (75%) and freezing (76%) in presence of 1.5M DMSO only when protocol 1 was used. The increase of the concentration from 1.5 to 3.0M, for all cryoprotectants, reduced significantly the percentage of viable preantral follicles after freezing. Ultra-structural analysis has confirmed trypan blue results, showing that not only basement membrane, but also organelles, were intact in viable preantral follicles. In conclusion, ovarian tissue cooling at 4 degrees C for 24h before cryopreservation (protocol 2) does not affect the viability of bovine preantral follicles when 1.5M EG is present in the cryopreservation medium.
authors.affiliation: Laboratory of Manipulation of Oocytes and Ovarian Preantral Follicles-LAMOFOPA, Faculty of Veterinary, Ceará State University, Fortaleza, CE, Brazil.
authors.forename: Juliana Jales de Hollanda
authors.initials: JJ
authors.lastname: Celestino
authors.forename: Regiane Rodrigues
authors.initials: RR
authors.lastname: dos Santos
authors.forename: Cláudio Afonso Pinho
authors.initials: CA
authors.lastname: Lopes
authors.forename: Fabrício Sousa
authors.initials: FS
authors.lastname: Martins
authors.forename: Maria Helena Tavares
authors.initials: MH
authors.lastname: Matos
authors.forename: Mônica Aline Parente
authors.initials: MA
authors.lastname: Melo
authors.forename: Sônia Nair
authors.initials: SN
authors.lastname: Báo
authors.forename: Ana Paula Ribeiro
authors.initials: AP
authors.lastname: Rodrigues
authors.forename: José Roberto Viana
authors.initials: JR
authors.lastname: Silva
authors.forename: José Ricardo
authors.initials: JR
authors.lastname: de Figueiredo
mesh_terms: D000818:Animals; D002417:Cattle / Q000502:physiology*; D016009:Chi-Square Distribution; D015925:Cryopreservation / Q000379:methods / Q000662:veterinary*; D003451:Cryoprotective Agents / Q000494:pharmacology*; D004121:Dimethyl Sulfoxide / Q000494:pharmacology; D019855:Ethylene Glycol / Q000494:pharmacology; D005260:Female; D005990:Glycerol / Q000494:pharmacology; D046529:Microscopy, Electron, Transmission / Q000662:veterinary; D006080:Ovarian Follicle / Q000502:physiology* / Q000648:ultrastructure; D011409:Propylene Glycols / Q000494:pharmacology; D014343:Trypan Blue / Q000737:chemistry
chemical_list: D003451:Cryoprotective Agents; D011409:Propylene Glycols; D019855:Ethylene Glycol; D014343:Trypan Blue; D005990:Glycerol; D004121:Dimethyl Sulfoxide