Preservation of bovine preantral follicle viability and ultra-structure after cooling and freezing of ovarian tissue

• Published in: Animal reproduction science Vol. 108; no. 3; pp. 309 - 318
• Main Authors: Celestino, Juliana Jales de Hollanda, Santos, Regiane Rodrigues dos, Lopes, Cláudio Afonso Pinho, Martins, Fabrício Sousa, Matos, Maria Helena Tavares, Melo, Mônica Aline Parente, Báo, Sônia Nair, Rodrigues, Ana Paula Ribeiro, Silva, José Roberto Viana, Figueiredo, José Ricardo de
• Format: Journal Article
• Language: English
• Published: Netherlands Elsevier B.V 01.11.2008; [Amsterdam]: Elsevier Science
• Subjects: Animals; assisted reproductive technologies; Bovine; Cattle - physiology; Chi-Square Distribution; Cooling; cows; cryopreservation; Cryopreservation - methods; Cryopreservation - veterinary; cryoprotectants; Cryoprotective Agents - pharmacology; cytotoxicity; differential staining; dimethyl sulfoxide; Dimethyl Sulfoxide - pharmacology; ethylene glycol; Ethylene Glycol - pharmacology; Female; female fertility; freeze-thaw cycles; Freezing; germplasm conservation; glycerol; Glycerol - pharmacology; Microscopy, Electron, Transmission - veterinary; Ovarian Follicle - physiology; Ovarian Follicle - ultrastructure; ovarian follicles; Preantral follicles; propanediols; Propylene Glycols - pharmacology; storage temperature; Trypan Blue - chemistry; ultrastructure; Viability; Preantral follicles; Cooling; Bovine; Viability; Freezing
• ISSN: 0378-4320; 1873-2232
• DOI: 10.1016/j.anireprosci.2007.08.016

Bovine preantral follicles within ovarian fragments were exposed and cryopreserved in absence or presence of 1.5 M glycerol (GLY), ethylene glycol (EG), propanediol (PROH) or dimethyl sulfoxide (DMSO), undergoing a previous cooling at 20 °C for 1 h (protocol 1) or at 4 °C for 24 h (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 °C for 1 h (78%) and 4 °C for 24 h (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%), 1.5 M EG (78 and 71%), as well as frozen in 1.5 M EG (74 and 77%). Percentages of viable follicles in control were similar to those observed after exposure (75%) and freezing (76%) in presence of 1.5 M DMSO only when protocol 1 was used. The increase of the concentration from 1.5 to 3.0 M, 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 °C for 24 h before cryopreservation (protocol 2) does not affect the viability of bovine preantral follicles when 1.5 M EG is present in the cryopreservation medium.