Preservation of bovine preantral follicle viability and ultra-structure after cooling and freezing of ovarian tissue.
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.
Celestino
Juliana Jales de Hollanda
JJ
Laboratory of Manipulation of Oocytes and Ovarian Preantral Follicles-LAMOFOPA, Faculty of Veterinary, Ceará State University, Fortaleza, CE, Brazil.
dos Santos
Regiane Rodrigues
RR
Lopes
Cláudio Afonso Pinho
CA
Martins
Fabrício Sousa
FS
Matos
Maria Helena Tavares
MH
Melo
Mônica Aline Parente
MA
Báo
Sônia Nair
SN
Rodrigues
Ana Paula Ribeiro
AP
Silva
José Roberto Viana
JR
de Figueiredo
José Ricardo
JR
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
D003451:Cryoprotective Agents; D011409:Propylene Glycols; D019855:Ethylene Glycol; D014343:Trypan Blue; D005990:Glycerol; D004121:Dimethyl Sulfoxide