Examination of viability and quality of ovarian tissue after cryopreservation using simple laboratory methods in ewe.

The objective of the present study is to assess viability tests and to evaluate follicle ovarian tissue quality after freezing-thawing procedures. Ewe's ovaries were harvested at the slaughterhouse, after dissection each ovarian specimen was divided into two groups: fresh tissue (control group) and frozen tissue.In the first part of the study, the follicles viability was assessed by trypan blue staining, calcein AM/ethidium homodimer-1 staining (LIVE/DEAD viability/cytotoxicity kit, Molecular Probes) and morphology in the two groups. In the second part of the study the quality of the whole ovarian tissue was evaluated by the quantification of the release of lactate dehydrogenase measurement (Cytotoxicity Detection kit ROCHE), DNA fragmentation by terminal deoxynucleotidyl transferase-mediated dUTP-biotin nick end labelling (TUNEL) in primordial and primary follicles (ApopDETEK Kit system Enzo) and morphology in the two groups. 100 Follicles (primordial and primary) were counted on both fresh and frozen hemiovary to assess this various tests. Ovarian follicle viability assessment was similar using trypan blue or calcein/ethidium staining. Follicles showed a decreased viability after freezing-thawing.After cryopreservation, a significant correlation between the percentage of normal follicles and viability rate was found using trypan blue (r=0.82, p<0.05) or calcein AM/ethidium homodimer-1 staining (r=0.76, p<0.05). Increased cytotoxicity showed by enhancement of LDH release was found after cryopreservation (21.60+/-1.1% vs 52.2+/-7.7%). A significant negative correlation between the percentage of morphologically normal follicles and cytotoxicity was observed. No significant difference in DNA fragmentation rate between frozen and control groups was found (26±8.2% vs 38±4.5%). We suggest the use of trypan blue staining for the histological assessment of viability, the use of LDH assay for the cytotoxicity assessement and finally the use of DNA fragmentation assessment to valid different freezing-thawing protocols.