407. Effects of vitrification and slow-cooling on the structure of lamb and mouse ovarian tissues

Abstract

Follicular dynamics of ovarian tissue varies with species and age. The morphology and distribution of primordial, primary and secondary follicles were examined using mouse and lamb ovarian tissue before and after cryopreservation. Ovarian cortical tissue was processed from mouse (C57BL6J × CBA) and abattoir-sourced lamb ovaries and sliced into fragments (1 mm3). The fragments were randomly divided into three experimental groups. For vitrification, the tissue fragments were equilibrated (10% v/v ethylene glycol (EG) and DMSO, 20 min) and transferred to vitrification solution (17% v/v e.g. and DMSO and 0.75M sucrose, 3 min), and loaded onto a Fibreplug (CVM kit, Cryologic). For slow-cooling (SC), up to 10 ovarian fragments were placed in slow cooling solution (10% v/v DMSO and 0.1M sucrose, 5 min, RT), and then loaded in straws before placed in a programmable freezer (Cryologic CL8800i). For histological comparison, fresh and cryopreserved-thawed ovarian tissues were fixed, embedded and sectioned (5µm) and stained with haematoxylin and eosin. Primordial, primary and secondary follicles were evaluated and the normality of follicular structures was scored. Differences between treatment groups were examined using a Chi Square test. In lamb, 83% of follicles assessed were primordial, 16.4% primary and 0.6% secondary whereas in mouse the ratios were 60%, 27% and 13% respectively. In lamb, proportion of follicles with normal morphology after thawing for vitrification (77.2%) and SC (79.3%) was not significantly different from fresh controls (83.1%). Similarly, in mouse, the proportion of normal follicles after warming for vitrification (92.3%) and SC (90.5%) was not significantly different from fresh controls (91.4%). No obvious detrimental effects on morphology of follicles derived from either vitrification or slow-cooling of lamb and mouse ovarian tissue was observed.