Reproduction Abstracts

Introduction: The ovarian follicle has a three-dimensional structure in which the oocyte is surrounded by tightly connected follicle cells that mediate the action of external signals and nourish the gamete during its maturation. In vitro follicle techniques provide a tool to model follicle development in order to investigate basic biology and are further being developed as a technique to preserve human fertility or to product transgenic animals for cell therapies. Main objective of this work was to establish an effective method for retrieving viable, preantral pig follicles from ovarian medulla.

Material and methods: Medulla was collected from 30, 4–5 months old prepubertal gilts. Primordial and primary follicles were isolated from ovarian medulla using different types of collagenase (types I, II, and IV) and Liberase (DH, TM, and TH). Different treatment protocols have been also used. Follicles quality was assessed by evaluating their morphology and viability after H&E and fluorescent staining.

Results and discussion: The number of fully isolated follicles recovered from medullas treated by Liberase TH was always higher than from collagenase group (independently of an enzyme type). Very high proportion of follicles were viable after Liberase TH isolation, and most of follicles were of good morphology with a complete granulosa cell layer. TUNEL method indicated that Liberase-isolated follicles showed signs of atresia only occasionally and that the oolemma-follicular cell interface was well preserved. It seems, Liberase TH preparation is promising alternative allowing use of isolating primordial and primary pig follicles for future reproductive applications.

Supported by grant 2013/09/B/NZ9/00226.