412 VIABILITY OF ENZYMATICALLY ISOLATED BOVINE PRIMORDIAL AND PRIMARY OVARIAN FOLLICLES COLLECTED BY THE BIOPSY PICK-UP TECHNIQUE

Our institution recently developed a new tool for transvaginal, ultrasound-guided collection of ovarian biopsies from living donor cows (Aerts et al. 2005 Theriogenology 64, 947–957). The biopsy pickup (BPU) device consists of a modified needle guidance system, which is equipped with a trocar needle and carries a 60-cm-long true-cut biopsy needle. In a previous experiment, the presence of primordial and preantral follicles in BPU-derived biopsies was demonstrated. As follicular integrity is a prerequisite for further culture or research, the aim of the present study was to assess the viability of enzymatically isolated primordial and primary ovarian follicles collected by the BPU instrument. Four cows were subjected to a one-time BPU procedure. Four cortical biopsies were collected per ovary from each animal. Follicle viability was determined by the dual fluorescent probe technique described by Cortvrindt and Smitz (2001 Fertil. Steril. 75, 588–593). In live cells, calcein-AM is converted into calcein by intracellular esterase enzymes, producing an intense green fluorescence. In dead cells, ethidium homodimer-I is able to penetrate the damaged plasma membrane and, upon binding to nucleic acids, undergo a 40-fold enrichment of fluorescence, thereby producing an intense red signal. Upon retrieval, the biopsies were immersed in HEPES-buffered minimum essential medium (GIBCO, Grand Island, NY, USA) at 4�C. In the laboratory, the tissue samples were transferred to 50-mL conical tubes containing 20 mL PBS supplemented with 1 mg mL-1 collagenase type IA (Sigma-Aldrich NV/SA, Bornem, Belgium) and were incubated in a water bath at 37�C for 60 min. The digestion was terminated by adding an equal volume of cold PBS. The resulting suspension was centrifuged at 300g for 10 min at 8�C. The pellet was resuspended in PBS and transferred to a Petri dish. Each Petri dish was examined under a phase-contrast inverted microscope (Olympus CHX41) equipped with an eyepiece micrometer, and the primordial and primary follicles were collected with a glass micropipette. Isolated follicles were mounted on a slide in 50-�L droplets of PBS containing 2 �mol L-1 calceine-AM and 5 �mol L-1 ethidium homodimer-I (Molecular Probes, Leiden, The Netherlands), and incubated in the dark for 45 min at 37�C. After incubation, the follicles were examined under a fluorescence microscope (Olympus BX61). A total of 95 enzymatically isolated follicles were analyzed for viability with fluorescent probes; 89 (93.7%) of these were viable. Viable follicles were retrieved from all animals. This experiment confirms that viable primordial and primary follicles can be retrieved from living donor cows by the BPU procedure, which makes the technique suited for further culture or research.