295 COMPARISON OF TWO METHODS TO AVOID MOVEMENT OF BOVINE OOCYTES	DURING IN VITRO MATURATION
Authors: Petersen M.M. , Avery B. , Greve T. , Bøgh I.B. 
Published: December 2004

Development of two-photon laser scanning microscopy (TPLSM) has made it possible to conduct several recordings over time of early stage embryos without compromising viability. To use TPLSM to study structures within the oocyte it is necessary to remove at least part of the cumulus cells to prevent emitted light from being blocked. Aspiration of cumulus oocyte complexes (COC) through a denudation pipette creates a “window” through which the emitted light can escape and be recorded. To allow repeated recordings of the same location within an object it is important to avoid movement of the object. Gelatine (Gel) and poly-l-lysine (PLL) have previously been used to promote adhesion of cells in culture. The aim of our study was to develop a method to avoid movement during IVM of partially denuded COCs without compromising oocyte viability. Previous experiments in our lab showed that partial denudation of COC had no effect on embryo development (unpublished). Bovine COCs were obtained from abattoir ovaries. In the control group COCs were placed in non-treated dishes. In the experimental groups, they were placed in Gel- or PLL-coated dishes, either intact or partially denuded, where the length of cumulus cell “tails” was shortened to around 200 μm on each side of the oocyte. The coated dishes were prepared 24 h prior to IVM with 200 μL of 0.1% Gel (Sigma, Copenhagen, Denmark, G2500) or 200 μL 0.01% PLL (Sigma, P-4832). Partial denudation of COCs was performed with a 127–129 μm diameter denudation pipette. Standard procedures were used for IVM (23 h in DMEM with 5% serum and eCG/hCG), IVF (23 h in TALP), and IVC (SOF with 10% serum); IVM and IVF were incubated at 38.5°C in 5% CO2 in air, and IVC at 5% CO2 in 5% O2. The study was based on a total of 1151 oocytes and 3 replicates. Day 8 blastocyst (BL) rates, BL kinetics, and morphology were used as endpoints to assess oocyte maturation. Kinetics/morphology were graded by a scoring system: hatched/excellent 3, expanded/good 2, non-expanded/poor 1. COCs placed in Gel- or PLL-coated dishes did not move during handling of the dishes. The BL rates in the Gel group were 37%, 25%, and 17%, and in the PLL group 24%, 21%, and 12%, for the control, intact, and partially denuded COCs, respectively. In the Gel group the BL rates showed a decreasing trend (P < 0.0036), whereas in only the PLL group the BL rates from the partially denuded COC differed from the control and the intact COCs (P < 0.008). No significant differences were seen between blastocyst kinetics (Gel/PLL 1.9/1.9, 1.8/1.9, 1.6/1.7) or morphology (Gel/PLL 2.2/2.4, 2.0/2.5, 2.2/2.1) in the control, intact or partially denuded groups. Fisher's exact test used. We conclude that it is possible to avoid movement of COCs during IVM without compromising oocyte maturation in dishes coated with Gel or PLL, if the cumulus layer is intact. The BL rates are compromised if COCs are partially denuded and the “cumulus tails” shortened before IVM in Gel or PLL coated dishes, whereas kinetics and morphology are unaffected.
This research was funded by the Danish Research Agency, no. 23-023-0133.