144 A comparative study using flat, round, and V-shaped 96-well plates during bovine oocyte in vitro maturation

Conventional IVM is performed on flat surfaces. Very few studies have evaluated different culture surface topographies, but not during bovine IVM and its potential influence on embryo development. We aimed to compare three multiwell plates with different bottom shapes during oocyte IVM on embryo development. First, a pilot study was performed to evaluate the ideal volume of maturation medium (MM) and the effect of paraffin oil covering in oocytes matured in multiwell plates. Cumulus–oocyte complexes (COCs) from slaughterhouse-derived ovaries were matured in different conditions in V-shaped 96-well plates (Corning®; Corning Inc.): five COCs in 30 μL of MM with (V-30) or without (OV-30) oil covering (30 μL), 10 COCs in 60 μL of MM with (V-60) or without (OV-60) oil covering (30 μL), and 20 COCs in 120 μL of MM with (V-120) or without (OV-120) oil covering (30 μL). The control group consisted of 60 COCs in 500 μL of MM in flat-bottomed 4-well dishes (ThermoFisher Scientific) without oil covering. After 22 h of IVM, fertilisation and culture were performed as previously described (Wydooghe et al. 2014 Reproduction 148, 519-529) (n = 1021 in 3 replicates). V-30 was excluded from the treatment groups because of excessive MM evaporation after IVM. No significant differences were observed in cleavage rate for all the treatment groups compared with the control (78.6 ± 2.6%; P > 0.3). Interestingly, the V-60 group showed a higher cleavage rate (84.5 ± 2.9%) than OV-120 (69.9 ± 3.8%; P = 0.03). The blastocyst rates at Day 8, which ranged from 28.1 ± 3.6 to 35.6 ± 3.8% in all groups, were not affected by the volume of MM or the presence of oil (P > 0.7). Although there were no significant differences (P < 0.05) among treatments, OV-60 had the greatest blastocyst rate at Day 8 (35.6 ± 3.8%). Therefore, we selected conditions as described for the OV-60 group for follow-up experiments. Consequently, we aimed to evaluate three different surface geometries during IVM: flat, ultra-low attachment round-bottom, and V-shaped 96-well plates (all from Corning Inc.). After IVM, nuclear maturation was assessed by Hoechst staining in randomly selected oocytes (n = 187 in 5 replicates). The rest of the oocytes were conventionally fertilised and cultured (n = 1276 zygotes in five replicates). Maturation, cleavage, and embryo development rates were fitted in generalised mixed-effects models, and the replicate was set as random. Most of the oocytes matured (metaphase II stage) in the three surface topographies and the control, with no significant differences (P > 0.7) among groups (84.9 ± 4.9–91.1 ± 4.2%). Similarly, there was no difference (P < 0.05) among groups in the proportion of oocytes in germinal vesicle (0–3%), germinal vesicle breakdown (4–11%), or metaphase I (0–3%) stages. Likewise, all plates and the control showed similar cleavage (75.8 ± 2.3–82.4 ± 2.3%; P > 0.6) and Day 8 blastocyst (34.0 ± 2.7%-41.4 ± 3.2%; P > 0.2) rates. In conclusion, none of the three different surface geometries influenced meiotic resumption nor embryo development. However, embryo quality parameters among treatments groups deserve further exploration.