188 Influence of duration of in vitro maturation in relation to COC morphology on nuclear and cytoplasmic maturation of wood bison oocytes

In vitro embryo production (IVEP) could play an important role in the conservation of North American wood bison, which is classified as a threatened species by the Canadian government. The study objective was to evaluate the influence of the duration of IVM in relation to the morphologic grade of the cumulus–oocyte complex (COC) on nuclear and cytoplasmic characteristics of bison oocytes. Ultrasound-guided COC collection was done at random stages of the follicular wave in adult wood bison (n = 27 bison, six replicates). A 2 × 2 factorial experiment was conducted to compare two IVM durations (24 vs. 30 h) and two morphologic grades of COC (good vs. poor). The COC graded as compact good (three or more layers of cumulus cells and homogeneous ooplasm), compact regular (one to three layers of cumulus cells and homogeneous ooplasm), and expanded good (expanded cumulus layer and homogeneous ooplasm) were combined as the good-quality COC group. Those graded as compact poor (more than one layer of cumulus cells and heterogeneous ooplasm), expanded poor (expanded cumulus layer and heterogeneous ooplasm), and denuded (oocyte without cumulus cells) were combined as the poor-quality COC group. After collection, the COC were matured individually in drops of 20 µL of commercial buffered medium (ART Lab Solutions Inc.) with oil overlay at 38.5°C. Oocytes matured for 0, 24, or 30 h were denuded and co-incubated in drops of buffered medium containing dyes to visualize mitochondria and cortical granules (Mitotracker Deep Red FM and FITC-PNA). Oocytes were then fixed in 1% paraformaldehyde and transferred to vectashield antifade mounting medium with DAPI. Oocytes were examined under epifluorescence wide-field microscopy, and nuclear status was used to classify oocytes as immature (germinal vesicle and MI) or mature (MII). For assessment of active mitochondria and cortical granule distribution pattern, oocytes were examined by confocal laser scanning microscopy to build 20-µm-thick three-dimensional image stacks. Groups were compared by two-way ANOVA using arcsine transformation of proportional data and are presented as mean ± s.e.m. As expected, a greater proportion of oocytes were of immature nuclear status after 0 h versus 24–30 h of IVM (97.1 ± 1.8% vs. 29.8 ± 7.1%; P < 0.01). There was no influence of the duration of IVM (24 vs. 30 h) on the proportion of oocytes exhibiting nuclear maturation (70.6 ± 10.6% vs. 69.7 ± 10.1%; P = 0.86), nor was there any interaction between main effects. However, a greater proportion of COCs in the good-quality group were of mature nuclear status than in the poor-quality group (95.8 ± 2.0% vs. 44.5 ± 4.9%; P < 0.01). Quantitative image analysis results regarding cytoplasmic maturation are pending. In conclusion, the morphological grade of the COCs at the time of collection was predictive of the ability of bison oocytes to undergo IVM regardless of the duration of maturation. The influence of morphologic grade and duration of IVM on cytoplasmic maturation remains to be determined but will be essential for the development of an effective bison IVEP protocol.

This research was supported by grants from Genome Canada and MITACS.