213 Nuclear assessment of bovine oocytes matured in cytokine-supplemented medium using microtubule and DNA staining

In vitro maturation of oocytes involves the reorganization of chromosomes during nuclear maturation, ending in polar body extrusion and metaphase II (MII) arrest. Before entering MII, oocytes undergo germinal vesicle breakdown (GVBD) and meiosis I, as well as form the meiotic spindle, which is a microtubule-based structure that is responsible for chromosome segregation. Recently, addition of FGF-2, LIF, and IGF-1 (FLI) in bovine maturation media (BMM) has improved nuclear maturation rates and increased blastocyst development following IVF (Keim et al. Reprod. Fertil. Dev. 35, 575–588). However, the effect of FLI supplementation on nuclear progression has not been investigated. Thus, the aim of this study was to assess meiotic spindle organisation at MII and to determine the stage at which immature oocytes arrest after IVM in FLI or BMM. Bovine oocytes, obtained from a local abattoir, underwent IVM for 21 h at 38.5°C in either FLI supplemented or BMM. For the assessment of meiotic spindle organisation, oocytes at the MII stage, presenting with a polar body after cumulus cell stripping with hyaluronidase, were incubated in 10 µM paclitaxel to stabilise microtubules, had zona removed, and were fixed in 4% paraformaldehyde. The MII oocytes were further permeabilized and blocked, then microtubules were stained with anti α-tubulin primary and AF488 secondary antibodies. DNA was counter-stained using Hoechst 33342. To determine the stages at which immature oocytes arrested, oocytes were fixed, permeabilized, and incubated in 5 µg mL⁻¹ Hoechst 33342. All oocytes were mounted then imaged using a Zeiss fluorescent microscope. ImageJ was used to categorize the meiotic spindle as normal (barrel-shaped) or abnormal (dispersed) (Da Broi et al. Hum. Reprod. 29, 315–323), as well as to categorize immature oocytes in one of five nuclear stages: GV, GVBD, metaphase I, anaphase I, and telophase I. All experiments had at least four replicates. In Jamovi, maturation rates were analysed with analysis of variance, spindle organisation in MII oocytes was analysed with a logistic generalized linear model, and immature nuclear stage was analysed with a multinomial generalized linear model. Statistical differences were considered significant when P < 0.05. In total, 64 MII oocytes (FLI:32, BMM:32) and 286 immature oocytes (FLI:133, BMM:153) were evaluated. Maturation rates were higher in FLI compared with BMM (75% vs 67%, P < 0.001). In MII oocytes, the FLI group had a higher percentage of normal microtubules than the BMM group (68.8% vs 50%, P < 0.001). In immature oocytes, a lower percentage of GV oocytes and a higher percentage of telophase I oocytes were observed in FLI than in BMM (3% vs 13.7%, P = 0.009; 34.6% vs 17.6%, P = 0.011). In conclusion, FLI-supplemented IVM aids in proper meiotic spindle configuration at MII in bovine oocytes and promotes nuclear progression through meiosis I, which may explain improved nuclear maturation rates.