219 Evaluation of mitochondrial quantity and distribution in bovine oocytes matured in cytokine-supplemented medium

In vitro maturation (IVM) of oocytes involves the reorganisation of chromosomes during nuclear maturation and organelles during cytoplasmic maturation. Mitochondria play an important role both in oocyte maturation as well as early embryo development, and they must reorganise during IVM to keep their metabolic activity. Recently, the addition of FGF-2, LIF, and IGF-1 (FLI) in oocyte maturation media (MM) has shown improved nuclear maturation rates and blastocyst development in pigs (2017 P.N.A.S. 114, E5796–E5804) and cattle (2018 Reprod. Fertil. Dev. 31, 213). However, there have not been any investigations into the mitochondrial activity, as one of the indicators of cytoplasmic maturation, in bovine oocytes matured in FLI-supplemented media. Thus, the aim of this study was to determine the mitochondrial distribution and mass in FLI matured versus control matured oocytes. Grade I bovine oocytes were matured in an incubator at 38.5°C in either FLI-supplemented MM or standard bovine MM. After 21 h of maturation, oocytes were divided into four groups: control metaphase II (CM), control not MII (CNM), FLI MII (FM), and FLI not MII (FNM); and incubated at 38.5°C in PBS with 400 nM MitoTracker Green for 30 min. After washing three times in PBS + 0.1% polivinyl alcohol, each oocyte was mounted on a glass slide to preserve the oocyte’s cytoskeletal integrity and imaged using a Zeiss fluorescent microscope. ImageJ (National Institutes of Health) was used to categorise mitochondrial distribution on a 2-D image as diffuse (linked to higher developmental competence), aggregates or cortical (linked to lower developmental competence), and to analyse fluorescent intensity, which is indicative of the mitochondrion mass present (2008 Cytotechnology 56, 145–149). The depth and manner of assessment were consistent for all groups. All experiments were replicated at least three times and statistical analyses were performed using Jamovi. A multinomial generalised linear model was used to assess the difference in mitochondrial distribution between treatment groups. The difference in fluorescent intensity between treatment groups was compared by one-way ANOVA with a post hoc Tukey test. Statistical differences were considered significant when P < 0.05. In total, 279 oocytes were evaluated in this experiment (FM: 96, FNM: 42, CM: 96, CNM: 45). The percentage of cortical distribution was significantly lower in FM (4%) and CNM (1%) than FNM (18%, P = 0.043 and P = 0.01) and the percentage of diffuse distribution was trending higher, but not significantly, in FM (82%) than in FNM (64%, P = 0.089). Additionally, fluorescent intensity was significantly higher in FM and CM than FNM (P = 0.013 and P = 0.043). In conclusion, FLI supplemented-IVM medium, in addition to improving nuclear maturation, could also improve cytoplasmic maturation in bovine oocytes, as indicated by increased mitochondrial mass and lower percentage of cortical distribution.

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