Thesis
Effect of polymethoxylated flavone Nobiletin in bovine oocyte maturation and subsequent development in vitro [2018]
Ladrón de Guevara, M.;
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The ability of an oocyte to undergo successful cytoplasmic and nuclear maturation is fundamental for fertilization and embryo development. The aim of this study was to evaluate the effect of Nobiletin supplementation during In vitro maturation (IVM) of bovine oocytes on nuclear and cytoplasmic maturation and their developmental competence. Nobiletin is a polymethoxylated flavonoid isolated from citrus fruits exhibiting a wide biological effect in cell adhesion, cell migration, cell cycle regulation and inhibition of reactive oxygen species (ROS) production; important factors for oocyte IVM. Immature cumulus oocytes complexes (COCs) were aspirated from ovaries of slaughtered heifers. Selected COCs were in vitro matured in TCM-199 plus 10 per cent foetal calf serum (FCS) and 10 ng per ml epidermal growth factor (EGF) (Control) supplemented either with 10, 25, 50 and 100 μM of Nobiletin (N10, N25, N50 and N100 respectively) or 0.01 per cent dimethyl sulfoxide (CDMSO), vehicle for nobiletin dilution. After 24 h of IVM at 5 per cent CO2 in air at 38.5 degreesC, a representative number of oocytes from each group were fixed and stained to evaluate nuclear and cytoplasmic maturation. In addition, oocytes were stained to measure oocyte metabolism in terms of ROS and glutathione (GSH) content. The remaining oocytes were fertilized and cultured in vitro to evaluate their developmental competence by cleavage rate and blastocyst yield. Significantly higher percentage o
f matured oocytes were observed in metaphase II when N25 (87 plus minus 0.6 per cent) or N50 (89.3 plus minus 0.3 per cent) were added to the IVM medium compared to N10 (72.9 plus minus 0.3 per cent), N100 (71.5 plus minus 0.8 per cent), Control (71.7 plus minus 0.7 per cent) and CDMSO (70.5 plus minus 0.5 per cent) groups. Furthermore, N25 and N50 showed higher rate of oocytes with peripheral migration of cortical granules (85.7 plus minus 0.3 per cent and 89.9 plus minus 2.2 per cent respectively) and mitochondria (86.7 plus minus 0.6 per cent and 88.9 plus minus 1.2 per cent respectively) compared to the remaining groups (P less than 0.05). In addition, the supplementation of N25 and N50 showed a significant reduction (P less than 0.05) in the ROS (2.53 plus minus 0.8; 2.62 plus minus 1.2 a.u. respectively), and GSH (2.84 plus minus 0.4; 3.09 plus minus 0.1 a.u. respectively) content in comparison with all other groups. Cleavage rate was significantly higher (P less than 0.05) for N25 (89.9 plus minus 0.3 per cent) and N50 (91.3 plus minus 0.3 per cent) compared to all other groups (N10: 75.6 plus minus 0.3 per cent; N100: 74.0 plus minus 0.6 per cent; Control: 74.2 plus minus 0.4 per cent; and CDMSO: 73.6 plus minus 0.4 per cent). Similarly, cumulative blastocyst yield at D8 was significantly higher (P less than 0.05) for N25 (32.1 plus minus 0.8 per cent) and N50 (35.5 plus minus 0.8 per cent) compared to N10 (23.1 plus minus 0.7 per cent), N100 (24.5 plus minus 0.9 per cent), Control (25.9 plus minus 0.4 per cent) and CDMSO (26.1 plus minus 0.6 per cent) groups. In conclusion, supplementation of 25 μM or 50 μM of Nobiletin to the IVM medium improves oocyte nuclear and cytoplasmic maturation, reduces oxidative stress and improve embryo development.
