Effects of anethole supplementation on bovine embryo production and quality

Improvements are necessary to further expand the potential of in vitro embryo production (IVP). Anethole, a natural antioxidant, was shown to be a low-cost alternative with an important role in the gene modulation of oxidative stress, and it can be used to produce higher quality embryos. The aim of this study was to evaluate the anethole effect during in vitro maturation (IVM) on the oocyte nuclear maturation, and then, during IVM and/or in vitro culture (IVC) on the production and quality of bovine embryos. In Experiment 1, cumulus-oocyte complexes (COCs) were matured in IVM medium in the following groups: control group (MC); supplemented with 300 µg/ml anethole (M300); and supplemented with 3,000 µg/ml anethole (M3000). The three groups were matured for 24 h to assess nuclear maturation (metaphase II) and gene expression of cumulus cells (CCs). The group that obtained the best response was M300, which was chosen for Experiment 2, together with MC. In this experiment, maturation was carried out either in IVM medium with 300 µg/ml anethole (M300) or not (MC). Subsequently, the COCs were submitted to in vitro fertilization and then divided into four groups for IVC: MC–CC (IVM and IVC controls); MC–C300 (control IVM and IVC with 300 µg/ml); M300-CC (IVM with 300 µg/ml and control IVC); and M300-C300 (IVM and IVC with 300 µg/ml anethole each). On the third day (D3) of the culture, cleavage was evaluated, and on D8, the production and classification of blastocysts were assessed. It was observed that 3,000 µg/ml anethole reduced the percentage of COCs that had reached nuclear maturation. The genes GREM1, GSTA1, SOD1, and CAT, related to oxidative stress resistance, and COX2 and HAS, involved in cell metabolism, showed increased abundance in CCs in the anethole presence. Regarding embryo production, the use of 300 µg/ml anethole during IVM and IVC improved the percentage of cleaved embryos. In addition, anethole treatment during IVM and IVC increased the expression of GPX1 gene while decreasing IFNT2α gene in the embryos produced. In conclusion, the addition of 300 µg/ml anethole during IVM modulated the expression of genes related to oxidative stress and oocyte development, and its use during IVM and IVC modulated the gene expression related to embryo quality. However, further studies need to be performed to better understand anethole's impact on embryo development.