Anethole supplementation during in vitro maturation increases in vitro goat embryo production in a concentration-dependent manner.

During in vitro maturation (IVM) cumulus-oocyte complexes (COCs) are exposed to conditions that can trigger oxidative stress, thus, reducing oocyte maturation and viability. Aiming to mitigate these detrimental conditions, the effects of IVM medium supplementation with anethole have been tested. Anethole, also known as trans-anethole (1-methoxy-4 [1-propenyl]-benzene), is a naturally occurring phenylpropanoid with various pharmacological properties, including antioxidant effects. However, no study has examined anethole effect on goat COCs during IVM. Thus, the aim of this study was to evaluate the effects of different anethole concentrations on oocyte maturation, oxidative stress, and in vitro development of caprine embryos after parthenogenetic activation. Goat COCs were selected and randomly distributed into the following treatments: TCM-199+ medium (control), or TCM-199+ medium supplemented with 30 µg/mL (AN30); 300 µg/mL (AN300) or 2000 µg/mL (AN2000) of anethole. After IVM, part of the COCs was chosen for oocyte viability and chromatin configuration, intracellular reactive oxygen species levels, and mitochondrial membrane potential assessment. Another part of COCs was parthenogenetically activated, and presumptive zygotes were cultured for 7 days. Results demonstrated that anethole at 30 µg/mL increased oocyte maturation and cleavage rates when compared to the other treatments (P < 0.05), as well as oocyte viability and in vitro embryo production when compared to the control treatment (P < 0.05). Additionally, treatment with anethole at 2000 µg/mL decreased oocyte nuclear maturation and cleavage rates when compared to other treatments (P < 0.05) and embryo production if compared to control and AN30 treatments (P < 0.05). Moreover, anethole at 2000 µg/mL increased mitochondrial membrane potential when compared to the other treatments (P < 0.05). In conclusion, anethole exerts a concentration-dependent effect during goat COCs IVM. For a more desirable outcome of oocyte viability and maturation, and in vitro embryo production, the use of anethole at 30 µg/mL is recommended.

Oocyte in vitro maturation with eugenol improves the medium antioxidant capacity and total cell number per blastocyst.

This study investigates the impact of eugenol (EU) supplementation on bovine oocyte in vitro maturation (IVM) and antioxidant capacity, as well as in vitro embryo production and quality after conventional in vitro fertilization (IVF). A total of 1077 cumulus oocyte complexes were cultured in TCM-199+ without EU supplementation (control treatment) or supplemented with EU at the concentrations of 10 µM (EU-10), 20 µM (EU-20), or 40 µM (EU-40). After IVM, the oocytes were subjected to IVF and embryo culture. The addition of EU at 40 µM to the IVM medium improved (P < 0.05) the antioxidant capacity and cleavage rate when compared to the control treatment. Moreover, a positive correlation (r = 0.61, P < 0.03) was observed between cleavage rate and EU concentration. The addition of EU at concentrations of 10 and 20 µM decreased (P < 0.05) the calreticulin (CALR) levels in expanded blastocysts when compared to the control treatment and EU-40 treatment. However, the EU-10 and EU-20 treatments had a greater (P < 0.05) mean total cell number (TCN) per expanded blastocyst when compared to the control treatment and EU-40 treatment. In conclusion, the addition of EU to the enriched culture medium during IVM of bovine oocytes improved the antioxidant capacity of the spent medium, as well as the cleavage rate and embryonic quality (i.e., TCN/expanded blastocyst), and reduced the endoplasmic reticulum stress (i.e., CALR levels) in the embryos. Thus, we recommend enriching the IVM medium with 10 µM EU for in vitro bovine embryo production.

Eugenol Improves Follicular Survival and Development During in vitro Culture of Goat Ovarian Tissue.

This study evaluated the effects of different concentrations (10, 20, or 40 µM) of eugenol (EUG 10, EUG 20, or EUG 40), ascorbic acid (50 µg/mL; AA) or anethole (300 µg/mL; ANE 300) on the in-vitro survival and development of goat preantral follicles and oxidative stress in the cultured ovarian tissue. Ovarian fragments from five goats were cultured for 1 or 7 days in Alpha Minimum Essential Medium (α-MEM+) supplemented or not with AA, ANE 300, EUG 10, EUG 20 or EUG 40. On day 7 of culture, when compared to MEM, the addition of EUG 40 had increased the rate of follicular development, as observed by a decrease in the proportion of primordial follicles alongside with an increase in the rate of normally developing follicles. Furthermore, EUG 40 significantly increased both follicular and oocyte diameters. Subsequently, ovarian fragments from three goats were cultured for 1 or 7 days in α-MEM+ supplemented or not with AA, ANE 300 or EUG 40. All tested antioxidants, except ANE 300, were able to significantly decrease the levels of reactive oxygen species in the ovarian tissue, but EUG 40 could most efficiently neutralize free radicals. All ovarian tissues cultured in the presence of antioxidants, especially EUG 40, presented a significant decrease in H3K4me3 labeling, indicating a silencing of genes that play a role in the inhibition of follicular activation and apoptosis induction. When compared to cultured control tissues, both EUG 40 and ANE 300 significantly increased the intensity of calreticulin labeling in growing follicles. The mRNA relative expression of ERP29 and KDM3A was significantly increased when the culture medium was supplemented with EUG 40, indicating a response to ER stress experienced during culture. In conclusion, EUG 40 improved in-vitro follicle survival, activation and development and decreased ROS production, ER stress and histone lysine methylation in goat ovarian tissue.