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.

Impact of ethanol and heat stress-dependent effect of ultra-diluted Arnica montana 6 cH on in vitro embryo production in cattle.

This study evaluated the effect of adding ultra-diluted and dynamized Arnica montana 6 cH, and its vehicle (0.3% ethanol) to the in vitro maturation (IVM) medium, in the absence (experiment 1) or presence (experiment 2) of heat stress (HS), on bovine oocyte maturation and in vitro embryo production (IVEP). In experiment 1 (n = 902 cumulus oocyte complexes, COCs), the treatments were 1) IVM medium (Control treatment), 2) IVM medium + 0.3% ethanol, and 3) IVM medium + Arnica montana 6 cH. In experiment 2 (n = 1064 COCs), the treatments were 1) IVM medium without HS, 2) IVM medium under HS, 3) IVM medium + ethanol under HS, and 4) IVM medium + Arnica montana under HS. In the absence of HS (experiment 1), the addition of Arnica montana to the IVM medium had a deleterious effect on the IVEP (cleavage and blastocyst rates) and the total cell number/blastocysts. On the other hand, ethanol (0.3%) increased IVEP in relation to the Control and Arnica montana treatments. However, in the presence of HS during IVM (experiment 2), the addition of ethanol or Arnica montana increased IVEP when compared to the HS treatment alone, and the Arnica montana treatment resulted in greater total cell number/blastocysts compared to the other treatments. In conclusion, this study showed for the first time that the negative or positive effect of Arnica montana 6 cH on IVEP depends on the culture condition (i.e., absence or presence of HS during IVM). On the other hand, ethanol showed beneficial and consistent results on IVEP regardless of exposure to HS.

Anethole improves the in vitro development of isolated caprine secondary follicles.

The aim of this study was to investigate the effect of three concentrations of anethole (30, 300, and 2000 µg/mL) on survival, antrum formation, follicular diameter, and oocyte maturation in the caprine species. The study also evaluated the effects of anethole on transcripts of ICAM-1, CAV-1, TIMP-2, and PAI-1 genes and levels of reactive oxygen species (ROS) in isolated goat preantral ovarian follicles before and after in vitro culture for 18 days. Preantral follicles were isolated from goat ovaries and individually cultured in alpha minimum essential medium modified (α-MEM+), defined as the control treatment, α-MEM+ supplemented with ascorbic acid at a concentration of 100 µg/mL (AA), or α-MEM+ supplemented with three different concentrations of anethole (30, 300, 2000 µg/mL) for a period of 18 days. Treatments were named as α-MEM+, AA, AN30, AN300, and AN2000, respectively. After culture, the follicles were opened, the cumulus oocytes complex (COCs) were removed and matured in vitro. The walls of the follicles were used for the quantitation of mRNA by quantitative real-time polymerase chain reaction. Finally, the medium collected at the end of culture was used for the measurements of ROS. After 18 days of culture, the AA treatment showed the percentage of intact follicles and follicular diameter significantly higher compared with the other treatments. However, daily growth rate, antrum formation, and also oocyte diameter were similar among the treatments. In addition, compared with AA, the rate of oocytes for in vitro maturation (diameter ≥ 110 µm) and the meiosis resumption rate were significantly higher in the treatments AN30 and AN2000, respectively. When assessing gene related to remodeling of the basement membrane, significant differences in mRNA levels for ICAM-1, CAV-1, TIMP-2, and PAI-1 were observed in comparison with Day 0, i.e., in the noncultured control. In addition, the ROS from Day 12, all treatments with the addition of anethole have significantly lower values of ROS than α-MEM+ and AA. In conclusion, the addition of anethole to the in vitro culture medium was able to improve the development of goat preantral follicles by reducing concentrations of ROS and increasing the percentage of oocytes able to resume meiosis.