Experimental Design

Tauroursodeoxycholic acid (Selleckchem, Houston, TX, United States) was dissolved in sterile distilled water to prepare a 100 mM stock solution that was stored at −80°C. This stock solution was freshly diluted with IVM media to obtain 50 μM (T50), 200 μM (T200), and 1,000 μM (T1000) solutions of TUDCA (Kim et al., 2012; Zhang et al., 2012; Mochizuki et al., 2018; Figure 1). In all experiments, we included the control group (without TUDCA). The first series of experiments aimed to evaluate the influences of control, T50, T200, and T1000 TUDCA on the following oocyte quality parameters during IVM of bovine oocytes: nuclear maturation (experiment 1), ROS production (experiment 2), mitochondrial activity (experiment 3), and the abundance of target transcripts in oocytes (experiment 4). Other experiments aimed to evaluate the influence of supplementation with control, T50, and T200 TUDCA during IVM on fertilization and embryonic development, particularly on pronucleus formation in presumptive zygotes (experiment 5), developmental competence of embryos (experiment 6), and transcript abundance of embryonic quality markers (experiment 7).

Illustrative experimental design. IVM, in vitro maturation; IVF, in vitro fertilization; IVC, in vitro culture; IO, immature oocyte; MO, mature oocyte; PZ, presumptive zygote; BL, blastocyst and hatched blastocyst.