Micro-vibration results in vitro-derived bovine blastocysts with greater cryotolerance, epigenetic abnormalities, and a massive transcriptional change.

Much effort has been employed to improve the quality of embryos obtained by in vitro production (IVP) given the relevance of this technology to current livestock systems. In this context, dynamic IVP systems have proved beneficial to the embryo once they mimic fluid flows and mechanical forces resulting from the movement of ciliated cells and muscle contraction in the reproductive tract. In the present study, we sought to confirm these initial findings as well as assess potential molecular consequences to the embryo by applying micro-vibration (45 Hz for 5 s once per 60 min) during both oocyte maturation and embryo culture in cattle. As a result, micro-vibration led to lower incidence of apoptosis in blastocysts following vitrification-thawing. Further analyses revealed epigenetic and transcriptional changes in blastocysts derived from the micro-vibration treatment, with a total of 502 differentially expressed genes. Enrichment analyses linked differentially expressed genes to 'Oxidative phosphorylation', 'Cytokine-cytokine receptor interaction', and 'Signaling pathways regulating pluripotency of stem cells'. Yet, a meta-analysis indicated that the transcriptional changes induced by micro-vibration were not toward that of in vivo-derived embryos. In conclusion, micro-vibration increases the cryoresistance of bovine embryos, but caution should be taken given the unclear consequences of epigenetic and transcriptional abnormalities induced by the treatment.

Effects of melatonin during IVM in defined medium on oocyte meiosis, oxidative stress, and subsequent embryo development.

Melatonin may have beneficial effects when used in oocyte maturation and embryo development culture. The effect of melatonin during IVM on meiosis resumption and progression in bovine oocytes and on expression of antioxidant enzymes, nuclear fragmentation and free radicals, as well as on embryo development were assessed. Cumulus-oocyte complexes were matured in vitro with melatonin (10(-9) and 10(-6) M), FSH (positive control), or without hormones (negative control) in defined medium. Maturation rates were evaluated at 6, 12, 18, and 24 hours. Transcripts for antioxidant enzymes (CuZnSOD, MnSOD, and glutathione peroxidase 4 (GPX4)) in oocytes and cumulus cells, nuclear fragmentation in cumulus cells (TUNEL) and reactive oxygen species levels in oocytes (carboxy-H2 difluorofluorescein diacetate) were determined at 24 hours IVM. Effect of treatments on embryo development was determined after in vitro fertilization and culture. At 12 hours, meiosis resumption rates in FSH and melatonin-treated groups were similar (69.6%-81.8%, P > 0.05). At 24 hours, most oocytes were in metaphase II, with FSH showing highest rates (90.0%, P < 0.05) compared with the other groups (51.6%-69.1%, P > 0.05). In cumulus cells, MnSOD expression was higher in FSH group (P < 0.05) whereas Cu,ZnSOD transcripts were more abundant in melatonin group (10(-6)M; P < 0.05). Nuclear fragmentation in cumulus cells was highest in controls (37.4%/10,000 cells; P < 0.05) and lower in FSH and 10(-6)M melatonin (29.4% and 25.6%/10,000 cells, respectively). Reactive oxygen species levels were lower in oocytes matured with 10(-6)M melatonin than in control and FSH groups (P < 0.05). Embryo development from oocytes matured only with melatonin was similar to those matured in complete medium (P > 0.05). In conclusion, although melatonin during IVM in a defined medium does not stimulate nuclear maturation progression it does stimulate meiosis resumption and such treated oocytes support subsequent embryo development. Melatonin also shows cytoprotective effects on cumulus-oocyte complexes.

The interval between the emergence of pharmacologically synchronized ovarian follicular waves and ovum pickup does not significantly affect in vitro embryo production in Bos indicus, Bos taurus, and Bubalus bubalis.

The aim of the present study was to determine the optimal phase of the follicular wave to perform ovum pickup (OPU) for in vitro embryo production (IVEP) in various genetic groups. For this purpose, 27 heifers-nine Bos taurus (Holstein), nine Bos indicus (Nelore), and nine Bubalus bubalis (Mediterranean)-were maintained under the same nutritional, management, and environmental conditions. Heifers within each genetic group were submitted to six consecutive OPU trials with 14-day intersession intervals, at three different phases of the pharmacologically synchronized follicular wave (Day 1, 3, or 5 after follicular wave emergence), in a 3 × 3 crossover design. When OPU was performed at different phases of the pharmacologically synchronized follicular wave (Day 1, 3, or 5), no differences were found in the percent of oocytes recovered (70.5 ± 3.1%, 75.0 ± 3.1%, 76.0 ± 3.2%, respectively; P = 0.41) or blastocyst production rates (19.4 ± 2.9%, 16.6 ± 2.9%, 15.9 ± 2.6%, respectively; P = 0.36). Comparing genetic groups, B indicus showed a higher blastocyst rate (28.3(a) ± 2.8%; P < 0.01) than B taurus and B bubalis (14.1(b) ± 2.9% and 10.2(b) ± 2.0%, respectively). However, only B indicus heifers showed a variation in the number of visualized follicles and the total and viable oocytes along consecutive OPU sessions. In conclusion, different phases of the pharmacologically synchronized ovarian follicular wave did not affect OPU-IVEP in B indicus, B taurus, and B bubalis heifers. Additionally, B indicus heifers showed greater OPU-IVEP efficiency than did the other genetic groups, under the same management conditions.