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.

Number of oocytes retrieved per donor during OPU and its relationship with in vitro embryo production and field fertility following embryo transfer

The association of OPU-IVEP is an important instrument to drive genetic progress. In vitro embryo production (IVEP) has remarkably expanded in the last decade compared to in vivo embryo production. Because of the high repeatability of oocyte retrieval within oocyte-donors, studies exploring the relationship between the number of oocytes recovered per OPU section with IVEP efficiency, as well as with field fertility (pregnancy results following embryo transfer; P/ET) are extremely important to guide cow-donor selection and optimize field reproduction efficiency and the herds genetic gain. Based on this rationale, our group conducted a retrospective analysis of a large database comprising IVEP records from several cattle breeds, including Bos indicus and Bos taurus for either beef or dairy purposes. A total of 205,140 oocytes recovered from 7,906 OPU procedures of 6,902 donors (5,227 beef and 1,675 dairy) of Brazilian farms were analyzed. Beef breeds analyzed were Nelore (Bos indicus) and Senepol (Bos taurus) and dairy breeds Were Gyr (Bos indicus) and Holstein (Bos taurus). According to our analysis, the IVEP in beef cattle had a great improvement throughout the last years, with a remarkable increase in numbers of pregnancies per OPU compared to late 90s (averaging only 1 pregnancy per OPU in 1998 vs 2,4 in 2014). As for the distribution of oocytes retrieved, both Bos indicus beef (Nelore = 27.2) and dairy (Gyr = 23.8) breeds seem to yield greater average numbers of oocytes per OPU compared to Bos taurus (Senepol = 21.8; Holstein = 19.3).(AU)

Number of oocytes retrieved per donor during OPU and its relationship with in vitro embryo production and field fertility following embryo transfer

The association of OPU-IVEP is an important instrument to drive genetic progress. In vitro embryo production (IVEP) has remarkably expanded in the last decade compared to in vivo embryo production. Because of the high repeatability of oocyte retrieval within oocyte-donors, studies exploring the relationship between the number of oocytes recovered per OPU section with IVEP efficiency, as well as with field fertility (pregnancy results following embryo transfer; P/ET) are extremely important to guide cow-donor selection and optimize field reproduction efficiency and the herd’s genetic gain. Based on this rationale, our group conducted a retrospective analysis of a large database comprising IVEP records from several cattle breeds, including Bos indicus and Bos taurus for either beef or dairy purposes. A total of 205,140 oocytes recovered from 7,906 OPU procedures of 6,902 donors (5,227 beef and 1,675 dairy) of Brazilian farms were analyzed. Beef breeds analyzed were Nelore (Bos indicus) and Senepol (Bos taurus) and dairy breeds Were Gyr (Bos indicus) and Holstein (Bos taurus). According to our analysis, the IVEP in beef cattle had a great improvement throughout the last years, with a remarkable increase in numbers of pregnancies per OPU compared to late 90’s (averaging only 1 pregnancy per OPU in 1998 vs 2,4 in 2014). As for the distribution of oocytes retrieved, both Bos indicus beef (Nelore = 27.2) and dairy (Gyr = 23.8) breeds seem to yield greater average numbers of oocytes per OPU compared to Bos taurus (Senepol = 21.8; Holstein = 19.3).

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.

Reference gene selection for gene expression analysis of oocytes collected from dairy cattle and buffaloes during winter and summer.

Oocytes from dairy cattle and buffaloes have severely compromised developmental competence during summer. While analysis of gene expression is a powerful technique for understanding the factors affecting developmental hindrance in oocytes, analysis by real-time reverse transcription PCR (RT-PCR) relies on the correct normalization by reference genes showing stable expression. Furthermore, several studies have found that genes commonly used as reference standards do not behave as expected depending on cell type and experimental design. Hence, it is recommended to evaluate expression stability of candidate reference genes for a specific experimental condition before employing them as internal controls. In acknowledgment of the importance of seasonal effects on oocyte gene expression, the aim of this study was to evaluate the stability of expression levels of ten well-known reference genes (ACTB, GAPDH, GUSB, HIST1H2AG, HPRT1, PPIA, RPL15, SDHA, TBP and YWHAZ) using oocytes collected from different categories of dairy cattle and buffaloes during winter and summer. A normalization factor was provided for cattle (RPL15, PPIA and GUSB) and buffaloes (YWHAZ, GUSB and GAPDH) based on the expression of the three most stable reference genes in each species. Normalization of non-reference target genes by these reference genes was shown to be considerably different from normalization by less stable reference genes, further highlighting the need for careful selection of internal controls. Therefore, due to the high variability of reference genes among experimental groups, we conclude that data normalized by internal controls can be misleading and should be compared to not normalized data or to data normalized by an external control in order to better interpret the biological relevance of gene expression analysis.