In vitro production of bovine embryos derived from individual donors in the Corral® dish

Background: Since the identity of the embryo is of outmost importance during commercial in vitro embryo production, bovine oocytes and embryos have to be cultured strictly per donor. Due to the rather low yield of oocytes collected after ovum pick-up (OPU) per individual cow, oocyte maturation and embryo culture take place in small groups, which is often associated with inferior embryo development. The objective of this study was to improve embryonic development in small donor groups by using the Corral (R) dish. This commercial dish is designed for human embryo production. It contains two central wells that are divided into quadrants by a semi-permeable wall. In human embryo culture, one embryo is placed per quadrant, allowing individual follow-up while embryos are exposed to a common medium. In our study, small groups of oocytes and subsequently embryos of different bovine donors were placed in the Corral (R) dish, each donor group in a separate quadrant. Results: In two experiments, the Corral (R) dish was evaluated during in vitro maturation (IVM) and/or in vitro culture (IVC) by grouping oocytes and embryos of individual bovine donors per quadrant. At day 7, a significantly higher blastocyst rate was noted in the Corral (R) dish used during IVM and IVC than when only used during IVM (12.9% +/- 2.10 versus 22.8% +/- 2.67) (P < 0.05). However, no significant differences in blastocyst yield were observed anymore between treatment groups at day 8 post insemination. Conclusions: In the present study, the Corral (R) dish was used for in vitro embryo production (IVP) in cattle; allowing to allocate oocytes and/or embryos per donor. As fresh embryo transfers on day 7 have higher pregnancy outcomes, the Corral (R) dish offers an added value for commercial OPU/IVP, since a higher blastocyst development at day 7 is obtained when the Corral (R) dish is used during IVM and IVC

Photons and neutral mesons from hot hadronic matter

Results from the experimental program with light ion beams and heavy target nuclei at the CERN SPS could demonstrate the occurrence of an unprecedented state of high density in hadronic matter. The thermal nature of the hadronic system has been investigated by analyzing spectra and production ratios of hadrons which reveal a large degree of rescattering of primary and secondary hadrons.Thermal photons from elementary quark-gluon interactions are considered a promising signal for the occurrence of a phase transition to the quark-gluon plasma. The predictions for thermal photons from elementary parton interactions are discussed and compared to the thermal emission rate of photons from a hot hadronic gas. Recent results from the photon spectrometers in heavy ion experiments are presented. Production cross sections of pi0 and eta mesons are determined and the projectile and target mass dependence is discussed. An upper limit for the single photon yield was determined for central O+Au reactions. Recent S+Au reactions exhibit an excess of photons over the yield expected from hadronic decays. The spectral shape of the expected single photon signal is discussed which might reveal the temperature of hot matter and indicate a phase transition.</p

111. Regulatory microRNA enrichment and degradation in Granulosa cells during bovine follicular recruitment and dominance

Follicular development is a result of complex hormonal and biochemical synergies that could be activated or deactivated in a spatiotemporal manner in oocytes and surrounding cells including theca, granulosa, and cumulus cells. The microRNA (miRNA), 19 to 22 nucleotides noncoding RNA, are one of the molecular cues that could play a role in posttranscriptional regulation of genes involved in follicular development. Here we aimed to understand the availability and abundance of miRNA in bovine granulosa cells (GC) derived from subordinate (SF) and leading or dominant (DF) follicles during bovine follicular recruitment and dominance at Day 3 and 7 of the oestrus cycle, respectively. For this, Simmental heifers (n = 15) were oestrus synchronized and slaughtered at 3 (n = 6) and 7 (n = 7) days after the onset of the oestrous. The SF and DF were retrieved from each animal to obtain the corresponding GC, which were subjected to miRNA-enriched total RNA isolation using the miRNeasy mini kit (Qiagen GmbH, Hilden, Germany). The integrity and quality of RNA was determined using Agilent 2100 Bioanalyzer (Agilent Technologies Inc., Santa Clara, CA, USA) and Nanodrop 8000 Spectrophotometer (Thermo Fisher Scientific Inc., DE, USA), respectively. The RNA was then subjected to miRNA deep sequencing using the Illumina HISEqn 2000. Raw sequence data were further processed and analysed using miRDeep2 software package. Quantification of differentially expressed (DE) miRNA was done using R software and DESEqn 2 package. MiRNA with log2 fold change difference ≥1, P-value ≤0.05, and false discovery rate ≤1 were considered to be significant. Data analysis revealed that 291 and 311 miRNA were detected in GC of SF and 312 and 314 were detected in GC of DF at Days 3 and 7, respectively. A total of 17 miRNA were DE in GC from SF compared with the DF at Day 3, of which 15 miRNA were enriched and the remaining 2 were down-regulated in SF. Similarly, at Day 7 a total of 136 miRNA was altered with 51 miRNA showed to be enriched, whereas 85 others remained low in SF compared with the DF. Nine miRNA (bta-miR-21–3p, bta-miR-221, bta-miR-708, bta-miR-214, bta-miR-335, bta-miR-155, bta-miR-199a-5p, bta-miR-21–5p and bta-miR-222) were commonly differentially expressed both at Day 3 and 7 between SF and DF. Interestingly, all of the commonly DE miRNA, except bta-miR-335, were enriched in GC of SF at both days. Gene ontological analysis indicated that majority of the DE miRNA were found to be involved in regulation of programmed cell death, cell projection morphogenesis, regulation of cell proliferation, and macromolecule biosynthesis. Therefore, the temporal abundance of mature miRNAs in GC during bovine follicular development may suggest their potential role in regulation of follicular development in general and follicular recruitment and dominance in particular

Expression profiling of noncoding microRNAs in bovine granulosa cells of preovulatory dominant follicle using deep sequencing

In cattle, follicles grow in a wave-like pattern, with typically 2 or 3 waves per oestrous cycle. During each wave, one follicle of a cohort becomes dominant (DF), whereas the remaining subordinate follicles (SF) in the cohort undergo atresia. If the endocrine conditions are appropriate (low progesterone), the dominant follicle goes on to ovulate. In order to unravel the molecular mechanisms associated with ovulation and follicular atresia, here we aimed to investigate the expression of short regulatory microRNA (miRNA) in granulosa cells of DF and SF using deep sequencing. For this, Simmental heifers (n = 7) were synchronized according to standard protocols and slaughtered at Day 19 of the oestrous cycle. Follicles were categorized as DF (≥12 mm; n = 5) and SF (≤10 mm; n = 78). Granulosa cells from both follicle groups were used for total RNA (enriched with miRNA) isolation using miRNeasy mini kit (Qiagen GmbH, Hilden, Germany). The RNA concentration and integrity were measured using Nano Drop 8000 spectrophotometer (Nano Drop, Wilmington, DE, USA) and Agilent, 2100 Bioanalyzer (Agilent Technologies, Santa Clara, CA), respectively. Libraries were constructed by GATC BioTech AG (Konstanz, Germany) and sequenced on Illumina HISEqn 2000. Prediction of both known and novel miRNA was done using miRDeep2 software packages. Quantification of differentially expressed miRNA was done using R software and DESEqn 2 packages. The MiRNA with log2 fold change difference ≥1, P-value ≤0.05, and false discovery rate of ≤0.1 were considered to be significant. Results showed that 318 and 322 known miRNA were detected in DF and SF, respectively. It was shown that 28 miRNA including bta-miR-122, bta-miR-139, and bta-miR-375, and 35 others including bta-miR-138, bta-miR-20b, and bta-miR-33a were uniquely detected in DF and SF, respectively. In addition to the known annotated miRNA, 20 and 24 novel miRNA were detected in DF and SF, respectively. Expression analysis revealed that 65 miRNA were differentially expressed in granulosa cells of SF compared with DF. Thirty miRNA including bta-miR-409a (involved in cell death by targeting genes BCL2l11, BIRC5, and PTEN) and bta-miR-335 (involved in cell proliferation, migration, and differentiation) are up-regulated in SF, whereas 35 miRNA including the miR-183 cluster (bta-mir-183, bta-miR-182, and bta-mir-96) involved in apoptosis inhibition are down-regulated in SF. The pathway analysis of potential target genes of differentially expressed miRNA is found to be involved in pathways, namely Wnt signalling, MAPK signalling, TGF-β signaling, and other pathways related to cell proliferation and apoptosis. In conclusion, the presence of stage-specific miRNA in granulosa cells support the potential role of miRNA in posttranscriptional regulation of genes during follicular development, mainly ovulation and follicular atresia