The mitochondrial respiration signature of the bovine blastocyst reflects both environmental conditions of development as well as embryo quality.

The major limitation of the widespread use of IVP derived embryos is their consistent deficiencies in vitality when compared with their ex vivo derived counterparts. Although embryo metabolism is considered a useful metric of embryo quality, research connecting mitochondrial function with the developmental capacity of embryos is still lacking. Therefore, the aim of the present study was to analyse bovine embryo respiration signatures in relation to developmental capacity. This was achieved by taking advantage of two generally accepted metrics for developmental capacity: (I) environmental conditions during development (vivo vs. vitro) and (II) developmental kinetics (day 7 vs. day 8 blastocysts). Our study showed that the developmental environment affected total embryo oxygen consumption while different morphokinetics illustrating the embryo qualities correlate with maximal mitochondrial respiration, mitochondrial spare capacity, ATP-linked respiration as well as efficiency of ATP generation. This respiration fingerprint for high embryo quality is reflected by relatively lower lipid contents and relatively higher ROS contents. In summary, the results of the present study extend the existing knowledge on the relationship between bovine embryo quality and the signature of mitochondrial respiration by considering contrasting developmental environments as well as different embryo morphokinetics.

Endometrial DNA methylation signatures during the time of breeding in relation to the pregnancy outcome in postpartum dairy cows fed a control diet or supplemented with rumen-protected methionine.

Post calving metabolic stress reduces the fertility of high producing dairy cows possibly by altering the expression of genes in the maternal environment via epigenetic modifications. Therefore, this study was conducted to identify endometrial DNA methylation marks that can be associated with pregnancy outcomes in postpartum cows at the time of breeding. For this, twelve days post-calving, cows were either offered a control diet or supplemented daily with rumen-protected methionine. Cows showing heat 50-64 days postpartum were artificially inseminated. Endometrial cytobrush samples were collected 4-8 h after artificial insemination and classified based on the pregnancy out comes as those derived from cows that resulted in pregnancy or resulted in no pregnancy. The DNAs isolated from endometrial samples were then subject to reduced representative bisulfite sequencing for DNA methylation analysis. Results showed that in the control diet group, 1,958 differentially methylated CpG sites (DMCGs) were identified between cows that resulted in pregnancy and those that resulted in no pregnancy of which 890 DMCGs were located on chr 27: 6217254-6225600 bp. A total of 537 DMCGs were overlapped with 313 annotated genes that were involved in various pathways including signal transduction, signalling by GPCR, aldosterone synthesis and secretion. Likewise, in methionine supplemented group, 3,430 CpG sites were differentially methylated between the two cow groups of which 18.7% were located on Chr27: 6217254-6225600 bp. A total of 1,781 DMCGS were overlapped with 890 genes which involved in developmental and signalling related pathways including WNT-signalling, focal adhesion and ECM receptor interaction. Interestingly, 149 genes involved in signal transduction, axon guidance and non-integrin membrane-ECM interactions were differentially methylated between the two cow groups irrespective of their feeding regime, while 453 genes involved in axon guidance, notch signalling and collagen formation were differentially methylated between cows that received rumen protected methionine and control diet irrespective of their fertility status. Overall, this study indicated that postpartum cows that could potentially become pregnant could be distinguishable based on their endometrial DNA methylation patterns at the time of breeding.

The cell cycle stage of bovine zygotes electroporated with CRISPR/Cas9-RNP affects frequency of Loss-of-heterozygosity editing events.

At the embryonic level, CRISPR technologies have been used to edit genomes reliably and efficiently in various mammalian models, with Ribonucleoprotein (RNP) electroporation potentially representing a superior delivery method into mammalian zygotes. However, detailed insights of the interactions between varying technical settings as well as the time point of electroporation in a bovine zygote's cell cycle on developmental metrics and the frequency and type of editing events are largely unknown. The present study uncovers that increasing pulse lengths result in higher Full Edit rates, with Mosaicism in Full-Edit embryos being significantly affected by adjusting RNP-electroporation relative to zygote cell cycle. A considerable proportion of Full Edit embryos demonstrated loss-of-heterozygosity after RNP-electroporation prior to S-phase. Some of these loss-of-heterozygosity events are a consequence of chromosomal disruptions along large sections of the target chromosomes making it necessary to check for their presence prior use of this technique in animal breeding. One out of 2 of these loss-of-heterozygosity events, however, was not associated with loss of an entire chromosome or chromosomal sections. Whether analysed loss-of-heterozygosity in these cases, however, was a false negative result due to loss of PCR primer sequences after INDEL formation at the target side or indeed due to interhomolog recombination needs to be clarified in follow up studies since the latter would for sure offer attractive options for future breeding schedules.

The global gene expression outline of the bovine blastocyst: reflector of environmental conditions and predictor of developmental capacity.

BACKGROUND: Morphological evaluation of embryos has been used to screen embryos for transfer. However, the repeatability and accuracy of this method remains low. Thus, evaluation of an embryo's gene expression signature with respect to its developmental capacity could provide new opportunities for embryo selection. Since the gene expression outline of an embryo is considered as an aggregate of its intrinsic characteristics and culture conditions, we have compared transcriptome profiles of in vivo and in vitro derived blastocysts in relation to pregnancy outcome to unravel the discrete effects of developmental competence and environmental conditions on bovine embryo gene expression outlines. To understand whether the gene expression patterns could be associated with blastocyst developmental competency, the global transcriptome profile of in vivo (CVO) and in vitro (CVT) derived competent blastocysts that resulted in pregnancy was investigated relative to that of in vivo (NVO) and in vitro (NVT) derived blastocysts which did not establish initial pregnancy, respectively while to unravel the effects of culture condition on the transcriptome profile of embryos, the transcriptional activity of the CVO group was compared to the CVT group and the NVO group was compared to the NVT ones. RESULTS: A total of 700 differentially expressed genes (DEGs) were identified between CVO and NVO blastocysts. These gene transcripts represent constitutive regions, indel variants, 3'-UTR sequence variants and novel transcript regions. The majority (82%) of these DEGs, including gene clusters like ATP synthases, eukaryotic translation initiation factors, ribosomal proteins, mitochondrial ribosomal proteins, NADH dehydrogenase and cytochrome c oxidase subunits were enriched in the CVO group. These DEGs were involved in pathways associated with glycolysis/glycogenesis, citrate acid cycle, pyruvate metabolism and oxidative phosphorylation. Similarly, a total of 218 genes were differentially expressed between CVT and NVT groups. Of these, 89%, including TPT1, PDIA6, HSP90AA1 and CALM, were downregulated in the CVT group and those DEGs were overrepresented in pathways related to protein processing, endoplasmic reticulum, spliceasome, ubiquitone mediated proteolysis and steroid biosynthesis. On the other hand, although both the CVT and CVO blastocyst groups resulted in pregnancy, a total of 937 genes were differential expressed between the two groups. Compared to CVO embryos, the CVT ones exhibited downregulation of gene clusters including ribosomal proteins, mitochondrial ribosomal protein, eukaryotic translation initiation factors, ATP synthases, NADH dehydrogenase and cytochrome c oxidases. Nonetheless, downregulation of these genes could be associated with pre and postnatal abnormalities observed after transfer of in vitro embryos. CONCLUSION: The present study provides a detailed inventory of differentially expressed gene signatures and pathways specifically reflective of the developmental environment and future developmental capacities of bovine embryos suggesting that transcriptome activity observed in blastocysts could be indicative of further pregnancy success but also adaptation to culture environment.

Effect of methionine supplementation in chicken feed on the quality and shelf life of fresh poultry meat.

The aim of this study was to investigate the influence of different methionine sources and concentrations on the quality and spoilage process of broiler meat. The trial was comprised of 7 treatment groups: one basal group (suboptimal in Methionine+Cysteine; i.e., 0.89, 0.74, 0.69% in DM SID Met+Cys in starter, grower, and finisher diets, respectively) and 3 doses (0.10, 0.25, and 0.40%) of either DL-Methionine (DLM) or DL-2-hydroxy-4-methylthio butanoic acid (DL-HMTBA) on an equimolar basis of the DLM-supplemented groups. The broilers were fed the diets for 35 d, then slaughtered and processed. The filets were aerobically packed and stored under temperature controlled conditions at 4°C. Meat quality investigations were comprised of microbial investigations (total viable count and Pseudomonas spp.), pH and drip loss measurements of the filets. The shelf life of the meat samples was determined based on sensory parameters. After slaughtering, all supplemented meat samples showed a high quality, whereby no differences between the 2 methionine sources could be detected for the microbial load, pH, and drip loss. In comparison to the control group, the supplemented samples showed a higher sensory quality, characterized by a fresh smell and fresh red color. Methionine supplementation had a significant influence on meat quality parameters during storage. The microbial load, pH and drip loss of the chicken filets were positively correlated to the methionine concentration. Additionally, the microbial load at the end of storage was positively correlated to pH and drip loss values. Nevertheless, the microbial parameters were in a normal range and the positive correlation to methionine concentration did not affect the sensory shelf life. The mean sensory shelf life of the broiler filets varied between 7 to 9 d. During storage, no difference in the development of sensory parameters was observed between the supplemented groups, while the spoilage process of the basal group occurred slightly faster. In conclusion, methionine concentration, but not methionine source, effected meat quality parameters in breast muscles of broilers.