A trial with different concentrations of DL-methionine (DLM) and DL-2-hydroxy-4-methylthiobutanoic acid (DL-HMTBA) in broiler feed was performed to investigate their effect on the meat quality parameters and the shelf life of breast fillet. In total, fillets from 210 male broiler chickens (Ross 308) were tested in seven groups with 30 animals each. Three different concentrations (0.04, 0.12, and 0.32%; on an equimolar basis) of either DLM or DL-HMTBA were added to a basal diet, summing up to seven treatment groups. After slaughter, fillets were packed aerobically and stored at 4°C. The investigated parameters comprised measurements of microbial as well as physicochemical parameters, such as pH, drip loss, cooking loss, and color measurements. Additionally, sensory investigations were conducted and shelf life was calculated. Mean pH values were between 6.1 and 6.4. Drip loss values were low, with mean values below 0.4%. The cooking loss ranged between 22% and 28% on average. The fillets showed a normal initial microbial quality (2.5 log10 cfu/g) and spoilage process with microbial counts of 8.5 log10 cfu/g at the end of storage. The study revealed a significant influence of methionine supplementation on the quality of broiler breast meat in comparison with the basal group. Methionine supplementation led to higher pH values and a higher water binding. Higher concentrations of methionine had a positive influence on the water-holding capacity by lowering the cooking loss. The L∗ value showed a significant negative correlation to the methionine concentration supplemented. No differences in physicochemical as well as sensory parameters could be detected between both methionine sources. The fillets showed a normal sensory spoilage process and a shelf life of 6 d. White striping was positively correlated to fillet weight as well as color values and significantly affected the Purchase Decision, the sensory investigation, and thus the shelf life of the samples.

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.

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.

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In recent years, CRISPR/Cas9 has been used to efficiently edit the genomes of embryos in many animal models. Due to smaller anatomy, lower costs, and multiple ovulations, it is relatively simple to derive large numbers of invivo fertilized zygotes for gene editing experiments in small mammal models. In cattle, however, harvesting invivo fertilized zygotes generally requires a highly invasive surgical procedure. Here, we use the combination of a minimally invasive endoscopic method for harvesting invivo fertilized zygotes by oviductal flushing of superovulated heifers and the subsequent electroporation of zygotes with CRISPR/Cas9 ribonucleoproteins (RNP). After superstimulation of 21 heifers, on average 12 zygotes were flushed per animal with fetal bovine serum, then stored in synthetic oviductal fluid (SOFaa) before electroporation. Targeting exon 1 of the tyrosinase (Tyr) gene, zygotes were electroporated in 1-mm gap cuvettes (Biorad) in groups of ~20 in 20μL of OptiMEM media containing 3μM Cas9 RNP (IDT Cas9 protein pre-incubated with anti-Tyr guide RNA). Electroporation was performed in 3 replicates of 3 electrical potentials, namely 20, 25, and 30V using a Biojet CF 50. The other electroporation parameters were fixed at 5 repetitions of 2-ms square wave pulses at 100-ms intervals. The zygotes were than cultured under standard embryo culture conditions (SOFaa + 0.3% bovine serum albumin, 5% CO2, 5% O2, 39°C, humidified air). Embryo survival, cleavage, and developmental rates to the blastocyst stage were tracked. Statistical significance between groups was determined by pairwise one-way ANOVA using Sidak correction for multiple comparisons. Electroporation of invivo-derived zygotes using 20V yielded significantly higher survival (83.6% vs. 42.8% vs. 20.7% for 20, 25, and 30V, respectively), cleavage (65.6% vs. 37.9% vs. 40.0%), and developmental rates (47.5% vs. 21.4% vs. 16.5%) than 25 or 30V. There was no statistical difference between 25 and 30V. Subsequently, editing rates were determined using the T7 mismatch assay and verified with Sanger sequencing followed by sequence alignment and analysis using Tracking of Indels by Decomposition (TIDE) software (https://tide.nki.nl/). Although there was high variance between electroporation groups, blastocyst editing rates of up to 80.0% were achieved using 30V. To our knowledge, these are the first confirmed gene-edited bovine embryos produced from invivo fertilized zygotes. This method offers the ability to utilise the embryos of high-value cows or cows with known genotypes for genetic engineering experiments. In addition, given that electroporated bovine zygotes can be transferred back to the oviduct endoscopically, our future attempts will focus on genome editing in bovine embryos developed nearly completely within the physiological invivo environment.