An Optimized Trichloroacetic Acid/Acetone Precipitation Method for Two-Dimensional Gel Electrophoresis Analysis of Qinchuan Cattle Longissimus Dorsi Muscle Containing High Proportion of Marbling.

Longissimus dorsi muscle (LD) proteomics provides a novel opportunity to reveal the molecular mechanism behind intramuscular fat deposition. Unfortunately, the vast amounts of lipids and nucleic acids in this tissue hampered LD proteomics analysis. Trichloroacetic acid (TCA)/acetone precipitation is a widely used method to remove contaminants from protein samples. However, the high speed centrifugation employed in this method produces hard precipitates, which restrict contaminant elimination and protein re-dissolution. To address the problem, the centrifugation precipitates were first grinded with a glass tissue grinder and then washed with 90% acetone (TCA/acetone-G-W) in the present study. According to our result, the treatment for solid precipitate facilitated non-protein contaminant removal and protein re-dissolution, ultimately improving two-dimensional gel electrophoresis (2-DE) analysis. Additionally, we also evaluated the effect of sample drying on 2-DE profile as well as protein yield. It was found that 30 min air-drying did not result in significant protein loss, but reduced horizontal streaking and smearing on 2-DE gel compared to 10 min. In summary, we developed an optimized TCA/acetone precipitation method for protein extraction of LD, in which the modifications improved the effectiveness of TCA/acetone method

The Effect of FATP1 on Adipocyte Differentiation in Qinchuan Beef Cattle

FATP1 plays an important role in the regulation of fatty acid metabolism and lipid accumulation. In this study, we investigated the patterns of FATP1 expression in various tissues obtained from calf and adult Qinchuan cattle, and in differentiating adipocytes. Next, we investigated the effect of FATP1 expression on preadipocyte differentiation in Qinchuan cattle using overexpression and interference assays. We also identified the differentially expressed genes (DEGs) and pathways associated with FATP1 overexpression/interference. Our results reveal that FATP1 was broadly expressed in heart, kidney, muscle, small intestine, large intestine, and perirenal fat tissues. While FATP1 overexpression promoted preadipocyte differentiation, fat deposition, and the expression of several genes involved in fat metabolism, FATP1 interference had the opposite effects on adipocyte differentiation. Following FATP1 overexpression and FATP1 interference in adipocytes, RNA-seq analysis was performed to identify DEGs related to fat metabolism. The DEGs identified include SLPI, STC1, SEMA6A, TNFRSF19, SLN, PTGS2, ADCYP1, FADS2, and SCD. Pathway analysis revealed that the DEGs were enriched in the PPAR signaling pathway, AMPK signal pathway, and Insulin signaling pathway. Our results provide an in-depth understanding of the function and regulation mechanism of FAPT1 in fat metabolism

Data_Sheet_1_Integrated metabolome and microbiome analysis reveals the effect of rumen-protected sulfur-containing amino acids on the meat quality of Tibetan sheep meat.docx

IntroductionThis study investigated the effects of rumen-protected sulfur-containing amino acids (RPSAA) on the rumen and jejunal microbiota as well as on the metabolites and meat quality of the longissimus lumborum (LL) in Tibetan sheep.MethodsBy combining 16S rDNA sequencing with UHPLC-Q-TOF MS and Pearson correlation analysis, the relationship between gastrointestinal microbiota, muscle metabolites and meat quality was identified.ResultsThe results showed that feeding RPSAA can increase the carcass weight, abdominal fat thickness (AP-2 group), and back fat thickness (AP-2 and AP-3 group) of Tibetan sheep. The water holding capacity (WHC), texture, and shear force (SF) of LL in the two groups also increased although the fatty acids content and brightness (L*) value significantly decreased in the AP-2 group. Metabolomics and correlation analysis further showed that RPSAA could significantly influence the metabolites in purine metabolism, thereby affecting L* and SF. In addition, RPSAA was beneficial for the fermentation of the rumen and jejunum. In both groups, the abundance of Prevotella 1, Lachnospiraceae NK3A20 group, Prevotella UCG-003, Lachnospiraceae ND3007 group in the rumen as well as the abundance of Eubacterium nodatum group and Mogibacterium group in the jejunum increased. In contrast, that of Turicibacter pathogens in the jejunum was reduced. The above microorganisms could regulate meat quality by regulating the metabolites (inosine, hypoxanthine, linoleic acid, palmitic acid, etc.) in purine and fatty acids metabolism.DiscussionOverall, reducing the levels of crude proteins in the diet and feeding RPSAA is likely to improve the carcass quality of Tibetan sheep, with the addition of RPMET (AP-2) yielding the best edible quality, possibly due to its ability to influence the gastrointestinal microbiota to subsequently regulate muscle metabolites.</p

Effects of dietary forage-to-concentrate ratio on fat deposition, fatty acid composition, oxidative stability and mRNA expression of sirtuins genes of subcutaneous fat in sheep (Ovis aries)

ABSTRACTThis study was carried out to evaluate the effects of dietary concentrate: forage (C: F) ratio on fat deposition, fatty acid composition, oxidative stability and mRNA expression levels of sirtuins genes associated with adipose tissue metabolism of subcutaneous fat in Black Tibetan sheep. Three diets with different C: F (HC: 70:30, IC:50:50 and LC: 30:70) were fed to fifteen weaned male lambs (2-month-old, 10.05 ± 0.96 Kg). The experiment lasted for 120 d. Five lambs from each group were randomly selected and slaughtered at the end of the experiment. The subcutaneous fat thicknesses increased with increasing concentrate level (P < 0.05). The concentration of polyunsaturated fatty acid (PUFA), C15:1 and C18:2n decreased by feeding the HC diet (P < 0.05). Both glutathione peroxidase (GSH-Px) and superoxide dismutase (SOD) activities significantly increased as dietary concentration levels decreased (P < 0.05). Additionally, SIRT1 and SIRT2 expression level was downregulated (P < 0.05) with increasing concentration supplementation (P < 0.05 and P < 0.05, respectively). In conclusion, the addition of 70% concentration supplementation is not recommended in Black Tibetan sheep, considering that no benefits were observed for nutrient utilization, oxidative stability or economic returns, while the supplementation of C: F at 50:50 proved to be suitable for finishing lambs

Overlay analysis of total protein spots with different methods.

<p>A: comparison of TCA/acetone-G-W to direct extraction; B: comparison of TCA/acetone-G-W to two standard TCA/acetone procedures. U represented the number of unique spots from each method; C represented the number of spots common to all the methods.</p

Representative 2-DE maps of LD protein extracted using different methods.

<p>A: one-step extraction by lysis buffer; B: TCA/acetone; C: TCA/acetone-B; D: TCA/acetone-G-W. The modified procedure of TCA/acetone-G-W gave the best 2-DE gel with a clear background and minimal streaking and smearing.</p

Protein yields associated to the different protein extraction protocols.

<p>Values followed by different letters are significantly different in a row (capital: p<0.01, lowercase: p<0.05).</p><p>Protein yields associated to the different protein extraction protocols.</p

Performance Measurement and Comparative Transcriptome Analysis Revealed the Efforts on Hybrid Improvement of Qinchuan Cattle

<p>Crossbreeding can provide productive gains through heterosis, however, surveys about the effects of crossbreeding through global transcriptomic sequencing are few. This study revealed that Angus × Qinchuan cattle (AQF) have improved performance characteristics compared to Qinchuan cattle (QCF). We performed RNA-seq on the subcutaneous fat tissue of QCF and AQF. More than 42.2 million clean reads were obtained in each sample. We detected 40 and 21 breed-specific highly expressed genes (FPKM > 500) in QCF and AQF, respectively. Furthermore, a total of 353 differentially expressed genes (DEGs, |log2 ratio| ≥ 1 and Probability ≥ 0.8) were found between these two groups, of which 227 genes were upregulated in AQF and 126 genes were upregulated in QCF. Functional enrichment analyses showed that breed-specific highly expressed genes and DEGs were closely related to terms such as development in AQF, and adaption or immune in QCF. In addition, we also identified the novel transcript units, alternative splicing events, single-nucleotide polymorphisms and Indels. Our results revealed differences in inherent characteristics and genetic differences when comparing QCF with AQF.</p

Exploring the effects of palm kernel meal feeding on the meat quality and rumen microorganisms of Qinghai Tibetan sheep

Abstract Palm kernel meal (PKM) has been shown to be a high‐quality protein source in ruminant feeds. This study focused on the effects of feed, supplemented with different amounts of PKM (ZL‐0 as blank group, and ZL‐15, ZL‐18, and ZL‐21 as treatment group), on the quality and flavor profile of Tibetan sheep meat. Furthermore, the deposition of beneficial metabolites in Tibetan sheep and the composition of rumen microorganisms on underlying regulatory mechanisms of meat quality were studied based on ultra‐performance liquid chromatography coupled with quadrupole time‐of‐flight mass spectrometry as well as 16S rDNA sequencing. The results of the study showed that Tibetan sheep in the ZL‐18 group exhibited superior eating quality and flavor profile while depositing more protein and fat relative to the other groups. The ZL‐18 group also changed significantly in terms of the concentration and metabolic pathways of meat metabolites, as revealed by metabolomics. Metabolomics and correlation analyses finally showed that PKM feed mainly affected carbohydrate metabolism in muscle, which in turn affects meat pH, tenderness, and flavor. In addition, 18% of PKM increased the abundance of Christensenellaceae R‐7 group, Ruminococcaceae UCG‐013, Lachnospiraceae UCG‐002, and Family XIII AD3011 group in the rumen but decreased the abundance of Prevotella 1; the above bacteria groups regulate meat quality by regulating rumen metabolites (succinic acid, DL‐glutamic acid, etc.). Overall, the addition of PKM may improve the quality and flavor of the meat by affecting muscle metabolism and microorganisms in the rumen