Transcriptome analysis of adipose tissue in grazing cattle: Identifying key regulators of fat metabolism.

The taste and tenderness of meat are the main determinants of carcass quality in many countries. This study aimed to discuss the mechanisms of intramuscular fat deposition in grazing and house-breeding cattle. We performed transcriptome analysis to characterize messenger RNA and microRNA (miRNA) expression profiles. A total of 456 and 66 differentially expressed genes (DEGs) and differentially expressed (DE) miRNAs were identified in the adipose tissue of grazing and house-breeding cattle. Kyoto Encyclopedia of Genes and Genomes pathway analysis identified the association of DEGs with fatty acid metabolism, fatty acid degradation, peroxisome proliferator-activated receptors signaling pathway, adenosine monophosphate-activated protein kinase signaling pathway, adipocytokine signaling pathway, and the association of DE miRNAs with mitogen-activated protein kinase signaling pathway. Apolipoprotein L domain containing 1, pyruvate dehydrogenase kinase 4, and sphingosine-1-phosphate lyase 1 genes may be the key regulators of fat metabolism in grazing cattle. Finally, we found that miR-211 and miR-331-5p were negatively correlated with the elongation of very long-chain fatty acids protein 6 (ELOVL6), and miR-331-5p might be the new regulator involved in fat metabolism. The results indicated that ELOVL6 participated in various functions and pathways related to fat metabolism. Meanwhile, miR-331-5p, as a new regulator, might play an essential role in this process. Our findings laid a more in-depth and systematic research foundation for the formation mechanism and characteristics of adipose tissue in grazing cattle.

The Characterization of Subcutaneous Adipose Tissue in Sunit Sheep at Different Growth Stages: A Comprehensive Analysis of the Morphology, Fatty Acid Profile, and Metabolite Profile.

Adipose tissue is a crucial economically significant trait that significantly influences the meat quality and growth performance of domestic animals. To reveal the changes in adipose tissue metabolism during the growth of naturally grazing sheep, we evaluated the thickness, adipocyte morphology, fatty acid profile, and metabolite profile of subcutaneous adipose tissue (SAT) from naturally grazing Sunit sheep at 6, 18, and 30 months of age (referred to as Mth-6, Mth-18, and Mth-30, respectively). The fat thickness and adipocyte number were significantly increased with the growth of the sheep (p < 0.05), and the increase of which from Mth-18 to Mth-30 was less than that from Mth-6 to Mth-18. Additionally, the alpha-linolenic acid metabolism was enhanced and fatty acid (FA) elongation increased with growth. The metabolomic analysis revealed 76 differentially expressed metabolites (DEMs) in the SAT in different growth stages. Interestingly, we observed elongation of FAs in lipids correlated with sheep growth. Furthermore, the expression of acylcarnitines was downregulated, and fatty acid amides, aspartic acid, acetic acid and phosphocholine were upregulated in Mth-18 and Mth-30 compared to Mth-6. Altogether, the study found that the difference in SAT in Mth-6 was great compared to Mth-18 and Mth-30. An increase in fat deposition via adipocyte proliferation with the growth of the sheep in naturally grazing. The DEMs of acylcarnitines, fatty acid amides, aspartic acid, acetic acid, and phosphocholine emerged as potential key regulators of adipose tissue metabolism. These findings illustrate the variation in and metabolic mechanism of sheep adipose tissue development under natural grazing, thus providing valuable insights into improving the edible quality of sheep meat and developing the mutton sheep industry.

Integrated Transcriptome Analysis of miRNAs and mRNAs in the Skeletal Muscle of Wuranke Sheep.

MicroRNAs (miRNAs) are regarded as important regulators in skeletal muscle development. To reveal the regulatory roles of miRNAs and their target mRNAs underlying the skeletal muscle development of Wuranke sheep, we investigated the miRNA and mRNA expression profiles in the biceps femoris of these sheep at the fetal (3 months of gestation) and 3- and 15-month-old postnatal stages. Consequently, a total of 1195 miRNAs and 24,959 genes were identified. Furthermore, 474, 461, and 54 differentially expressed miRNAs (DEMs) and 6783, 7407, and 78 differentially expressed genes (DEGs) were detected among three comparative groups. Functional analysis demonstrated that the target mRNAs of the DEMs were enriched in multiple pathways related to muscle development. Moreover, the interactions among several predicted miRNA-mRNA pairs (oar-miR-133-HDAC1, oar-miR-1185-5p-MYH1/HADHA/OXCT1, and PC-5p-3703_578-INSR/ACTG1) that potentially affect skeletal muscle development were verified using dual-luciferase reporter assays. In this study, we identified the miRNA and mRNA differences in the skeletal muscle of Wuranke sheep at different developmental stages and revealed that a series of candidate miRNA-mRNA pairs may act as modulators of muscle development. These results will contribute to future studies on the function of miRNAs and their target mRNAs during skeletal muscle development in Wuranke sheep.

Characterization of Type I and Type III Collagen in the Intramuscular Connective Tissue of Wuzhumuqin Sheep.

Intramuscular connective tissue (IMCT) collagen is an important factor in meat quality. This study analyzed the characteristics of type I and III collagen in the IMCT of the semitendinosus (SD) and longissimus dorsi (LD) of Wuzhumuqin sheep at different growth stages (6, 9, 12, and 18 months). Utilizing sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and Fourier transform infrared spectroscopy (FTIR), collagen types I and III were successfully isolated and shown to contain an intact triple helix structure. Immunofluorescence revealed that these collagens were located in the endomysium and perimysium. Collagen-related genes were significantly expressed in sheep aged 9 and 12 months. The amino acid content increased with age in type I collagen whereas it decreased in type III collagen. Furthermore, type III collagen contained more hydroxyproline (Hyd) than type I collagen. Differential scanning calorimetry (DSC) revealed that the thermal stability of collagen increased with age, accompanied by a decrease in solubility. Semitendinosus muscle had more collagen cross-linkages than LD muscle due to the high pyridinoline (Pyr) content in the endomysium. Finally, a correlation analysis highlighted the multiple correlations between characteristics in different types of collagen during sheep growth. In summary, the collagen characteristics in the IMCT of sheep were impacted by collagen type, muscle type, and age. Furthermore, the various correlations between these characteristics may play an important role in the development of IMCT.

Effects of the Expressions and Variants of the CAST Gene on the Fatty Acid Composition of the Longissimus Thoracis Muscle of Grazing Sonid Sheep.

Fatty acid (FA) composition has an important impact on the nutrition and flavor of meat, and on consumer health, and is receiving more attention in the sheep industry. This study aimed to evaluate the relationship between the expression levels of the CAST gene and the FA composition in the longissimus thoracis (LL) muscle, to identify novel variants of CAST, and to perform association analysis with the FA composition in grazing Sonid lambs. The correlation results showed that high expression levels of CAST are correlated with better FA compositions and classes in LL. For association studies, the results showed that c.1210C>T and c.1437G>A in LD-M, and c.2097C>T mutations are associated with some compositions and classes of FA in the LL of grazing Sonid sheep. Two missense c.646G>C (G216R) and c.1210C>T (R404C) mutations were predicted to influence the Calpain_inhib domains of CAST. Thus, the correlation results and associated mutations are expected to be genetic selection markers for the FA composition and meat quality of grazing Sonid lamb muscle and provide new insights into sheep meat quality traits influenced by the ovine CAST gene.

Anti-inflammatory effects of the prostaglandin D2/prostaglandin DP1 receptor and lipocalin-type prostaglandin D2 synthase/prostaglandin D2 pathways in bacteria-induced bovine endometrial tissue.

Dairy cows often develop different degrees of endometritis after calving and this is attributed to pathogenic bacterial infections such as by Escherichia coli and Staphylococcus aureus. Infection of the bovine endometrium causes tissue damage and increases the expression of prostaglandin D2 (PGD2), which exerts anti-inflammatory effects on lung inflammation. However, the roles of PGD2 and its DP1 receptor in endometritis in cows remain unclear. Here, we examined the anti-inflammatory roles of the lipocalin-type prostaglandin D2 synthase (L-PGDS)/PGD2 and DP1 receptor regulatory pathways in bovine endometritis. We evaluated the regulatory effects of PGD2 on inflammation and tissue damage in E. coli- and S. aureus-infected bovine endometrial cells cultured in vitro. We found that the secretion of pro-inflammatory cytokines interleukin (IL)-6, IL-1ß, and tumour necrosis factor (TNF)-α as well as expression of matrix metalloproteinase (MMP)-2, platelet-activating factor receptor (PAFR), and high mobility group box (HMGB)-1 were suppressed after DP1 receptor agonist treatment. In contrast, IL-6, IL-1ß, and TNF-α release and MMP-2, PAFR, and HMGB-1 expression levels were increased after treatment of bovine endometrial tissue with DP1 receptor antagonists. DP1-induced anti-inflammatory effects were dependent on cellular signal transduction. The L-PGDS/PGD2 pathway and DP1 receptor induced anti-inflammatory effects in bovine endometrium infected with S. aureus and E. coli by inhibiting the mitogen-activated protein kinase and nuclear factor-κB signalling pathways, thereby reducing tissue damage. Overall, our findings provide important insights into the pathophysiological roles of PGD2 in bovine endometritis and establish a theoretical basis for applying prostaglandins or non-steroidal anti-inflammatory drugs for treating endometrial inflammatory infertility in bovines.

MicroRNA and circular RNA profiling in the deposited fat tissue of Sunite sheep.

As the most typical deposited fat, tail fat is an important energy reservoir for sheep adapted to harsh environments and plays an important role as a raw material in daily life. However, the regulatory mechanisms of microRNA (miRNA) and circular RNA (circRNA) in tail fat development remain unclear. In this study, we characterized the miRNA and circRNA expression profiles in the tail fat of sheep at the ages of 6, 18, and 30 months. We identified 219 differentially expressed (DE) miRNAs (including 12 novel miRNAs), which exhibited a major tendency to be downregulated, and 198 DE circRNAs, which exhibited a tendency to be upregulated. Target gene prediction analysis was performed for the DE miRNAs. Functional analysis revealed that their target genes were mainly involved in cellular interactions, while the host genes of DE circRNAs were implicated in lipid and fatty acid metabolism. Subsequently, we established a competing endogenous RNA (ceRNA) network based on the negative regulatory relationship between miRNAs and target genes. The network revealed that upregulated miRNAs play a leading role in the development of tail fat. Finally, the ceRNA relationship network with oar-miR-27a_R-1 and oar-miR-29a as the core was validated, suggesting possible involvement of these interactions in tail fat development. In summary, DE miRNAs were negatively correlated with DE circRNAs during sheep tail fat development. The multiple ceRNA regulatory network dominated by upregulated DE miRNAs may play a key role in this developmental process.

Effects of sheep slaughter age on myogenic characteristics in skeletal muscle satellite cells.

OBJECTIVE: The objective of this study was to investigate the effects of sheep slaughter age on myogenic characteristics in skeletal muscle satellite cells (SMSCs). METHODS: Primary SMSCs were isolated from hind leg biceps femoris muscles of Wurank lambs (slaughtered at three months, Mth-3) and adults (slaughtered at fifteen months, Mth-15). SMSCs were selected by morphological observation and fluorescence staining. Myogenic regulatory factors (MRF) and myosin heavy chain (MyHC) expressions of SMSCs were analyzed on days 1, 3, 4, and 5. RESULTS: The expressions of myogenic factor 5 (Myf5), myogenic differentiation (MyoD), Myf6, and myogenin (MyoG) in Mth-15 were significantly higher in Mth-15 than in Mth-3 on days 1, 3, and 4 (p<0.05). However, MyoG expression in Mth-15 was significantly lower than in Mth-3 on day 5 (p<0.05). The expressions of MyHC I, MyHC IIa, and MyHC IIx in Mth-15 were significantly higher than in Mth-3 on days 1 and 3 (p<0.05), and MyHC IIb were significantly lower than in Mth-3 on days 3 and 4 (p<0.05). In contrast, the expression of MyHC IIx in Mth-15 was significantly lower and MyHC IIb was significantly higher than in Mth-3 on days 5 (p<0.05). CONCLUSION: The slaughter age altered the expression of MRFs and MyHCs in SMSCs while differentiation, which caused the variation of myogenic characteristics, and thus may affect the meat quality of Wurank sheep.

Transcriptome analysis of messenger RNA and long noncoding RNA related to different developmental stages of tail adipose tissues of sunite sheep.

The tail fat of sheep is the most typical deposited fat, and it can be widely used in human daily life, such as diet, cosmetics, and industrial raw materials. To understand the potential regulatory mechanism of different growth stages of tail fat in Sunite sheep, we performed high-throughput RNA sequencing to characterize the long noncoding RNA (lncRNA) and messenger RNA (mRNA) expression profiles of the sheep tail fat at the age of 6, 18, and 30 months. A total of 223 differentially expressed genes (DEGs) and 148 differentially expressed lncRNAs were found in the tail fat of 6-, 18-, and 30-month-old sheep. Based on functional analysis, we found that fat-related DEGs were mainly expressed at 6 months of age and gradually decreased at 18 and 30 months of age. The target gene prediction analysis shows that most of the lncRNAs target more than 20 mRNAs as their transregulators. Further, we obtained several fat-related differentially expressed target genes; these target genes interact with different differentially expressed lncRNAs at various ages and play an important role in the development of tail fat. Based on the DEGs and differentially expressed lncRNAs, we established three co-expression networks for each comparison group. Finally, we concluded that the development of the sheep tail fat is more active during the early stage of growth and gradually decreases with the increase in age. The mutual regulation of lncRNAs and mRNAs may play a key role in this complex biological process.

Effects of feed intake restriction during late pregnancy on the function, anti-oxidation capability and acute phase protein synthesis of ovine liver.

OBJECTIVE: An experiment was conducted to investigate the effects of feed intake restriction during late pregnancy on the function, anti-oxidation capability and acute phase protein synthesis of ovine liver. METHODS: Eighteen time-mated ewes with singleton fetuses were allocated to three groups: restricted group 1 (RG1, 0.18 MJ ME/kg W0.75 d, n = 6), restricted group 2 (RG2, 0.33 MJ ME/kg W0.75 d), n = 6) and a control group (CG, ad libitum, 0.67 MJ ME/kg W0.75 d, n = 6). The feed restriction period was from 90 days to 140 days of pregnancy. RESULTS: The ewe's body weight, liver weights, water, and protein content of liver in the restricted groups were reduced compared with the CG group (p<0.05), but the liver fat contents in the RG1 group were higher than those of the CG group (p<0.05). The increased hepatic collagen fibers and reticular fibers were observed in the restricted groups with the reduction of energy intake. The concentrations of nonesterified free fatty acids in the RG1 and RG2 groups were higher than those of the CG group with the reduction of energy intake (p<0.05), but there were decreased concentrations of lipoprotein lipase and hepatic lipase in both restricted groups compared with the CG group (p<0.05). In addition, the increased concentrations of ß-hydroxybutyric acid, triglycerides, malondialdehyde, total antioxidant capacity and activities of superoxide dismutase activity and catalase were found in the RG1 group, and the concentrations of cholinesterase in the RG1 group were reduced compared with the CG group (p<0.05). For the concentrations of acute phase proteins, the C-reactive protein (CRP) in the RG1 group were reduced compared with the CG group, but there were no differences in haptoglobin relative to the controls (p>0.05). CONCLUSION: The fat accumulation, increased hepatic fibrosis, antioxidant imbalance and modified synthesis of acute phase proteins were induced in ewe's liver by maternal malnutrition during late pregnancy, which were detrimental for liver function to accommodate pregnancy.

Relationships among muscle fiber type composition, fiber diameter and MRF gene expression in different skeletal muscles of naturally grazing Wuzhumuqin sheep during postnatal development.

The aim of this study was to determine the relationships among muscle fiber-type composition, fiber diameter, and myogenic regulatory factor (MRF) gene expression in different skeletal muscles during development in naturally grazing Wuzhumuqin sheep. Three major muscles (i.e. the Longissimus dorsi (LD), Biceps femoris (BF) and Triceps brachii (TB)) were obtained from 20 Wuzhumuqin sheep and 20 castrated rams at each of the following ages: 1, 3, 6, 9, 12 and 18 months. Muscle fiber-type composition and fiber diameter were measured using histochemistry and morphological analysis, and MRF gene expression levels were determined using real-time PCR. In the LD muscle, changes in the proportion of each of different types of fiber (I, IIA and IIB) were relatively small. In the BF muscle, a higher proportion of type I and a 6.19-fold lower proportion of type IIA fibers were observed (P < 0.05). In addition, the compositions of type I and IIA fibers continuously changed in the TB muscle (P < 0.05). Moreover, muscle diameter gradually increased throughout development (P < 0.05). Almost no significant difference was found in MRF gene expression patterns, which appeared to be relatively stable. These results suggest that changes in fiber-type composition and increases in fiber size may be mutually interacting processes during muscle development.

Changes in the immunogold electron-microscopic localization of calpain in bovine skeletal muscle induced by conditioning and high-pressure treatment.

Changes in the localization of calpain in conditioned and pressurized bovine skeletal muscles were investigated by immunogold electron-microscopy. In the muscle immediately after thawing (control), the relative distributions of colloidal particles statistically calculated by counting the colloidal particles were about 65% and 35% in the I-band/Z-disk and A-band regions, respectively. In the muscle conditioned for 7 days, distribution of colloidal particles was more than two times greater in both the I-band/Z-disk and A-band regions than in the control muscle. Almost no change in either the absolute concentration or relative distribution of the colloidal particles was detectable during further storage. In the muscle exposed to a pressure of 100 MPa or 200 MPa, slightly more immunogold was detected in the sarcomere than in that of the control muscle. Increasing pressure up to 300 MPa enabled high-density particles to be seen throughout the sarcomere. Conversely, few particles were detected in the sarcomere of the muscle exposed to 400 MPa. These electron-microscopic observations were confirmed from the statistical analysis as with the conditioned muscles. It was clear from the results obtained that the pressure-induced changes in calpain localization were much more pronounced than those from conditioning.

Effects of a high-pressure treatment on the activity and structure of rabbit muscle proteasome.

The effects were assessed of high hydrostatic pressure on the activity and structure of rabbit skeletal muscle proteasome. The pressure effects on the activity were measured by the amount of fluorometric products released from synthetic substrates under pressure and from fluorescein isothiocyanate (FITC)-labeled casein after releasing the pressure. The effects on the structure were measured by fluorescene spectroscopy under pressure, and by circular dichroism (CD) spectroscopy and surface hydrophobicity after releasing the pressure. The optimal pressure for the hydrolyzing activity of synthetic peptides was 50 MPa. The degradation of FITC-labeled casein increased linearly with increasing pressure applied up to 200 MPa, and then markedly decreased up to at 400 MPa. The changes in the tertiary structure detected by fluorometric measurement were irreversible, whereas the changes in the secondary structure were small compared with those by heat treatment. The pressure-induced activation of proteasome therefore seems to have been due to a little unfolding of the active sites of proteasome.