Differentially Expressed miRNA-Gene Targets Related to Intramuscular Fat in Musculus Longissimus Dorsi of Charolais × Holstein F2-Crossbred Bulls.

Intramuscular fat (IMF) is a meat quality indicator associated with taste and juiciness. IMF deposition, influenced by genetic and non-genetic factors, occurs through a transcriptionally coordinated process of adipogenesis. MicroRNAs (miRNAs) are transcriptional regulators of vital biological processes, including lipid metabolism and adipogenesis. However, in bovines, limited data on miRNA profiling and association with divergent intramuscular fat content, regulated exclusively by genetic parameters, have been reported. Here, a microarray experiment was performed to identify and characterize the miRNA expression pattern in the Musculus longissimus dorsi of F2-cross (Charolais × German Holstein) bulls with high and low IMF. A total of 38 differentially expressed miRNAs (DE miRNAs), including 33 upregulated and 5 downregulated (corrected p-value ≤ 0.05, FC ≥ ±1.2), were reported. Among DE miRNAs, the upregulated miRNAs miR-105a/b, miR-695, miR-1193, miR-1284, miR-1287-5p, miR-3128, miR-3178, miR-3910, miR-4443, miR-4445 and miR-4745, and the downregulated miRNAs miR-877-5p, miR-4487 and miR-4706 were identified as novel fat deposition regulators. DE miRNAs were further analyzed, along with previously identified differentially expressed genes (DEGs) from the same samples and predicted target genes, using multiple bioinformatic approaches, including target prediction tools and co-expression networks, as well as Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment. We identified DE miRNAs and their gene targets associated with bovine intramuscular adipogenesis, and we provide a basis for further functional investigations.

Determination and Comparison of Physical Meat Quality Parameters of PERCIDAE and Salmonidae in Aquaculture.

Although aquaculture has been the fastest growing food sector for decades, there are no standardized parameters for most of the fish species regarding physical meat quality. Therefore, this study provides for the first time an overview of the physical meat characteristics of the most important fish species of the German Baltic Sea coast. Traditional farmed salmonids (rainbow trout (Oncorhynchus mykiss) and maraena whitefish (Coregonus maraena) as well as two percids (European perch, Perca fluviatilis and pikeperch, Sander lucioperca) were utilized for this comparison. The results demonstrate that the meat of the salmonids is very analogous. However, the post mortem degradation process starts faster in trout meat. In contrast, the meat quality characteristics of the percids are relatively different. The meat of pikeperch has comparatively low shear strength with a high water-holding capacity resulting in high meat tenderness. The opposite situation is present in European perch. The results indicate that it is not possible to establish the overall quality characteristics for fish or production form, as there is a high range of variability. Consequently, it is particularly important that meat quality characteristics are developed for important aquaculture species for further improvement through changes in husbandry conditions when necessary.

Increased expression of thyroid hormone responsive protein (THRSP) is the result but not the cause of higher intramuscular fat content in cattle.

Thyroid hormone responsive protein (THRSP) is known to be involved in lipogenic processes in rodents. In cattle, THRSP could be a potential molecular marker for intramuscular fat (IMF) deposition since mRNA abundance was frequently found to be increased in skeletal muscle with high IMF content compared to those with low IMF. The aim of this study was to elucidate the background of this differential expression and to evaluate the role of THRSP as candidate for increased IMF content in cattle. By combination of mRNA and protein analyses, we could demonstrate that THRSP is present mainly in nuclei of adipose tissue, in intramuscular fat cells and associated cells, and in cells of the portal triad of liver, whereas muscle cells did not express THRSP. Cell culture analyses revealed furthermore that THRSP is expressed in mature adipocytes rather than in early stages of adipogenesis. Collectively, our data support the putative role of THRSP as transcriptional regulator and demonstrate that an increased expression of THRSP in M. longissimus is a consequence of but not the reason for a higher number of intramuscular adipocytes in cattle with enhanced IMF deposition.

Transcriptome profiling of Musculus longissimus dorsi in two cattle breeds with different intramuscular fat deposition.

Intramuscular fat (IMF) deposition is a physiological process in cattle and is highly variable among breeds suggesting a large influence of genetic factors besides environmental factors. In order to elucidate molecular pathways underlying the genetic variation in this trait we compared transcriptomes of Musculus longissimus dorsi (MLD) in steers of Japanese Black and Holstein Friesian cattle breeds fed a high energy diet typically applied in Japan to achieve maximum IMF content. We identified a total of 569 differentially expressed genes (DEGs) with the majority (433) up-regulated in Japanese Black cattle. This breed is characterized by an extreme capacity for IMF deposition. Subsequent Ingenuity Pathway Analysis (IPA) revealed a gene network linking parameters of cell morphology and maintenance with lipid metabolism. The data from this study were deposited in NCBI's Gene Expression Omnibus and are accessible through GEO Series accession number GSE75348. We provide here a dataset which is of potential value to dissect molecular pathways influencing differences in fat deposition under high-energy nutrition.

Gene expression profile of Musculus longissimus dorsi in bulls of a Charolais × Holstein F2-cross with divergent intramuscular fat content.

Transcriptomes of Musculus longissimus dorsi (MLD) were compared between bulls from a F2-cross derived from Charolais and Holstein Friesian. Two groups of 10 bulls were selected which differed significantly in intramuscular fat (IMF) deposition despite standardized husbandry and feeding conditions and identical sires in both groups. Consequently, genetic factors underlying the different capability of IMF deposition should be identified. A total of 32 differentially expressed genes (DEGs) were found of which 11 were up-regulated and 21 were down-regulated in the high IMF group. Ingenuity Pathway Analysis (IPA) identified a gene network comprising DEGs with functions in carbohydrate metabolism, lipid metabolism and molecular transport. The data from this study were deposited in NCBI's Gene Expression Omnibus and are accessible through GEO Series accession number GSE75347. We provide here a dataset which is of potential value to dissect molecular pathways influencing differences in IMF deposition in crossbred cattle with standardized genetic background.

Localization and abundance of early markers of fat cell differentiation in the skeletal muscle of cattle during growth--are DLK1-positive cells the origin of marbling flecks?

Localization and abundance of early markers of adipogenic differentiation were investigated in bovine muscle tissue to verify their association with marbling development. Bovine skeletal muscle samples were used for immunohistochemical localization and measurement of mRNA and protein abundance of delta-like homolog 1 (DLK1) and CCAAT/enhancer-binding protein beta (CEBPB). The localization of DLK1-positive cells confirmed the position of small clusters of adipocytes which can be considered as the origin of marbling flecks. The results indicated higher DLK1 expression in the less marbled muscle of Holstein (HS) steers (P < 0.05) and a trend to higher CEBPB expression in Japanese Black (JB) steers (P < 0.1) at slaughter. The number of DLK1-positive cells and fat content were negatively correlated. The lower expression of DLK1 together with higher CEBPB abundance during fattening in JB may have contributed to the development of more adipocytes in the skeletal muscle of JB.

Molecular heterogeneities of adipose depots - potential effects on adipose-muscle cross-talk in humans, mice and farm animals.

Adipose tissue is considered as a major endocrine organ that secretes numerous proteins called adipokines. The heterogeneous nature of adipose tissue in different parts of the body suggests respective heterogeneity of proteomes and secretomes. This review consolidates knowledge from recent studies targeting the diversity of different adipose depots affecting the pattern of secreted adipokines and discusses potential consequences for the cross-talk between adipose and skeletal muscle in humans, rodent models and farm animals. Special attention is paid to muscle-associated fat depots like inter- and intramuscular fat that become focus of attention in the context of the rather new notion of skeletal muscle as a major endocrine organ. Understanding the complexity of communication between adipocytes and skeletal muscle cells will allow developing strategies for improvement of human health and for sustainable production of high quality meat.

Irisin is elevated in skeletal muscle and serum of mice immediately after acute exercise.

Recent findings regarding the response of fibronectin type III domain-containing protein 5 (Fndc5) and irisin to exercise are partly controversial. While the 25 kDa form of Fndc5 can be observed in muscle and serum of different species, the ~12 kDa irisin band was not detectable up to now. The present study aimed to clarify whether irisin exists in its theoretical size of ~12 kDa in mice and if it is affected by exercise. Male mice were randomly assigned to a sedentary control group (CO), a group with free access to running wheels (RW), and a treadmill group (TM). Blood and leg muscles were collected to investigate the regulatory cascade including peroxisome proliferator-activated receptor gamma co-activator 1-alpha (Ppargc1a) and Fndc5. In western blot analysis, antibodies were used capable of differentiation between full-length Fndc5 and irisin. This enabled us to demonstrate that irisin exists in muscle and serum of mice independent of exercise and that it is increased immediately after acute exercise. Different transcripts of Ppargc1a mRNA, but not Fndc5 mRNA, were up-regulated in the TM group. Furthermore, neither Fndc5 (25 kDa) nor Ppargc1a protein was elevated in muscle tissue. The Ppargc1a-Fndc5/irisin pathway did not clearly respond to mild exercise in the RW group. Our results provide evidence for the existence of irisin and for its immediate response to acute exercise in mice.

Locus characterization and gene expression of bovine FNDC5: is the myokine irisin relevant in cattle?

The transmembrane protein FNDC5 was recently characterized as precursor of an exercise induced myokine named irisin. Previous studies found a relationship between circulating irisin levels and muscle mass in humans. Consequently, we tested the hypothesis whether FNDC5/irisin is involved in the modulation of body composition in cattle. Since information on the bovine FNDC5 locus was scarce, we characterized the gene experimentally as prerequisite for these investigations. We provide here a revised and extended gene model for bovine FNDC5. Although similarly organized like the human and murine loci, a higher variability was observed at transcript level in the bovine locus. FNDC5 mRNA was abundant in bovine skeletal muscle and was detected at lower levels in adipose tissue and liver. There were no expression differences between two groups of bulls highly different in muscularity and adiposity. Full-length FNDC5 protein (25 kDa) was present in bovine skeletal muscle independent of muscularity. Neither FNDC5 nor its putatively secreted peptide irisin were found in circulation of bulls. In contrast, we demonstrated that FNDC5 (25 kDa) and irisin (12 kDa) were present in murine skeletal muscle and that irisin was circulating in murine serum. This indicates fundamental differences in the regulation of FNDC5 and irisin between rodents and cattle.

Healthy sheep that differ in scrapie associated PRNP genotypes exhibit significant differences of expression pattern associated with immune response and cell-to-cell signalling in retropharyngeal lymph nodes.

The present study was conducted to test the hypothesis whether prion protein gene (PRNP) associated scrapie susceptibility is connected with physiological changes in tissue involved in pathogen uptake, migration and propagation. Jejunum, ileal Peyer's patches, retropharyngeal lymph nodes, brain stem and liver of healthy and non scrapie-infected sheep with PRNP genotypes representing the scrapie risk class R1 (scrapie-resistant) and R5 (scrapie-susceptible), respectively, were comparatively analysed by microarray technology and quantitative reverse transcriptase polymerase chain reaction (RT qPCR). Significantly higher expression levels of genes involved in immune response and cell communication pathways in retropharyngeal lymph nodes of R1 sheep in comparison with R5 animals strongly suggest PRNP associated physiological processes with impact as an early barrier in pathogen defence. Equal expression patterns in brain stem suggest no physiological differences in brain of healthy R1 and R5 animals. In addition, similar expression pattern in liver indicates that there are no transcriptional differences in genes of the hepatic energy metabolism between animals of scrapie classes R1 and R5.

Agouti revisited: transcript quantification of the ASIP gene in bovine tissues related to protein expression and localization.

Beside its role in melanogenesis, the agouti signaling protein (ASIP) has been related to obesity. The potentially crucial role in adipocyte development makes it a tempting candidate for economic relevant, fat related traits in farm animals. The objective of our study was to characterize the mRNA expression of different ASIP transcripts and of putative targets in different bovine tissues, as well as to study consequences on protein abundance and localization. ASIP mRNA abundance was determined by RT-qPCR in adipose and further tissues of cattle representing different breeds and crosses. ASIP mRNA was up-regulated more than 9-fold in intramuscular fat of Japanese Black cattle compared to Holstein (p<0.001). Further analyses revealed that a transposon-derived transcript was solely responsible for the increased ASIP mRNA abundance. This transcript was observed in single individuals of different breeds indicating a wide spread occurrence of this insertion at the ASIP locus in cattle. The protein was detected in different adipose tissues, skin, lung and liver, but not in skeletal muscle by Western blot with a bovine-specific ASIP antibody. However, the protein abundance was not related to the observed ASIP mRNA over-expression. Immuno-histochemical analyses revealed a putative nuclear localization of ASIP additionally to the expected cytosolic signal in different cell types. The expression of melanocortin receptors (MCR) 1 to 5 as potential targets for ASIP was analyzed by RT-PCR in subcutaneous fat. Only MC1R and MC4R were detected indicating a similar receptor expression like in human adipose tissue. Our results provide evidence for a widespread expression of ASIP in bovine tissues at mRNA and, for the first time, at protein level. ASIP protein is detectable in adipocytes as well as in further cells of adipose tissue. We generated a basis for a more detailed investigation of ASIP function in peripheral tissues of various mammalian species.