Pikeperch muscle tissues: a comparative study of structure, enzymes, genes, and proteins in wild and farmed fish.

Pikeperch (Sander lucioperca) is a freshwater species and an internationally highly demanded fish in aquaculture. Despite intensive research efforts on this species, fundamental knowledge of skeletal muscle biology and structural characteristics is missing. Therefore, we conducted a comprehensive analysis of skeletal muscle parameters in adult pikeperch from two different origins, wild-caught specimens from a lake and those reared in a recirculating aquaculture system. The analyses comprised the biochemical characteristics (nucleic acid, protein content), enzyme activities (creatine kinase, lactate dehydrogenase, NADP-dependent isocitrate dehydrogenase), muscle-specific gene and protein expression (related to myofibre formation, regeneration and permanent growth, muscle structure), and muscle fibre structure. The findings reveal distinct differences between the skeletal muscle of wild and farmed pikeperch. Specifically, nucleic acid content, enzyme activity, and protein expression varied significantly. The higher enzyme activity observed in wild pikeperch suggests greater metabolically activity in their muscles. Conversely, farmed pikeperch indicated a potential for pronounced muscle growth. As the data on pikeperch skeletal muscle characteristics is sparse, the purpose of our study is to gain fundamental insights into the characteristics of adult pikeperch muscle. The presented data serve as a foundation for further research on percids' muscle biology and have the potential to contribute to advancements and adaptations in aquaculture practices.

Acute and persistent effects of oral glutamine supplementation on growth, cellular proliferation, and tight junction protein transcript abundance in jejunal tissue of low and normal birthweight pre-weaning piglets.

Breeding for higher fertility has resulted in a higher number of low birthweight (LBW) piglets. It has been shown that LBW piglets grow slower than normal birthweight (NBW) littermates. Differences in growth performance have been associated with impaired small intestinal development. In suckling and weaning piglets, glutamine (Gln) supplementation has been associated with improved growth and intestinal development. This study was designed to examine the effects of oral Gln supplementation on growth and small intestinal parameters in LBW and NBW suckling piglets. At birth (day 0), a total of 72 LBW (1.10 ± 0.06 kg) and 72 NBW (1.51 ± 0.06) male piglets were selected. At day 1, litters were standardized to 12 piglets, and experimental piglets supplemented daily with either Gln (1 g/kg BW) or isonitrogenous amounts of Alanine (Ala) as control (1.22 g/kg BW) until day 12. Creep feed was offered from day 14 onward. Subgroups of piglets were euthanized at days 5, 12, and 26 for the analyses of jejunal morphometry, cellular proliferation, glutathione concentration and transcript abundance of tight junction proteins. From age day 11 to 21, Gln supplemented LBW (LBW-Gln) piglets were heavier than Ala supplemented LBW (LBW-Ala) littermates (P = 0.034), while NBW piglets were heavier until age day 26 compared to LBW littermates. Villus height was higher in LBW-Gln compared to LBW-Ala on age day 12 (P = 0.031). Sporadic differences among supplementation and birthweight groups were detected for jejunal cellular proliferation, cellular population and glutathione concentration, whereas age was the most dominant factor. These results show that Gln supplementation improved the growth of LBW piglets compared to LBW-Ala beyond the termination of Gln supplementation, but this was not associated with consistent effects on selected parameters of jejunal development.

The effect of N-arachidonoylethanolamide administration on energy and fat metabolism of early lactating dairy cows.

The aim of the study was to investigate the effect of N-arachidonoylethanolamide (AEA), an endocannabinoid with orexigenic characteristics, on plasma endocannabinoid concentrations, feed intake, energy balance, lipomobilisation, and hepatic lipid metabolism of early-lactating dairy cows. The experiment involved 10 pairs of Holstein half-sibling cows (end of 2nd-3rd pregnancy). Half-sibs of each pair were randomly assigned to either AEA (n = 10) or control (CON) group (n = 10). From day 1 to 30 postpartum, the AEA group received 5 intraperitoneal injections per week of 3 µg/kg body weight AEA and the CON group 0.9% NaCl. In week 1-3 postpartum, AEA administration had no effect on dry matter intake, body weight, or lipomobilisation, but increased plasma triglyceride concentration on d 21 p.p. and mRNA abundances of genes related to hepatic triglyceride synthesis. In week 4 postpartum, the AEA group showed reduced feed intake and whole-body carbohydrate oxidation, but increased whole-body fat oxidation and hepatic lipid accumulation, likely as a result of a counter-regulatory leptin increase. In conclusion, the present study shows a tissue-specific AEA insensitivity and may point to a leptin-controlled regulation of the ECS in early-lactation.

Identification and Quantification of Proliferating Cells in Skeletal Muscle of Glutamine Supplemented Low- and Normal-Birth-Weight Piglets.

One way to improve the growth of low-birth-weight (LBW) piglets can be stimulation of the cellular development of muscle by optimized amino acid supply. In the current study, it was investigated how glutamine (Gln) supplementation affects muscle tissue of LBW and normal-birth-weight (NBW) piglets. Longissimus and semitendinosus muscles of 96 male piglets, which were supplemented with 1 g Gln/kg body weight or alanine, were collected at slaughter on day 5 or 26 post natum (dpn), one hour after injection with Bromodeoxyuridine (BrdU, 12 mg/kg). Immunohistochemistry was applied to detect proliferating, BrdU-positive cells in muscle cross-sections. Serial stainings with cell type specific antibodies enabled detection and subsequent quantification of proliferating satellite cells and identification of further proliferating cell types, e.g., preadipocytes and immune cells. The results indicated that satellite cells and macrophages comprise the largest fractions of proliferating cells in skeletal muscle of piglets early after birth. The Gln supplementation somewhat stimulated satellite cells. We observed differences between the two muscles, but no influence of the piglets' birth weight was observed. Thus, Gln supplements may not be considered as effective treatment in piglets with low birth weight for improvement of muscle growth.

Effects of the Probiotic Enterococcus faecium on Muscle Characteristics of Chickens.

The use of antibiotics in farm animals is one of the main reasons for the development of resistant bacterial strains (e.g., zoonotic pathogens). Therefore, save alternatives are needed. Here, we examined how post-hatch application (day one to seven of life) of the probiotic Enterococcus faecium AL41 (EF) affects the development and tissue properties of the broiler pectoralis major muscle (PM). Expression of regulators, namely IGF-1, PAX7, and MYF5, was also investigated. At day 1 (n = 6), and days 5, 8, and 12 (n = 10), muscle samples were taken from control and EF supplemented chicks. From day 5 on, myonuclei number per fiber was elevated in EF chicks. Improved capillarization (from day 8), larger myofibers, increased body and PM weights (day 12) were found in the EF group. Part of our findings is explainable by higher intramuscular expression of IGF-1 and lower MYF5 expression in EF birds. In both groups IGF-1 expression decreases with age, thereby increasing the cellular myogenic potential. However, a strong increase in PAX7 expression and more PAX7-positive nuclei were found in EF chicks at day 12. We conclude that EF supplementation improves PM growth and health due to positive effects on bioavailability and fusion capacity of SATC progeny and better tissue perfusion.

MicroRNA-100 Reduced Fetal Bovine Muscle Satellite Cell Myogenesis and Augmented Intramuscular Lipid Deposition by Modulating IGF1R.

Previously, microRNA-100 (miR-100) and its putative mRNA target, insulin-like growth factor receptor-1 (IGF1R) were identified as differentially and inversely expressed in bovine longissimus dorsi (LD) muscles with divergent intramuscular fat (IMF) content by our group. While IGF1R signaling is implicated in myogenesis and muscle lipid metabolism, the underlying regulatory mechanisms are poorly understood. In the present study, we aimed to investigate the regulation of IGF1R by miR-100 during bovine muscle satellite cell (BMSC) myogenesis and lipid deposition. MiR-100 was confirmed to target the IGF1R 3'-untranslated region (3'-UTR) by luciferase reporter assay. Furthermore, expression of miR-100 and IGF1R was reciprocal during BMSC differentiation, suggesting a crosstalk between the two. Correspondingly, miR-100 mimic (agomiR) suppressed the levels of IGF1R, PI3K/AKT pathway signaling, myogenic gene MYOG, muscle structural components MYH7 and MYH8, whereas the inhibitor (antagomiR) had no clear stimulating effects. The IGF1R inhibitor (BMS-754807) curtailed receptor levels and triggered atrophy in muscle myotubes but did not influence miR-100 expression. AgomiR increased oleic acid-induced lipid deposition in BMSC myotubes supporting its involvement in intramuscular fat deposition, while antagomiR had no effect. Moreover, mitochondrial beta-oxidation and long-chain fatty acid synthesis-related genes were modulated by agomiR addition. Our results demonstrate modulatory roles of miR-100 in BMSC development, lipid deposition, and metabolism and suggest a role of miR-100 in marbling characteristics of meat animals and fat oxidation in muscle.

Cellular detection of the chemokine receptor CXCR4 in bovine mammary glands and its distribution and regulation on bovine leukocytes.

Mastitis has a high incidence in dairy cows. Experimental infection with Escherichia coli increased the number of leukocytes in milk and the gene expression of the chemokine receptor CXCR4 in mammary gland tissues. A link between CXCR4 expression and lipopolysaccharide sensing was demonstrated in other species using in vitro models. The receptor that binds the chemokine stomal cell-derived factor 1 might be associated with the inflammatory response in bovine mammary glands. However, studies in cows are rare, and data on the localization of CXCR4 in bovine mammary glands and its distribution in bovine leukocytes are lacking. Fatty acids (FA) affect the inflammatory response. In human peripheral blood monocytes, exposure to conjugated linoleic acids (CLA) decreases the expression of CXCR4, leading to a decreased inflammatory response in these cells. In this study, we analyzed the expression of CXCR4 in the mammary glands of dairy cows by immunohistochemistry (n = 5) and laser capture microdissection followed by qualitative PCR (n = 3). We characterized the surface expression of CXCR4 on bovine leukocytes, including monocyte subpopulations, first by flow cytometry (n = 5) and then confirmed these results by Western blotting (n = 3). Rumen fistulated dairy cows (n = 4; 126 ± 4 d in milk) were fitted with abomasal infusion tubes, arranged in a 4 × 4 Latin square design, and supplemented for 6 wk twice daily with rising doses of FA followed by a 3-wk washout period. Then, CXCR4 expression on leukocytes was analyzed. The cows received a corn-based diet and were supplemented with coconut oil delivering medium-chain FA (38 g/d), linseed-safflower oil mix delivering n-3 FA (EFA, 39 g of linseed oil and 2 g of safflower oil per day), Lutalin (cis-9,trans-11 and trans-10,cis-12 CLA, 5 g/d; BASF), and EFA + CLA. In the bovine mammary gland, the epithelial cells of the lactiferous duct, but not alveolar epithelial cells, showed clear CXCR4 protein and mRNA signals. Among the leukocyte subsets, monocytes displayed the highest percentage of CXCR4-positive cells (87%), whereas circulating neutrophils showed almost no CXCR4 surface expression (3%) but stored the receptor intracellularly. The percentage of CXCR4-positive leukocytes was not affected by the different FA supplements, but FA supplementation reduced the receptor abundance per cell (40% on average). In conclusion, CXCR4 was clearly detected in the lactiferous duct cells of the mammary gland but not in the alveolar epithelial cells. Compared with other leukocytes, bovine monocytes showed the highest signal intensity of CXCR4 on their surface, whereas granulocytes stored CXCR4 intracellularly. Supplementation with all the FA reduced the surface expression of CXCR4 per leukocyte and could therefore potentially affect the inflammatory status associated with the surface expression of CXCR4. The importance of our observations should be verified in cows with mastitis in the future.

Metabogenomic analysis to functionally annotate the regulatory role of long non-coding RNAs in the liver of cows with different nutrient partitioning phenotype.

Long non-coding RNAs (lncRNAs) hold gene regulatory potential, but require substantial further functional annotation in livestock. Applying two metabogenomic approaches by combining transcriptomic and metabolomic analyses, we aimed to identify lncRNAs with potential regulatory function for divergent nutrient partitioning of lactating crossbred cows and to establish metabogenomic interaction networks comprising metabolites, genes and lncRNAs. Through correlation analysis of lncRNA expression with transcriptomic and metabolomic data, we unraveled lncRNAs that have a putative regulatory role in energy and lipid metabolism, the urea and tricarboxylic acid cycles, and gluconeogenesis. Especially FGF21, which correlated with a plentitude of differentially expressed genes, differentially abundant metabolites, as well as lncRNAs, suggested itself as a key metabolic regulator. Notably, lncRNAs in close physical proximity to coding-genes as well as lncRNAs with natural antisense transcripts appear to perform a fine-tuning function in gene expression involved in metabolic pathways associated with different nutrient partitioning phenotypes.

Distinct Roles of Perilipins in the Intramuscular Deposition of Lipids in Glutamine-Supplemented, Low-, and Normal-Birth-Weight Piglets.

Piglets with low birth weight (LBW) usually have reduced muscle mass and increased lipid deposition compared with their normal-birth-weight (NBW) littermates. Supplementation of piglets with amino acids during the first days of life may improve muscle growth and simultaneously alter the intramuscular lipid deposition. The aim of the current study was to investigate the influence of glutamine (Gln) supplementation during the early suckling period on lipid deposition in the longissimus muscle (MLD) and the role of different perilipin (PLIN) family members in this process. Four groups were generated consisting of 72 male LBW piglets and 72 NBW littermates. Piglets were supplemented with either 1 g Gln/kg body weight or an isonitrogenous amount of alanine (Ala) between days post natum (dpn) 1 and 12. Twelve piglets per group were slaughtered at 5, 12, and 26 dpn, and muscle tissue was collected. Perilipins were localized by immunohistochemistry in muscle sections. The mRNA and protein abundances of PLIN family members and related lipases were quantified by quantitative RT-PCR (qPCR) and western blots, respectively. While PLIN1 was localized around lipid droplets in mature and developing adipocytes, PLIN2 was localized at intramyocellular lipid droplets, PLIN3 and 4 at cell membranes of muscle fibers and adipocytes, and PLIN5 in the cytoplasm of undefined cells. The western blot results indicated higher protein abundances of PLIN2, 3, 4, and 5 in LBW piglets (p < 0.05) at 5 dpn compared with their NBW littermates independent of supplementation, while not directly reflecting the mRNA expression levels. The mRNA abundance of PLIN2 was lower while PLIN4 was higher in piglets at 26 dpn in comparison with piglets at 5 dpn (p < 0.01). Relative mRNA expression of LPL and CGI-58 was lowest in piglets at 5 dpn (p < 0.001). However, ATGL mRNA was not influenced by birth weight or supplementation, but the Spearman correlation coefficient analysis revealed close correlations with PLIN2, 4, and 5 mRNA at 5 and 26 dpn (r > 0.5, p < 0.001). The results indicated the importance of birth weight and age for intramuscular lipid deposition and different roles of PLIN family members in this process, but no clear modulating effect of Gln supplementation.

Glutamine supplementation stimulates cell proliferation in skeletal muscle and cultivated myogenic cells of low birth weight piglets.

Muscle growth of low birth weight (LBW) piglets may be improved with adapted nutrition. This study elucidated effects of glutamine (Gln) supplementation on the cellular muscle development of LBW and normal birth weight (NBW) piglets. Male piglets (n = 144) were either supplemented with 1 g Gln/kg body weight or an isonitrogeneous amount of alanine (Ala) between postnatal day 1 and 12 (dpn). Twelve piglets per group were slaughtered at 5, 12 and 26 dpn, one hour after injection with Bromodeoxyuridine (BrdU, 12 mg/kg). Muscle samples were collected and myogenic cells were isolated and cultivated. Expression of muscle growth related genes was quantified with qPCR. Proliferating, BrdU-positive cells in muscle sections were detected with immunohistochemistry indicating different cell types and decreasing proliferation with age. More proliferation was observed in muscle tissue of LBW-GLN than LBW-ALA piglets at 5 dpn, but there was no clear effect of supplementation on related gene expression. Cell culture experiments indicated that Gln could promote cell proliferation in a dose dependent manner, but expression of myogenesis regulatory genes was not altered. Overall, Gln supplementation stimulated cell proliferation in muscle tissue and in vitro in myogenic cell culture, whereas muscle growth regulatory genes were barely altered.

Heat stress during late pregnancy of sows influences offspring longissimus dorsi muscle growth at weaning.

In pregnant sows, heat stress (HS) not only affects sows, but also has long-term effects on offspring growth. However, it is still unclear how HS in pregnant sows influences offspring skeletal muscle development. In this study, 12 sows with similar body conditions were assigned into either a control (CON) or an HS group. The CON sows were housed at 18-22 ℃, and the sows in the HS group were housed at 28-32 ℃ from day 85 to 114 of pregnancy. The results showed that maternal HS decreased the total protein content (P < 0.05) and prolactin level (P < 0.05), yet increased the triglyceride content (P < 0.05) of milk. The piglets of both groups had similar body weight and longissimus dorsi (LD) muscle weight at birth, but body weight (P < 0.05) and LD weight (P < 0.05) was significantly lower at weaning age in the HS group. Increased expression of myostatin (MSTN) (P < 0.05) and its receptor (P < 0.05) in the LD of HS piglets was observed at weaning. The following decreased in HS piglets: expression of serine/threonine-specific protein kinase (P < 0.05), the mammalian target of rapamycin (P < 0.05), and glycogen synthase kinase-3ß (P < 0.05) signal pathway-involved proteins. The results indicated that maternal HS during late pregnancy influenced offspring LD muscle growth via the activated MSTN pathway. This effect may be related to sow's milk composition.

Effects of Oral Glutamine Supplementation on Early Postnatal Muscle Morphology in Low and Normal Birth Weight Piglets.

Adapted nutrition can improve the growth of low birth weight (LBW) piglets. Since maternal milk is thought to provide insufficient glutamine (Gln) for LBW piglets, the current study investigated the influence of Gln supplementation during the early suckling period on development and lipid deposition in skeletal muscle. The weight differences between LBW and normal birth weight (NBW) littermates persisted from birth to slaughter (p < 0.001). However, intramuscular Gln and Ala concentrations were altered in piglets according to the supplementation (p < 0.01). There were larger muscle fibers (p = 0.048) in Gln-supplemented piglets. Capillarization or nuclei number per muscle fiber was not influenced by birth weight (BiW) or Gln supplementation. Abundance of myosin heavy chain (MYH) isoforms was slightly altered by Gln supplementation. LBW piglets had more lipid droplets than NBW piglets at day 5 of life in both muscles (p < 0.01). The differences decreased with age. Adipocyte development increased with age, but was not influenced by BiW or supplementation. The results indicate that BiW differences were accompanied by differences in lipid deposition and muscle fiber structure, suggesting a delayed development in LBW piglets. Supplementation with Gln may support piglets to overcome those disadvantages.

Consequences of Maternal Essential Fatty Acid and Conjugated Linoleic Acid Supplementation on the Development of Calf Muscle and Adipose Tissue.

Common silage and concentrate-based diets in dairy and beef production may deliver insufficient amounts of essential fatty acids (EFA), thereby also reducing conjugated linoleic acids (CLA) in body tissues and milk. An impaired maternal EFA and CLA supply can have an important impact on calf postnatal development. The current study investigates how maternal supplementation with EFA and CLA affects muscle and adipose tissue development in neonatal calves. Holstein cows (n = 40) were abomasaly supplemented with coconut oil (control), CLA or EFA, or both combined during the transition period. Calves were fed their dam's colostrum until slaughter at day 5 of life. Fatty acid composition and tissue morphology were analyzed. In muscle and adipose tissues, EFA, CLA, and metabolites were elevated, indicating the effective transfer of maternally-supplemented FA to the offspring. Muscle fiber types, fiber nuclei, myosin heavy chain isoform distribution, capillarization, and fat cell size of intramuscular and other adipose tissues did not differ among groups. The results confirm that maternal nutrition during the transition period can alter the FA composition of the calf tissues. This could influence the offspring's development and health in the long-term, even though only minor effects were observed in the neonatal calves' tissue morphology.

Indication of Premelanosome Protein (PMEL) Expression Outside of Pigmented Bovine Skin Suggests Functions Beyond Eumelanogenesis.

The premelanosome protein (PMEL) is important for fibril formation within melanosomes during vertebrate melanogenesis. Fibrils form a matrix for pigment deposition within pigmented tissues such as skin and hair. PMEL mutations are known to modulate eumelanic pigmentation in vertebrates. However, in bovines, PMEL mutations were also found to alter pheomelanic pigmentation resulting in coat color dilution. Furthermore, epistatic effects of a mutated PMEL allele were detected in the phenotypic expression of the bovine hair defect "rat-tail syndrome" (RTS) characterized by charcoal coat color and hair deformation. Reports about PMEL gene expression in non-pigmented tissues raised the hypothesis that there may be unknown functions of the PMEL protein beyond eumelanin deposition to PMEL fibrils. In our study, we analysed the PMEL protein expression in pigmented skin and non-pigmented bovine tissues (non-pigmented skin, thyroid gland, rumen, liver, kidney, and adrenal gland cortex). We found that a processed form of the bovine PMEL protein is expressed in pigmented as well as in non-pigmented tissues, which is in line with gene expression data from targeted RT-PCR and whole transcriptome RNAseq analysis. The PMEL protein is located in membranes and within the cytosol of epithelial cells. Based on our data from bovine tissues, we concluded that at least in cattle PMEL potentially has additional, yet unexplored functions, which might contribute to effects of PMEL mutations on pheomelanin coat color dilution and charcoal coat color in RTS animals. However, indication of PMEL protein in unpigmented cells and tissues will require further confirmation in the future, because there have been no confirmed reports before, which had detected bovine PMEL protein with specific antibodies either in pigmented or unpigmented tissue.

Identification and Annotation of Potential Function of Regulatory Antisense Long Non-Coding RNAs Related to Feed Efficiency in Bos taurus Bulls.

Long non-coding RNAs (lncRNAs) can influence transcriptional and translational processes in mammalian cells and are associated with various developmental, physiological and phenotypic conditions. However, they remain poorly understood and annotated in livestock species. We combined phenotypic, metabolomics and liver transcriptomic data of bulls divergent for residual feed intake (RFI) and fat accretion. Based on a project-specific transcriptome annotation for the bovine reference genome ARS-UCD.1.2 and multiple-tissue total RNA sequencing data, we predicted 3590 loci to be lncRNAs. To identify lncRNAs with potential regulatory influence on phenotype and gene expression, we applied the regulatory impact factor algorithm on a functionally prioritized set of loci (n = 4666). Applying the algorithm of partial correlation and information theory, significant and independent pairwise correlations were calculated and co-expression networks were established, including plasma metabolites correlated with lncRNAs. The network hub lncRNAs were assessed for potential cis-actions and subjected to biological pathway enrichment analyses. Our results reveal a prevalence of antisense lncRNAs positively correlated with adjacent protein-coding genes and suggest their participation in mitochondrial function, acute phase response signalling, TCA-cycle, fatty acid ß-oxidation and presumably gluconeogenesis. These antisense lncRNAs indicate a stabilizing function for their cis-correlated genes and a putative regulatory role in gene expression.

Isolation and Identification of Bovine Preadipocytes and Screening of MicroRNAs Associated with Adipogenesis.

The elucidation of the mechanisms of preadipocyte differentiation and fat accumulation in adipocytes is a major work in beef cattle breeding. As important post-transcriptional regulators, microRNAs (miRNAs) take part in cell proliferation, differentiation, apoptosis, and fat metabolism through binding seed sites of targeting mRNAs. The aim of this study was to isolate and identify bovine preadipocytes and screen miRNAs associated with adipogenesis. Bovine preadipocytes were isolated from subcutaneous fatty tissue and induced to differentiate into adipocytes. Verification of preadipocytes and adipocytes was performed by qRT-PCR (real-time quantitative reverse transcription PCR), Oil Red O staining, and immunofluorescence staining. Total RNA was extracted for small RNA sequencing. The sequencing data showed that 131 miRNAs were highly expressed in adipocytes, and 119 miRNAs were highly expressed in preadipocytes. Stem-loop qPCR (stem-loop quantitative real-time PCR) results showed that the expression patterns of 11 miRNAs were consistent with the sequencing results (miR-149-5p, miR-24-3p, miR-199a-5p, miR-33a, etc.). According to KEGG pathway and Gene Ontology (GO) analyses, multiple predicted target genes were associated with lipid metabolism. In summary, this study provides a protocol of isolating bovine preadipocytes and screening various differently expressed miRNAs during preadipocyte differentiation.

Irisin: Still chasing shadows.

OBJECTIVE: Considerable uncertainty remains regarding the veracity of measuring myokine irisin more than seven years after its original description. Unresolved issues include the nature of transcription of the irisin precursor fibronectin type III domain containing 5 (FNDC5) gene across species, the reliability of irisin levels measured with commercial enzyme-linked immunosorbent assays (ELISAs), and the overall validity of the recently published reference values for human serum measured with quantitative mass spectrometry. We utilized multiple species and measures to evaluate the robustness of commonly used reagents and methods for reporting irisin. METHODS: Amplification of cDNA was used to assess the FNDC5 transcript patterns in humans and mice. The specificity and sensitivity of different irisin antibodies were examined via western blotting. Quantification of circulating native irisin was conducted with mass spectrometry using an absolute quantification peptide for irisin. RESULTS: We show that there is a greater transcript diversity of human FNDC5 than currently annotated, but no indication of the expression of transcripts leading to a truncated form of irisin. Available irisin antibodies still bind to patterns of unspecific serum proteins, which compromise reliable measurements of irisin with ELISAs. Absolute quantification of irisin with labeled peptides by mass spectrometry is an advanced method but requires a multi-step sample preparation introducing uncontrollable variations in the measurement. CONCLUSION: Our data represent an explicit warning against measuring circulating irisin using available methods. Measuring irisin is akin to chasing shadows.

Polymorphisms of the ASIP gene and the haplotype are associated with fat deposition traits and fatty acid composition in Chinese Simmental steers.

Unlike specific expression in the skin of wild mice, the agouti signaling protein (ASIP) is expressed widely in the tissue of cattle, including adipose and muscle tissue. Hence, it has been suggested that ASIP plays a role in bovine fat metabolism. An inserted L1-BT element was recently identified upstream of the ASIP locus which led to an ectopic expression of ASIP mRNA in cattle. In this study, we detected the indel of the L1-BT element at g.  - 14 643  nt and three SNPs in introns of the ASIP gene (g.  - 568 A  >  G, g.  - 554 A  >  T, and g. 4805A  >  T) in a Chinese Simmental steer population. The association analysis between variants of ASIP and economic traits showed that the homozygous genotype of L1-BT element insertion, AA genotype of g.  - 568 A  >  G, and AT genotype of g. 4805A  >  T were significantly correlated with carcass and fat-related traits, such as live weight and back fat thickness. Moreover, three haplotypes (H1: AT; H2: AA; H3: GT) were identified by linkage disequilibrium analysis and formed six combined genotypes. Results indicated that Chinese Simmental steers with an H1H2 combined genotype had a higher measured value of fat-deposition-related traits ( p < 0.05 ), including thickness of back fat and percentage of carcass fat coverage, but a lower content of linoleic acid and α -linolenic acid ( p < 0.05 ). Individuals of an H3H3 combination had a lower marbling score, perirenal fat weight, and carcass weight ( p < 0.05 ). This suggests that these three SNPs and two combined haplotypes might be molecular markers for beef cattle breeding selection.

Biological Network Approach for the Identification of Regulatory Long Non-Coding RNAs Associated With Metabolic Efficiency in Cattle.

Background: Genomic regions associated with divergent livestock feed efficiency have been found predominantly outside protein coding sequences. Long non-coding RNAs (lncRNA) can modulate chromatin accessibility, gene expression and act as important metabolic regulators in mammals. By integrating phenotypic, transcriptomic, and metabolomic data with quantitative trait locus data in prioritizing co-expression network analyses, we aimed to identify and functionally characterize lncRNAs with a potential key regulatory role in metabolic efficiency in cattle. Materials and Methods: Crossbred animals (n = 48) of a Charolais x Holstein F2-population were allocated to groups of high or low metabolic efficiency based on residual feed intake in bulls, energy corrected milk in cows and intramuscular fat content in both genders. Tissue samples from jejunum, liver, skeletal muscle and rumen were subjected to global transcriptomic analysis via stranded total RNA sequencing (RNAseq) and blood plasma samples were used for profiling of 640 metabolites. To identify lncRNAs within the indicated tissues, a project-specific transcriptome annotation was established. Subsequently, novel transcripts were categorized for potential lncRNA status, yielding a total of 7,646 predicted lncRNA transcripts belonging to 3,287 loci. A regulatory impact factor approach highlighted 92, 55, 35, and 73 lncRNAs in jejunum, liver, muscle, and rumen, respectively. Their ensuing high regulatory impact factor scores indicated a potential regulatory key function in a gene set comprising loci displaying differential expression, tissue specificity and loci overlapping with quantitative trait locus regions for residual feed intake or milk production. These were subjected to a partial correlation and information theory analysis with the prioritized gene set. Results and Conclusions: Independent, significant and group-specific correlations (|r| > 0.8) were used to build a network for the high and the low metabolic efficiency group resulting in 1,522 and 1,732 nodes, respectively. Eight lncRNAs displayed a particularly high connectivity (>100 nodes). Metabolites and genes from the partial correlation and information theory networks, which each correlated significantly with the respective lncRNA, were included in an enrichment analysis indicating distinct affected pathways for the eight lncRNAs. LncRNAs associated with metabolic efficiency were classified to be functionally involved in hepatic amino acid metabolism and protein synthesis and in calcium signaling and neuronal nitric oxide synthase signaling in skeletal muscle cells.

Retinol binding protein 4 abundance in plasma and tissues is related to body fat deposition in cattle.

Retinol binding protein 4 (RBP4) facilitates the transport of retinol in the body but is also an adipokine and fatty acid transporter. Our study was aimed at investigating the associations between RBP4 abundance and fat deposition in cattle. Blood samples of 246 crossbred bulls were taken at 8 months of age and at slaughter at 18 months of age for the determination of RBP4, hormone levels, and fatty acid composition. Significant correlations between plasma RBP4 abundance at 8 months of age and carcass traits at 18 months of age were detected (e.g., r = 0.3; P < 0.001 to carcass fat). Furthermore, RBP4 abundances in the plasma and subcutaneous fat were higher (P < 0.05) in bulls with increased fat deposition, whereas the liver RBP4 expression was not (P > 0.05). Retinol binding protein 4 was immunohistochemically localized in or close to adipocytes within muscle and adipose tissue and in liver stellate cells but not in hepatocytes. Overall, our results indicate that increased RBP4 levels were associated with increased fat deposition and altered fatty acid composition, but not with altered glucose tolerance, in crossbred bulls. Moreover, our results suggest that adipose-tissue-derived RBP4 may contribute to the circulating RBP4 level.