Masaaki Taniguchi

Effect of dietary forage to concentrate ratio on volatile fatty acid absorption and the expression of genes related to volatile fatty acid absorption and metabolism in ruminal tissue.

The objective of the study was to investigate the fractional rate of volatile fatty acid (VFA) absorption and the expression of genes encoding for transporters and enzymes involved in the absorption and metabolism of VFA in ruminal tissue when cattle were fed high or low concentrate diets. Twelve ruminally cannulated Holstein cows were used in a randomized complete block design. The low concentrate (LC) and high concentrate (HC) diets contained 8 and 64% dietary concentrate (dry matter basis), respectively. Cows were fed their respective diet for at least 28 d, following which data and samples were collected over 6 d. Ruminal pH was measured continuously for 72 h, and the in vivo VFA absorption and passage rates were measured using Co-EDTA and n-valeric acid as markers. Ruminal tissue was collected postslaughter from the ventral sac of the rumen, and gene expression was evaluated using quantitative real-time PCR. Dry matter intake was not affected by treatment, averaging 14.9 kg/d, but cows fed HC had lower mean ruminal pH (6.03 vs. 6.48), and a greater duration (376 vs. 10 min/d) that ruminal pH was <5.8. Ruminal VFA concentration was 24 mM higher for cows fed HC compared with LC; however, the fractional rate of VFA absorption and passage from the rumen was not affected by dietary treatment, averaging 23.4 and 9.6%/h, respectively. The expression of genes encoding for enzymes involved in VFA activation and ketogenesis were not affected by treatment. Cows fed HC tended to have a relative abundance of pyruvate dehydrogenase lipoamide alpha 1 mRNA transcripts that was 1.4 times lower than that of cows fed LC, but other enzymes involved in pyruvate metabolism or regulation of the citric acid cycle were not affected. Collectively, these results suggest that the dietary forage to concentrate ratio does not affect the fractional rate of VFA absorption in vivo, but potentially alters energy metabolism in ruminal tissue.

Genotype of bovine sterol regulatory element binding protein-1 (SREBP-1) is associated with fatty acid composition in Japanese Black cattle

To investigate genetic factors that affect fatty acid composition in beef carcass, we previously investigated genetic profiles of stearoyl-CoA desaturase (SCD) and their effect on fatty acid composition in fat tissue of cattle. It has been known that sterol regulatory element binding protein (SREBP) is a transcription factor that regulates gene expression levels of SCD and other genes relevant to lipid and fatty acid metabolism in tissue. Therefore, we determined the full-length sequence of bovine SREBP-1 cDNA and then surveyed polymorphisms in whole exons and introns in the bovine genome. Large 84-bp insertion (long type: L) and deletion (short type: S) were found in intron 5 of bovine SREBP-1 in Japanese Black cattle, although there was no notable mutation in exon regions. The associations between the SREBP-1 genotypes and fatty acid compositions/fat melting points were analyzed by using genomic DNA with carcass trait information from 606 Japanese Black cattle. The S type contributed to 1.3% higher monounsaturated fatty acid (MUFA) proportion and 1.6°C lower melting point in intramuscular fat. Genotyping of bovine SREBP-1 is considered to reflect a genetic variation which is associated with physiologic characteristics of fat tissue in Japanese black cattle.

Association between fatty acid compositions and genotypes of FABP4 and LXR-alpha in Japanese Black cattle

BackgroundFatty acid composition has become an important trait in the beef industry in terms of beef flavor and decreasing the circulating concentration of LDL cholesterol. In this study, we examined the association between polymorphisms of six genes, adipocytes-type fatty acid binding protein (FABP4), liver X receptor α (LXRα), cytochrome b5 (Cyt b5), long-chain acyl-CoA synthetase (ACSL) 1, ACSL4 and diacylglycerol acyltransferase 2 (DGAT2) and fatty acid composition.ResultsSequence comparisons revealed 14 single nucleotide polymorphisms in six genes. Four of them, I74V and V110M in FABP4 and G51E and V133I in LXRα, were nonsynonymous substitutions. The associations between the genotypes and fatty acid compositions were analyzed by using 234 Japanese Black cattle. The genotypes of FABP4 I74V and LXRα V133I were significantly associated with palmitoleic acids (C16:1, P = 0.0086) and linoleic acid (C18:2, P = 0.0121) content in intramuscular fat, respectively.ConclusionOur findings suggest that the two polymorphisms of FABP4 I74V and LXRα V133I might be genetic factors in part associated with palmitoleic acid (FABP4 I74V) and linoleic acid (LXRα V133I) composition in intramuscular fat of Japanese Black cattle, respectively. Especially, FABP4 I74V had highly significant effect (P < 0.01) on C16:1 proportion, indicating that the I/I homozygote exhibited 0.5% higher percentage than V/V homozygote.

Effect of SCD and SREBP genotypes on fatty acid composition in adipose tissue of Japanese Black cattle herds.

Fatty acid composition of beef adipose tissue is one of important traits because high proportions of monounsaturated fatty acid are related to favorable beef flavor and tenderness. In this study, we investigated effects of genetic factors such as stearoyl-CoA desaturase (SCD) and sterol regulatory element binding protein (SREBP) on beef carcass traits including fatty acid composition using two cattle populations. Sire effect was significantly related to almost all traits except BMS, suggesting that the trait examined in this study is highly controlled by genetic factors. The effect of SCD genotype on fatty acid composition was detected remarkably in both cattle groups, especially on stearic and oleic acids. This result was consistent with our previous studies and suggests that SCD is associated with fatty acid composition. Unlike SCD genotyping, the effect of SREBP genotype was not identified in this study. Our results suggested that SCD genotype would contribute to improving beef quality in field populations. Further studies about the relationship among these factors will bring an insight into the molecular mechanism of fatty acid metabolism in cattle.

Effect of DNA polymorphisms related to fatty acid composition in adipose tissue of Holstein cattle

Fatty acid composition of adipose tissue has been recognized as an important carcass trait because of its relationship with eating quality such as favorable beef flavor and tenderness. Therefore, we investigated the effects of genetic polymorphisms of liver X receptor, alpha (LXR), stearoyl-CoA desaturase (SCD), Fatty acid synthase (FASN), and Fatty acid binding protein 4 (FABP4) on fatty acid composition in intramuscular fat tissue of Holstein steers. The major allele frequencies were 0.705 in SCD, 0.518 in FABP4, 0.888 in FASN, and 0.984 in LXR. Genotyping of SCD showed significant effect on C14:0, C14:1, C18:0 and saturated fatty acid (P < 0.05). In addition, the result suggested that SCD genotype possibly had effect on composition of C18:1 and monounsaturated fatty acid. Genotype of FABP4 had significant effect on composition of C16:0. Effect of LXR genotypes could not be analyze because of extremely biased genotype frequencies. Our results suggest that genotypes of SCD and FABP4 may in part affect meat quality in Holstein.

Comparative analysis on gene expression profiles in cattle subcutaneous fat tissues.

Fat related carcass traits are important to the beef industry due to their association with value of the meat. In this study, we attempted to discover the genes that are associated with fat metabolism by identification of differentially expressed genes in subcutaneous adipose tissues of beef steers with different backfat thicknesses. Microarray analysis was performed using a bovine specific oligo-platform containing 8329 probes. In total, 360 differentially expressed genes were identified and their functions were characterized by bioinformatical tools to elucidate molecular pathways. 45 out of 360 differentially expressed genes were found to be involved in 82 KEGG pathways. Validation of 6 selected differentially expressed genes by quantitative real-time PCR revealed correlations between backfat thickness and their expression levels. Our results suggest that expression differences of novel genes and the genes that have been known as genetic markers for fat related traits may be associated with backfat development in beef cattle. Moreover, the gene expression differences were also compared between two cattle crossbreds. The observed different association between the expression of selected genes and breed types suggested that the mechanisms of fat metabolism may differ in response to genotypes.

Comparative analysis of gene expression profiles in ruminal tissue from Holstein dairy cows fed high or low concentrate diets.

Cattle are often fed high concentrate (HC) diets to increase productivity, although HC diets cause changes in ruminal environment such as pH reduction. Despite those well-documented changes in cattle fed HC diets, there is currently a paucity of data describing the molecular events regulating the ruminal environment. Our objective was to gain an understanding of which genes are differentially expressed in ruminal tissue from Holstein cows fed a HC comparing to low concentrate (LC) diet using microarray analysis using a bovine 24 k microarray. A total of 5,200 differentially expressed genes (DEG) were detected for cows fed HC relative to LC. The DEG were firstly annotated with gene ontology (GO) and Kyoto encyclopedia of Genes and Genomes (KEGG), indicating that the DEG were associated with catalytic activity and MAPK pathway, respectively. Further characterization using GeneCodis identified patterns of interrelated annotations for the DEG to elucidate the relationships among annotation groups revealed that a cAMP-dependent protein kinase A catalytic subunit beta (PRKACB), may be associated with ruminal tissue maintenance. The results contributed to understanding of the regulatory mechanisms at the mRNA level for Holstein cows fed at different concentrate ratio diets.

Microarray analysis of differentially expressed genes from Peyer's patches of cattle orally challenged with bovine spongiform encephalopathy.

The most likely route of entry of infection following oral exposure to transmissible spongiform encephalopathies (TSE) is via the immunologically active Peyers patches (PP). These secondary lymphoid organs appear to be the potential route for prion neuroinvasion. However, the molecular mechanisms involved in the uptake of the infectious prion agent and progression of disease remain still unclear. This investigation examined the changes in gene expression in PP following oral exposure of cattle to bovine spongiform encephalopathy (BSE) agents. The gene expression patterns in PP from cows 12 mo after BSE challenge were compared with controls using a microarray platform containing 24,000 oligonucleotides representing 16,846 unique gene loci and 5943 Expressed Sequence Tag (EST) from bovine genome. Between the challanged and control animals, 90 genes and 16 EST were identified as significantly differentially, expressed (>2.0-fold change): 36 were upregulated and 70 were downregulated. Of these genes, five were found to be related to immune function. Major histocompatibility complex (MHC) class II, MHC class II DQ alpha, L-RAP, and two hypothetical proteins. Differentially expressed genes related to cellular and metabolic processes including development and maturation of cells in the PP were also identified. In this context, the potential impacts of these gene expression changes in PP on BSE development are discussed.

Gene Expression in the Medulla Following Oral Infection of Cattle with Bovine Spongiform Encephalopathy

The identification of variations in gene expression in response to bovine spongiform encephalopathy (BSE) may help to elucidate the mechanisms of neuropathology and prion replication and discover biomarkers for disease. In this study, genes that are differentially expressed in the caudal medulla tissues of animals infected with different doses of PrPBSE at 12 and 45 mo post infection were compared using array containing 24,000 oligonucleotide probes. Data analysis identified 966 differentially expressed (DE) genes between control and infected animals. Genes identified in at least two of four experiments (control versus 1-g infected animals at 12 and 45-mo; control versus 100-g infected animals at 12 and 45 mo) were considered to be the genes that may be associated with BSE disease. From the 176 DE genes associated with BSE, 84 had functions described in the Gene Ontology (GO) database. Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analysis of 14 genes revealed that prion infection may cause dysfunction of several different networks, including extracellular matrix (ECM), cell adhesion, neuroactive ligand–receptor interaction, complement and coagulation cascades, MAPK signaling, neurodegenerative disorder, SNARE interactions in vesicular transport, and the transforming growth factor (TGF) beta signaling pathways. The identification of DE genes will contribute to a better understanding of the molecular mechanisms of neuropathology in bovine species. Additional studies on larger number of animals are in progress in our laboratory to investigate the roles of these DE genes in pathogenesis of BSE.

MicroRNA-33b downregulates the differentiation and development of porcine preadipocytes.

Sterol regulatory element binding transcription factor (SREBF) is a key transcription regulator for lipid homeostasis. MicroRNA-33b (miR-33b) is embedded in intron 16 of porcine SREBF1 and is conserved among most mammals. Here, we investigated the effect of miR-33b on adipocyte differentiation and development in porcine subcutaneous pre-adipocytes (PSPA). PSPA were transiently transfected with miR-33b, and adipose differentiation was then induced. Delayed adipose differentiation and decreased lipid accumulation were observed in miR-33b-transfected PSPA. Computational predictions suggested that miR-33b may target early B cell factor 1 (EBF1), an adipocyte activator of lipogenesis regulators such as CCAAT-enhancer binding protein alpha (C/EBPα) and peroxisome proliferator-activated receptor gamma (PPARγ). Both gene and protein expression of EBF1 were downregulated in miR-33b-transfected PSPA, followed by considerable decreases in the expression of C/EBPα and PPARγ and their downstream lipogenic genes. However, miR-33b transfection did not markedly affect mRNA and protein expression of SREBF1. We also investigated differences in the expression of miR-33b and lipogenic genes in subcutaneous fat tissues between 5-month-old crossbred gilts derived from Landrace (lean-type) and Meishan (fatty-type) boars. Landrace-derived crossbred gilts expressed more miR-33b and less lipogenic genes than did gilts derived from Meishan. Our results suggest that miR-33b affected the differentiation and development of PSPA by attenuating the lipogenic gene expression cascade through EBF1 to C/EBPα and PPARγ. The differential expression of miR-33b observed in crossbred gilts may in part account for differences in lipogenic gene expression and the fat:lean ratio between pig breeds.