Identification of co-expression gene networks and regulatory mechanisms related to mineral composition and meat quality in bovine

Meat quality and mineral composition are complex traits genetically regulated. Growing evidence suggests that minerals play a central role in regulatory and physiological functions related to meat tenderness and fat deposition in cattle. Furthermore, miRNAs and genes have been identified as potential regulators of these traits. However, there is a lack of knowledge regarding the interplay among miRNA-gene expression-mineral metabolism in cattle. Thus, to explore regulatory pathways, candidate genes and miRNAs, and their relationships to muscle and mineral metabolism in Nelore steers, we integrated gene and miRNA expression data, eQTLs, mineral content, and meat quality traits based on coexpression networks. To this end, the muscle genome-wide expression profiles of mRNAs (n = 200) and miRNAs (n = 50), obtained by RNA-Seq, were used separately to construct co-expression networks using the WGCNA (Weighted gene co-expression network analysis) R package. Phenotypic data of macro (Ca, K, Mg, Na, P, S) and micro minerals (Co, Cr, Cu, Fe, Mn, Se, Zn), as well as meat quality (intramuscular fat, meat pH, and tenderness) were also integrated to identify gene/miRNAs modules associated to these traits. By clustering 11,996 genes and 343 miRNAs, we identified, based on a linear model, 15 and nine modules, respectively, associated with at least one trait (p < 0.05). We identified 82 potential candidate genes based on the module-phenotype association analysis. From the functional analysis, we identified as over-represented biological pathways related to energy and protein metabolism, such as AMPK, mTOR, insulin, and thyroid hormone. We also integrated the gene and miRNA modules, and pointed out 1,815 unique genes targets of 41 miRNAs. Among the minerals, Ca and Fe were strongly regulated, mainly by the miR-29 family. In addition to the pathways previously mentioned, the target genes functional over-representation analysis highlighted signaling pathways such as hypoxia-inducible factor 1, ferroptosis, and p53. Key genes involved in Fe homeostasis, such as transferrin receptor (TFRC), iron responsive element binding protein 2 (IREB2), and transferrin (TF) were identified, as well as those underlying lipid metabolism. Overall, there is a complex relationship between meat quality and mineral metabolism, as well as the fundamental role of miRNAs regulating target genes. Further studies are needed to investigate the effect of different levels of mineral supplementation in gene expression and meat quality traits.Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES)Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)De caráter complexo, as características de qualidade de carne e composição mineral são reguladas geneticamente. Evidências crescentes sugerem que os minerais têm papel central em funções regulatórias e fisiológicas relacionadas à maciez e deposição de gordura em bovinos. Além disso, miRNAs e genes têm sido apontados como potenciais reguladores desses fenótipos. No entanto, ainda pouco se sabe a respeito dessa interação e seu efeito sobre a qualidade da carne em bovinos. Desse modo, a fim de explorar vias regulatórias, genes e miRNAs candidatos e suas relações com o metabolismo muscular e mineral em novilhos Nelore, nós integramos dados de expressão de genes e miRNAs, eQTLs, conteúdo de minerais e características de qualidade de carne baseados em redes de coexpressão. Para tanto, os perfis de expressão global de mRNAs (n = 200) e miRNAs (n = 50), obtidos por meio de RNA-Seq, foram utilizados separadamente para construir redes de coexpressão utilizando o pacote do R WGCNA (Weighted gene co-expression network analysis). Os dados fenotípicos de macro (Ca, K, Mg, Na, P, S) e micro minerais (Co, Cr, Cu, Fe, Mn, Se, Zn), assim como de qualidade de carne (gordura intramuscular, maciez, e pH da carne) foram também integrados a fim de identificar módulos de genes/miRNAs associados a estas características. Mediante a clusterização de 11.996 genes e 343 miRNAs, nós identificamos, com base em um modelo linear, 15 módulos de genes e nove de miRNAs associados a pelo menos um dos fenótipos (p < 0,05). Nós identificamos 82 genes candidatos potenciais reguladores dos módulos associados aos fenótipos avaliados. A partir da análise funcional, nós identificamos vias biológicas enriquecidas relacionadas ao metabolismo energético e protéico, tais como AMPK, mTOR, insulina e hormônio da tireoide. Nós também integramos os módulos gênicos com os de miRNAs e apontamos 1.815 genes únicos, alvos de 41 miRNAs. Ca e Fe mostraram-se fortemente regulados, principalmente pelos miRNAs da família miR-29. Além das vias previamente apontadas, o enriquecimento funcional dos genes alvo também indicou a participação de vias como o fator 1 induzido por hipóxia, ferroptose e p53. Genes chave envolvidos na homeostase do Fe, tais como receptor da transferrina (TFRC), proteina de ligação do elemento responsivo ao ferro (IREB2) e transferrina (TF) foram identificados, bem como envolvidos com metabolismo de lipídeos. De modo geral, existe uma intricada relação entre qualidade de carne e metabolismo mineral, bem como destaca-se o papel fundamental dos miRNAs regulando os genes alvo. Estudos adicionais são necessários para investigar o efeito de diferentes níveis de suplementação mineral na expressão gênica e na qualidade de carne.FAPESP: 2017/20761-7FAPESP: 2015/09158-1CAPES: Código de Financiamento 00

Expressão gênica diferencial relacionada ao conteúdo de ferro no músculo em animais nelore

Iron (Fe) is an essential micronutrient for cellular homeostasis. Structural component of proteins or enzyme cofactor, Fe has participation in important metabolic pathways that include oxidative metabolism, oxygen transport, cell proliferation and immune system function. Despite of its essentiality, Fe has a toxic potential to cells when in excess. So, a sophisticated system is needed to coordinate the process of absorption, recycling, use and storage. Mutations in genes related to homeostasis of this mineral may potentially alter the cellular distribution and storage. Furthermore, the Fe levels affect biological pathways such as carbohydrate and lipid metabolism. Iron content in cattle muscle has been associated with many sensory and technological parameters of meat quality. However, to date, studies that evaluate how the iron levels in the muscle can alter gene expression and the consequences for the metabolism in cattle are still absent. Therefore, this study aims to identify differentially expressed genes, metabolic pathways, gene interactions and potential regulatory biological mechanisms of physiological processes related to meat quality parameters. Longissimus dorsi (LD) muscle were collected at slaughter for total RNA extraction and determination of CFe by optical emission spectrometry (ICP OES). Eight Nelore steers, who are representatives of extreme value for Genetic Genomic Estimate (GEBV) for iron content (CFe), were selected from a reference population of 373 animals. The sequencing of the total mRNA of extreme animals was carried out from the next generation Illumina technology, which resulted in average l9.13 million of reads per sample after quality control and trimming. Data analysis carried out by Tuxedo Suite pipeline identified 49 annotated and differentially expressed genes (DE) (FDR <0.05) between groups of extremes for GEBV value for CFe. From the DE genes, 18 genes were up-regulated and 31 down-regulated for animals of low GEBV for CFe. Candidate genes for meat quality traits were identified in this study and they are related to transport and lipid metabolism. Other pathways identified through functional enrichment analysis include cell growth and development, function of the hematological system, among others. Canonical signaling pathways (interferon signaling, thyroid receptor activation (TR/RXR) and complement system) and canonical metabolic pathways (biosynthesis of stearate, fatty acid biosynthesis and palmitate biosynthesis) were also identified. Although this study did not identify genes with direct role in the regulation of Fe content, our results suggest biological pathways influenced by this mineral and contribute with information to the understanding of their participation in processes affecting quality of meat. This information will be useful in developing strategies that contribute to the production of better quality meat, healthy and nutritionally rich. In addition, this information may help in understanding of metabolic disorders in other species, including humans.Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)O ferro (Fe) é um micronutriente essencial à homeostase celular. Necessário como componente estrutural de proteínas ou cofator enzimático, o Fe participa de vias metabólicas importantes que incluem metabolismo oxidativo, transporte de oxigênio, proliferação celular e funcionamento do sistema imune. Apesar de essencial, apresenta um potencial tóxico às células quando em excesso. Por isso, é necessário um sofisticado sistema que coordene os processos de absorção, reciclagem, uso e armazenamento. Mutações em genes relacionados à homeostase desse mineral podem potencialmente alterar a sua distribuição e armazenamento celular. Ademais, os níveis de Fe afetam vias biológicas, tais como metabolismo de carboidratos e lipídeos. O conteúdo de Fe no músculo em bovinos tem sido associado a diversos parâmetros sensoriais e tecnológicos de qualidade de carne. Entretanto, até a presente data, são escassos estudos que avaliem como os níveis de ferro no músculo podem alterar a expressão gênica e quais as consequências para o metabolismo em bovinos. Portanto, o presente estudo tem como objetivo principal identificar genes diferencialmente expressos, vias metabólicas, interações gênicas e potenciais mecanismos biológicos que participam de processos fisiológicos relacionados à regulação do ferro e de parâmetros de qualidade da carne. Amostras do músculo Longissimus dorsi (LD) foram coletadas no momento do abate para extração de RNA total e determinação do conteúdo de ferro (CFe) por espectrometria de emissão óptica (ICP OES). Oito machos castrados da raça Nelore, representantes dos extremos para Valor Genético Genômico Estimado (GEBV) para CFe foram selecionados a partir de uma população referência de 373 animais. O equenciamento do mRNA total dos animais extremos foi realizado a partir da tecnologia de nova geração Illumina, o qual resultou em média 9,13 milhões de reads por amostra após o controle de qualidade. Por meio da análise de dados realizada pelo Tuxedo Suíte pipeline foram identificados 49 genes anotados diferencialmente expressos (DE) (FDR <0,05) entre os grupos de extremos para o valor de GEBV para CFe. Dentre os genes DE, 18 genes apresentaram-se up-regulated e 31 down-regulated para os animais do grupo de baixo GEBV para CFe. Genes candidatos para características de qualidade de carne foram identificados no presente estudo e estão relacionados ao transporte e metabolismo de lipídeos. Outras vias identificadas por meio das análises de enriquecimento funcional incluem crescimento e desenvolvimento celular, função do sistema hematológico, entre outras. Vias canônicas de sinalização (sinalização do interferon, ativação do receptor da tireóide (TR/RXR) e sistema complemento) e metabólicas (biossíntese do estearato, biossíntese de ácidos graxos e biossíntese do palmitato) foram também identificadas. Embora o presente estudo não tenha identificado genes com papel direto na regulação do conteúdo de Fe, nossos resultados apontam rotas biológicas influenciadas por esse mineral e contribui com informações para o entendimento da sua participação em vias que afetem a qualidade da carne. Essas informações serão úteis no desenvolvimento de estratégias que contribuam para a produção de carne de qualidade, saudável e nutricionalmente rica. Além disso, essas informações poderão auxiliar no entendimento de distúrbios metabólicos em outras espécies, inclusive a humana

Comparative study of morphometric proportions among Campolina´s stallions and gelded ones

A estreita relação entre as características de exterior e a classificação funcional dos equinos tem contribuído consideravelmente no processo seletivo a partir da identificação de indivíduos morfologicamente uperiores, permitindo a eliminação do processo reprodutivo de indivíduos portadores de caracteres indesejáveis. Objetivou-se avaliar as medidas lineares dos garanhões e castrados registrados no livro definitivo da ABCCCampolina, bem como compará-las e determinar as correlações existentes. Para tal, foram analisadas 15 medidas lineares de 4.837 garanhões e 1.371 castrados a partir da estatística descritiva, correlação de Pearson e análise de variância. De forma geral, as correlações encontradas apresentaram valores maiores para garanhões que para os castrados. Para aqueles a maioria das medidas foram significativamente influenciadas (P<0,01) pelo ano de nascimento, estado do criatório e pelagem, enquanto que para os castrados a pelagem foi a variável de menor influência (P<0,01) sobre as medidas. Destaca-se que a castração não comprometeu o desenvolvimento final dos animais, entretanto a falta de critérios dos produtores no momento da seleção e a preferência pessoal podem contribuir para a fixação de caracteres raciais em detrimento dos funcionais. Isso pode ser evidenciado pelo fato de os machos castrados da raça Campolina mostraram-se, em média, melhores proporcionados quando comparados aos garanhões.In the equine species, the surface characteristics, present a close relationship with its functional classification. The scientific use of linear measurements can contribute decisively in the selective process of many breeds, by identifying the higher morphologically individuals and excluding from reproduction those that are not closed to desirable. The present study objected to evaluate and compare, the linear measures from stallions and gelded horses registered in ABCCCampolina’s CP6 and CP8 books, and to determinate the existing correlations. In order to reach so, 15 linear measures from 4.837 stallions and 1.371 gelded were evaluated from descriptive statistic, Pearson’s correlation and analysis of variance. Generally, higher values of correlations were observed for stallions than to gelded ones. To stallions, most of the measures were significantly influenced (P<0,01) by the year of birth, state of birth and coat color, while to gelded ones the coat color showed lower influence (P<0,01) upon the measures. It’s important to notice that castration did not committed the animal’s final growth, however the breeders non sense the lack of appropriated criteria during the selection process and personal preferences can contribute towards stablishing breed characters rather than functionals. This can be highlighted by the fact that Campolina’s gelded horses showed, in average, better proportioned than stallions.

Iron Content Affects Lipogenic Gene Expression in the Muscle of Nelore Beef Cattle

<div><p>Iron (Fe) is an essential mineral for metabolism and plays a central role in a range of biochemical processes. Therefore, this study aimed to identify differentially expressed (DE) genes and metabolic pathways in <i>Longissimus dorsi</i> (LD) muscle from cattle with divergent iron content, as well as to investigate the likely role of these DE genes in biological processes underlying beef quality parameters. Samples for RNA extraction for sequencing and iron, copper, manganese, and zinc determination were collected from LD muscles at slaughter. Eight Nelore steers, with extreme genomic estimated breeding values for iron content (Fe-GEBV), were selected from a reference population of 373 animals. From the 49 annotated DE genes (FDR<0.05) found between the two groups, 18 were up-regulated and 31 down-regulated for the animals in the low Fe-GEBV group. The functional enrichment analyses identified several biological processes, such as lipid transport and metabolism, and cell growth. Lipid metabolism was the main pathway observed in the analysis of metabolic and canonical signaling pathways for the genes identified as DE, including the genes <i>FASN</i>, <i>FABP4</i>, and <i>THRSP</i>, which are functional candidates for beef quality, suggesting reduced lipogenic activities with lower iron content. Our results indicate metabolic pathways that are partially influenced by iron, contributing to a better understanding of its participation in skeletal muscle physiology.</p></div

DNA methylation may affect beef tenderness through signal transduction in Bos indicus

BackgroundBeef tenderness is a complex trait of economic importance for the beef industry. Understanding the epigenetic mechanisms underlying this trait may help improve the accuracy of breeding programs. However, little is known about epigenetic effects on Bos taurus muscle and their implications in tenderness, and no studies have been conducted in Bos indicus.ResultsComparing methylation profile of Bos indicus skeletal muscle with contrasting beef tenderness at 14&nbsp;days after slaughter, we identified differentially methylated cytosines and regions associated with this trait. Interestingly, muscle that became tender beef had higher levels of hypermethylation compared to the tough group. Enrichment analysis of predicted target genes suggested that differences in methylation between tender and tough beef may affect signal transduction pathways, among which G protein signaling was a key pathway. In addition, different methylation levels were found associated with expression levels of GNAS, PDE4B, EPCAM and EBF3 genes. The differentially methylated elements correlated with EBF3 and GNAS genes overlapped CpG islands and regulatory elements. GNAS, a complex imprinted gene, has a key role on G protein signaling pathways. Moreover, both G protein signaling pathway and the EBF3 gene regulate muscle homeostasis, relaxation, and muscle cell-specificity.ConclusionsWe present differentially methylated loci that may be of interest to decipher the epigenetic mechanisms affecting tenderness. Supported by the previous knowledge about regulatory elements and gene function, the methylation data suggests EBF3 and GNAS as potential candidate genes and G protein signaling as potential candidate pathway associated with beef tenderness via methylation

Ranking of the biological processes based on the terms of Gene Ontology (GO-Slim) for the differentially expressed genes identified in Nelore steers with high and low genomic breeding value for iron content in the <i>Longissimus dorsi</i> muscle.

<p>The bar represents each category of the biological process. The number of genes per category is indicated above the bar.</p

Biological processes significantly enriched by WebGestalt for the differentially expressed genes in the <i>Longissimus dorsi</i> muscle, comparing high and low genomic breeding value groups for iron content in Nelore steers.

<p>Biological processes significantly enriched by WebGestalt for the differentially expressed genes in the <i>Longissimus dorsi</i> muscle, comparing high and low genomic breeding value groups for iron content in Nelore steers.</p

Significantly down-regulated genes identified in the Nelore steers <i>Longissimus dorsi</i> muscle with low genomic breeding value compared to animals with high genomic breeding value for iron content.

<p>Significantly down-regulated genes identified in the Nelore steers <i>Longissimus dorsi</i> muscle with low genomic breeding value compared to animals with high genomic breeding value for iron content.</p