BACKGROUND
beef is characterized by its high nutritional value, being an important source of protein, essential amino acids, vitamins , and minerals such as iron, zinc and selenium  <cit> .
the fats present in beef are rich in long chain polyunsaturated fatty acid, which participate in several biological processes relevant to human health.
in addition, beef fatty acids  composition plays an important role in the oxidative stability during the cooking process, affecting beef’s tenderness, flavor and juiciness  <cit> .
additionally, beef is a natural source of essential fas, such as linoleic acid and conjugated linoleic acid  isomers, in particular the cis  <dig>  trans  <dig> isomer and oleic acid .
fatty acids type in beef, however, can have detrimental effect on human health if consumed in large quantities due to lipid composition, which is predominantly composed by saturated fatty acids  and has been associated with obesity, cardiovascular diseases and high cholesterol rate  <cit> .
the major factors that influence beef fas composition are animal age, diet, and breed type.
several studies have demonstrated that intramuscular fat from bos indicus breeds is less saturated than those from bos taurus .
in this regard,  <cit>  pointed out that nellore beef is nutritionally healthier than angus beef, since it has lower percentages of cholesterol and higher amounts of ω <dig> fa and cla precursor .
bressan et al.
<cit>  comparing bos taurus and bos indicus animals showed that the production system has an important role on beef’s fas composition.
these authors reported that bos taurus animals had lower percentage of sfa and higher percentage for monounsaturated fatty acids  than bos indicus animals finished in feedlot.
according to them, bos taurus cattle finished under feedlot conditions have higher ability to desaturate sfa than bos indicus cattle.
the intramuscular and subcutaneous adipose tissues are the most important fat deposits associated with meat quality traits in cattle.
the expression level of adipogenic and lipogenic genes in the adipose tissue is regulated by several transcription factors  <cit> .
fatty acid metabolism is a complex process, which includes lipolysis of dietary fat and its biohydrogenation in the rumen, de novo synthesis of fas by rumen bacteria, absorption and transport of fas by the host animal, de novo synthesis in the host’s tissues, elongation and desaturation in the animal’s tissues, hydrolysis of triglycerides and esterification, and the oxidation of fa or its metabolization into other components .
up to date, there are few studies using rna-seq technique to identify differentially expressed genes  associated with intramuscular fas composition in domestic animals.
ramayo-caldas et al.
<cit>  identified deg in the liver of crossbred swine  for groups with extreme values for intramuscular fas composition.
costa et al.
<cit>  used bulls from different genetic groups of alentejana and barrosã breeds with divergent diets, high and low concentration of silage, to identify deg associated with lipid metabolism in subcutaneous adipose tissue and in the longissimus lumborum muscle.
recently,  <cit>  studied the gene expression pattern in taurine cattle finished in different diets with extreme phenotypes for fa profile in the intramuscular fat.
transcriptomic studies should contribute to elucidate the genetic and non-genetic mechanisms that determine beef fas composition in the intramuscular fat.
these studies could also identify genomic regions and metabolic pathways involved in those mechanisms, aiming to improve the biological knowledge associated with beef fas composition.
due to the limited number of studies and the implications of intramuscular fas composition on beef palatability and on human health, it is essential the ongoing study of gene expression for beef fas composition in nellore cattle.
moreover, livestock production in brazil is one of the world’s most important food commerce.
in addition, the brazilian beef production is the second largest in the planet, with 80 % of the herds having the influence of zebu cattle  on its composition  <cit> .
thus, this study aimed to identify deg in nellore cattle finished in feedlot conditions with extreme phenotypes for intramuscular fas composition in longissimus thoracis  muscle by rna-seq technique.
methods
animals and information management
samples were obtained from a total of  <dig> nellore young bulls, sons of six sires, belonged to a capivara farm located in são paulo state, brazil, which participates in the nellore qualitas breeding program.
animals were selected based on growth, finishing and sexual precocity traits.
animals were raised on grazing conditions using brachiaria sp.
and panicum sp.
forages, and free access to mineral salt.
after yearling, the breeding animals were selected and the remaining was kept in feedlots for a period of 90 days.
the diet was based on whole-plant silage and mix of sorghum grain, soybean meal or sunflower seeds were used as concentrate, with a concentrate/roughage ratio from 50/ <dig> to 70/ <dig>
animals were slaughtered with an average age of 24 months and 550 kg of liveweight in commercial slaughterhouses, in accordance with the brazilian federal inspection service procedures.
after 48 h post mortem at 0–2 °c, the samples were removed from the longissimus thoracis muscle , from between the 12–13th ribs from each animal .
samples were placed in airtight plastic bags and stored at −80 °c for the analyses described below.
extraction of lipids
the total lipid concentration was quantified at the animal product technology laboratory in the technology department of fcav/unesp according to the method described by  <cit> .
raw and ground meat samples from longissimus thoracis muscle with approximate  <dig>  g were transferred into a 250 ml erlenmeyer flask, where 10 ml of chloroform, 20 ml of methanol and 8 ml of distilled water was added.
after homogenizing the samples with glass rods, the flasks were placed on a horizontal shaker table  for 30 min.
later, 10 ml of chloroform and 10 ml of a  <dig>  % aqueous sodium sulfate solution were added and the samples were shaken for more two minutes, transferred to 50 ml falcon tubes and then centrifuged at  <dig>  × g for two minutes at room temperature.
after centrifugation, the supernatant was discarded and the remainder was passed through filter paper to separate the meat fragments from the solution that contained the extracted lipids.
the samples were filtered into 25 ml measuring cylinders.
the filtrate value was kept to be used in the total lipid calculation and 5 ml was transferred to a 50 ml pre-weighed beaker, oven-dried, cooled in a desiccator for at least 24 h, placed in an oven at 110 °c until complete solvent evaporation, cooled in a desiccator  and weighed once again.
differences in the initial weight of the beaker  and final weight  were used to determine the total lipid concentration of samples.
fatty acids composition
fatty acid composition was determined for each sample using the extraction method described by  <cit> .
muscle samples  were collected and grounded for fas composition.
the lipids were extracted by homogenizing the sample with a chloroform and methanol  solution.
nacl at  <dig>  % was added and so that the lipids were isolated.
the isolated lipids were methylated and the methyl esters were formed according to  <cit> .
the fas composition was quantified using a gas chromatography  with a sp- <dig> capillary column .
the initiating temperature was 70 °c with gradual warming  up to 175 °c, holding for 27 min, and later a further increase of 4 °c/min until 215 °c was reached and held for 31 min.
the fas were identified by comparison of retention time of methyl esters of the samples with standards of c4–c <dig> , vaccenic acid c18: <dig> trans- <dig>  c18: <dig> trans- <dig> cis- <dig> , cla e c18: <dig> cis- <dig>  trans- <dig> ,  and tricosanoic acid .
the fas were quantified by normalizing the area under the curve of methyl esters using software gs solution  <dig> .
the fas were expressed in percentage of total fa methyl ester.
the fa composition in meat was performed at the meat science laboratory  in the department of animal nutrition and production at fmvz/usp.
based on the identified acids,  <dig> fas  were selected due to their importance in human health.
the following fas were determined: myristic , palmitic , stearic , myristoleic , oleic , linoleic , conjugated linoleic acid  c18: <dig> cis <dig> trans <dig>  alfa linolenic , sum of saturated fatty acid , sum of mufa , sum of polyunsaturated fatty acid , ratio between pufa and sfa , sum of ω <dig> , ω <dig>  and ratio between ω <dig> and ω <dig>
rna-seq quantification
total rna was extracted for each sample with trizol® reagent  from 100 mg of frozen lt muscle.
rna integrity was verified by agilent  <dig> bioanalyzer® , where only samples with rin >  <dig> were used.
a total of 2 μg of rna from each sample was used for library preparation according to the protocol described in truseq rna sample preparation kit® v <dig> guide .
the resultant libraries were quantified using a kapa library quantification kit® , according to illumina's library quantification protocol.
finally, libraries were pooled  to perform multiplexing sequencing process, which adds an individual barcode sequences to each sample allowing that each one can be distinguished and analyzed separately during the data analysis.
six lanes of a sequencing flowcell, using the truseq pe cluster kit v3-cbot-hs kit , were clustering and sequenced using hiseq  with a truseq sbs v3-hs kit , according to manufacturer’s instructions.
paired-end reads of 2 × 100 bp were produced.
the sequencing analyses were performed at the genome center at esalq, piracicaba, são paulo, brazil.
alignment of sequence reads and transcript assembly
the sequencing data for each sample generated by hiseq system platform was converted to fastq format, and separated by libraries  through casava software available at https://support.illumina.com/sequencing/sequencing_software/casava.html.
the tuxedo pipeline  <cit> , which includes fastqc , tophat <dig>  and cuffdiff  program were performed in this transcriptomic study using the iplant collaborative platform  <cit> .
the fastqc program was used to analyze the sequencing data quality, subsequently, the tophat <dig> package was performed to align the reads against the bos taurus virtual transcriptome internally built by tophat using the umd <dig>  reference genome, containing  <dig>  genes.
this program was also used to identify the splice junctions of exons transcripts showing the potential exons.
for each library, a file was generated with extension ".bam" containing the aligned reads in relation to the reference genome.
cufflinks  was used to assemble the aligned read for each sample individually, providing a parsimonious set of transcripts and to estimate transcript abundances in fpkm  which normalizes transcript expression for transcript length and the total number of sequence reads per sample.
deg analysis and functional enrichment
fatty acid concentration was classified into two extreme phenotype values groups  to identify deg for each fa in  <dig> samples.
ten animals or biological replicates composed each fa concentration group.
different animals composed those two groups for each beef fa, since the same animal was not necessary extreme for different beef fa.
cuffdiff program included in tuxedo pipeline performed differential expression analysis.
the false discovery rate  threshold used in this analysis was 10 %.
database for annotation, visualization, and integrated discovery  v <dig>   <cit>  was used for functional enrichment analyses using the list of deg for each fa and the bos taurus annotation file as background.
RESULTS
phenotypic variation between groups
the descriptive statistics and the analysis of variance for the fa concentration  for high and low groups are described in table  <dig>  the coefficient of variation ranged from  <dig>  to  <dig>  %, indicating a high homogeneity within each group.
there were significant differences  between the high and low groups for the concentration of all beef fas measured .table  <dig> descriptive statistics and analysis of variance for the fatty acidsa for groups of animals with extreme phenotypes
athe concentration of fatty acids are expressed as a percentage of total fatty acid methyl esters ; blow group: ten lowest extreme phenotypes; chigh group: ten highest extreme phenotypes
throughput sequencing, read mapping and assembly
the table  <dig> presents the sequencing throughput and mapping statistics for each high and low groups.
the sequence quality was assessed through the distribution of transcript abundance for each fa and gene expressed as a box-plot of the log of fragments per kilobase of exon per million fragments mapped  values .
for each fa, similar median and quartiles values for fpkm estimates were obtained between the high and low groups.table  <dig> average number of pair-end reads, number of mapped reads and concordant pair alignment rate  for high and lowa groups for each beef fatty acid
ahigh group: ten highest extreme phenotypes; blow group: ten lowest extreme phenotypes
the principal component analyses of fpkm values for all genes indicated that there were sufficient number of deg to differentiate high and low groups for most of the fas .
moreover, the expression profiles of selected housekeeping genes were evaluated, such as the hypoxanthine phosphoribosyltransferase  <dig>  and tyrosine 3-monooxygenase /tryptophan 5-monooxygenase activation protein, zeta .
for both genes, the expression patterns were similar between high and low groups for all beef fas evaluated.
differential expression genes
differential expression analysis between the high and low groups identified  <dig> deg for c14: <dig>   <dig> for c16: <dig>   <dig> for c18: <dig>   <dig> for c18: <dig> cis- <dig>   <dig> for the c18: <dig> cis- <dig> trans- <dig> ,  <dig> for c18: <dig> cis- <dig> cis − <dig> n <dig> and  <dig> for c18: <dig> n <dig> fa.
for the respective sums of the individual fas,  <dig> deg for sfa,  <dig> for mufa,  <dig> for pufa,  <dig> for ω <dig>  and  <dig> for ω <dig> were identified.
for pufa/sfa and ω6/ ω <dig> ratio,  <dig> and  <dig> deg were identified, respectively.
the list of the deg identified between groups with different fas composition is described in table  <dig>  .table  <dig> description of differentially expressed genes identified between groups of nellore bulls with different fatty acid composition in longissimus thoracis muscle
fabp7
loc782922
slc27a6
acat1
pafah2
scd5_bovin
tnxb
ptgs1
slc1a4
cd74
fabp3
gpam
mgll
lipe
acox2
ppara
ppard
ptpla
plod2
acsm1
adipoq
fabp4
dgat2
c16:0
<dig>
<dig>
slc16a7
c18:0
<dig>
<dig>
ucp3
mufa
<dig>
<dig>
lbp
c16:0
<dig>
<dig>
lpl
c18:1cis-9
pufa/sfa
− <dig>
− <dig>
<dig>
<dig>
anxa1
c18:1cis-9
mufa
pufa
− <dig>
− <dig>
<dig>
<dig>
<dig>
<dig>
acsm3
mufa
pufa
sfa
<dig>
− <dig>
− <dig>
<dig>
<dig>
<dig>
acss1
c16:0
c18:0
c18:1cis-9
sfa
<dig>
<dig>
− <dig>
<dig>
<dig>
<dig>
<dig>
<dig>
cpe
c16:0
c18:1cis-9
sfa
mufa
<dig>
− <dig>
<dig>
− <dig>
<dig>
<dig>
<dig>
<dig>
bdh1
c16:0
c18:0
sfa
c18:2cis-9cis-12
mufa
<dig>
<dig>
<dig>
− <dig>
− <dig>
<dig>
<dig>
<dig>
<dig>
<dig>
the acsm <dig>  gene, that was differentially expressed for linoleic, mufa, pufa, sfa and ω <dig> acids, participates in the metabolism of lipids and in metabolic pathways that involves the precursor acetyl-coa metabolism .
following the process of fas synthesis, the acss <dig>  gene acts in the transformation of acetyl-coa into fas through chemical reactions and metabolic pathways involving acetyl-coa .
this gene was differentially expressed , upregulated for sfa such as palmitic, stearic, oleic and sfa sum, and downregulated for unsaturated acids, such as ω <dig> fig.
<dig> metabolic pathways of acsm <dig> gene   <cit>
fig.
<dig> metabolic pathways of acss <dig> gene   <cit>
other important deg  identified was the slc16a <dig> , member 7), which was upregulated for sfa sum, myristic and stearic acids, and downregulated for ω <dig>  this gene is responsible for catalyzing the transfer of monocarboxylic acids from one cell to another.
other genes such as anxa <dig> , upregulated for stearic acids, ω <dig> and pufa sum, and downregulated for oleic and mufa; and the lbp  gene upregulated for the myristic, palmitic and sfa sum, are also responsible for the transport of fas between cells through pores of a carrier or agent.
the slc27a <dig> , member 6) gene was upregulated for sfa and acts as a carrier of fas.
the acsm <dig> gene was downregulated for ω <dig> and ω <dig>  and it participates directly in fas synthesis.
this gene is responsible for the chemical reactions and metabolic pathways that involve fas and aliphatic monocarboxylic acids of open chain that are naturally released by hydrolysis of the fats and oils.
the gpam  and ucp <dig>  genes have similar functions, in which the first one was found upregulated for oleic acid and mufa and downregulated for ω <dig>  while the second gene was downregulated for pufa/sfa ratio.
the stearoyl-coa desaturase is an integral membrane protein of the endoplasmic reticulum, which catalyzes the synthesis of mufa from sfa, which can also be a key regulator of energy metabolism.
the adipoq , plod <dig> , and lpl  genes were differentially expressed for several fas, and participate in several processes related to fas synthesis.
in this sense, the adipoq gene was downregulated for ω <dig> and ω <dig>  this gene participates directly in the metabolic pathways related to fas production, such lipids and organic acids and is also involved in the regulation of cellular ketone metabolic process  and in fas oxidation and beta-oxidation.
the plod <dig> gene acts in the binding of carboxylic acids and in other organic acid containing one or more carboxyl group  or anions  and was upregulated for linolenic fas and pufa sum, indicating that its expression may promote the synthesis of pufa.
finally, the lpl gene was differentially expressed for palmitic and oleic acids, ω <dig> and pufa/sfa ratio.
there was downregulated expression of all unsaturated fas, indicating that high expression of this gene is associated with a low concentration of these acids in the samples analyzed.
the loc <dig>  gene was downregulated for mufa, which can act in the metabolism of prostaglandins and participates in the chemical reactions and metabolic pathways of unsaturated fas synthesis or other fas containing one or more double bonds between the carbon atoms.
the cpe  gene is responsible for insulin synthesis through proteolysis of its precursor , which was upregulated for c14: <dig>  c16: <dig>  c18: <dig> and sfa sum, and downregulated for c18: <dig> cis- <dig>  mufa and ω <dig>  while the bdh <dig>  gene was differentially expressed  for c14: <dig>  c16: <dig>  c18: <dig>  ω <dig>  mufa and sfa sums.
functional analysis
gene ontology  and pathway enrichment analysis were perfomed to gain insight into the predicted gene network.
the most significant go terms were focused on cellular components, molecular functions and biological processes .
molecular functions controlling fas metabolism are highly interconnected and linked with related pathways, such as lipid, carbohydrate metabolism and energy homeostasis pathway.
the essential metabolic network for homeostatic control and organism development is constituted by these pathways and its interactions  <cit> .
in this study, molecular functions related to recognize  glycosaminoglycan, polysaccharide and carbohydrate molecules were identified .table  <dig> gene ontology  terms enriched with differentially expressed genes
p
cellular components
molecular functions
biological process
anumber of differentially expressed genes
the biologicals processes identified are related mainly with extracellular structure and organization, response to wounding, inflammatory response, embryonic development, skeletal and muscle developments .
four kegg  pathways were identified over represented for deg by david tool.
these pathways were related with ecm-receptor interaction , focal adhesion , ppar signaling pathway , and tgf-beta signaling pathway .
discussion
phenotypic variation between groups
evaluating longissimus muscle of bos indicus,  <cit>  observed similar concentrations of sfa as described in this study, and different for the pufa, mufa, pufa/sfa, ω <dig>  ω <dig> and ω6/ω <dig> ratio.
cesar et al.
<cit>  identified genomic regions associated with fas composition and fat deposition in nellore steers and found concentrations of fas that are close to the average obtained for the high groups in the present study.
differential expression genes
in ruminants, the fas synthesis occurs mainly in the adipose tissue, except during the lactation, when the mammary gland becomes the predominant organ  <cit> .
the main point about fas synthesis control is the acetyl-coa carboxylase, and it seems that the endocrine control is very similar in, at least, adipose tissue  of ruminants and non-ruminants  <cit> .
acetate is the principal precursor of fas synthesis in ruminants, and must be converted to acetyl-coa by the action of acetyl-coa synthetase and then incorporated into fas.
the adipose tissue is largely responsible for the conversion of acetate into acetyl-coa, and consequently, the greatest synthesizer of fas in ruminants  <cit> .
from the results obtained in this study, it was possible to highlight some important genes related to biologicals processes involved in beef’s fas synthesis, such as those involved in the transport of essential components in animal tissues.
the myristic and palmitic fas are considered to be hypercholesterolemic, and are responsible for increasing the amount of low density lipoproteins , which expand the risk of heart diseases  <cit> .
other genes that also operate in the transport of fas were identified, but the deg were only for a single fa, such as fabp <dig>  and fabp <dig>  genes, which appeared upregulated for linolenic acid and pufa/sfa ratio, respectively.
these genes produce proteins that apparently play a role in intracellular transport of long chain fas and their acyl-coa esters.
the intracellular fas binding proteins  belong to a multigene family.
the fabps are divided into at least three distinct groups: hepatic, intestinal, and cardiac.
these form 14–15 kda protein and participate in the absorption, metabolism and/or intracellular transport of long chain fas, and may also be responsible for growth modulation and cell proliferation .
regarding the fabp <dig>  gene, it works in fas binding proteins and it was downregulated for ω <dig> and ω <dig>  the fabps are often associated with lipid metabolism by acting as intracellular transport of hydrophobic intermediates and lipids metabolites trough the membranes.
the pafah <dig> gene, downregulated for oleic acid, participates in chemical reactions and pathways that break lipids.
the mgll  gene, downregulated for pufa/sfa ratio, operates in the chemical reactions for lipids synthesis and acts as a catalyst in fas synthesis reactions.
our results showed some genes directly associated with fas synthesis.
in this sense, the dgat <dig>  gene, upregulated for palmitic and downregulated for linoleic acid and pufa/sfa ratio, is essential for the triglycerides synthesis and intracellular storage  <cit>  found negative correlations between marbling and concentrations of stearic, linoleic acid, and pufa  <cit>  reported a positive and moderate correlation between the level of marbling and the expression of dgat <dig> gene.
the dgat <dig> gene is an important contributor to the triacylglycerol synthesis through their acyltransferase activity.
as the amount of triglyceride within the adipocyte increases, the total proportion of sfa also increases in relation to other ones  <cit> .
an increase in dgat <dig> gene expression was previously demonstrated to be associated with an increase in the amount of intramuscular fat  <cit> .
thus, these results demonstrate that dgat <dig> gene contributed to the accumulation of sfa in the intramuscular tissue during the finishing phase .fig.
<dig> metabolic pathway of dgat <dig>  and lpl  genes  <cit>
the lpl gene plays a role in chemical reactions and metabolic pathways that result in fas synthesis and open-chain monocarboxylic acids, which can be released by hydrolysis that occur in fats and oils.
the activity of this gene in the adipose tissue and the subsequent increase in deposition of triglycerides are promoted by insulin  <cit> .
some studies have suggested that the fas synthesis in the subcutaneous adipose tissue on beef is not sensitive to insulin levels  <cit> .
in this sense, the activity of lpl gene in the muscle tissue appears not to be insulin dependent  <cit> , however, many authors have provided evidence to support the opposite  <cit> .
the bdh <dig> gene was upregulated for sfa and downregulated for unsaturated fas, indicating a higher gene expression as the degree of saturation of the sample is increased.
this gene is responsible for the synthesis and degradation of ketone bodies, which allows transporting the energy obtained by the oxidation of fas to the peripheral tissues, then to be used in the atp synthesis in the absence of carbohydrates in the diet .
this fact justifies the greatest expression of this gene in the presence of sfa for the synthesis of ketone bodies from sfa, since it is less complex when compared to the degradation of unsaturated fas.
the acat <dig>  gene has the same function of bdh <dig> gene, and was upregulated for ω <dig> .fig.
<dig> metabolic pathway for bdh <dig>  and acat <dig>  genes  <cit>
the results showed an evident antagonism for the expression of some deg related to fas synthesis.
in this sense, some of those genes that were upregulated for sfa group were also downregulated for mufa and pufa groups.
animals with high degree of fatness or deposition of intramuscular fat in lt muscle presented higher concentrations of sfa and less concentrations of pufa.
de smet et al.
<cit>  and wood et al.
<cit>  showed that when the proportion of animal fat increases, the proportion of pufa in meat decreases drastically.
ruminants incorporate essential fas, especially phospholipids, in muscle lipids rather than storing them in fat  <cit> .
recently, more emphasis has been dedicated on muscle due to its importance as a protein source and as a growing aversion to visible fat at retail.
the muscle also contains higher concentrations of ω <dig> and ω <dig> acids, whose importance to human nutrition have been recently recognized.
in recent years, the procedures for separation and identification of low levels of unsaturated fas in muscle have been greatly improved  <cit> .
thus, the development of molecular genetics, particularly high throughput sequencing methods, provides a unique opportunity to identify genes and pathways associated with diseases and complex traits  <cit> .
however, recent studies revealed a limitation of genome-wide association study  to identify loci with significant effects on different populations of the same breed, since many discordant genomic regions have been identified  <cit> .
tizioto et al.
<cit>  observed deg using rna-seq method and also applied gwas to identify genomic regions for feed efficiency in nellore cattle.
these authors found several biological mechanisms and attributed the differences in the candidate regions/genes to the specific modulation of mrna.
in a recent gwas study,  <cit>  used a population with  <dig> nellore bulls, which contained  <dig> animals used in this study, and identified several genomic regions which explained more than 1 % of the additive genetic variance for beef fas composition.
the authors reported some regions near to deg identified in this study.
in this regard, the acsm <dig> and acsm <dig> genes were differentially expressed for ω <dig> acids and were located in a region  which explained more than 1 % of the additive variance genetic for ω <dig>  as reported by  <cit> .
those authors also reported qtl detected by gwas in the same region that the tnxb  and ppard  genes were differentially expressed in this study.
these genes are related with fas metabolism, transport and oxidation.
these results showed some degree of equivalence, since it was identified common regions between the results of structural analysis  and functional analysis  for beef fas composition.
the results obtained in this study indicated that beef’s fas composition in nellore cattle is influenced by many genes and complex metabolic pathways.
furthermore, it identified genes that contribute to fas metabolism, through intra or extra-cellular transport of fas synthesis precursors in intramuscular fat of longissimus muscle.
among those genes, some of them must be highlighted, such as: acsm <dig> and acss <dig> genes, since they work in the fas precursor synthesis and their subsequent transformation into fas, respectively.
in addition, the dgat <dig> gene that assists the deposition of saturated fat tissue; gpp and lpl genes that support the insulin synthesis, which stimulates the glucose synthesis as the amino acids entry into the cells; and the bdh <dig> gene which is responsible for the synthesis and degradation of ketone bodies, used in the atp synthesis.
functional analysis
several gde related to biological processes associated with inflammatory response were identified in the present study.
in these sense, stated that the fatty acids influenced inflammatory response acting via cell surface and intracellular receptors/sensors that control gene expression patterns and inflammatory cell signaling.
some effects of fas on inflammatory cells appear to be mediated or associated with changes in fas composition of cell membranes  <cit> .
extracellular matrix  consists of a complex of structural and functional macromolecules with an important role in tissue, organ morphogenesis, cell maintenance, and tissue structure and function.
it can directly or indirectly influence specific cellular activities such as cell adhesion, proliferation, differentiation, and migration  <cit> .
in muscle tissue, cells are tightly bound together, and the extracellular spaces containing the extracellular matrix are limited.
these results corroborates with previous study of transcriptome profile of nellore steers with different genomic breeding value of intramuscular fat deposition  <cit> .
jiang et al.
<cit>  studying transcriptome comparison between porcine subcutaneous and intramuscular stromal vascular cells during adipogenic differentiation speculated that the ecm-receptor interaction pathway might participate in intramuscular stromal vascular cell differentiation process.
lee et al.
<cit>  studied the difference of the depot specific gene expression from different adipose tissues of omental, subcutaneous and intramuscular tissues in cattle, and identified the ecm-receptor interaction with the one of commonly enriched pathways in all adipose tissues and also functioned as a sub-pathway of other enriched pathways.
these authors suggested that the interactions between ecm components and transmembrane receptors of fat cells depend on the depot specific adipogenesis.
the most overexpressed genes identified in this study related to muscle and skeletal developments could be good candidates for nellore breeding programs in which the main goal is to enhance meat and carcass quality.
studying a hanwoo beef cattle population,  <cit>  identified pathways related to cell adhesion regulation, structure, integrity, and chemokine signaling pathway upregulated in intramuscular adipose but downregulated in the muscle.
cui et al.
<cit>  also proposed that these pathways play an important role in the intramuscular fat deposition in chicken.
cánovas et al.
<cit>  identified the ecm-receptor interaction and tgf-beta signaling pathways as the most relevant metabolic pathways represented in the list of deg related with meat composition in pig longissimus dorsi muscle.
the transforming growth factor tgf-beta signaling pathway is involved in many cellular processes including cell growth, differentiation and apoptosis, cellular homeostasis in both the adult organism and in the developing embryo.
mehla et al.
<cit>  identified the tgf-beta signaling pathway related to deg genes in zebu cattle due to heat stress effects.
peroxisome proliferator-activated receptors  are nuclear hormone receptors that are activated by fas and their derivatives, and play an essential physiological role in the regulation of adipocyte tissue development lipogenesis and skeletal muscle lipid metabolism .
there are three members of the ppar family  with different expression patterns in vertebrates.
ppar alpha plays a role in lipid metabolism in the liver and in the skeletal muscle, and in the modulation of the inflammatory response.
ppar beta/delta is involved in lipid oxidation and cell proliferation, and acts on embryo implantation, cell proliferation and apoptosis.
ppar gamma is related to cell cycle withdrawal and promotes myocyte/adipocyte differentiation to enhance blood glucose uptake .
doran et al.
<cit>  studying gwas in holstein-friesan cattle identified the ppar signaling pathway as the most significantly overrepresented biological pathway involved in carcass trait performance, suggesting that ppar would also play a key role in controlling carcass weight, carcass fat and carcass conformation traits.
he et al.
<cit>  identified an association between genes and snps in the ppar signaling pathway with porcine meat quality traits.
carcass and meat traits, especially those obtained through beef fas composition of intramuscular fat analyses, are not used by the industry as a criterion for determining the animal’s value for slaughter.
however, there is a growing trend in the international meat market to provide technical and scientific guarantees to certify food safety, product quality and its benefits to human health.
therefore, the production of the required information is essential and needed to improve the marketing of beef products.
there are few studies about transcriptome in zebu animals, in which  <cit>  is the unprecedented as it is the first study of gene expression for beef feed efficiency in bos indicus animals.
thus, it provides subsidies to improve the beef quality of zebu cattle under tropical conditions, producing a healthier food for consumers.
CONCLUSIONS
several genes related to lipids metabolism and beef fas composition were found in this study.
the identification of such candidate genes must contribute to the elucidation of the genetic basis that determines the beef fas composition of intramuscular fat in nellore cattle.
this information would contribute to the improvement of meat quality through selection processes, since the molecular processes that control fas composition and metabolism are not completely understood yet.
moreover, the deg identified can be used in future studies of fine mapping whose primary function is to search for functional mutations and can be useful to identify some specific variants.
additional files
additional file 1: boxplot of the transcript abundance distribution for each beef fatty acid and gene expressed.
additional file 2: principal component analysis  of gene fpkm values between the high and low groups with fatty acids profile:  pca for sum of sfa;  pca for sum of mufa;  pca for sum of pufa;  pca for sum of pufa/sfa ratio;  pca for sum of n3;.
pca for sum of n6;  pca for n6/n <dig> ratio; pca for myristic acid ;  pca for palmitic acid ;  pca for stearic acid ; pca for oleic acid ;  pca for conjugated linoleic acid ;  pca for linoleic acid ;  pca for α-linolenic .
additional file 3: table s <dig>  differentially expressed genes for c14: <dig>  table s <dig>  differentially expressed genes for c16: <dig>  table s <dig>  differentially expressed genes for c18: <dig>  table s <dig>  differentially expressed genes for c18: <dig> cis- <dig>  table s <dig> differentially expressed genes for c18: <dig> cis-9trans- <dig>  table s <dig>  differentially expressed genes for c18: <dig> cis- <dig> cis - <dig> n <dig>  table s <dig>  differentially expressed genes for c18: <dig> n <dig>  table s <dig>  differentially expressed genes for sfa.
table s <dig>  differentially expressed genes for mufa.
table s <dig>  differentially expressed genes for pufa.
table s <dig>  differentially expressed genes for relationship between pufa and sfa.
table s <dig>  differentially expressed genes.
table s <dig>  differentially expressed genes for n <dig>  table s <dig>  differentially expressed genes for the ratio of ω6/ω <dig>
abbreviations
claconjugated linoleic acid
degdifferentially expressed genes
fafatty acids
gogene ontology
highthe group with highest phenotype
lowthe group with lowest phenotype
mufamonounsaturated fatty acids
pufapolyunsaturated fatty acids
sfasaturated fatty acids
ω3omega 3
ω6omega 6

 beef is characterized by its high nutritional value, being an important source of protein, essential amino acids, vitamins, and minerals such as iron, zinc and selenium. the fats present in beef are rich in long chain polyunsaturated fatty acid, which participate in several biological processes relevant to human health. in addition, beef fatty acids composition plays an important role in the oxidative stability during the cooking process, affecting beef’s tenderness, flavor and juiciness. additionally, beef is a natural source of essential fas, such as linoleic acid and conjugated linoleic acid isomers, in particular the cis trans isomer and oleic acid. fatty acids type in beef, however, can have detrimental effect on human health if consumed in large quantities due to lipid composition, which is predominantly composed by saturated fatty acids and has been associated with obesity, cardiovascular diseases and high cholesterol rate. the major factors that influence beef fas composition are animal age, diet, and breed type. several studies have demonstrated that intramuscular fat from bos indicus breeds is less saturated than those from bos taurus. in this regard, pointed out that nellore beef is nutritionally healthier than angus beef, since it has lower percentages of cholesterol and higher amounts of ω fa and cla precursor. bressan et al. comparing bos taurus and bos indicus animals showed that the production system has an important role on beef’s fas composition. these authors reported that bos taurus animals had lower percentage of sfa and higher percentage for monounsaturated fatty acids than bos indicus animals finished in feedlot. according to them, bos taurus cattle finished under feedlot conditions have higher ability to desaturate sfa than bos indicus cattle. the intramuscular and subcutaneous adipose tissues are the most important fat deposits associated with meat quality traits in cattle. the expression level of adipogenic and lipogenic genes in the adipose tissue is regulated by several transcription factors. fatty acid metabolism is a complex process, which includes lipolysis of dietary fat and its biohydrogenation in the rumen, de novo synthesis of fas by rumen bacteria, absorption and transport of fas by the host animal, de novo synthesis in the host’s tissues, elongation and desaturation in the animal’s tissues, hydrolysis of triglycerides and esterification, and the oxidation of fa or its metabolization into other components. up to date, there are few studies using rna-seq technique to identify differentially expressed genes associated with intramuscular fas composition in domestic animals. ramayo-caldas et al. identified deg in the liver of crossbred swine for groups with extreme values for intramuscular fas composition. costa et al. used bulls from different genetic groups of alentejana and barrosã breeds with divergent diets, high and low concentration of silage, to identify deg associated with lipid metabolism in subcutaneous adipose tissue and in the longissimus lumborum muscle. recently, studied the gene expression pattern in taurine cattle finished in different diets with extreme phenotypes for fa profile in the intramuscular fat. transcriptomic studies should contribute to elucidate the genetic and non-genetic mechanisms that determine beef fas composition in the intramuscular fat. these studies could also identify genomic regions and metabolic pathways involved in those mechanisms, aiming to improve the biological knowledge associated with beef fas composition. due to the limited number of studies and the implications of intramuscular fas composition on beef palatability and on human health, it is essential the ongoing study of gene expression for beef fas composition in nellore cattle. moreover, livestock production in brazil is one of the world’s most important food commerce. in addition, the brazilian beef production is the second largest in the planet, with 80 % of the herds having the influence of zebu cattle on its composition. thus, this study aimed to identify deg in nellore cattle finished in feedlot conditions with extreme phenotypes for intramuscular fas composition in longissimus thoracis muscle by rna-seq technique. methods animals and information management samples were obtained from a total of nellore young bulls, sons of six sires, belonged to a capivara farm located in são paulo state, brazil, which participates in the nellore qualitas breeding program. animals were selected based on growth, finishing and sexual precocity traits. animals were raised on grazing conditions using brachiaria sp. and panicum sp. forages, and free access to mineral salt. after yearling, the breeding animals were selected and the remaining was kept in feedlots for a period of 90 days. the diet was based on whole-plant silage and mix of sorghum grain, soybean meal or sunflower seeds were used as concentrate, with a concentrate/roughage ratio from 50/ to 70/ animals were slaughtered with an average age of 24 months and 550 kg of liveweight in commercial slaughterhouses, in accordance with the brazilian federal inspection service procedures. after 48 h post mortem at 0–2 °c, the samples were removed from the longissimus thoracis muscle, from between the 12–13th ribs from each animal. samples were placed in airtight plastic bags and stored at −80 °c for the analyses described below. extraction of lipids the total lipid concentration was quantified at the animal product technology laboratory in the technology department of fcav/unesp according to the method described by. raw and ground meat samples from longissimus thoracis muscle with approximate  g were transferred into a 250 ml erlenmeyer flask, where 10 ml of chloroform, 20 ml of methanol and 8 ml of distilled water was added. after homogenizing the samples with glass rods, the flasks were placed on a horizontal shaker table for 30 min. later, 10 ml of chloroform and 10 ml of a  % aqueous sodium sulfate solution were added and the samples were shaken for more two minutes, transferred to 50 ml falcon tubes and then centrifuged at  × g for two minutes at room temperature. after centrifugation, the supernatant was discarded and the remainder was passed through filter paper to separate the meat fragments from the solution that contained the extracted lipids. the samples were filtered into 25 ml measuring cylinders. the filtrate value was kept to be used in the total lipid calculation and 5 ml was transferred to a 50 ml pre-weighed beaker, oven-dried, cooled in a desiccator for at least 24 h, placed in an oven at 110 °c until complete solvent evaporation, cooled in a desiccator and weighed once again. differences in the initial weight of the beaker and final weight were used to determine the total lipid concentration of samples. fatty acids composition fatty acid composition was determined for each sample using the extraction method described by. muscle samples were collected and grounded for fas composition. the lipids were extracted by homogenizing the sample with a chloroform and methanol solution. nacl at  % was added and so that the lipids were isolated. the isolated lipids were methylated and the methyl esters were formed according to. the fas composition was quantified using a gas chromatography with a sp- capillary column. the initiating temperature was 70 °c with gradual warming up to 175 °c, holding for 27 min, and later a further increase of 4 °c/min until 215 °c was reached and held for 31 min. the fas were identified by comparison of retention time of methyl esters of the samples with standards of c4–c, vaccenic acid c18: trans- c18: trans- cis-, cla e c18: cis- trans-, and tricosanoic acid. the fas were quantified by normalizing the area under the curve of methyl esters using software gs solution. the fas were expressed in percentage of total fa methyl ester. the fa composition in meat was performed at the meat science laboratory in the department of animal nutrition and production at fmvz/usp. based on the identified acids, fas were selected due to their importance in human health. the following fas were determined: myristic, palmitic, stearic, myristoleic, oleic, linoleic, conjugated linoleic acid c18: cis trans alfa linolenic, sum of saturated fatty acid, sum of mufa, sum of polyunsaturated fatty acid, ratio between pufa and sfa, sum of ω, ω and ratio between ω and ω rna-seq quantification total rna was extracted for each sample with trizol® reagent from 100 mg of frozen lt muscle. rna integrity was verified by agilent bioanalyzer®, where only samples with rin >  were used. a total of 2 μg of rna from each sample was used for library preparation according to the protocol described in truseq rna sample preparation kit® v guide. the resultant libraries were quantified using a kapa library quantification kit®, according to illumina's library quantification protocol. finally, libraries were pooled to perform multiplexing sequencing process, which adds an individual barcode sequences to each sample allowing that each one can be distinguished and analyzed separately during the data analysis. six lanes of a sequencing flowcell, using the truseq pe cluster kit v3-cbot-hs kit, were clustering and sequenced using hiseq with a truseq sbs v3-hs kit, according to manufacturer’s instructions. paired-end reads of 2 × 100 bp were produced. the sequencing analyses were performed at the genome center at esalq, piracicaba, são paulo, brazil. alignment of sequence reads and transcript assembly the sequencing data for each sample generated by hiseq system platform was converted to fastq format, and separated by libraries through casava software available at https://support.illumina.com/sequencing/sequencing_software/casava.html. the tuxedo pipeline, which includes fastqc, tophat and cuffdiff program were performed in this transcriptomic study using the iplant collaborative platform. the fastqc program was used to analyze the sequencing data quality, subsequently, the tophat package was performed to align the reads against the bos taurus virtual transcriptome internally built by tophat using the umd reference genome, containing genes. this program was also used to identify the splice junctions of exons transcripts showing the potential exons. for each library, a file was generated with extension ".bam" containing the aligned reads in relation to the reference genome. cufflinks was used to assemble the aligned read for each sample individually, providing a parsimonious set of transcripts and to estimate transcript abundances in fpkm which normalizes transcript expression for transcript length and the total number of sequence reads per sample. deg analysis and functional enrichment fatty acid concentration was classified into two extreme phenotype values groups to identify deg for each fa in samples. ten animals or biological replicates composed each fa concentration group. different animals composed those two groups for each beef fa, since the same animal was not necessary extreme for different beef fa. cuffdiff program included in tuxedo pipeline performed differential expression analysis. the false discovery rate threshold used in this analysis was 10 %. database for annotation, visualization, and integrated discovery v was used for functional enrichment analyses using the list of deg for each fa and the bos taurus annotation file as background.  phenotypic variation between groups the descriptive statistics and the analysis of variance for the fa concentration for high and low groups are described in table  the coefficient of variation ranged from to  %, indicating a high homogeneity within each group. there were significant differences between the high and low groups for the concentration of all beef fas measured.table descriptive statistics and analysis of variance for the fatty acidsa for groups of animals with extreme phenotypes athe concentration of fatty acids are expressed as a percentage of total fatty acid methyl esters; blow group: ten lowest extreme phenotypes; chigh group: ten highest extreme phenotypes throughput sequencing, read mapping and assembly the table  presents the sequencing throughput and mapping statistics for each high and low groups. the sequence quality was assessed through the distribution of transcript abundance for each fa and gene expressed as a box-plot of the log of fragments per kilobase of exon per million fragments mapped values. for each fa, similar median and quartiles values for fpkm estimates were obtained between the high and low groups.table average number of pair-end reads, number of mapped reads and concordant pair alignment rate for high and lowa groups for each beef fatty acid ahigh group: ten highest extreme phenotypes; blow group: ten lowest extreme phenotypes the principal component analyses of fpkm values for all genes indicated that there were sufficient number of deg to differentiate high and low groups for most of the fas. moreover, the expression profiles of selected housekeeping genes were evaluated, such as the hypoxanthine phosphoribosyltransferase and tyrosine 3-monooxygenase /tryptophan 5-monooxygenase activation protein, zeta. for both genes, the expression patterns were similar between high and low groups for all beef fas evaluated. differential expression genes differential expression analysis between the high and low groups identified deg for c14: for c16: for c18: for c18: cis- for the c18: cis- trans-, for c18: cis- cis − n and for c18: n fa. for the respective sums of the individual fas, deg for sfa, for mufa, for pufa, for ω and for ω were identified. for pufa/sfa and ω6/ ω ratio, and deg were identified, respectively. the list of the deg identified between groups with different fas composition is described in table .table description of differentially expressed genes identified between groups of nellore bulls with different fatty acid composition in longissimus thoracis muscle fabp7 loc782922 slc27a6 acat1 pafah2 scd5_bovin tnxb ptgs1 slc1a4 cd74 fabp3 gpam mgll lipe acox2 ppara ppard ptpla plod2 acsm1 adipoq fabp4 dgat2 c16:0 slc16a7 c18:0 ucp3 mufa lbp c16:0 lpl c18:1cis-9 pufa/sfa − − anxa1 c18:1cis-9 mufa pufa − − acsm3 mufa pufa sfa − − acss1 c16:0 c18:0 c18:1cis-9 sfa − cpe c16:0 c18:1cis-9 sfa mufa − − bdh1 c16:0 c18:0 sfa c18:2cis-9cis-12 mufa − − the acsm gene, that was differentially expressed for linoleic, mufa, pufa, sfa and ω acids, participates in the metabolism of lipids and in metabolic pathways that involves the precursor acetyl-coa metabolism. following the process of fas synthesis, the acss gene acts in the transformation of acetyl-coa into fas through chemical reactions and metabolic pathways involving acetyl-coa. this gene was differentially expressed, upregulated for sfa such as palmitic, stearic, oleic and sfa sum, and downregulated for unsaturated acids, such as ω fig. metabolic pathways of acsm gene fig. metabolic pathways of acss gene other important deg identified was the slc16a, member 7), which was upregulated for sfa sum, myristic and stearic acids, and downregulated for ω this gene is responsible for catalyzing the transfer of monocarboxylic acids from one cell to another. other genes such as anxa, upregulated for stearic acids, ω and pufa sum, and downregulated for oleic and mufa; and the lbp gene upregulated for the myristic, palmitic and sfa sum, are also responsible for the transport of fas between cells through pores of a carrier or agent. the slc27a, member 6) gene was upregulated for sfa and acts as a carrier of fas. the acsm gene was downregulated for ω and ω and it participates directly in fas synthesis. this gene is responsible for the chemical reactions and metabolic pathways that involve fas and aliphatic monocarboxylic acids of open chain that are naturally released by hydrolysis of the fats and oils. the gpam and ucp genes have similar functions, in which the first one was found upregulated for oleic acid and mufa and downregulated for ω while the second gene was downregulated for pufa/sfa ratio. the stearoyl-coa desaturase is an integral membrane protein of the endoplasmic reticulum, which catalyzes the synthesis of mufa from sfa, which can also be a key regulator of energy metabolism. the adipoq, plod, and lpl genes were differentially expressed for several fas, and participate in several processes related to fas synthesis. in this sense, the adipoq gene was downregulated for ω and ω this gene participates directly in the metabolic pathways related to fas production, such lipids and organic acids and is also involved in the regulation of cellular ketone metabolic process and in fas oxidation and beta-oxidation. the plod gene acts in the binding of carboxylic acids and in other organic acid containing one or more carboxyl group or anions and was upregulated for linolenic fas and pufa sum, indicating that its expression may promote the synthesis of pufa. finally, the lpl gene was differentially expressed for palmitic and oleic acids, ω and pufa/sfa ratio. there was downregulated expression of all unsaturated fas, indicating that high expression of this gene is associated with a low concentration of these acids in the samples analyzed. the loc gene was downregulated for mufa, which can act in the metabolism of prostaglandins and participates in the chemical reactions and metabolic pathways of unsaturated fas synthesis or other fas containing one or more double bonds between the carbon atoms. the cpe gene is responsible for insulin synthesis through proteolysis of its precursor, which was upregulated for c14: c16: c18: and sfa sum, and downregulated for c18: cis- mufa and ω while the bdh gene was differentially expressed for c14: c16: c18: ω mufa and sfa sums. functional analysis gene ontology and pathway enrichment analysis were perfomed to gain insight into the predicted gene network. the most significant go terms were focused on cellular components, molecular functions and biological processes. molecular functions controlling fas metabolism are highly interconnected and linked with related pathways, such as lipid, carbohydrate metabolism and energy homeostasis pathway. the essential metabolic network for homeostatic control and organism development is constituted by these pathways and its interactions. in this study, molecular functions related to recognize glycosaminoglycan, polysaccharide and carbohydrate molecules were identified.table gene ontology terms enriched with differentially expressed genes p cellular components molecular functions biological process anumber of differentially expressed genes the biologicals processes identified are related mainly with extracellular structure and organization, response to wounding, inflammatory response, embryonic development, skeletal and muscle developments. four kegg pathways were identified over represented for deg by david tool. these pathways were related with ecm-receptor interaction, focal adhesion, ppar signaling pathway, and tgf-beta signaling pathway. discussion phenotypic variation between groups evaluating longissimus muscle of bos indicus, observed similar concentrations of sfa as described in this study, and different for the pufa, mufa, pufa/sfa, ω ω and ω6/ω ratio. cesar et al. identified genomic regions associated with fas composition and fat deposition in nellore steers and found concentrations of fas that are close to the average obtained for the high groups in the present study. differential expression genes in ruminants, the fas synthesis occurs mainly in the adipose tissue, except during the lactation, when the mammary gland becomes the predominant organ. the main point about fas synthesis control is the acetyl-coa carboxylase, and it seems that the endocrine control is very similar in, at least, adipose tissue of ruminants and non-ruminants. acetate is the principal precursor of fas synthesis in ruminants, and must be converted to acetyl-coa by the action of acetyl-coa synthetase and then incorporated into fas. the adipose tissue is largely responsible for the conversion of acetate into acetyl-coa, and consequently, the greatest synthesizer of fas in ruminants. from the results obtained in this study, it was possible to highlight some important genes related to biologicals processes involved in beef’s fas synthesis, such as those involved in the transport of essential components in animal tissues. the myristic and palmitic fas are considered to be hypercholesterolemic, and are responsible for increasing the amount of low density lipoproteins, which expand the risk of heart diseases. other genes that also operate in the transport of fas were identified, but the deg were only for a single fa, such as fabp and fabp genes, which appeared upregulated for linolenic acid and pufa/sfa ratio, respectively. these genes produce proteins that apparently play a role in intracellular transport of long chain fas and their acyl-coa esters. the intracellular fas binding proteins belong to a multigene family. the fabps are divided into at least three distinct groups: hepatic, intestinal, and cardiac. these form 14–15 kda protein and participate in the absorption, metabolism and/or intracellular transport of long chain fas, and may also be responsible for growth modulation and cell proliferation. regarding the fabp gene, it works in fas binding proteins and it was downregulated for ω and ω the fabps are often associated with lipid metabolism by acting as intracellular transport of hydrophobic intermediates and lipids metabolites trough the membranes. the pafah gene, downregulated for oleic acid, participates in chemical reactions and pathways that break lipids. the mgll gene, downregulated for pufa/sfa ratio, operates in the chemical reactions for lipids synthesis and acts as a catalyst in fas synthesis reactions. our results showed some genes directly associated with fas synthesis. in this sense, the dgat gene, upregulated for palmitic and downregulated for linoleic acid and pufa/sfa ratio, is essential for the triglycerides synthesis and intracellular storage found negative correlations between marbling and concentrations of stearic, linoleic acid, and pufa reported a positive and moderate correlation between the level of marbling and the expression of dgat gene. the dgat gene is an important contributor to the triacylglycerol synthesis through their acyltransferase activity. as the amount of triglyceride within the adipocyte increases, the total proportion of sfa also increases in relation to other ones. an increase in dgat gene expression was previously demonstrated to be associated with an increase in the amount of intramuscular fat. thus, these results demonstrate that dgat gene contributed to the accumulation of sfa in the intramuscular tissue during the finishing phase.fig. metabolic pathway of dgat and lpl genes the lpl gene plays a role in chemical reactions and metabolic pathways that result in fas synthesis and open-chain monocarboxylic acids, which can be released by hydrolysis that occur in fats and oils. the activity of this gene in the adipose tissue and the subsequent increase in deposition of triglycerides are promoted by insulin. some studies have suggested that the fas synthesis in the subcutaneous adipose tissue on beef is not sensitive to insulin levels. in this sense, the activity of lpl gene in the muscle tissue appears not to be insulin dependent, however, many authors have provided evidence to support the opposite. the bdh gene was upregulated for sfa and downregulated for unsaturated fas, indicating a higher gene expression as the degree of saturation of the sample is increased. this gene is responsible for the synthesis and degradation of ketone bodies, which allows transporting the energy obtained by the oxidation of fas to the peripheral tissues, then to be used in the atp synthesis in the absence of carbohydrates in the diet. this fact justifies the greatest expression of this gene in the presence of sfa for the synthesis of ketone bodies from sfa, since it is less complex when compared to the degradation of unsaturated fas. the acat gene has the same function of bdh gene, and was upregulated for ω.fig. metabolic pathway for bdh and acat genes the results showed an evident antagonism for the expression of some deg related to fas synthesis. in this sense, some of those genes that were upregulated for sfa group were also downregulated for mufa and pufa groups. animals with high degree of fatness or deposition of intramuscular fat in lt muscle presented higher concentrations of sfa and less concentrations of pufa. de smet et al. and wood et al. showed that when the proportion of animal fat increases, the proportion of pufa in meat decreases drastically. ruminants incorporate essential fas, especially phospholipids, in muscle lipids rather than storing them in fat. recently, more emphasis has been dedicated on muscle due to its importance as a protein source and as a growing aversion to visible fat at retail. the muscle also contains higher concentrations of ω and ω acids, whose importance to human nutrition have been recently recognized. in recent years, the procedures for separation and identification of low levels of unsaturated fas in muscle have been greatly improved. thus, the development of molecular genetics, particularly high throughput sequencing methods, provides a unique opportunity to identify genes and pathways associated with diseases and complex traits. however, recent studies revealed a limitation of genome-wide association study to identify loci with significant effects on different populations of the same breed, since many discordant genomic regions have been identified. tizioto et al. observed deg using rna-seq method and also applied gwas to identify genomic regions for feed efficiency in nellore cattle. these authors found several biological mechanisms and attributed the differences in the candidate regions/genes to the specific modulation of mrna. in a recent gwas study, used a population with nellore bulls, which contained animals used in this study, and identified several genomic regions which explained more than 1 % of the additive genetic variance for beef fas composition. the authors reported some regions near to deg identified in this study. in this regard, the acsm and acsm genes were differentially expressed for ω acids and were located in a region which explained more than 1 % of the additive variance genetic for ω as reported by. those authors also reported qtl detected by gwas in the same region that the tnxb and ppard genes were differentially expressed in this study. these genes are related with fas metabolism, transport and oxidation. these results showed some degree of equivalence, since it was identified common regions between the results of structural analysis and functional analysis for beef fas composition. the results obtained in this study indicated that beef’s fas composition in nellore cattle is influenced by many genes and complex metabolic pathways. furthermore, it identified genes that contribute to fas metabolism, through intra or extra-cellular transport of fas synthesis precursors in intramuscular fat of longissimus muscle. among those genes, some of them must be highlighted, such as: acsm and acss genes, since they work in the fas precursor synthesis and their subsequent transformation into fas, respectively. in addition, the dgat gene that assists the deposition of saturated fat tissue; gpp and lpl genes that support the insulin synthesis, which stimulates the glucose synthesis as the amino acids entry into the cells; and the bdh gene which is responsible for the synthesis and degradation of ketone bodies, used in the atp synthesis. functional analysis several gde related to biological processes associated with inflammatory response were identified in the present study. in these sense, stated that the fatty acids influenced inflammatory response acting via cell surface and intracellular receptors/sensors that control gene expression patterns and inflammatory cell signaling. some effects of fas on inflammatory cells appear to be mediated or associated with changes in fas composition of cell membranes. extracellular matrix consists of a complex of structural and functional macromolecules with an important role in tissue, organ morphogenesis, cell maintenance, and tissue structure and function. it can directly or indirectly influence specific cellular activities such as cell adhesion, proliferation, differentiation, and migration. in muscle tissue, cells are tightly bound together, and the extracellular spaces containing the extracellular matrix are limited. these results corroborates with previous study of transcriptome profile of nellore steers with different genomic breeding value of intramuscular fat deposition. jiang et al. studying transcriptome comparison between porcine subcutaneous and intramuscular stromal vascular cells during adipogenic differentiation speculated that the ecm-receptor interaction pathway might participate in intramuscular stromal vascular cell differentiation process. lee et al. studied the difference of the depot specific gene expression from different adipose tissues of omental, subcutaneous and intramuscular tissues in cattle, and identified the ecm-receptor interaction with the one of commonly enriched pathways in all adipose tissues and also functioned as a sub-pathway of other enriched pathways. these authors suggested that the interactions between ecm components and transmembrane receptors of fat cells depend on the depot specific adipogenesis. the most overexpressed genes identified in this study related to muscle and skeletal developments could be good candidates for nellore breeding programs in which the main goal is to enhance meat and carcass quality. studying a hanwoo beef cattle population, identified pathways related to cell adhesion regulation, structure, integrity, and chemokine signaling pathway upregulated in intramuscular adipose but downregulated in the muscle. cui et al. also proposed that these pathways play an important role in the intramuscular fat deposition in chicken. cánovas et al. identified the ecm-receptor interaction and tgf-beta signaling pathways as the most relevant metabolic pathways represented in the list of deg related with meat composition in pig longissimus dorsi muscle. the transforming growth factor tgf-beta signaling pathway is involved in many cellular processes including cell growth, differentiation and apoptosis, cellular homeostasis in both the adult organism and in the developing embryo. mehla et al. identified the tgf-beta signaling pathway related to deg genes in zebu cattle due to heat stress effects. peroxisome proliferator-activated receptors are nuclear hormone receptors that are activated by fas and their derivatives, and play an essential physiological role in the regulation of adipocyte tissue development lipogenesis and skeletal muscle lipid metabolism. there are three members of the ppar family with different expression patterns in vertebrates. ppar alpha plays a role in lipid metabolism in the liver and in the skeletal muscle, and in the modulation of the inflammatory response. ppar beta/delta is involved in lipid oxidation and cell proliferation, and acts on embryo implantation, cell proliferation and apoptosis. ppar gamma is related to cell cycle withdrawal and promotes myocyte/adipocyte differentiation to enhance blood glucose uptake. doran et al. studying gwas in holstein-friesan cattle identified the ppar signaling pathway as the most significantly overrepresented biological pathway involved in carcass trait performance, suggesting that ppar would also play a key role in controlling carcass weight, carcass fat and carcass conformation traits. he et al. identified an association between genes and snps in the ppar signaling pathway with porcine meat quality traits. carcass and meat traits, especially those obtained through beef fas composition of intramuscular fat analyses, are not used by the industry as a criterion for determining the animal’s value for slaughter. however, there is a growing trend in the international meat market to provide technical and scientific guarantees to certify food safety, product quality and its benefits to human health. therefore, the production of the required information is essential and needed to improve the marketing of beef products. there are few studies about transcriptome in zebu animals, in which is the unprecedented as it is the first study of gene expression for beef feed efficiency in bos indicus animals. thus, it provides subsidies to improve the beef quality of zebu cattle under tropical conditions, producing a healthier food for consumers.  several genes related to lipids metabolism and beef fas composition were found in this study. the identification of such candidate genes must contribute to the elucidation of the genetic basis that determines the beef fas composition of intramuscular fat in nellore cattle. this information would contribute to the improvement of meat quality through selection processes, since the molecular processes that control fas composition and metabolism are not completely understood yet. moreover, the deg identified can be used in future studies of fine mapping whose primary function is to search for functional mutations and can be useful to identify some specific variants. additional files additional file 1: boxplot of the transcript abundance distribution for each beef fatty acid and gene expressed. additional file 2: principal component analysis of gene fpkm values between the high and low groups with fatty acids profile: pca for sum of sfa; pca for sum of mufa; pca for sum of pufa; pca for sum of pufa/sfa ratio; pca for sum of n3;. pca for sum of n6; pca for n6/n ratio; pca for myristic acid; pca for palmitic acid; pca for stearic acid; pca for oleic acid; pca for conjugated linoleic acid; pca for linoleic acid; pca for α-linolenic. additional file 3: table s differentially expressed genes for c14: table s differentially expressed genes for c16: table s differentially expressed genes for c18: table s differentially expressed genes for c18: cis- table s differentially expressed genes for c18: cis-9trans- table s differentially expressed genes for c18: cis- cis - n table s differentially expressed genes for c18: n table s differentially expressed genes for sfa. table s differentially expressed genes for mufa. table s differentially expressed genes for pufa. table s differentially expressed genes for relationship between pufa and sfa. table s differentially expressed genes. table s differentially expressed genes for n table s differentially expressed genes for the ratio of ω6/ω abbreviations claconjugated linoleic acid degdifferentially expressed genes fafatty acids gogene ontology highthe group with highest phenotype lowthe group with lowest phenotype mufamonounsaturated fatty acids pufapolyunsaturated fatty acids sfasaturated fatty acids ω3omega 3 ω6omega 6 
several genes related to lipid metabolism and fatty acid composition were identified. these findings must contribute to the elucidation of the genetic basis to improve nellore meat quality traits, with emphasis on human health. the aim of this study was to identify differentially expressed genes in longissimus thoracis muscle of nellore young bulls with extreme phenotypes for fatty acid composition of intramuscular fat by rna-seq technique. functional annotation analyses identified several genes associated with fatty acid metabolism, such as those involved in intra and extra-cellular transport of fatty acid synthesis precursors in intramuscular fat of longissimus thoracis muscle. some of them must be highlighted, such as: acsm and acss genes, which work as a precursor in fatty acid synthesis; dgat gene that acts in the deposition of saturated fat in the adipose tissue; gpp and lpl genes that support the synthesis of insulin, stimulating both the glucose synthesis and the amino acids entry into the cells; and the bdh gene, which is responsible for the synthesis and degradation of ketone bodies used in the synthesis of atp. fatty acid type in beef can be detrimental to human health and has received considerable attention in recent years. bos indicushuman healthlipid compositionrna-seqtranscriptomeissue-copyright-statement© the author 2016 for the respective sums of the individual fatty acids, differentially expressed genes for saturated fatty acids, for monounsaturated, for polyunsaturated, for pufa/sfa ratio, for ω for ω and for ω6/ω ratio were identified. differential expression analyses between animals with extreme phenotype for fatty acid composition showed a total of differentially expressed genes for myristic, for palmitic, for stearic, for oleic, for conjugated linoleic, for linoleic, and genes for α-linolenic fatty acids.
 fatty acid type in beef can be detrimental to human health and has received considerable attention in recent years. the aim of this study was to identify differentially expressed genes in longissimus thoracis muscle of nellore young bulls with extreme phenotypes for fatty acid composition of intramuscular fat by rna-seq technique.  differential expression analyses between animals with extreme phenotype for fatty acid composition showed a total of differentially expressed genes for myristic, for palmitic, for stearic, for oleic, for conjugated linoleic, for linoleic, and genes for α-linolenic fatty acids. for the respective sums of the individual fatty acids, differentially expressed genes for saturated fatty acids, for monounsaturated, for polyunsaturated, for pufa/sfa ratio, for ω for ω and for ω6/ω ratio were identified. functional annotation analyses identified several genes associated with fatty acid metabolism, such as those involved in intra and extra-cellular transport of fatty acid synthesis precursors in intramuscular fat of longissimus thoracis muscle. some of them must be highlighted, such as: acsm and acss genes, which work as a precursor in fatty acid synthesis; dgat gene that acts in the deposition of saturated fat in the adipose tissue; gpp and lpl genes that support the synthesis of insulin, stimulating both the glucose synthesis and the amino acids entry into the cells; and the bdh gene, which is responsible for the synthesis and degradation of ketone bodies used in the synthesis of atp.  several genes related to lipid metabolism and fatty acid composition were identified. these findings must contribute to the elucidation of the genetic basis to improve nellore meat quality traits, with emphasis on human health. additionally, it can also contribute to improve the knowledge of fatty acid biosynthesis and the selection of animals with better nutritional quality. electronic supplementary material the online version of this article contains supplementary material, which is available to authorized users. keywords bos indicushuman healthlipid compositionrna-seqtranscriptomeissue-copyright-statement© the author 2016 