Expression of calpain system transcripts responds inversely to beef tenderization after vitamin D3 supplementation in Nellore cattle / Expressão de transcritos do sistema calpaína responde de forma inversa ao amaciamento após suplementação com vitamina D3 em bovinos Nelore

Uma vez que o sistema calpaína é central para o amaciamento da carne e dada a importância da atividade de calpastatina na determinação da maciez de bifes de bovinos Bos taurus indicus, o objetivo do presente estudo foi determinar se a expressão do gene de µ-calpaína (CAPN1), calpastatina total (CAST T), e suas variantes (CAST I e II) foi induzido pela inclusão de vitamina D3 na dieta. Os animais receberam nenhuma ou 2 × 106 UI dose de vitamina D3 por 2 ou 8 dias antes do abate, e foram submetidos a diferentes condições durante o confinamento: exposição solar ou sombreamento artificial. Bifes do Longissimus lumborum foram fabricados e submetidos a maturação por 1, 7, e 21 dias post-mortem e posteriormente usados para determinação da força de cisalhamento e do índice de fragmentação miofibrilar. Vitamina D3 não influenciou a abundância de RNAm, exceto para maior abundância de transcritos de CAST II em animais que foram suplementados por 8 dias antes do abate. Foi encontrada associação negativa entre a abundância de CAST II e a força de cisalhamento. Essa contradição revela uma importante modulação da expressão do sistema calpaína resultado da suplementação com vitamina D que pode ser importante na determinação de estratégias para melhorar a maciez da carne.(AU)

The calpain system is the central player for meat tenderization and the calpastatin activity plays an important role in beef tenderness of Bos taurus indicus cattle. This study investigated whether dietary vitamin D3 induced gene expression of µ-calpain (CAPN1), total calpastatin (CAST T), and their variants (CAST I and II). Animals received none or 2 × 106 IU of vitamin D3 for either 2 or 8 days before slaughter and were submitted to different conditions during feedlot: sun exposure or artificial shade. Steaks from Longissimus lumborum were fabricated, aged for 1, 7, and 21 days post-mortem, and later used for the analyses of shear force and the myofibrillar fragmentation index. Vitamin D3 did not influence mRNA abundance; however, it induced a greater CAST II transcript in animals supplemented 8 days before slaughter. There was a negative association between CAST II abundance and the shear force, which revealed an important modulation of the calpain system expression due to vitamin D supplementation. This result is an important tool for strategies to improve beef tenderness.(AU)

MiRNAs differentially expressed in skeletal muscle of animals with divergent estimated breeding values for beef tenderness.

BACKGROUND: MicroRNAs (miRNAs) are small noncoding RNAs of approximately 22 nucleotides, highly conserved among species, which modulate gene expression by cleaving messenger RNA target or inhibiting translation. MiRNAs are involved in the regulation of many processes including cell proliferation, differentiation, neurogenesis, angiogenesis, and apoptosis. Beef tenderness is an organoleptic characteristic of great influence in the acceptance of meat by consumers. Previous studies have shown that collagen level, marbling, apoptosis and proteolysis are among the many factors that affect beef tenderness. Considering that miRNAs can modulate gene expression, this study was designed to identify differentially expressed miRNAs that could be modulating biological processes involved with beef tenderness. RESULTS: Deep sequence analysis of miRNA libraries from longissimus thoracis muscle allowed the identification of 42 novel and 308 known miRNAs. Among the known miRNAs, seven were specifically expressed in skeletal muscle. Differential expression analysis between animals with high (H) and low (L) estimated breeding values for shear force (EBVSF) revealed bta-mir-182 and bta-mir-183 are up-regulated (q value < 0.05) in animals with L EBVSF, and bta-mir-338 is up-regulated in animals with H EBVSF. The number of bovine predicted targets for bta-mir-182, bta-mir-183 and bta-mir-338 were 811, 281 and 222, respectively, which correspond to 1204 unique target genes. Among these, four of them, MEF2C, MAP3K2, MTDH and TNRC6B were common targets of the three differentially expressed miRNAs. The functional analysis identified important pathways related to tenderness such as apoptosis and the calpain-calpastatin system. CONCLUSION: The results obtained indicate the importance of miRNAs in the regulatory mechanisms that influence muscle proteolysis and meat tenderness and contribute to our better understanding of the role of miRNAs in biological processes associated with beef tenderness.

Gene Co-expression Analysis Indicates Potential Pathways and Regulators of Beef Tenderness in Nellore Cattle.

Beef tenderness, a complex trait affected by many factors, is economically important to beef quality, industry, and consumer's palatability. In this study, RNA-Seq was used in network analysis to better understand the biological processes that lead to differences in beef tenderness. Skeletal muscle transcriptional profiles from 24 Nellore steers, selected by extreme estimated breeding values (EBVs) for shear force after 14 days of aging, were analyzed and 22 differentially expressed transcripts were identified. Among these were genes encoding ribosomal proteins, glutathione transporter ATP-binding cassette, sub-family C (CFTR/MRP), member 4 (ABCC4), and synaptotagmin IV (SYT4). Complementary co-expression analyses using Partial Correlation with Information Theory (PCIT), Phenotypic Impact Factor (PIF) and the Regulatory Impact Factor (RIF) methods identified candidate regulators and related pathways. The PCIT analysis identified ubiquitin specific peptidase 2 (USP2), growth factor receptor-bound protein 10 (GBR10), anoctamin 1 (ANO1), and transmembrane BAX inhibitor motif containing 4 (TMBIM4) as the most differentially hubbed (DH) transcripts. The transcripts that had a significant correlation with USP2, GBR10, ANO1, and TMBIM4 enriched for proteasome KEGG pathway. RIF analysis identified microRNAs as candidate regulators of variation in tenderness, including bta-mir-133a-2 and bta-mir-22. Both microRNAs have target genes present in the calcium signaling pathway and apoptosis. PIF analysis identified myoglobin (MB), enolase 3 (ENO3), and carbonic anhydrase 3 (CA3) as potentially having fundamental roles in tenderness. Pathways identified in our study impacted in beef tenderness included: calcium signaling, apoptosis, and proteolysis. These findings underscore some of the complex molecular mechanisms that control beef tenderness in Nellore cattle.

Genome-wide characterization of genetic variants and putative regions under selection in meat and egg-type chicken lines.

BACKGROUND: Meat and egg-type chickens have been selected for several generations for different traits. Artificial and natural selection for different phenotypes can change frequency of genetic variants, leaving particular genomic footprints throghtout the genome. Thus, the aims of this study were to sequence 28 chickens from two Brazilian lines (meat and white egg-type) and use this information to characterize genome-wide genetic variations, identify putative regions under selection using Fst method, and find putative pathways under selection. RESULTS: A total of 13.93 million SNPs and 1.36 million INDELs were identified, with more variants detected from the broiler (meat-type) line. Although most were located in non-coding regions, we identified 7255 intolerant non-synonymous SNPs, 512 stopgain/loss SNPs, 1381 frameshift and 1094 non-frameshift INDELs that may alter protein functions. Genes harboring intolerant non-synonymous SNPs affected metabolic pathways related mainly to reproduction and endocrine systems in the white-egg layer line, and lipid metabolism and metabolic diseases in the broiler line. Fst analysis in sliding windows, using SNPs and INDELs separately, identified over 300 putative regions of selection overlapping with more than 250 genes. For the first time in chicken, INDEL variants were considered for selection signature analysis, showing high level of correlation in results between SNP and INDEL data. The putative regions of selection signatures revealed interesting candidate genes and pathways related to important phenotypic traits in chicken, such as lipid metabolism, growth, reproduction, and cardiac development. CONCLUSIONS: In this study, Fst method was applied to identify high confidence putative regions under selection, providing novel insights into selection footprints that can help elucidate the functional mechanisms underlying different phenotypic traits relevant to meat and egg-type chicken lines. In addition, we generated a large catalog of line-specific and common genetic variants from a Brazilian broiler and a white egg layer line that can be used for genomic studies involving association analysis with phenotypes of economic interest to the poultry industry.

Oviductal transcriptional profiling of a bovine fertility model by next-generation sequencing.

In cattle, the oviduct plays a fundamental role in the reproductive process. Oviductal functions are controlled by the ovarian sex steroids: estradiol and progesterone. Here, we tested the hypothesis that the exposure to contrasting sex steroid milieus differentially impacts the oviductal transcriptional profile. We manipulated growth of the pre-ovulatory follicle to obtain cows that ovulated a larger (LF group) or a smaller (SF group) follicle. The LF group presented greater proestrus/estrus concentrations of estradiol and metaestrus concentrations of progesterone (Gonella-Diaza et al. 2015 [1], Mesquita et al. 2014 [2]). Also, the LF group was associated with greater fertility in timed-artificial insemination programs (Pugliesi et al. 2016 [3]). Cows were slaughtered on day 4 of the estrous cycle and total RNA was extracted from ampulla and isthmus fragments and analyzed by RNAseq. The resulting reads were mapped to the bovine genome (Bos taurus UMD 3.1, NCBI). The differential expression analyses revealed that 325 and 367 genes in ampulla and 274 and 316 genes in the isthmus were up-regulated and down-regulated in LF samples, respectively. To validate the RNAseq results, transcript abundance of 23 genes was assessed by qPCR and expression patterns were consistent between the two techniques. A functional enrichment analysis was performed using Database for Annotation, Visualization and Integrated Discovery (DAVID) software. Processes enriched in the LF group included tissue morphology changes (extracellular matrix remodeling), cellular changes (proliferation), and secretion changes (growth factors, ions and metal transporters). An overview of the gene expression data was deposited in the NCBI's Gene Expression Omnibus (GEO) and is accessible through the accession number GSE65681. In conclusion, differences in the peri-ovulatory sex steroid milieu modify the oviductal gene expression profiles. Such differences may be associated with the greater fertility of the LF cows. This dataset is useful for further investigations of the oviductal biology and the impact of sex-steroid on the female reproductive tract.

Size of the Ovulatory Follicle Dictates Spatial Differences in the Oviductal Transcriptome in Cattle.

In cattle, molecular control of oviduct receptivity to the embryo is poorly understood. Here, we used a bovine model for receptivity based on size of the pre-ovulatory follicle to compare oviductal global and candidate gene transcript abundance on day 4 of the estrous cycle. Growth of the pre-ovulatory follicle (POF) of Nelore (Bos indicus) cows was manipulated to produce two groups: large POF large corpus luteum (CL) group (LF-LCL; greater receptivity) and small POF-small CL group (SF-SCL). Oviductal samples were collected four days after GnRH-induced ovulation. Ampulla and isthmus transcriptome was obtained by RNA-seq, regional gene expression was assessed by qPCR, and PGR and ERa protein distribution was evaluated by immunohistochemistry. There was a greater abundance of PGR and ERa in the oviduct of LF-LCL animals thus indicating a greater availability of receptors and possibly sex steroids stimulated signaling in both regions. Transcriptomic profiles indicated a series of genes associated with functional characteristics of the oviduct that are regulated by the periovulatory sex steroid milieu and that potentially affect oviductal receptivity and early embryo development. They include tissue morphology changes (extra cellular matrix remodeling), cellular changes (proliferation), and secretion changes (growth factors, ions and metal transporters), and were enriched for the genes with increased expression in the LF-LCL group. In conclusion, differences in the periovulatory sex steroid milieu lead to different oviductal gene expression profiles that could modify the oviductal environment to affect embryo survival and development.

Gene expression profiling by high throughput sequencing to determine signatures for the bovine receptive uterus at early gestation.

The uterus plays a central role among the reproductive tissues in the context of early embryo-maternal communication and a successful pregnancy depends on a complex series of endometrial molecular and cellular events. The factors responsible for the initial interaction between maternal and embryonic tissues, leading to the establishment of pregnancy, remain poorly understood. In this context, Illumina's next-generation sequencing technology has been used to discover the uterine transcriptome signature that is favourable for ongoing pregnancy. More specifically, the present report documents on a retrospective in vivo study in which data on pregnancy outcome were linked to uterine gene expression signatures on day 6 (bovine model). Using the RNA-Seq method, 14.654 reference genes were effectively analysed for differential expression between pregnant and non-pregnant uterine tissue. Transcriptome data revealed that 216 genes were differently expressed when comparing uterine tissue from pregnant and non-pregnant cows. All read sequences were deposited in the Sequence Read Archive (SRA) of the NCBI (http://www.ncbi.nlm.nih.gov/sra). An overview of the gene expression data has been deposited in NCBI's Gene Expression Omnibus (GEO) and is accessible through GEO Series accession number GSE65117. This allows the research community to enhance reproducibility and allows for new discoveries by comparing datasets of signatures linked to receptivity and/or pregnancy success. The resulting information can serve as tool to identify valuable and urgently needed biomarkers for scoring maternal receptivity and even for accurate detection of early pregnancy, which is a matter of cross-species interest. Beyond gene expression analysis as a marker tool, the RNA-Seq information on pregnant uterine tissue can be used to gain novel mechanistic insights, such as by identifying alternative splicing events, allele-specific expression, and rare and novel transcripts that might be involved in the onset of maternal receptivity. This concept is unique and provides a new approach towards strategies that are highly needed to improve efficiency of fertility treatments.

Putative regulatory factors associated with intramuscular fat content.

Intramuscular fat (IMF) content is related to insulin resistance, which is an important prediction factor for disorders, such as cardiovascular disease, obesity and type 2 diabetes in human. At the same time, it is an economically important trait, which influences the sensorial and nutritional value of meat. The deposition of IMF is influenced by many factors such as sex, age, nutrition, and genetics. In this study Nellore steers (Bos taurus indicus subspecies) were used to better understand the molecular mechanisms involved in IMF content. This was accomplished by identifying differentially expressed genes (DEG), biological pathways and putative regulatory factors. Animals included in this study had extreme genomic estimated breeding value (GEBV) for IMF. RNA-seq analysis, gene set enrichment analysis (GSEA) and co-expression network methods, such as partial correlation coefficient with information theory (PCIT), regulatory impact factor (RIF) and phenotypic impact factor (PIF) were utilized to better understand intramuscular adipogenesis. A total of 16,101 genes were analyzed in both groups (high (H) and low (L) GEBV) and 77 DEG (FDR 10%) were identified between the two groups. Pathway Studio software identified 13 significantly over-represented pathways, functional classes and small molecule signaling pathways within the DEG list. PCIT analyses identified genes with a difference in the number of gene-gene correlations between H and L group and detected putative regulatory factors involved in IMF content. Candidate genes identified by PCIT include: ANKRD26, HOXC5 and PPAPDC2. RIF and PIF analyses identified several candidate genes: GLI2 and IGF2 (RIF1), MPC1 and UBL5 (RIF2) and a host of small RNAs, including miR-1281 (PIF). These findings contribute to a better understanding of the molecular mechanisms that underlie fat content and energy balance in muscle and provide important information for the production of healthier beef for human consumption.

The transcriptome signature of the receptive bovine uterus determined at early gestation.

Pregnancy success is critical to the profitability of cattle operations. However, the molecular events driving the uterine tissue towards embryo receptivity are poorly understood. This study aimed to characterize the uterine transcriptome profiles of pregnant (P) versus non-pregnant (NP) cows during early pregnancy and attempted to define a potential set of marker genes that can be valuable for predicting pregnancy outcome. Therefore, beef cows were synchronized (n=51) and artificially inseminated (n=36) at detected estrus. Six days after AI (D6), jugular blood samples and a biopsy from the uterine horn contralateral to the ovary containing the corpus luteum were collected. Based on pregnancy outcome on D30, samples were retrospectively allocated to the following groups: P (n=6) and NP (n=5). Both groups had similar plasma progesterone concentrations on D6. Uterine biopsies were submitted to RNA-Seq analysis in a Illumina platform. The 272,685,768 million filtered reads were mapped to the Bos Taurus reference genome and 14,654 genes were analyzed for differential expression between groups. Transcriptome data showed that 216 genes are differently expressed when comparing NP versus P uterine tissue (Padj ≤ 0.1). More specifically, 36 genes were up-regulated in P cows and 180 are up-regulated in NP cows. Functional enrichment and pathway analyses revealed enriched expression of genes associated with extracellular matrix remodeling in the NP cows and nucleotide binding, microsome and vesicular fraction in the P cows. From the 40 top-ranked genes, the transcript levels of nine genes were re-evaluated using qRT-PCR. In conclusion, this study characterized a unique set of genes, expressed in the uterus 6 days after insemination, that indicate a receptive state leading to pregnancy success. Furthermore, expression of such genes can be used as potential markers to efficiently predict pregnancy success.

Heat shock and structural proteins associated with meat tenderness in Nellore beef cattle, a Bos indicus breed.

Nellore beef cattle, a Bos indicus (Zebu) breed, is well adapted to tropical conditions and has allowed Brazil to become one of the largest producers of red meat. Nevertheless, B. indicus breeds are reported to have less tender meat than Bos taurus. This study was designed to identify genes associated with meat tenderness and thus provides important information for breeding programs. A group of 138 animals was evaluated for longissimus thoracis muscle shear force (SF). Animals with the highest and lowest SF values (six animals each) were then selected for protein abundance studies. Samples were subjected to two-dimensional gel electrophoresis (2-DE) followed by peptide sequencing through mass spectrometry (MS) to identify differentially expressed proteins associated with SF values. Seventeen differentially expressed spots were observed (p<0.05) between the two groups. The 13 proteins identified included structural proteins (alpha actin-1, MLC1, MLC3, MLC2F and tropomyosin), related to cell organization (HSPB1 and HSP70), metabolism (beta-LG, ACBD6 and Complex III subunit I) and some uncharacterized proteins. Results confirm the existence of differentially expressed proteins associated with SF, which can lead to a better understanding of mechanisms involved in meat tenderness.

Association of CSSM066 and ILSTS011 microsatellite markers and thyroglobulin gene SNP with backfat in Canchim cattle

Canchim, a synthetic breed of cattle derived from the Charolais and Zebu group has been used in the beef-cattle industry in Brazil as an alternative for intensifying production. One of the main concerns with this breed is its poor fat deposition and consequently, there is an effort to increase the performance for this trait. The thyroglobulin gene is located in a QTL region for fat deposition, and reports describe the influence of a polymorphism in the 5´ leader sequence of that gene on marbling and subcutaneous fat thickness. This study analyzed the association of this polymorphism in the thyroglobulin gene, as well as of two flanking microsatellite markers, CSSM066 and ILSTS011, with backfat thickness in 987 Canchim beef cattle. The CSSM066 and ILSTS011 microsatellite markers have a effect on fat thickness in the studied populations. However, this trait did not have association with the polymorphism of the thyroglobulin gene, which suggests that other genes of bovine chromosome 14 may be responsible for the variation in this trait.

Association of CSSM066 and ILSTS011 microsatellite markers and thyroglobulin gene SNP with backfat in Canchim cattle

Canchim, a synthetic breed of cattle derived from the Charolais and Zebu group has been used in the beef-cattle industry in Brazil as an alternative for intensifying production. One of the main concerns with this breed is its poor fat deposition and consequently, there is an effort to increase the performance for this trait. The thyroglobulin gene is located in a QTL region for fat deposition, and reports describe the influence of a polymorphism in the 5´ leader sequence of that gene on marbling and subcutaneous fat thickness. This study analyzed the association of this polymorphism in the thyroglobulin gene, as well as of two flanking microsatellite markers, CSSM066 and ILSTS011, with backfat thickness in 987 Canchim beef cattle. The CSSM066 and ILSTS011 microsatellite markers have a effect on fat thickness in the studied populations. However, this trait did not have association with the polymorphism of the thyroglobulin gene, which suggests that other genes of bovine chromosome 14 may be responsible for the variation in this trait.

Genome wide scan for quantitative trait loci affecting tick resistance in cattle (Bos taurus x Bos indicus).

BACKGROUND: In tropical countries, losses caused by bovine tick Rhipicephalus (Boophilus) microplus infestation have a tremendous economic impact on cattle production systems. Genetic variation between Bos taurus and Bos indicus to tick resistance and molecular biology tools might allow for the identification of molecular markers linked to resistance traits that could be used as an auxiliary tool in selection programs. The objective of this work was to identify QTL associated with tick resistance/susceptibility in a bovine F2 population derived from the Gyr (Bos indicus) x Holstein (Bos taurus) cross. RESULTS: Through a whole genome scan with microsatellite markers, we were able to map six genomic regions associated with bovine tick resistance. For most QTL, we have found that depending on the tick evaluation season (dry and rainy) different sets of genes could be involved in the resistance mechanism. We identified dry season specific QTL on BTA 2 and 10, rainy season specific QTL on BTA 5, 11 and 27. We also found a highly significant genome wide QTL for both dry and rainy seasons in the central region of BTA 23. CONCLUSIONS: The experimental F2 population derived from Gyr x Holstein cross successfully allowed the identification of six highly significant QTL associated with tick resistance in cattle. QTL located on BTA 23 might be related with the bovine histocompatibility complex. Further investigation of these QTL will help to isolate candidate genes involved with tick resistance in cattle.

Quantitative trait loci (QTL) mapping for growth traits on bovine chromosome 14

Quantitative trait loci (QTL) mapping in livestock allows the identification of genes that determine the genetic variation affecting traits of economic interest. We analyzed the birth weight and weight at 60 days QTL segregating on bovine chromosome BTA14 in a F2 resource population using genotypes produced from seven microsatellite markers. Phenotypes were derived from 346 F2 progeny produced from crossing Bos indicus Gyr x Holstein Bos taurus F1 parents. Interval analysis to detect QTL for birth weight revealed the presence of a QTL (p < 0.05) at 1 centimorgan (cM) from the centromere with an additive effect of 1.210 ± 0.438 kg. Interval analysis for weight at 60 days revealed the presence of a QTL (p < 0.05) at 0 cM from the centromere with an additive effect of 2.122 ± 0.735 kg. The region to which the QTL were assigned is described in the literature as responsible for some growth traits, milk yield, milk composition, fat deposition and has also been related to reproductive traits such as daughter pregnancy rate and ovulation rate. The effects of the QTL described on other traits were not investigated.

Quantitative trait locus affecting birth weight on bovine chromosome 5 in a F2 Gyr x Holstein population

Segregation between a genetic marker and a locus influencing a quantitative trait in a well delineated population is the basis for success in mapping quantitative trait loci (QTL). To detect bovine chromosome 5 (BTA5) birth weight QTL we genotyped 294 F2 Gyr (Bos indicus) x Holstein (Bos taurus) crossbreed cattle for five microsatellite markers. A linkage map was constructed for the markers and an interval analysis for the presence of QTL was performed. The linkage map indicated differences in the order of two markers relative to the reference map (http://www.marc.usda.gov). Interval analysis detected a QTL controlling birth weight (p < 0.01) at 69 centimorgans (cM) from the most centromeric marker with an effect of 0.32 phenotypic standard-error. These results support other studies with crossbred Bos taurus x Bos indicus populations