RESUMO
BACKGROUND: Testicular descent is a physiological process regulated by many factors. Eventually, disturbances in the embryological/fetal development path facilitate the occurrence of scrotal hernia, a congenital malformation characterized by the presence of intestinal portions within the scrotal sac due to the abnormal expansion of the inguinal ring. In pigs, some genes have been related to this anomaly, but the genetic mechanisms involved remain unclear. This study aimed to investigate the expression profile of a set of genes potentially involved with the manifestation of scrotal hernia in the inguinal ring tissue. METHODS AND RESULTS: Tissue samples from the inguinal ring/canal of normal and scrotal hernia-affected male pigs with approximately 30 days of age were used. Relative expression analysis was performed using qPCR to confirm the expression profile of 17 candidate genes previously identified in an RNA-Seq study. Among them, the Myosin heavy chain 1 (MYH1), Desmin (DES), and Troponin 1 (TNNI1) genes were differentially expressed between groups and had reduced levels of expression in the affected animals. These genes encode proteins involved in the formation of muscle tissue, which seems to be important for increasing the resistance of the inguinal ring to the abdominal pressure, which is essential to avoid the occurrence of scrotal hernia. CONCLUSIONS: The downregulation of muscular candidate genes in the inguinal tissue clarifies the genetic mechanisms involved with this anomaly in its primary site, providing useful information for developing strategies to control this malformation in pigs and other mammals.
Assuntos
Regulação para Baixo , Escroto , Animais , Masculino , Suínos/genética , Escroto/metabolismo , Escroto/anormalidades , Escroto/patologia , Regulação para Baixo/genética , Hérnia Inguinal/genética , Hérnia Inguinal/metabolismo , Hérnia Inguinal/veterinária , Perfilação da Expressão Gênica/métodos , Doenças dos Suínos/genética , Doenças dos Suínos/metabolismo , Cadeias Pesadas de Miosina/genética , Cadeias Pesadas de Miosina/metabolismoRESUMO
BACKGROUND: Umbilical Hernia (UH) is characterized by the passage of part of the intestine through the umbilical canal forming the herniary sac. There are several potential causes that can lead to the umbilical hernia such as bacterial infections, management conditions and genetic factors. Since the genetic components involved with UH are poorly understood, this study aimed to identify polymorphisms and genes associated with the manifestation of umbilical hernia in pigs using exome and transcriptome sequencing in a case and control design. RESULTS: In the exome sequencing, 119 variants located in 58 genes were identified differing between normal and UH-affected pigs, and in the umbilical ring transcriptome, 46 variants were identified, located in 27 genes. Comparing the two methodologies, we obtained 34 concordant variants between the exome and transcriptome analyses, which were located in 17 genes, distributed in 64 biological processes (BP). Among the BP involved with UH it is possible to highlight cell adhesion, cell junction regulation, embryonic morphogenesis, ion transport, muscle contraction, within others. CONCLUSIONS: We have generated the first exome sequencing related to normal and umbilical hernia-affected pigs, which allowed us to identify several variants possibly involved with this disorder. Many of those variants present in the DNA were confirmed with the RNA-Seq results. The combination of both exome and transcriptome sequencing approaches allowed us to better understand the complex molecular mechanisms underlying UH in pigs and possibly in other mammals, including humans. Some variants found in genes and other regulatory regions are highlighted as strong candidates to the development of UH in pigs and should be further investigated.
Assuntos
Hérnia Umbilical , Animais , Exoma/genética , Hérnia Umbilical/genética , Hérnia Umbilical/veterinária , Polimorfismo de Nucleotídeo Único , Suínos/genética , Transcriptoma , Sequenciamento do ExomaRESUMO
BACKGROUND: Copy number variations (CNVs) are a major type of structural genomic variants that underlie genetic architecture and phenotypic variation of complex traits, not only in humans, but also in livestock animals. We identified CNVs along the chicken genome and analyzed their association with performance traits. Genome-wide CNVs were inferred from Affymetrix® high density SNP-chip data for a broiler population. CNVs were concatenated into segments and association analyses were performed with linear mixed models considering a genomic relationship matrix, for birth weight, body weight at 21, 35, 41 and 42 days, feed intake from 35 to 41 days, feed conversion ratio from 35 to 41 days and, body weight gain from 35 to 41 days of age. RESULTS: We identified 23,214 autosomal CNVs, merged into 5042 distinct CNV regions (CNVRs), covering 12.84% of the chicken autosomal genome. One significant CNV segment was associated with BWG on GGA3 (q-value = 0.00443); one significant CNV segment was associated with BW35 (q-value = 0.00571), BW41 (q-value = 0.00180) and BW42 (q-value = 0.00130) on GGA3, and one significant CNV segment was associated with BW on GGA5 (q-value = 0.00432). All significant CNV segments were verified by qPCR, and a validation rate of 92.59% was observed. These CNV segments are located nearby genes, such as KCNJ11, MyoD1 and SOX6, known to underlie growth and development. Moreover, gene-set analyses revealed terms linked with muscle physiology, cellular processes regulation and potassium channels. CONCLUSIONS: Overall, this CNV-based GWAS study unravels potential candidate genes that may regulate performance traits in chickens. Our findings provide a foundation for future functional studies on the role of specific genes in regulating performance in chickens.
Assuntos
Galinhas , Variações do Número de Cópias de DNA , Animais , Galinhas/genética , Genoma , Estudo de Associação Genômica Ampla , Humanos , Fenótipo , Polimorfismo de Nucleotídeo ÚnicoRESUMO
BACKGROUND: Bacterial chondronecrosis with osteomyelitis (BCO) develops in the growth plate (GP) of the proximal femur and tibia and is initiated by damage to the less mineralized chondrocytes followed by colonization of opportunistic bacteria. This condition affects approximately 1% of all birds housed, being considered one of the major causes of lameness in fast growing broilers. Although several studies have been previously performed aiming to understand its pathogenesis, the molecular mechanisms involved with BCO remains to be elucidated. Therefore, this study aimed to generate a profile of global differential gene expression involved with BCO in the tibia of commercial broilers, through RNA sequencing analysis to identity genes and molecular pathways involved with BCO in chickens. RESULTS: Our data showed 192 differentially expressed (DE) genes: 63 upregulated and 129 downregulated in the GP of the tibia proximal epiphysis of BCO-affected broilers. Using all DE genes, six Biological Processes (BP) were associated with bone development (connective tissue development, cartilage development, skeletal system development, organ morphogenesis, system development and skeletal system morphogenesis). The analyses of the upregulated genes did not indicate any significant BP (FDR < 0.05). However, with the downregulated genes, the same BP were identified when using all DE genes in the analysis, with a total of 26 coding genes explaining BCO in the tibia: ACAN, ALDH1A2, CDH7, CHAD, CHADL, COL11A1, COMP, CSGALNACT1, CYR61, FRZB, GAL3ST1, HAPLN1, IHH, KIF26B, LECT1, LPPR1, PDE6B, RBP4A, SERINC5, SFRP1, SOX8, SOX9, TENM2, THBS1, UCHL1 and WFIKKN2. In addition, seven transcription factors were also associated to BCO: NFATC2, MAFB, HIF1A-ARNT, EWSR1-FLI1, NFIC, TCF3 and NF-KAPPAB. CONCLUSIONS: Our data show that osteochondral downregulated genes are potential molecular causes of BCO in broilers, and the bacterial process seems to be, in fact, a secondary condition. Sixteen genes responsible for bone and cartilage formation were downregulated in BCO-affected broilers being strong candidate genes to trigger this disorder.
Assuntos
Infecções Bacterianas/veterinária , Galinhas/genética , Osteogênese/genética , Osteomielite/veterinária , Doenças das Aves Domésticas/genética , Tíbia/patologia , Animais , Infecções Bacterianas/genética , Condrócitos , Regulação para Baixo , Expressão Gênica , Ontologia Genética , Masculino , Osteomielite/genética , Osteomielite/microbiologia , Doenças das Aves Domésticas/microbiologia , RNA-SeqRESUMO
The normalization with proper reference genes is a crucial step to obtain accurate mRNA expression levels in quantitative PCR (qPCR) studies. Therefore, in this study, 10 reference candidate genes were evaluated to determine their stability in normal pectoralis major muscle of broilers and those counterparts affected with White Striping (WS) myopathy at 42 days age. Four different tools were used for ranking the most stable genes: GeNorm, NormFinder, BestKeeper and Comparative Ct (ΔCt), and a general ranking was performed using the RankAggreg tool to select the best reference genes among all tools. From the 10 genes evaluated in the breast muscle of broilers, 8 were amplified. Most of the algorithms/tools indicated the same two genes, RPL30 and RPL5, as the most stable in the broilers breast muscle. In addition, there was agreement among the tools for the least stable genes: MRPS27, GAPDH and RPLP1 in the broilers breast muscle. Therefore, it is interesting to note that even with different tools for evaluating gene expression, there was consensus on the most and least stable genes. These results indicate that the Ribosomal protein L30 (RPL30) and Ribosomal protein L5 (RPL5) can be recommended for accurate normalization in qPCR studies with chicken pectoralis major muscle affected with White Striping and other myopathies.
Assuntos
Galinhas/genética , Perfilação da Expressão Gênica/normas , Genes Essenciais/genética , Doenças Musculares/genética , Músculos Peitorais/metabolismo , Reação em Cadeia da Polimerase em Tempo Real/normas , Animais , Expressão Gênica , Perfilação da Expressão Gênica/métodos , Regulação da Expressão Gênica , Doenças Musculares/metabolismo , Doenças Musculares/veterinária , Doenças das Aves Domésticas/genética , Doenças das Aves Domésticas/metabolismo , Reação em Cadeia da Polimerase em Tempo Real/métodos , Padrões de ReferênciaRESUMO
BACKGROUND: Excess fat content in chickens has a negative impact on poultry production. The discovery of QTL associated with fat deposition in the carcass allows the identification of positional candidate genes (PCGs) that might regulate fat deposition and be useful for selection against excess fat content in chicken's carcass. This study aimed to estimate genomic heritability coefficients and to identify QTLs and PCGs for abdominal fat (ABF) and skin (SKIN) traits in a broiler chicken population, originated from the White Plymouth Rock and White Cornish breeds. RESULTS: ABF and SKIN are moderately heritable traits in our broiler population with estimates ranging from 0.23 to 0.33. Using a high density SNP panel (355,027 informative SNPs), we detected nine unique QTLs that were associated with these fat traits. Among these, four QTL were novel, while five have been previously reported in the literature. Thirteen PCGs were identified that might regulate fat deposition in these QTL regions: JDP2, PLCG1, HNF4A, FITM2, ADIPOR1, PTPN11, MVK, APOA1, APOA4, APOA5, ENSGALG00000000477, ENSGALG00000000483, and ENSGALG00000005043. We used sequence information from founder animals to detect 4843 SNPs in the 13 PCGs. Among those, two were classified as potentially deleterious and two as high impact SNPs. CONCLUSIONS: This study generated novel results that can contribute to a better understanding of fat deposition in chickens. The use of high density array of SNPs increases genome coverage and improves QTL resolution than would have been achieved with low density. The identified PCGs were involved in many biological processes that regulate lipid storage. The SNPs identified in the PCGs, especially those predicted as potentially deleterious and high impact, may affect fat deposition. Validation should be undertaken before using these SNPs for selection against carcass fat accumulation and to improve feed efficiency in broiler chicken production.
Assuntos
Tecido Adiposo/metabolismo , Galinhas/genética , Galinhas/metabolismo , Estudo de Associação Genômica Ampla , Genômica , Animais , Fenótipo , Polimorfismo de Nucleotídeo Único , Locos de Características Quantitativas/genéticaRESUMO
Femoral head separation (FHS) is usually a subclinical condition characterized by the detachment of articular cartilage from the bone. In this study, a comprehensive analysis identifying shared and exclusive expression profiles, biological processes (BP) and variants related to FHS in the femoral articular cartilage and growth plate in chickens was performed through RNA sequencing analysis. Thirty-six differentially expressed (DE) genes were shared between femoral articular cartilage (AC) and growth plate (GP) tissues. Out of those, 23 genes were enriched in BP related to ion transport, translation factors and immune response. Seventy genes were DE exclusively in the AC and 288 in the GP. Among the BP of AC, the response against bacteria can be highlighted, and for the GP tissue, the processes related to chondrocyte differentiation and cartilage development stand out. When the chicken DE genes were compared to other datasets, eight genes (SLC4A1, RHAG, ANK1, MKNK2, SPTB, ADA, C7 and EPB420) were shared between chickens and humans. Furthermore, 89 variants, including missense in the SPATS2L, PRKAB1 and TRIM25 genes, were identified between groups. Therefore, those genes should be more explored to validate them as candidates to FHS/FHN in chickens and humans.
RESUMO
Femoral head necrosis (FHN) is one of the most common conditions in fast growing broilers, being characterized by separation of articular cartilage from epiphysis and classified as femoral head separation (FHS) or FHS with laceration (FHSL) depending on severity. Although molecular mechanisms involved with this disorder have been observed, its etiology is still unclear. Therefore, the expression of 15 candidate genes, chosen based on previous transcriptomic studies, was evaluated in the articular cartilage (AC) of normal and FHS-affected broilers at 21 days of age. Samples were collected based on the absence or presence of FHS for physical-chemical and qPCR analysis. The AvBD2, RHAG, COL28A1, ADA and ANGPTL7 were upregulated in FHS-affected broilers compared to the healthy group. These genes are involved in immune response, defense against pathogens, inflammation, cellular migration and adhesion, indicating different molecular mechanisms to control FHS progression at early age. Our results can contribute to improve the knowledge on FHN etiology in chickens and other species, such as horse and pigs that are severely affected by bone disorders.
Assuntos
Cartilagem Articular , Necrose da Cabeça do Fêmur , Doenças dos Cavalos , Doenças das Aves Domésticas , Doenças dos Suínos , Animais , Cartilagem Articular/metabolismo , Galinhas/genética , Galinhas/metabolismo , Cabeça do Fêmur/metabolismo , Necrose da Cabeça do Fêmur/genética , Necrose da Cabeça do Fêmur/metabolismo , Necrose da Cabeça do Fêmur/veterinária , Doenças dos Cavalos/metabolismo , Cavalos , Doenças das Aves Domésticas/genética , Suínos , Doenças dos Suínos/metabolismoRESUMO
Femoral head separation (FHS) is characterized by the detachment of growth plate (GP) and articular cartilage, occurring in tibia and femur. However, the molecular mechanisms involved with this condition are not completely understood. Therefore, genes and biological processes (BP) involved with FHS were identified in 21-day-old broilers through RNA sequencing of the femoral GP. 13,487 genes were expressed in the chicken femoral head transcriptome of normal and FHS-affected broilers. From those, 34 were differentially expressed (DE; FDR ≤0.05) between groups, where all of them were downregulated in FHS-affected broilers. The main BP were enriched in receptor signaling pathways, ossification, bone mineralization and formation, skeletal morphogenesis, and vascularization. RNA-Seq datasets comparison of normal and FHS-affected broilers with 21, 35 and 42 days of age has shown three shared DE genes (FBN2, C1QTNF8, and XYLT1) in GP among ages. Twelve genes were exclusively DE at 21 days, where 10 have already been characterized (SHISA3, FNDC1, ANGPTL7, LEPR, ENSGALG00000049529, OXTR, ENSGALG00000045154, COL16A1, RASD2, BOC, GDF10, and THSD7B). Twelve SNPs were associated with FHS (p < 0.0001). Out of those, 5 were novel and 7 were existing variants located in 7 genes (RARS, TFPI2, TTI1, MAP4K3, LINK54, and AREL1). We have shown that genes related to chondrogenesis and bone differentiation were downregulated in the GP of FHS-affected young broilers. Therefore, these findings evince that candidate genes pointed out in our study are probably related to the onset of FHS in broilers.
RESUMO
Chicken feed efficiency (FE) traits are the most important economic traits in broiler production. Several studies evaluating genetic factors affecting food consumption in chickens are available. However, most of these studies identified genomic regions containing putative quantitative trait loci for each trait separately. It is still a challenge to find common gene networks related to these traits. Therefore, here, a genome-wide association study (GWAS) was conducted to explore candidate genomic regions responsible for Feed Intake (FI), Body Weight Gain (BWG) and Feed Conversion Ratio (FCR) traits and their gene networks. A total of 1430 broilers from an experimental population was genotyped with the high density Affymetrix 600K SNP array. A total of 119 associated SNPs located in 20 chromosomes were identified, where some of them were common in more than one FE trait. In addition, novel genomic regions were prospected considering the SNPs dominance effects and sex interaction, identifying putative candidate genes only when these effects were fit in the model. Relevant candidate genes such as ATRNL1, PIK3C2A, PTPRN2, SORCS3 and gga-mir-1759 were highlighted in this study helping to elucidate the genomic architecture of feed efficiency traits. These results provide new insights on the mechanisms underlying the consumption and utilization of food in chickens.
Assuntos
Galinhas/fisiologia , Comportamento Alimentar , Animais , Galinhas/genética , Estudo de Associação Genômica Ampla/veterinária , Aumento de Peso/genéticaRESUMO
Hernia is one of the most common defects in pigs. The most prevalent are the scrotal (SH), inguinal (IH) and umbilical (UH) hernias. We compared the inguinal ring transcriptome of normal and SH-affected pigs with the umbilical ring transcriptome of normal and UH-affected pigs to discover genes and pathways involved with the development of both types of hernia. A total of 13,307 transcripts was expressed in the inguinal and 13,302 in the umbilical ring tissues with 94.91% of them present in both tissues. From those, 35 genes were differentially expressed in both groups, participating in 108 biological processes. A total of 67 polymorphisms was identified in the inguinal ring and 76 in the umbilical ring tissue, of which 11 and 14 were novel, respectively. A single nucleotide polymorphism (SNP) with deleterious function was identified in the integrin α M (ITGAM) gene. The microtubule associated protein 1 light chain 3 γ (MAP1LC3C), vitrin (VIT), aggrecan (ACAN), alkaline ceramidase 2 (ACER2), potassium calcium-activated channel subfamily M α 1 (KCNMA1) and synaptopodin 2 (SYNPO2) genes are highlighted as candidates to trigger both types of hernia. We generated the first comparative study of the pig umbilical and inguinal ring transcriptomes, contributing to the understanding of the genetic mechanism involved with these two types of hernia in pigs and probably in other mammals.
Assuntos
Predisposição Genética para Doença , Hérnia Umbilical/veterinária , Hérnia Ventral/veterinária , Polimorfismo de Nucleotídeo Único , Escroto/patologia , Doenças dos Suínos/genética , Animais , Biópsia , Mapeamento Cromossômico , Perfilação da Expressão Gênica , Redes Reguladoras de Genes , Imuno-Histoquímica , Masculino , Suínos , TranscriptomaRESUMO
The use of reference genes is required for relative quantification in gene expression analysis and the stability of these genes can be variable depending on the experimental design. Therefore, it is indispensable to test the reliability of endogenous genes previously to their use. This study evaluated nine candidate reference genes to select the most stable genes to be used as reference in gene expression studies with the femoral cartilage of normal and epiphysiolysis-affected broilers. The femur articular cartilage of 29 male broilers with 35 days of age was collected, frozen and further submitted to RNA extraction and quantitative PCR (qPCR) analysis. The candidate reference genes evaluated were GAPDH, HMBS, HPRT1, MRPS27, MRPS30, RPL30, RPL4, RPL5, and RPLP1. For the gene stability evaluation, three software were used: GeNorm, BestKeeper and NormFinder, and a global ranking was generated using the function RankAggreg. In this study, the RPLP1 and RPL5 were the most reliable endogenous genes being recommended for expression studies with femur cartilage in broilers with epiphysiolysis and possible other femur anomalies.
Assuntos
Doenças das Aves/genética , Cartilagem Articular/metabolismo , Galinhas/genética , Epifise Deslocada/veterinária , Algoritmos , Animais , Doenças das Aves/metabolismo , Galinhas/metabolismo , Epifise Deslocada/genética , Epifise Deslocada/metabolismo , Fêmur , Expressão Gênica , Perfilação da Expressão Gênica/estatística & dados numéricos , Perfilação da Expressão Gênica/veterinária , Masculino , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Reação em Cadeia da Polimerase em Tempo RealRESUMO
Umbilical hernia (UH) is one of the most frequent defects affecting pig production, however, it also affects humans and other mammals. UH is characterized as an abnormal protrusion of the abdominal contents to the umbilical region, causing pain, discomfort and reduced performance in pigs. Some genomic regions associated to UH have already been identified, however, no study involving RNA sequencing was performed when umbilical tissue is considered. Therefore, here, we have sequenced the umbilical ring transcriptome of five normal and five UH-affected pigs to uncover genes and pathways involved with UH development. A total of 13,216 transcripts were expressed in the umbilical ring tissue. From those, 230 genes were differentially expressed (DE) between normal and UH-affected pigs (FDR <0.05), being 145 downregulated and 85 upregulated in the affected compared to the normal pigs. A total of 68 significant biological processes were identified and the most relevant were extracellular matrix, immune system, anatomical development, cell adhesion, membrane components, receptor activation, calcium binding and immune synapse. The results pointed out ACAN, MMPs, COLs, EPYC, VIT, CCBE1 and LGALS3 as strong candidates to trigger umbilical hernias in pigs since they act in the extracellular matrix remodeling and in the production, integrity and resistance of the collagen. We have generated the first transcriptome of the pig umbilical ring tissue, which allowed the identification of genes that had not yet been related to umbilical hernias in pigs. Nevertheless, further studies are needed to identify the causal mutations, SNPs and CNVs in these genes to improve our understanding of the mechanisms of gene regulation.
Assuntos
Hérnia Umbilical/veterinária , Doenças dos Suínos/genética , Animais , Perfilação da Expressão Gênica/veterinária , Predisposição Genética para Doença/genética , Hérnia Umbilical/genética , Reação em Cadeia da Polimerase , Locos de Características Quantitativas/genética , Análise de Sequência de DNA/veterinária , Suínos/genéticaRESUMO
Scrotal hernias (SH) are common congenital defects in commercial pigs, characterized by the presence of abdominal contents in the scrotal sac, leading to considerable production and animal welfare losses. Since the etiology of SH remains obscure, we aimed to identify the biological and genetic mechanisms involved in its occurrence through the whole transcriptome analysis of SH affected and unaffected pigs' inguinal rings. From the 22,452 genes annotated in the pig reference genome, 13,498 were expressed in the inguinal canal tissue. Of those, 703 genes were differentially expressed (DE, FDR < 0.05) between the two groups analyzed being, respectively, 209 genes upregulated and 494 downregulated in the SH-affected group. Thirty-seven significantly overrepresented GO terms related to SH were enriched, and the most relevant biological processes were muscular system, cell differentiation, sarcome reorganization, and myofibril assembly. The calcium signaling, hypertrophic cardiomyopathy, dilated cardiomyopathy, and cardiac muscle contraction were the major pathways possibly involved in the occurrence of the scrotal hernias. The expression profile of the DE genes was associated with the reduction of smooth muscle differentiation, followed by low calcium content in the cell, which could lead to a decreased apoptosis ratio and diminished muscle contraction of the inguinal canal region. We have demonstrated that genes involved with musculature are closely linked to the physiological imbalance predisposing to scrotal hernia. According to our study, the genes MYBPC1, BOK, SLC25A4, SLC8A3, DES, TPM2, MAP1CL3C, and FGF1 were considered strong candidates for future evaluation.
Assuntos
Hérnia Inguinal/genética , Canal Inguinal/fisiopatologia , Transcriptoma/genética , Animais , Sequência de Bases/genética , Perfilação da Expressão Gênica/métodos , Genoma/genética , Hérnia Inguinal/fisiopatologia , Canal Inguinal/fisiologia , Masculino , Escroto/metabolismo , Escroto/fisiopatologia , Análise de Sequência de RNA/métodos , Suínos , Doenças dos Suínos , Sequenciamento do Exoma/métodosRESUMO
A new methodology is proposed for ractopamine residue analysis in pork. It consists of enzyme-mediated digestion and deconjugation steps; modified quick, easy, cheap, effective, rugged, and safe (QuEChERS) extraction; and liquid chromatography-tandem mass spectrometry (LC-MS/MS). In brief, the samples were digested with protease and then deconjugated with ß-glucuronidase enzyme; they were then subjected to extraction and cleanup by QuEChERS and underwent sequential analysis by LC-MS/MS. The method performance was evaluated in accordance to the validation guidelines regulated by the Brazilian Ministry of Agriculture and Food Supply. The limit of detection was 0.15 µg/kg and limit of quantification was 0.5 µg/kg. When the method was applied to real samples, ractopamine residue was found in concentrations (up to 7.86 µg/kg) below international recommendation limits up to 10 µg/kg. The method is sensitive, accurate, quick, simple, and suitable for routine analysis; therefore, it is a monitoring tool that may be adopted by laboratories to achieve compliance levels.
Assuntos
Cromatografia Líquida/métodos , Resíduos de Drogas/análise , Fenetilaminas/isolamento & purificação , Carne Vermelha , Espectrometria de Massas em Tandem , Animais , Brasil , Cromatografia Líquida de Alta Pressão , Limite de Detecção , Carne Vermelha/análise , Reprodutibilidade dos Testes , Sensibilidade e Especificidade , Suínos , Espectrometria de Massas em Tandem/métodosRESUMO
The use of reference genes is required for relative quantification in gene expression analysis and since the stability of these genes could be variable depending on the experimental design, it has become indispensable to test the reliability of endogenous genes. Therefore, this study evaluated 10 reference candidate genes in two different experimental conditions in order to obtain stable genes to be used as reference in expression studies related to scrotal hernias in pigs. Two independent experiments were performed: one with 30 days-old MS115 pigs and the other with 60 days-old Landrace pigs. The inguinal ring/canal was collected, frozen and further submitted to real-time PCR analysis (qPCR). For the reference genes stability evaluation, four tools were used: GeNorm in the SLqPCR, BestKeeper, NormFinder and Comparative CT. A general ranking was generated using the BruteAggreg function of R environment. In this study, the RPL19 was one of the most reliable endogenous genes for both experiments. The breed/age effects influenced the expression stability of candidate reference genes evaluated in the inguinal ring of pigs. Therefore, this study reinforces the importance of evaluating the stability of several endogenous genes previous their use, since a consensual set of reference genes is not easily obtained. Here, two sets of genes are recommended: RPL19, RPL32 and H3F3A for 30-days MS115 and PPIA and RPL19 for the 60 days-old Landrace pigs. This is the first study using the inguinal ring tissue and the results can be useful as an indicative for other studies working with gene expression in this tissue.
Assuntos
Perfilação da Expressão Gênica/normas , Hérnia/genética , Canal Inguinal/patologia , Animais , Padrões de Referência , SuínosRESUMO
Apolipoprotein B (APOB) and Adiponectin Receptor 1 (ADIPOR1) are related to the regulation of feed intake, fat metabolism and protein deposition and are candidate genes for genomic studies in birds. In this study, associations of two single nucleotide polymorphisms (SNPs) g.102A>T (APOB) and g.729C>T (ADIPOR1) with carcass, bone integrity and performance traits in broilers were investigated. Genotyping was performed on a paternal line of 1,454 broilers. The SNP detection was carried out by PCR-RFLP technique using the restriction enzymes HhaI for the SNP g.729C>T and MslI for the SNP g.102A>T. The association analyses of the two SNPs with 85 traits were performed using the restricted maximum likelihood (REML) and Generalized Quasi-Likelihood Score (GQLS) methods. For REML the model included the random additive genetic effect of animal and fixed effects of sex, hatch and SNP genotypes. In the GQLS method, a logistic regression was used to associate the genotypes with phenotypes adjusted for fixed effects of sex and hatch. The SNP g.729C>T in the ADIPOR1 gene was associated with thickness of the femur and breast skin yield. Thus, the ADIPOR1 gene seems implicated in the metabolism and/or fat deposition and bone integrity in broilers.
Assuntos
Tecido Adiposo/anatomia & histologia , Apolipoproteínas B/genética , Distribuição da Gordura Corporal , Peso Corporal/genética , Galinhas/anatomia & histologia , Galinhas/genética , Locos de Características Quantitativas , Receptores de Adiponectina/genética , Animais , Galinhas/metabolismo , Fêmur/anatomia & histologia , Frequência do Gene/genética , Marcadores Genéticos/genética , Polimorfismo de Nucleotídeo Único/genéticaRESUMO
A genome-wide association study for immune response to influenza vaccination in a crossbred swine population was conducted. Swine influenza is caused by influenza A virus (FLUAV) which is considered one of the most prevalent respiratory pathogens in swine worldwide. The main strategy used to control influenza in swine herds is through vaccination. However, the currently circulating FLUAV subtypes in swine are genetically and antigenically diverse and their interaction with the host genetics poses a challenge for the production of efficacious and cross-protective vaccines. In this study, 103 pigs vaccinated with an inactivated H1N1 pandemic virus were genotyped with the Illumina PorcineSNP60V2 BeadChip for the identification of genetic markers associated with immune response efficacy to influenza A virus vaccination. Immune response was measured based on the presence or absence of HA (hemagglutinin) and NP (nucleoprotein) antibodies induced by vaccination and detected in swine sera by the hemagglutination inhibition (HI) and ELISA assays, respectively. The ELISA test was also used as a measurement of antibody levels produced following the FLUAV vaccination. Associations were tested with x(2) test for a case and control data and using maximum likelihood method for the quantitative data, where a moderate association was considered if p<5×10(-5). When testing the association using the HI results, three markers with unknown location and three located on chromosomes SSCX, SSC14 and SSC18 were identified as associated with the immune response. Using the response to vaccination measured by ELISA as a qualitative and quantitative phenotype, four genomic regions were associated with immune response: one on SSC12 and three on chromosomes SSC1, SSC7, and SSC15, respectively. Those regions harbor important functional candidate genes possibly involved with the degree of immune response to vaccination. These results show an important role of host genetics in the immune response to influenza vaccination. Genetic selection for pigs with better response to FLUAV vaccination might be an alternative to reduce the impact of influenza virus infection in the swine industry. However, these results should to be validated in additional populations before its use.
Assuntos
Estudo de Associação Genômica Ampla , Interações Hospedeiro-Patógeno , Vírus da Influenza A Subtipo H1N1/imunologia , Vacinas contra Influenza/imunologia , Animais , Anticorpos Antivirais/sangue , Ensaio de Imunoadsorção Enzimática , Feminino , Testes de Inibição da Hemaglutinação , Glicoproteínas de Hemaglutininação de Vírus da Influenza/imunologia , Vacinas contra Influenza/administração & dosagem , Masculino , Proteínas do Nucleocapsídeo , Proteínas de Ligação a RNA/imunologia , Suínos , Vacinas de Produtos Inativados/administração & dosagem , Vacinas de Produtos Inativados/imunologia , Proteínas do Core Viral/imunologiaRESUMO
The PSS genotypes of 596 F2 pigs produced by initial mating of Brazilian native boars commercial sows and were characterized by PCR-RFLP and their carcass and performance traits were evaluated. Among the 596 animals analyzed, 493 (82.72 percent) were characterized as NN and 103 (17.28 percent) as Nn. With respect to carcass traits, Nn animals presented higher (p < 0.05) right half carcass weight, left half carcass weight, loin depth and loin eye area, and lower shoulder backfat thickness, backfat thickness between last and next to last but one lumbar vertebrae and backfat thickness after last rib at 6.5 cm from the midline compared to NN animals. Nn animals also showed (p < 0.05) higher values for most of the cut yields, indicating higher cutting yields for animals carrying the n allele and lower values for bacon depth, confirming lower fat deposition in carcass. In addition, Nn animals presented (p < 0.05) lower values for the performance trait weight at 105 days of age. These results indicate that animals carrying the PSS gene generate leaner carcasses, higher cut yields, and that the effects of the gene can be observed even in divergent crosses.