Development of a sandwich enzyme-linked immunosorbent assay based on single-domain antibody for detecting goose parvovirus infection / [Desenvolvimento de um ensaio imunoadsorvente ligado a enzima sandwich baseado em anticorpo de domínio único para detecção de infecção por parvovírus de ganso]

A infecção por parvovírus de ganso (GPV) é uma doença infecciosa altamente patogênica em gansinhos e patinhos de Muscovy. Para detectar o antígeno GPV, desenvolvemos um novo ensaio imunoenzimático (ELISA) sanduíche usando um anticorpo de domínio único de cadeia pesada específico contra a proteína GPV NS1 como anticorpo de detecção. Os limites de detecção da proteína GST-NS1 e do título do vírus da cepa GPV H1 foram 5 ng/mL e 102,9 TCID50/mL, respectivamente. O ELISA sanduíche foi específico para GPV sem reatividade cruzada com outros vírus comuns de ganso, incluindo vírus Tembusu, circovírus de ganso, adenovírus de aves, vírus Newcastle ou vírus H9 da gripe aviária. Um total de 118 amostras de swab cloacal foram usadas para detectar o antígeno GPV usando o ELISA sanduíche e reação em cadeia da polimerase com uma taxa de coincidência de 91,5%. A sensibilidade e especificidade do ELISA sanduíche foram de 91,6% e 91,4%, respectivamente. Esses resultados sugerem que este ELISA sanduíche pode ser aplicado para detectar GPV.

Study on the Major Genes Related with Fat Deposition in Liver and Abdominal Fat of Different Breeds of Chicken

Fat deposition is higher in fast growing chickens than in slow growing chickens. The liver is the major organ for lipogenesis and fat deposition in chickens, although genetic background, age, and gender also influence fat deposition. In the present study, we aimed to explore the molecular mechanisms underlying fat deposition in liver and abdominal fat. We determined the expression abundances of the key genes regulating fat metabolism in fast-growing (FG) broilers (Cobb) and slow-growing (SG) broilers (HS1) and found that ACC, FAS, PGC-1α, PPARγ, SREBP-1c and PLIN1genes were expressed in the abdominal fat and liver tissues of FG and SG. ANOVA analysis showed that the breed, age, and tissue factors influenced the expressions of ACC, FAS, PGC-1α, PPARγ, SREBP-1c, and PLIN1 genes in the liver and abdominal fat of FG and SG. Also, the expressions of PPARγ and PLIN1 in the liver of SG were higher than that of FG. The results suggest that the differences in adipocyte development and adipose deposition between breeds are due to genetic factors.(AU)

Expression Pattern of Sulf1 and Sulf2 in Chicken Tissues and Characterization of Their Expression During Different Periods in Skeletal Muscle Satellite Cells

Heparan sulfate proteoglycans (HSPGs) are present on the cell surface and in the extracellular matrix in all metazoans. HSPGs interact with growth factors and receptors through heparan sulfate (HS) chains. The sulfation pattern of heparan sulfate chains influences signaling events mediated by heparan sulfate proteoglycans located on the cell surface. SULF1 and SULF2 are two endo-sulfatases that can cleave specific 6-O-sulfate groups within the heparan chains. To determine their possible roles in tissues and satellite cells in vitro, their expression pattern was examined in tissues from 40-day-old chickens and in satellite cells from the breast muscles of 1-week-old and 2-week-old chickens using RT-PCR and immunocytochemistry analyses. The SULF1 and SULF2 transcripts were widely distributed in various tissues. Upon increasing culture times in chicken´s primary skeletal muscle satellite cells, SULF1 and SULF2 expression in 1-week-old chickens was significantly higher than in 2-week-old chickens, suggesting that sulfatases play a key role in satellite cell development. Therefore, our findings increase our knowledge of sulfatase expression diversity and provide a solid basis for further research concerning this molecular mechanism.(AU)

Expression Pattern of Sulf1 and Sulf2 in Chicken Tissues and Characterization of Their Expression During Different Periods in Skeletal Muscle Satellite Cells

Heparan sulfate proteoglycans (HSPGs) are present on the cell surface and in the extracellular matrix in all metazoans. HSPGs interact with growth factors and receptors through heparan sulfate (HS) chains. The sulfation pattern of heparan sulfate chains influences signaling events mediated by heparan sulfate proteoglycans located on the cell surface. SULF1 and SULF2 are two endo-sulfatases that can cleave specific 6-O-sulfate groups within the heparan chains. To determine their possible roles in tissues and satellite cells in vitro, their expression pattern was examined in tissues from 40-day-old chickens and in satellite cells from the breast muscles of 1-week-old and 2-week-old chickens using RT-PCR and immunocytochemistry analyses. The SULF1 and SULF2 transcripts were widely distributed in various tissues. Upon increasing culture times in chicken´s primary skeletal muscle satellite cells, SULF1 and SULF2 expression in 1-week-old chickens was significantly higher than in 2-week-old chickens, suggesting that sulfatases play a key role in satellite cell development. Therefore, our findings increase our knowledge of sulfatase expression diversity and provide a solid basis for further research concerning this molecular mechanism.

Expression of Myostatin (Mstn) and Myogenin (Myog) Genes in Zi And Rhine Goose and Their Correlation with Carcass Traits

The aim of this study was to investigate the effects of Myostatin (MSTN) and MyoGenin (MyoG) on goose skeletal muscle growth. In this study, MSTN and MyoG gene expression in breast and leg muscle of Zi and Rhine goose were detected by Real-time Polymerase Chain Reaction (PCR), and the correlations between genes expression levels and carcass traits were investigated. The results showed that the breast muscle weight and breast muscle percentage of Rhine goose were significantly higher than Zi goose (p 0.01). MSTN mRNA and MyoG mRNA expression in breast muscle of Zi goose were significantly higher than that of Rhine goose and the level of MSTN in leg muscle of Rhine was significantly higher than that of Zi goose (p 0.01). There was a significant difference between MSTN mRNA expression in breast muscle and in leg muscle of Zi goose (p 0.01). MSTN mRNA expression in leg muscle was significantly higher than that of breast muscle of Rhine goose (p 0.05). There was a significant difference between MyoG mRNA expression in breast muscle and in leg muscle of Zi goose and Rhine goose (p 0.01). There was a negative correlation between MSTN mRNA expression in breast muscle and body weight, breast muscle weight and breast muscle percentage.(AU)

Expression of Myostatin (Mstn) and Myogenin (Myog) Genes in Zi And Rhine Goose and Their Correlation with Carcass Traits

The aim of this study was to investigate the effects of Myostatin (MSTN) and MyoGenin (MyoG) on goose skeletal muscle growth. In this study, MSTN and MyoG gene expression in breast and leg muscle of Zi and Rhine goose were detected by Real-time Polymerase Chain Reaction (PCR), and the correlations between genes expression levels and carcass traits were investigated. The results showed that the breast muscle weight and breast muscle percentage of Rhine goose were significantly higher than Zi goose (p 0.01). MSTN mRNA and MyoG mRNA expression in breast muscle of Zi goose were significantly higher than that of Rhine goose and the level of MSTN in leg muscle of Rhine was significantly higher than that of Zi goose (p 0.01). There was a significant difference between MSTN mRNA expression in breast muscle and in leg muscle of Zi goose (p 0.01). MSTN mRNA expression in leg muscle was significantly higher than that of breast muscle of Rhine goose (p 0.05). There was a significant difference between MyoG mRNA expression in breast muscle and in leg muscle of Zi goose and Rhine goose (p 0.01). There was a negative correlation between MSTN mRNA expression in breast muscle and body weight, breast muscle weight and breast muscle percentage.

Detection of Snps in the Melanocortin 1-Receptor (MC1R) and Its Association with Shank Color Trait in Hs Chicken

The melanocortin 1 receptor (MC1R) gene plays a key role in controlling the deposition of melanin. In mammals, the MC1Rgene is regarded as a major candidate gene in the control of melanin formation. In domestic animals, the MC1R gene mainly controls the expression of coat, skin, and plumage color in mammals and birds. In order to breed chickens with dark-green shank faster, we screened the molecular markers for shank color in a HS chicken population by exploring the relationship between polymorphism of the MC1R gene and three different shank colors (light green, dark green and yellow). Two primer pairs for code region of the MC1R gene were designed in the basic of chicken genomic sequence. DNA sequencing was performed to detect the polymorphisms and PCR was used to amplify DNA fragment. Sequences analysis indicated that 7 SNPs were predominant the three HS chicken populations with different shank color, including g.18,287,945C>T, g.18,288,088T>C, g.18,288,150G>A, g.18,288,303A>G, g.18,288,512G>A, g.18,288,513T>C, and g.18,288,520A>C. Association analysis revealed that the dark-green shank population showed moderate polymorphism, whereas the light-green shank population showed low polymorphism among overall 7 SNPs and that SNP6 (g.18,288,513T>C) may be significantly associated with three different shank colors in HS chickens. The haplotype CTGGACA had the largest haplotype frequencies, accounting for 56.22%, and the haplotype combination H1H1 is mainly distributed in the dark-green shank population, and may be used as molecular maker for marker-assisted selection of shank color in HS chickens.(AU)

Detection of Snps in the Melanocortin 1-Receptor (MC1R) and Its Association with Shank Color Trait in Hs Chicken

The melanocortin 1 receptor (MC1R) gene plays a key role in controlling the deposition of melanin. In mammals, the MC1Rgene is regarded as a major candidate gene in the control of melanin formation. In domestic animals, the MC1R gene mainly controls the expression of coat, skin, and plumage color in mammals and birds. In order to breed chickens with dark-green shank faster, we screened the molecular markers for shank color in a HS chicken population by exploring the relationship between polymorphism of the MC1R gene and three different shank colors (light green, dark green and yellow). Two primer pairs for code region of the MC1R gene were designed in the basic of chicken genomic sequence. DNA sequencing was performed to detect the polymorphisms and PCR was used to amplify DNA fragment. Sequences analysis indicated that 7 SNPs were predominant the three HS chicken populations with different shank color, including g.18,287,945C>T, g.18,288,088T>C, g.18,288,150G>A, g.18,288,303A>G, g.18,288,512G>A, g.18,288,513T>C, and g.18,288,520A>C. Association analysis revealed that the dark-green shank population showed moderate polymorphism, whereas the light-green shank population showed low polymorphism among overall 7 SNPs and that SNP6 (g.18,288,513T>C) may be significantly associated with three different shank colors in HS chickens. The haplotype CTGGACA had the largest haplotype frequencies, accounting for 56.22%, and the haplotype combination H1H1 is mainly distributed in the dark-green shank population, and may be used as molecular maker for marker-assisted selection of shank color in HS chickens.

Molecular authentication of meats from three terrestrial birds based on PCR-RFLP analysis of the mitochondrial 12S rRNA gene

In this study, a method utilizing PCR-restriction fragment length polymorphism (PCR-RFLP) of a mitochondrial gene was developed for the identification of chicken (Gallus gallus), quail (Coturnix coturnix), and common pigeon (Columba livia) meat. PCR products of ~440 bp were obtained from the 12S rRNA gene of these three birds using a pair of universal primers. The three terrestrial birds can be distinguished using one restriction endonuclease, Alu I, which was selected based on species-specific variations in the mt 12S rRNA gene sequence using 9 newly-obtained and 44 published chicken, quail and pigeon sequences. This method was also successfully used to identify commercial quail and pigeon meat products, which were found to be adulterated with chicken meat. Additionally, our method had relatively high sensitivity for detecting a meat mixture. Ten percent of chicken meat in the mixed quail and pigeon sample was detectable. This assay can be useful for the accurate identification of meats from terrestrial birds, avoiding mislabeling or fraudulent species substitution in meat products.(AU)

Molecular authentication of meats from three terrestrial birds based on PCR-RFLP analysis of the mitochondrial 12S rRNA gene

In this study, a method utilizing PCR-restriction fragment length polymorphism (PCR-RFLP) of a mitochondrial gene was developed for the identification of chicken (Gallus gallus), quail (Coturnix coturnix), and common pigeon (Columba livia) meat. PCR products of ~440 bp were obtained from the 12S rRNA gene of these three birds using a pair of universal primers. The three terrestrial birds can be distinguished using one restriction endonuclease, Alu I, which was selected based on species-specific variations in the mt 12S rRNA gene sequence using 9 newly-obtained and 44 published chicken, quail and pigeon sequences. This method was also successfully used to identify commercial quail and pigeon meat products, which were found to be adulterated with chicken meat. Additionally, our method had relatively high sensitivity for detecting a meat mixture. Ten percent of chicken meat in the mixed quail and pigeon sample was detectable. This assay can be useful for the accurate identification of meats from terrestrial birds, avoiding mislabeling or fraudulent species substitution in meat products.

Effect of Feeding Conditions on the Methylation Status of Fatp1 Gene in Chicken Breast Muscle

The objective of this work was to investigate the effect of feeding conditions on methylation status of FATP1 gene, which is an important candidate gene of Intramuscular fat and important indicator of chicken meat quality. We selected Daninghe (DNH) and Qingjiaoma (QJM) chickens under scatter-feeding and captivity-feeding conditions as experimental animals, and detected the methylation status of FATP1 genes in chicken breast muscle using Bisulfite Sequencing PCR method. The results showed that the methylation level of FATP1 in scatter-fed chicken was lower than in captivity-fed conditions in DNH and QIM chicken breast tissues; DNA methylation in the promoter and exon1 region was demonstrated to negatively regulate the expression of the FATP1 gene. These results suggested that feeding conditions affect the methylation status and expression level of FATP1, thereby affecting the Intramuscular fat content in DNH and QJM chicken breast muscle.(AU)

Effect of Feeding Conditions on the Methylation Status of Fatp1 Gene in Chicken Breast Muscle

The objective of this work was to investigate the effect of feeding conditions on methylation status of FATP1 gene, which is an important candidate gene of Intramuscular fat and important indicator of chicken meat quality. We selected Daninghe (DNH) and Qingjiaoma (QJM) chickens under scatter-feeding and captivity-feeding conditions as experimental animals, and detected the methylation status of FATP1 genes in chicken breast muscle using Bisulfite Sequencing PCR method. The results showed that the methylation level of FATP1 in scatter-fed chicken was lower than in captivity-fed conditions in DNH and QIM chicken breast tissues; DNA methylation in the promoter and exon1 region was demonstrated to negatively regulate the expression of the FATP1 gene. These results suggested that feeding conditions affect the methylation status and expression level of FATP1, thereby affecting the Intramuscular fat content in DNH and QJM chicken breast muscle.

Detection of SNPs in the BMP6 Gene and Their Association with Carcass and Bone Traits in Chicken

BMP6, a member of the subfamilies of the morphogenetic proteins (BMPs), plays a crucial role in osteogenic and chondrocyte differentiation in vitro and stimulates chondrogenesis, making chondrocytes differen-tiate on their terminal stage. The objective of this study is to explore the relationship between polymorphism of BMP6 gene and slaughter traits in chicken respectively. We screened the exonic and intronic regions of BMP6 gene by DNA pool construction and amplified DNA fragment by PCR, and finally, we got nine SNPs. Association analysis revealed that BMP6 had no significant association among all slaughter traits in Yellow bantam chicken. However, BMP6 had a significant difference with femur weight, tibia weight, femur length (p 0.05), and was extremely significant with tibia length (p 0.01) in Avian chicken. Moreover, femur perimeter also had significant correlation with BMP6 in Avian chicken. These results provide useful information for further investigation on the function of chicken BMP6 gene.(AU)

Detection of SNPs in the BMP6 Gene and Their Association with Carcass and Bone Traits in Chicken

BMP6, a member of the subfamilies of the morphogenetic proteins (BMPs), plays a crucial role in osteogenic and chondrocyte differentiation in vitro and stimulates chondrogenesis, making chondrocytes differen-tiate on their terminal stage. The objective of this study is to explore the relationship between polymorphism of BMP6 gene and slaughter traits in chicken respectively. We screened the exonic and intronic regions of BMP6 gene by DNA pool construction and amplified DNA fragment by PCR, and finally, we got nine SNPs. Association analysis revealed that BMP6 had no significant association among all slaughter traits in Yellow bantam chicken. However, BMP6 had a significant difference with femur weight, tibia weight, femur length (p 0.05), and was extremely significant with tibia length (p 0.01) in Avian chicken. Moreover, femur perimeter also had significant correlation with BMP6 in Avian chicken. These results provide useful information for further investigation on the function of chicken BMP6 gene.

Single Nucleotide Polymorphisms in IGFBP-2 Gene and Their Associations with Body Weight Traits on Jinghai Yellow Chicken

Insulin-like growth factor binding protein-2 (IGFBP-2) regulates a broad spectrum of biological activities involved in growth, development, and differentiation. This study aimed at comparing polymorphisms in intron2 of the IGFBP-2 gene among four chicken breeds and at analyzing the associations between its genotypes and body weight in Jinghai Yellow chicken by using PCR-SSCP technique. For primer P2, three genotypes (AA, AB and BB) were observed in the four chicken breeds. Gene sequencing revealed one insertion/deletion (the inserted/deleted TC after position 552bp) in the intron 2 of IGFBP-2 gene. For primer P5, three genotypes were identified in Jinghai Yellow chickens, and named CC, CD and DD. Gene sequencing revealed two SNPs (C1107G, C1130T) and one inserted/deleted GCCAGGT after 1115bp in the intron 2 of IGFBP-2 gene. The results of the linear model analysis showed that Jinghai Yellow chickens with AA genotype had significantly heavier body weight, at hatch and 12 weeks of age, than those of the AB genotype (p 0.05). The A allele had a positive effect on body weight. We speculate that mutations in intron 2 could be used as genetic markers for body weight in Jinghai Yellow chicken. This study provides valuable information for the protection of genetic resources and for breeding of Jinghai Yellow chicken.

Single Nucleotide Polymorphisms in IGFBP-2 Gene and Their Associations with Body Weight Traits on Jinghai Yellow Chicken

Insulin-like growth factor binding protein-2 (IGFBP-2) regulates a broad spectrum of biological activities involved in growth, development, and differentiation. This study aimed at comparing polymorphisms in intron2 of the IGFBP-2 gene among four chicken breeds and at analyzing the associations between its genotypes and body weight in Jinghai Yellow chicken by using PCR-SSCP technique. For primer P2, three genotypes (AA, AB and BB) were observed in the four chicken breeds. Gene sequencing revealed one insertion/deletion (the inserted/deleted TC after position 552bp) in the intron 2 of IGFBP-2 gene. For primer P5, three genotypes were identified in Jinghai Yellow chickens, and named CC, CD and DD. Gene sequencing revealed two SNPs (C1107G, C1130T) and one inserted/deleted GCCAGGT after 1115bp in the intron 2 of IGFBP-2 gene. The results of the linear model analysis showed that Jinghai Yellow chickens with AA genotype had significantly heavier body weight, at hatch and 12 weeks of age, than those of the AB genotype (p 0.05). The A allele had a positive effect on body weight. We speculate that mutations in intron 2 could be used as genetic markers for body weight in Jinghai Yellow chicken. This study provides valuable information for the protection of genetic resources and for breeding of Jinghai Yellow chicken.(AU)