Complete genome sequence and phylogenetic analysis of a goose astrovirus isolate in China.

Astroviruses are considered the cause of gastroenteritis in humans and animals. Studies in recent years show avian astroviruses are also associated with duckling hepatitis, gosling gout, and chicken nephritis. In this study, a GAstV strain, designated as JS2019/China, was detected in dead goslings from a commercial goose farm in Jiangsu province of China. Viral strain was proliferated in goose embryos and sequence analysis showed the isolated strain had a classical structure arrangement and a series of conserved regions compared with other GAstVs. Sequence comparison and phylogenetic analysis of whole genome and ORF2 revealed that JS2019/China belongs to the GAstV-1 group, which consists of most of the GAstV strains. Amino acid analysis indicated that some mutants might have an impact on viral protease capacity, such as V505I and K736E of ORF1a and T107I, F342S, and S606P of ORF2. Taken together, a novel GAstV strain was isolated and genomic analysis and protein polymorphism analysis indicated that some amino acid mutants might affect the viral virulence.

Facile, ultrasensitive, and highly specific diagnosis of goose astrovirus via reverse transcription-enzymatic recombinase amplification coupled with a CRISPR-Cas12a system detection.

Fatal gout in geese caused by goose astrovirus (GAstV) has been spreading rapidly in China since 2018, causing serious economic losses in the goose breeding industry. To achieve simple, convenient and sensitive detection of GAstV, a novel diagnostic test was developed by combining reverse transcription-enzymatic recombinase amplification (RT-ERA) and CRISPR-Cas12a technologies. RT-ERA primers were designed to pre-amplify the conserved region of the ORF2 gene of GAstV and the predefined target sequence detected using the Cas12a/crRNA complex at 37℃ for 30 min. Specific detection of GAstV was achieved with no cross-reaction with non-GAstV templates and a sensitivity detection limit of 2 copies. The experimental procedure could be completed within 1 h, including RNA extraction (15 min), RT-ERA reaction (20 min), CRISPR-Cas12a/crRNA detection (5 min) and result readout (within 2 min) steps. In conclusion, the combination of RT-ETA and CRISPR-Cas12a provides a rapid and specific method that should be effective for the control and surveillance of GAstV infections in farms from remote locations.

Molecular characterization and expression analysis of the chicken-type and goose-type lysozymes from totoaba (Totoaba macdonaldi).

Lysozymes play a key role in innate immune response to bacterial pathogens, catalyzing the hydrolysis of the peptidoglycan layer of bacterial cell walls. In this study, the genes encoding the c-type (TmLyzc) and g-type (TmLyzg) lysozymes from Totoaba macdonaldi were cloned and characterized. The cDNA sequences of TmLyzg and TmLyzc were 582 and 432 bp, encoding polypeptides of 193 and 143 amino acids, respectively. Amino acid sequences of these lysozymes shared high identity (60-90%) with their counterparts of other teleosts and showed conserved functional-structural signatures of the lysozyme superfamily. Phylogenetic analysis indicated a close relationship with their vertebrate homologues but distinct evolutionary paths for each lysozyme. Expression analysis by qRT-PCR revealed that TmLyzc was expressed in stomach and pyloric caeca, while TmLyzg was highly expressed in stomach and heart. These results suggest that both lysozymes play important roles in defense of totoaba against bacterial infections or as digestive enzyme.

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.

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)

Molecular Cloning and Mrna Expression Analysis of Sichuan White Goose (Anser Cygnoides) Chrebp Gene

The carbohydrate response element-binding protein (ChREBP) is an important nuclear factor that regulates glycolysis and de novo lipogenesis. However, the role of ChREBP in fatty liver development in geese remains unclear. In order to understand the function of ChREBP in lipid metabolism of geese, we first cloned the complete cDNA of the ChREBP of the Sichuan White goose (Anser cygnoides) using RT-PCR, 5 RACE and 3 RACE, and analyzed goose ChREBP expression in nine different tissues using real-time PCR technology. The results showed that the goose ChREBP CDS consists of 945bp nucleotides that encode 314 amino acids, and the sequence has high similarities with the swan goose (Anser cygnoides domesticus) and duck (Anas platyrhynchos) sequences, both at the nucleotide and amino acid levels. The predicted ChREBP protein had a molecular mass of 35.64 kDa with pI value of 5.36. The phylogenetic analysis indicated its evolutionary relationships with corresponding orthologous sequences in swan geese and ducks. The qPCR assays revealed that ChREBP is highly expressed in liver in the Sichuan White goose. Together, these results indicate that goose ChREBP may play an important role in the development of hepatic steatosis.

Molecular Cloning and Mrna Expression Analysis of Sichuan White Goose (Anser Cygnoides) Chrebp Gene

The carbohydrate response element-binding protein (ChREBP) is an important nuclear factor that regulates glycolysis and de novo lipogenesis. However, the role of ChREBP in fatty liver development in geese remains unclear. In order to understand the function of ChREBP in lipid metabolism of geese, we first cloned the complete cDNA of the ChREBP of the Sichuan White goose (Anser cygnoides) using RT-PCR, 5 RACE and 3 RACE, and analyzed goose ChREBP expression in nine different tissues using real-time PCR technology. The results showed that the goose ChREBP CDS consists of 945bp nucleotides that encode 314 amino acids, and the sequence has high similarities with the swan goose (Anser cygnoides domesticus) and duck (Anas platyrhynchos) sequences, both at the nucleotide and amino acid levels. The predicted ChREBP protein had a molecular mass of 35.64 kDa with pI value of 5.36. The phylogenetic analysis indicated its evolutionary relationships with corresponding orthologous sequences in swan geese and ducks. The qPCR assays revealed that ChREBP is highly expressed in liver in the Sichuan White goose. Together, these results indicate that goose ChREBP may play an important role in the development of hepatic steatosis.(AU)

Gene Expression Patterns of Geese Expression Patterns of L-FABP, Spot 14, OB and APO A1 Genes in Different Tissues of Overfed and Control Geese

Ten-week-old Langde geese in similar body weight were randomly selected, four for overfeeding and four for routinly feeding. The abundance of liver fatty acid-binding protein (L-FABP), thyroid hormone-responsive (THRSP or Spot 14), obese (OB), and apolipoprotein A1 (Apo A1) genes in goose were detected by quantitative RT-PCR. L-FABP was higher expressed in liver and intestine than other tissues, but no expression was detected in the pancreas or brain. The other three genes were widely expressed in different tissues, OB was higher expressed in pancreas and abdominal fat, Spot 14 and Apo A1 was higher expressed in sebum and abdominal fat. Spot 14 and Apo A1 genes were up-regulated in overfed goose livers compared with that in the control. Thus, Spot 14 and Apo A1 genes may play important roles in lipid metabolism in goose fat liver.

Gene Expression Patterns of Geese Expression Patterns of L-FABP, Spot 14, OB and APO A1 Genes in Different Tissues of Overfed and Control Geese

Ten-week-old Langde geese in similar body weight were randomly selected, four for overfeeding and four for routinly feeding. The abundance of liver fatty acid-binding protein (L-FABP), thyroid hormone-responsive (THRSP or Spot 14), obese (OB), and apolipoprotein A1 (Apo A1) genes in goose were detected by quantitative RT-PCR. L-FABP was higher expressed in liver and intestine than other tissues, but no expression was detected in the pancreas or brain. The other three genes were widely expressed in different tissues, OB was higher expressed in pancreas and abdominal fat, Spot 14 and Apo A1 was higher expressed in sebum and abdominal fat. Spot 14 and Apo A1 genes were up-regulated in overfed goose livers compared with that in the control. Thus, Spot 14 and Apo A1 genes may play important roles in lipid metabolism in goose fat liver.(AU)

Sex identification of domestic geese (Anser anser): comparison of cytogenetic analysis technique and post anesthetic inspection of the cloaca / Identificação do sexo de gansos domésticos (Anser anser): comparação da técnica de sexagem cromossômica e a inspeção pós-anestésica da cloaca

The domestic geese used in this experiment were free-living, from a lake located in an urban area and its population had increased dramatically. The need for a project proposing the sterilization of some males to restrain population growth, led to the need for sexing these animals. As domestic geese present minimal sexual dimorphism, two techniques were tested: cytogenetic analysis and post anesthetic inspection of the cloaca. There were used eight adults geese randomly chosen from a group of 80 animals. They were caught with nets or by hand, were individually transported and housed in appropriate place waiting for the cytogenetic analysis (three to four days). For the chromosomal sexing, was collected 2 mL of blood and the peripheral blood lymphocyte culture technique was used. The sample was treated with hypotonic KCl, fixed in three changes of methanol/acetic acid solution. The slides were prepared with the standard technique and examined under an optical microscope to observe the metaphase. To the technique of cloaca eversion, was performed injectable anesthesia with tiletamine-zolazepam 5%. Ten minutes after anesthesia, the cloaca has been everted to visualize the internal structures and sex determination. The techniques were performed in blinded trial to avoidinaccurate results. From the eight animals submitted to chromosomal sexing technique, five were identified as male and three as females, reaching 100% agreement when compared with the cloaca eversion technique. Sexing chromosomal technique appeared to cause the least discomfort in the animals evaluated, proving to be a reliable technique for sexualdetermination and favorable to animal welfare. (AU)

Os gansos domésticos utilizados neste experimento eram de vida livre, pertencentes a um lago localizado em uma área urbana e sua população havia aumentado dramaticamente. A necessidade de um projeto com foco na esterilização de alguns machos, para reduzir o crescimento populacional, levou à necessidade de sexagem desses animais. como os gansos domésticos apresentam mínimo dimorfismo sexual, duas técnicas foram testadas: análise citogenética e inspeção da cloaca pós-anestesia. Foram utilizados oito gansos adultos, escolhidos aleatoriamente de um grupo de 80 animais. Eles foram capturados com redes ou manualmente, transportados individualmente e alojados em local apropriado a espera da análise citogenética (três a quatro dias). Para a sexagem cromossomal, foram coletados 2 ml de sangue e a técnica de cultura de linfócitos periféricos foi empregada. As amostras resultantes foram tratadas com kcl hipotônico, fixadas em três trocas de solução de metanol e ácido acético. As lâminas foram preparadas com a técnica padrão e examinadas ao microscópio óptico para observação das metáfases. para a técnica de eversão da cloaca, foi realizada a anestesia injetável com tiletamina-zolazepam 5%. Dez minutos após a anestesia, a cloaca foi evertida para a visualização das estruturas internas e a determinação sexual. AAs técnicas foram conduzidas em duplo cego, para melhor acurácia dos resultados. Dos oito animais submetidos à técnica de sexagem cromossomal, cinco foram identificados como machos e três como fêmeas, alcançando 100% de concordância quando comparada com a técnica de eversão da cloaca. A técnica de sexagem cromossomal aparentou causar o menor desconforto nos animais avaliados, mostrando-se uma técnicaconfiável para determinação sexual e favorável ao bem-estar animal. (AU)

Sex identification of domestic geese (Anser anser): comparison of cytogenetic analysis technique and post anesthetic inspection of the cloaca / Identificação do sexo de gansos domésticos (Anser anser): comparação da técnica de sexagem cromossômica e a inspeção pós-anestésica da cloaca

The domestic geese used in this experiment were free-living, from a lake located in an urban area and its population had increased dramatically. The need for a project proposing the sterilization of some males to restrain population growth, led to the need for sexing these animals. As domestic geese present minimal sexual dimorphism, two techniques were tested: cytogenetic analysis and post anesthetic inspection of the cloaca. There were used eight adults geese randomly chosen from a group of 80 animals. They were caught with nets or by hand, were individually transported and housed in appropriate place waiting for the cytogenetic analysis (three to four days). For the chromosomal sexing, was collected 2 mL of blood and the peripheral blood lymphocyte culture technique was used. The sample was treated with hypotonic KCl, fixed in three changes of methanol/acetic acid solution. The slides were prepared with the standard technique and examined under an optical microscope to observe the metaphase. To the technique of cloaca eversion, was performed injectable anesthesia with tiletamine-zolazepam 5%. Ten minutes after anesthesia, the cloaca has been everted to visualize the internal structures and sex determination. The techniques were performed in blinded trial to avoidinaccurate results. From the eight animals submitted to chromosomal sexing technique, five were identified as male and three as females, reaching 100% agreement when compared with the cloaca eversion technique. Sexing chromosomal technique appeared to cause the least discomfort in the animals evaluated, proving to be a reliable technique for sexualdetermination and favorable to animal welfare.

Os gansos domésticos utilizados neste experimento eram de vida livre, pertencentes a um lago localizado em uma área urbana e sua população havia aumentado dramaticamente. A necessidade de um projeto com foco na esterilização de alguns machos, para reduzir o crescimento populacional, levou à necessidade de sexagem desses animais. como os gansos domésticos apresentam mínimo dimorfismo sexual, duas técnicas foram testadas: análise citogenética e inspeção da cloaca pós-anestesia. Foram utilizados oito gansos adultos, escolhidos aleatoriamente de um grupo de 80 animais. Eles foram capturados com redes ou manualmente, transportados individualmente e alojados em local apropriado a espera da análise citogenética (três a quatro dias). Para a sexagem cromossomal, foram coletados 2 ml de sangue e a técnica de cultura de linfócitos periféricos foi empregada. As amostras resultantes foram tratadas com kcl hipotônico, fixadas em três trocas de solução de metanol e ácido acético. As lâminas foram preparadas com a técnica padrão e examinadas ao microscópio óptico para observação das metáfases. para a técnica de eversão da cloaca, foi realizada a anestesia injetável com tiletamina-zolazepam 5%. Dez minutos após a anestesia, a cloaca foi evertida para a visualização das estruturas internas e a determinação sexual. AAs técnicas foram conduzidas em duplo cego, para melhor acurácia dos resultados. Dos oito animais submetidos à técnica de sexagem cromossomal, cinco foram identificados como machos e três como fêmeas, alcançando 100% de concordância quando comparada com a técnica de eversão da cloaca. A técnica de sexagem cromossomal aparentou causar o menor desconforto nos animais avaliados, mostrando-se uma técnicaconfiável para determinação sexual e favorável ao bem-estar animal.

Characterization of HSP70 and its expression in tissue: correlation with physiological and immune indices in goose (Anser cygnoides) serum.

We cloned the goose heat shock protein 70 gene (HSP70), to determine its sequence variation and elucidate its mRNA expression. We designed primers to amplify the entire goose HSP70 sequence. We used 10 commercial Wuzong goslings in a heat-stress experiment. We collected tissue samples for RNA extraction and quantitative real-time polymerase chain reaction (qRT-PCR). We analyzed the variation in expression of goose HSP70 before and after heat stress. We constructed a DNA pool from six different species, for single nucleotide polymorphism (SNP) screening. We detected 18 SNPs and selected three of these SNPs for correlation analysis with biological and immune traits in 200 Wuzong geese. We showed that T+237C was significantly correlated with the serum corticosterone level, whereas T+1122C was significantly correlated with the heterophil to lymphocyte ratio. Goose HSP70 contained no introns. The results of qRT-PCR analysis revealed significant gender differences in the expression of goose HSP70 at 40°C but not at 25°C; moreover, in general, expression was significantly higher at 40°C than at 25°C. With the exception of the leg muscle and cerebellum, HSP70 expression was significantly higher in male geese than in female geese. Our results indicate that goose HSP70 plays an important role in response to severe heat stress.

Gene expression profiles in the pituitary glands of Sichuan White geese during prelaying and laying periods.

To better understand the molecular mechanism(s) underlying egg-laying in Sichuan white geese, the profiles of genes in the pituitary gland were investigated during the prelaying and laying periods. Total RNA was extracted from the pituitary glands of geese during prelaying or laying periods and cDNA was generated. After sequencing and annotation, 54 upregulated and 84 downregulated genes were obtained from gene libraries. These genes were related primarily to biosynthetic processes, cellular nitrogen metabolic processes, transport, cell differentiation, cellular protein modification processes, signal transduction, and small molecule metabolic processes. Eleven genes were selected for further analyses using quantitative real-time PCR, and the results were generally consistent with the profiling results. Among these genes, levels of gonadotropin-releasing hormone, gonadotropin-inhibitory hormone, vasoactive intestinal peptide and its receptor, follistatin, estrogen receptor beta, and the progesterone receptor were differentially overexpressed during the prelaying period compared with the laying period. These results provide a solid foundation for elucidating the molecular mechanism of egg-laying performance in Sichuan white geese.

Differential gene expression in pre-laying and laying period ovaries of Sichuan White geese (Anser cygnoides).

Geese are an economically important poultry species worldwide. Their superior meat production performance and meat qual-ity make them a popular food. However, they are not bred worldwide because their poor laying capacity increases farming costs. To gain a global view of the genes that are differentially expressed between pre-laying (P) and laying (L) periods and to develop a database for further studies, we performed large-scale transcriptome sequencing of ovarian tissue collected from Anser cygnoides. In total, 30,151,422 raw reads, with an average length of 151 bp and a total length of 4,552,864,722 bp, were obtained. After primers and adaptors were removed, 19,167,132 clean reads, with an average length of 134.5 bp and a total length of 2,577,297,281 bp, were obtained, among which 1,268,906,694 bp and 1,308,390,587 bp were from L and P ovarian tissue, respectively. The 16,605 assembled sequences were further functionally annotated by comparing their sequences to different protein and functional domain databases and assigning gene ontology (GO) terms. Of these, 511 as-sembled sequences were considered differentially expressed based on the 2-fold method, among which 396 were assigned at least one GO term. Digital expression analysis using the Kyoto encyclopedia of genes and genomes annotation identified 121 genes that were differ-entially expressed in the P vs L periods. Five of these are of special interest for further investigation of their roles in determining high re-productive performance. This study provides valuable information and sequence resources for uncovering genes determining high egg-laying performance and for future functional genomics analysis of geese.

Molecular characterization, tissue expression, and polymorphism analysis of liver-type fatty acid binding protein in Landes geese.

Liver weight is an important economic trait in the fatty goose liver industry. Liver-type fatty acid binding protein (L-FABP) is involved in the formation and metabolism of fatty acids. Thus, we hypothesized that sequence polymorphisms in L-FABP were associated with fatty liver weight in goose. We first isolated, sequenced, and characterized the goose L-FABP gene, which had not been previously reported. The goose L-FABP gene was 2490 bp and included 4 exons coding for a 126-amino acid protein. Analysis of expression levels of the goose L-FABP gene in different tissues showed that the expression level in the liver tissue was higher than in other tissues, and was significantly higher in the liver tissue of overfed geese than in control geese. Moreover, a single nucleotide polymorphism located at 774 bp in the gene was identified in a Landes goose population. To test whether this single nucleotide polymorphism was associated with fatty liver production, liver weight and the ratio of liver to carcass weights were determined for the 3 genotypes with this single nucleotide polymorphism (TT, TG, GG) in overfed Landes geese. Our data indicate that individuals with the GG genotype had higher values for the variables measured than those with the other 2 genotypes, suggesting that L-FABP can be a selection marker for the trait of fatty liver production in goose.

Development of eight novel microsatellite markers for Huoyan geese.

In this study, we isolated microsatellite DNA from the Huoyan goose genome with magnetic beads. As a result, 150 positive clones were identified, and 148 microsatellites were found. Among the 148 microsatellites, 69.6% were perfect, 17.6% were imperfect, and the rest were compound type (12.8%). Twenty microsatellite primers were used to screen 90 individuals from 3 Huoyan goose populations. Eight loci were polymorphic with a low number of alleles (2 to 4). The observed and expected heterozygosities ranged from 0.3556 to 1 and from 0.2923 to 0.6868, respectively. All the 8 polymorphic loci were in Hardy-Weinberg equilibrium. These molecular markers will be useful for future studies on population genetic structure and conservation genetics in Huoyan geese.

Myf5 gene polymorphisms and production performance traits in Songliao white geese.

To explore the relationship between Myf5 gene polymorphisms and production performance traits in Songliao white geese, we used the chicken Myf5 sequence to design primers and amplified part of the exon 1 sequence of the Songliao white goose Myf5 gene. Results of single-strand conformation polymorphism polymerase chain reaction analysis revealed polymorphisms of the amplified fragment, including three genotypes (AA, AB, and BB). Three varieties were dominated by allele A and were mainly expressed in AA genotypes. We also identified that the Myf5 gene has one single nucleotide change (A→G) on exon 1 at locus 1344, and another (G→C) at locus 1410. Analysis of variance showed significant differences between genotypes before slaughter in live weight, carcass weight, eviscerated weight, leg muscle weight, weight of the wings, and slaughter rate. There were no significant differences with respect to other growth and carcass traits evaluated.

MHC class I loci of the Bar-Headed goose (Anser indicus)

MHC class I proteins mediate functions in anti-pathogen defense. MHC diversity has already been investigated by many studies in model avian species, but here we chose the bar-headed goose, a worldwide migrant bird, as a non-model avian species. Sequences from exons encoding the peptide-binding region (PBR) of MHC class I molecules were isolated from liver genomic DNA, to investigate variation in these genes. These are the first MHC class I partial sequences of the bar-headed goose to be reported. A preliminary analysis suggests the presence of at least four MHC class I genes, which share great similarity with those of the goose and duck. A phylogenetic analysis of bar-headed goose, goose and duck MHC class I sequences using the NJ method supports the idea that they all cluster within the anseriforms clade.