Genomic selection(GS) is a new approach for selection breeding using high-density single nucleotide polymorphisms (SNPs) across the whole genome, which can shorten the generation interval by early selection, accelerate genetic progress by improving the accuracy of breeding value estimation. GS has a good predictive effect, especially for the complex traits with low heritability and difficult to be measured, and which truly realizes the genomic technology guiding breeding program. With the development of chip and sequencing technology, the detection cost of high-density SNP chips is continuously reduced, the genome-wide selection model is continuously upgraded and optimized, and the prediction accuracy is continuously improved, the whole genome selection has become an important means and research hotspot in animal genetic improvement. At present, genomic selection has made significant progress and becomes a standard method for genetic evaluation in dairy cattle, and its application in other species is being carried out. In this paper, we reviewed the statistical models of genomic selection, summarized its applications in animal breeding, discussed the existing problems, and prospected its development direction and application prospects.

Genomic selection(GS) is a new approach for selection breeding using high-density single nucleotide polymorphisms (SNPs) across the whole genome, which can shorten the generation interval by early selection, accelerate genetic progress by improving the accuracy of breeding value estimation. GS has a good predictive effect, especially for the complex traits with low heritability and difficult to be measured, and which truly realizes the genomic technology guiding breeding program. With the development of chip and sequencing technology, the detection cost of high-density SNP chips is continuously reduced, the genome-wide selection model is continuously upgraded and optimized, and the prediction accuracy is continuously improved, the whole genome selection has become an important means and research hotspot in animal genetic improvement. At present, genomic selection has made significant progress and becomes a standard method for genetic evaluation in dairy cattle, and its application in other species is being carried out. In this paper, we reviewed the statistical models of genomic selection, summarized its applications in animal breeding, discussed the existing problems, and prospected its development direction and application prospects.

Thyroid hormone (TH) and its receptors play an important biological role in animal reproduction, in addition to regulating normal body development and metabolic balance. Thyroid hormone receptor alpha (TRα) and thyroid hormone receptor beta (TRβ) belong to the ligand-dependent nuclear receptor superfamily and are encoded by the THRA and THRB genes, respectively. Different alternative splicing or transcription start positions in the 2 genes can result in multiple receptor subtypes in different tissues. In this review, the basic characteristics of TRs were introduced firstly. Then using the hypothalamic-pituitary-gonadal axis (HPGA) as a clue, the regulation of TRs on the gonadal axis system by involving pulse release of gonadotropin-releasing hormone (GnRH), levels of gonadal hormones, gonadotropin receptor and sex hormone receptor expression was reviewed. Finally, this review summarizes the regulation of TH/TRs on seasonal reproduction from aspects of TH-GnRH pathway, the role of TH/TRs in seasonal reproduction of mammals and their role in seasonal bird reproduction in favor of understanding the molecular mechanism of seasonal reproduction of animals and providing new ideas for artificial regulation of seasonal estrus.

Thyroid hormone (TH) and its receptors play an important biological role in animal reproduction, in addition to regulating normal body development and metabolic balance. Thyroid hormone receptor alpha (TRα) and thyroid hormone receptor beta (TRβ) belong to the ligand-dependent nuclear receptor superfamily and are encoded by the THRA and THRB genes, respectively. Different alternative splicing or transcription start positions in the 2 genes can result in multiple receptor subtypes in different tissues. In this review, the basic characteristics of TRs were introduced firstly. Then using the hypothalamic-pituitary-gonadal axis (HPGA) as a clue, the regulation of TRs on the gonadal axis system by involving pulse release of gonadotropin-releasing hormone (GnRH), levels of gonadal hormones, gonadotropin receptor and sex hormone receptor expression was reviewed. Finally, this review summarizes the regulation of TH/TRs on seasonal reproduction from aspects of TH-GnRH pathway, the role of TH/TRs in seasonal reproduction of mammals and their role in seasonal bird reproduction in favor of understanding the molecular mechanism of seasonal reproduction of animals and providing new ideas for artificial regulation of seasonal estrus.

The spermatogenesis of mammals is regulated by Sertoli cells (SCs), which not only provide physical support and stable microenvironment to the spermatogenesis, but also regulate and control the proliferation, differentiation, apoptosis, phagocytosis and immune privilege of generative cells through the secretion of multiple proteins. However,the exact mechanism of SCs regulating spermatogenesis in physiological situations is unclear. Therefore, in this paper, the SCs and spermatogonial stem cells (SSCs) and a series of sperm cells special structure relationship and the regulation between the two were summarized, in order to promote the in-depth study of the regulatory mechanism between the two and to provide reference for improving the animal breeding efficiency.

The spermatogenesis of mammals is regulated by Sertoli cells (SCs), which not only provide physical support and stable microenvironment to the spermatogenesis, but also regulate and control the proliferation, differentiation, apoptosis, phagocytosis and immune privilege of generative cells through the secretion of multiple proteins. However,the exact mechanism of SCs regulating spermatogenesis in physiological situations is unclear. Therefore, in this paper, the SCs and spermatogonial stem cells (SSCs) and a series of sperm cells special structure relationship and the regulation between the two were summarized, in order to promote the in-depth study of the regulatory mechanism between the two and to provide reference for improving the animal breeding efficiency.

The objective of this study was to obtain the sequence of KLF15 gene of Tibetan chicken,clarify its tissue and temporal expression profiles and to analyze the relationship between its expression and the intramuscular fat (IMF) content. Ten healthy Tibetan chicken(5 cock and 5 hen) on 1, 81, 119, 154 and 210 days old were selected, respectively as experimental animals. RT-PCR technology was used to clone KLF15 gene of Tibetan chicken, and bioinformatics softwares were used to analyze the biological characteristics of KLF15. The expression profiles of KLF15 gene in different tissues and different developmental stages was detected by fluorescence quantitative PCR, and its correlation with IMF content was analyzed. The result showed that the cloned Tibetan chicken KLF15 gene sequence was 1 288 bp in length (GenBank accession number:KY747450), an open reading frame of 1 212 bp, encoding 403 amino acids, and it was a hydrophilic unstable basic protein with 3 zinc finger structures. The homology between the nucleotide and amino acid sequences of KLF15 gene of Tibetan chicken and Gallus gallus were 99%. KLF15 mRNA was widely expressed in all detected tissues of Tibetan chicken, and the expression level was the highest in lung, which is significantly higher than that in other tissues (P<0.01). The expression level of KLF15 gene in breast muscle of Tibetan chicken of 1 day old was higher than that of the other age groups, and declined with the increasing age. The expression level of KLF15 gene in leg muscle of Tibetan chicken of 119 days old was higher than that of the other age groups, and the change of the overall expression level showed a trend of decreasing first, then rising, finally falling. The expression level of KLF15 gene before 154 days old was positively correlated with IMF content in Tibetan cocks,after 154 days old, the correlation was reverse, and the Tibetan hens mainly showed a weak negative correlation in all age groups. These results provide the basic data for further research on the role of KLF15 in IMF deposition of Tibetan chicken.

The objective of this study was to obtain the sequence of KLF15 gene of Tibetan chicken,clarify its tissue and temporal expression profiles and to analyze the relationship between its expression and the intramuscular fat (IMF) content. Ten healthy Tibetan chicken(5 cock and 5 hen) on 1, 81, 119, 154 and 210 days old were selected, respectively as experimental animals. RT-PCR technology was used to clone KLF15 gene of Tibetan chicken, and bioinformatics softwares were used to analyze the biological characteristics of KLF15. The expression profiles of KLF15 gene in different tissues and different developmental stages was detected by fluorescence quantitative PCR, and its correlation with IMF content was analyzed. The result showed that the cloned Tibetan chicken KLF15 gene sequence was 1 288 bp in length (GenBank accession number:KY747450), an open reading frame of 1 212 bp, encoding 403 amino acids, and it was a hydrophilic unstable basic protein with 3 zinc finger structures. The homology between the nucleotide and amino acid sequences of KLF15 gene of Tibetan chicken and Gallus gallus were 99%. KLF15 mRNA was widely expressed in all detected tissues of Tibetan chicken, and the expression level was the highest in lung, which is significantly higher than that in other tissues (P<0.01). The expression level of KLF15 gene in breast muscle of Tibetan chicken of 1 day old was higher than that of the other age groups, and declined with the increasing age. The expression level of KLF15 gene in leg muscle of Tibetan chicken of 119 days old was higher than that of the other age groups, and the change of the overall expression level showed a trend of decreasing first, then rising, finally falling. The expression level of KLF15 gene before 154 days old was positively correlated with IMF content in Tibetan cocks,after 154 days old, the correlation was reverse, and the Tibetan hens mainly showed a weak negative correlation in all age groups. These results provide the basic data for further research on the role of KLF15 in IMF deposition of Tibetan chicken.

The purpose of this study was to investigate the effects of the methylation and SNPs in DCT gene promoter region on the coat color of goats, and to provide a theoretical basis for exploring the mechanism of DCT gene regulating the coat color of goats. Goats were used as experimental animals. The CpG island in the promoter region of goat DCT gene was predicted and two pairs of primers were designed for bisulfite methylation sequencing of the two CpG island enriched regions. Methylation level online analysis software, BISMA, was used to count the number of methylation sites and to compare the differences of methylation levels between Tangshan dairy goats(with white coat) and Nanjiang Huang goats(with black coat). Core promoter region sequence of DCT was cloned and SNPs were screened for goats with different coat colors. JASPAR and Nsite, the transcription factor online prediction software, were used to predict the transcription factor alterations with the mutation of SNPs, and promoter activity variation with the mutation of SNPs was detected. The methylation sequence and the core promoter region sequence(g.-1045--318) of goat DCT gene were successfully cloned. Six and 23 methylation sites were found in the g.-348--150 region and the g.+222-+502 region, respectively. The methylation levels of white goats at the g.+312, g.+352 and g.+400 sites and at the g.+389 and g.+404 sites were significantly and extremely significantly higher than those of black goats (P<0.05 and P<0.01), respectively. And the average methylation level of white goats in g.+222-+502 region was extremely significantly higher than that of black goats (P<0.01). The genotype compositions of the 3 SNPs in the core promoter region, g.-804 T > G, g.-705 C > T and g. -679 G > A, was different between white goats and the 3 colorful goats. SOX10 transcription factor binding site disappeared with the g.-804T > G mutation, and the promoter activity of the DCT gene was significantly decreased (P<0.05). The higher methylation level in the g.+222 -+502 region of DCT gene in white goats, and the 3 mutations at g.-804, g.-714 and g.-679 sites, especially the g.-804 T > G mutation resulted in the loss of the SOX10 transcription factor binding site and the promoter activity of DCT with G genotype was significantly decreased.In conclusion, the SNP mutation and the higher methylation level in promoter region may suppress the expression of DCT gene and form the white coat color of goat.

The purpose of this study was to investigate the effects of the methylation and SNPs in DCT gene promoter region on the coat color of goats, and to provide a theoretical basis for exploring the mechanism of DCT gene regulating the coat color of goats. Goats were used as experimental animals. The CpG island in the promoter region of goat DCT gene was predicted and two pairs of primers were designed for bisulfite methylation sequencing of the two CpG island enriched regions. Methylation level online analysis software, BISMA, was used to count the number of methylation sites and to compare the differences of methylation levels between Tangshan dairy goats(with white coat) and Nanjiang Huang goats(with black coat). Core promoter region sequence of DCT was cloned and SNPs were screened for goats with different coat colors. JASPAR and Nsite, the transcription factor online prediction software, were used to predict the transcription factor alterations with the mutation of SNPs, and promoter activity variation with the mutation of SNPs was detected. The methylation sequence and the core promoter region sequence(g.-1045--318) of goat DCT gene were successfully cloned. Six and 23 methylation sites were found in the g.-348--150 region and the g.+222-+502 region, respectively. The methylation levels of white goats at the g.+312, g.+352 and g.+400 sites and at the g.+389 and g.+404 sites were significantly and extremely significantly higher than those of black goats (P<0.05 and P<0.01), respectively. And the average methylation level of white goats in g.+222-+502 region was extremely significantly higher than that of black goats (P<0.01). The genotype compositions of the 3 SNPs in the core promoter region, g.-804 T > G, g.-705 C > T and g. -679 G > A, was different between white goats and the 3 colorful goats. SOX10 transcription factor binding site disappeared with the g.-804T > G mutation, and the promoter activity of the DCT gene was significantly decreased (P<0.05). The higher methylation level in the g.+222 -+502 region of DCT gene in white goats, and the 3 mutations at g.-804, g.-714 and g.-679 sites, especially the g.-804 T > G mutation resulted in the loss of the SOX10 transcription factor binding site and the promoter activity of DCT with G genotype was significantly decreased.In conclusion, the SNP mutation and the higher methylation level in promoter region may suppress the expression of DCT gene and form the white coat color of goat.

The aim of this study was to estimate the genetic parameters of reproductive diseases within 0-35 days after calving in Chinese Holsteins. Twenty five thousand and twenty-six reproductive diseases records within 0-35 days after calving from 27 farms from 1993 to 2017 were collected, threshold model and linear model were adopted to estimate the genetic parameters. Farm-year, parity and calving season were considered as fixed effects, additive genetic effect and permanent environmental effect as random effect to explore the genetic regularity of reproductive diseases within 0-35 days after calving in Chinese Holsteins. The genetic parameters of uterine diseases were estimated using the same model. The results showed that 54% reproductive diseases records of dairy cows happened within 0-35 days after calving comparing to total reproductive diseases records in the whole lactation. The estimated heritabilities of reproductive diseases within 0-35 days after calving using linear model and threshold model were 0.015 2(0.001 9) and 0.094 4(0.01), respectively. The rank correlation of estimated breeding value (EBV) derived from the two models for reproductive diseases was 95% (P<0.01). Uterine diseases accounted for 48% of reproductive diseases and mainly occurred during 0-35 days after calving, and the estimated heritability of uterine diseases within 0-35 days after calving by linear model was 0.010 7(0.001 6). There was a decrease tendency of EBV both male and female since 2000 based on the results of linear model. The study may provide a reference for the genetic selection and breeding for reproductive diseases in Chinese Holsteins.

The aim of this study was to estimate the genetic parameters of reproductive diseases within 0-35 days after calving in Chinese Holsteins. Twenty five thousand and twenty-six reproductive diseases records within 0-35 days after calving from 27 farms from 1993 to 2017 were collected, threshold model and linear model were adopted to estimate the genetic parameters. Farm-year, parity and calving season were considered as fixed effects, additive genetic effect and permanent environmental effect as random effect to explore the genetic regularity of reproductive diseases within 0-35 days after calving in Chinese Holsteins. The genetic parameters of uterine diseases were estimated using the same model. The results showed that 54% reproductive diseases records of dairy cows happened within 0-35 days after calving comparing to total reproductive diseases records in the whole lactation. The estimated heritabilities of reproductive diseases within 0-35 days after calving using linear model and threshold model were 0.015 2(0.001 9) and 0.094 4(0.01), respectively. The rank correlation of estimated breeding value (EBV) derived from the two models for reproductive diseases was 95% (P<0.01). Uterine diseases accounted for 48% of reproductive diseases and mainly occurred during 0-35 days after calving, and the estimated heritability of uterine diseases within 0-35 days after calving by linear model was 0.010 7(0.001 6). There was a decrease tendency of EBV both male and female since 2000 based on the results of linear model. The study may provide a reference for the genetic selection and breeding for reproductive diseases in Chinese Holsteins.

The molecular characteristics, the spatiotemporal expression patterns and the function of miR-21 during the proliferation and differentiation of duck skeletal muscle satellite cells were studied to provide support for exploring the mechanism of skeletal muscle development. One hundred and eighty eggs with the weight of (98 ±5) g were hatched under the same conditions. The embryo breast muscle samples were collected from 3 hatched ducks from 11^th day to 27^th day of hatching (E11, E12,E13……E26,E27) under sterile condition, respectively. The leg muscle, heart, liver, kidney, stomach, small intestine, abdominal fat and sebum were sampled from hatched eggs on E27 under sterile condition. The skeletal muscle satellite cells were isolated and purified by mixed enzyme digestion and differential adherence methods. The role of miR-21 was explored by fluorescence quantitative analysis, protein hybridization, EdU detection and flow cytometry during the proliferation and differentiation of duck skeletal muscle satellite cells transfected with Agomir and Antagomir of miR-21. The results of qRT-PCR showed that the expression of miR-21 increased gradually with the increase of incubation age, reached the highest level on E19, and then decreased gradually. In addition, the highest level of miR-21 expression was found in liver on E27. miR-21 was overexpressed in the proliferating duck skeletal muscle satellite cells, the mRNA expression levels of CDK2 and CyclinD1 significantly (P<0.05) and extremely significantly increased (P<0.01), respectively. The number of positive cells detected by EdU was significantly increased (P<0.05). The number of G2 phase cells was extremely significantly decreased (P<0.01), and the number of S phase cells was significantly increased (P<0.05) by flow cytometry analysis. When the expression of miR-21 was suppressed in the proliferating duck skeletal muscle satellite cells, the results were contrary to that of miR-21 overexpression in the proliferating duck skeletal muscle satellite cells. When the miR-21 was overexpressed in the differentiated duck skeletal muscle satellite cells, the mRNA expression of MyoG and MyHC were extremely significantly increased (P<0.01) by qRT-PCR. The protein expression of MyoG was siginificantly increased(P<0.05), and the protein expression of MyHC was extremely significantly increased (P<0.01) by Western blot (WB) analysis. Both the number of MyHC positive cells and the number of myotubes containing more than 10 nuclei were extremely significantly increased (P<0.01) detected by immunofluorescence assay. The results were contrary to that of miR-21 was suppressed in the differentiated duck skeletal muscle satellite cells. The results indicate that miR-21 can promote the proliferation and differentiation of skeletal muscle satellite cells of myoblasts of ducks.

The molecular characteristics, the spatiotemporal expression patterns and the function of miR-21 during the proliferation and differentiation of duck skeletal muscle satellite cells were studied to provide support for exploring the mechanism of skeletal muscle development. One hundred and eighty eggs with the weight of (98 ±5) g were hatched under the same conditions. The embryo breast muscle samples were collected from 3 hatched ducks from 11^th day to 27^th day of hatching (E11, E12,E13……E26,E27) under sterile condition, respectively. The leg muscle, heart, liver, kidney, stomach, small intestine, abdominal fat and sebum were sampled from hatched eggs on E27 under sterile condition. The skeletal muscle satellite cells were isolated and purified by mixed enzyme digestion and differential adherence methods. The role of miR-21 was explored by fluorescence quantitative analysis, protein hybridization, EdU detection and flow cytometry during the proliferation and differentiation of duck skeletal muscle satellite cells transfected with Agomir and Antagomir of miR-21. The results of qRT-PCR showed that the expression of miR-21 increased gradually with the increase of incubation age, reached the highest level on E19, and then decreased gradually. In addition, the highest level of miR-21 expression was found in liver on E27. miR-21 was overexpressed in the proliferating duck skeletal muscle satellite cells, the mRNA expression levels of CDK2 and CyclinD1 significantly (P<0.05) and extremely significantly increased (P<0.01), respectively. The number of positive cells detected by EdU was significantly increased (P<0.05). The number of G2 phase cells was extremely significantly decreased (P<0.01), and the number of S phase cells was significantly increased (P<0.05) by flow cytometry analysis. When the expression of miR-21 was suppressed in the proliferating duck skeletal muscle satellite cells, the results were contrary to that of miR-21 overexpression in the proliferating duck skeletal muscle satellite cells. When the miR-21 was overexpressed in the differentiated duck skeletal muscle satellite cells, the mRNA expression of MyoG and MyHC were extremely significantly increased (P<0.01) by qRT-PCR. The protein expression of MyoG was siginificantly increased(P<0.05), and the protein expression of MyHC was extremely significantly increased (P<0.01) by Western blot (WB) analysis. Both the number of MyHC positive cells and the number of myotubes containing more than 10 nuclei were extremely significantly increased (P<0.01) detected by immunofluorescence assay. The results were contrary to that of miR-21 was suppressed in the differentiated duck skeletal muscle satellite cells. The results indicate that miR-21 can promote the proliferation and differentiation of skeletal muscle satellite cells of myoblasts of ducks.

The study aimed to investigate the genetic polymorphisms of NCAPG-DCAF16 region in Dezhou donkeys and their associations with growth traits, which can help to mine significant genetic markers for molecular marker assisted selection of Dezhou donkeys. The polymorphisms of NCAPG-DCAF16 region in Dezhou donkeys were detected by DNA pool sequencing technology. Based on the results of DNA pool sequencing, mass-array system was used to genotype the detected SNPs in 227 Dezhou donkeys. Association analysis between the SNPs of NCAPG-DCAF16 region and the growth traits at birth, 3 months old and 6 months old were performed. The results showed that:1) Four SNPs were detected in NCAPG gene, rs1 (T>C) in the 9th intron, rs2 (A>G) in the 11th intron, rs4 (C>G) in the 19th intron, and rs5 (C>G) in the 8th intron. A synonymous mutation was found in the 2nd exon of DCAF16 gene, and the site was rs008 (A>G). 2) The correlation analysis at birth revealed that the average rump length of the individuals with CC genotype at the rs1 was significantly higher than those with CT genotype at birth (P<0.05), the phenotypic values of individuals with different genotypes at 3 months old had no significant difference (P>0.05). At 6 months old, the average chest width of individuals with TT genotype was extremely significantly different from those with CC genotype (P<0.01), and the average shin circumference of individuals with TT and CT genotypes was significantly higher than those with CC genotype (P<0.05). 3) The correlation analysis of rs4 locus indicated that the average chest depth of individuals with GG genotype was significantly higher than those with GC genotype at birth (P<0.05). At 3 months old, the average phenotypic values of individuals with different genotypes was not significantly different (P>0.05). The average chest width and shin circumference of individuals with CC genotype at the age of 6 months were significantly higher than those with GG genotype (P<0.05). 4) The results of rs008 locus correlation analysis showed that there was no significant difference in phenotypic values among individuals with different genotypes at birth (P>0.05). The body weight, body height and rump height of individuals with AA and GA genotypes were significantly higher than those with GG genotypes at the age of 3 months (P<0.05). At 6 months old, the body height and chest circumference of individuals with AA and GA genotypes were extremely significantly higher than those with GG genotype (P<0.01). The difference in chest depth and rump height between individuals with GA and GG genotypes was extremely significantly different (P<0.01). The rump width of individuals with GA genotype was significantly higher than those with GG and AA genotypes (P<0.05). 5) According to the result of linkage disequilibrium analysis, rs1, rs2, rs4 and rs5 had 5 combination genotypes in Dezhou donkeys. The results of association analysis displayed that the average chest width of individuals with TTAACCCC genotype was significantly higher than those with CCGGGGGG genotype at 6 months old (P<0.05). In summary, the 5 SNPs in the NCAPG-DCAF16 region of the Dezhou population have the significant effects on growth traits, especially the rs008 locus has a significant correlation with the important growth traits such as body weight and body height, which can be used as genetic markers to improve the breeding efficiency in Dezhou donkeys.

The study aimed to investigate the genetic polymorphisms of NCAPG-DCAF16 region in Dezhou donkeys and their associations with growth traits, which can help to mine significant genetic markers for molecular marker assisted selection of Dezhou donkeys. The polymorphisms of NCAPG-DCAF16 region in Dezhou donkeys were detected by DNA pool sequencing technology. Based on the results of DNA pool sequencing, mass-array system was used to genotype the detected SNPs in 227 Dezhou donkeys. Association analysis between the SNPs of NCAPG-DCAF16 region and the growth traits at birth, 3 months old and 6 months old were performed. The results showed that:1) Four SNPs were detected in NCAPG gene, rs1 (T>C) in the 9th intron, rs2 (A>G) in the 11th intron, rs4 (C>G) in the 19th intron, and rs5 (C>G) in the 8th intron. A synonymous mutation was found in the 2nd exon of DCAF16 gene, and the site was rs008 (A>G). 2) The correlation analysis at birth revealed that the average rump length of the individuals with CC genotype at the rs1 was significantly higher than those with CT genotype at birth (P<0.05), the phenotypic values of individuals with different genotypes at 3 months old had no significant difference (P>0.05). At 6 months old, the average chest width of individuals with TT genotype was extremely significantly different from those with CC genotype (P<0.01), and the average shin circumference of individuals with TT and CT genotypes was significantly higher than those with CC genotype (P<0.05). 3) The correlation analysis of rs4 locus indicated that the average chest depth of individuals with GG genotype was significantly higher than those with GC genotype at birth (P<0.05). At 3 months old, the average phenotypic values of individuals with different genotypes was not significantly different (P>0.05). The average chest width and shin circumference of individuals with CC genotype at the age of 6 months were significantly higher than those with GG genotype (P<0.05). 4) The results of rs008 locus correlation analysis showed that there was no significant difference in phenotypic values among individuals with different genotypes at birth (P>0.05). The body weight, body height and rump height of individuals with AA and GA genotypes were significantly higher than those with GG genotypes at the age of 3 months (P<0.05). At 6 months old, the body height and chest circumference of individuals with AA and GA genotypes were extremely significantly higher than those with GG genotype (P<0.01). The difference in chest depth and rump height between individuals with GA and GG genotypes was extremely significantly different (P<0.01). The rump width of individuals with GA genotype was significantly higher than those with GG and AA genotypes (P<0.05). 5) According to the result of linkage disequilibrium analysis, rs1, rs2, rs4 and rs5 had 5 combination genotypes in Dezhou donkeys. The results of association analysis displayed that the average chest width of individuals with TTAACCCC genotype was significantly higher than those with CCGGGGGG genotype at 6 months old (P<0.05). In summary, the 5 SNPs in the NCAPG-DCAF16 region of the Dezhou population have the significant effects on growth traits, especially the rs008 locus has a significant correlation with the important growth traits such as body weight and body height, which can be used as genetic markers to improve the breeding efficiency in Dezhou donkeys.

The purpose of this study was to investigate the effects of exogenous melatonin on in vitro maturation of buffalo oocytes and their receptor mediated mechanisms. The following tests:1)The expression of melatonin receptors MT1 and MT2 in cumulus cell and oocytes of buffalo were detected by immunofluorescence.2)The effects of different concentrations of melatonin (10^-9, 10^-8, 10^-7 mol·L^-1) added in the maturation medium on the efficiency of buffalo oocytes IVM and the development of the embryos after in vitro fertilization (IVF) were explored.3)According to the optimal concentration of melatonin, different treatment combinations were added into the mature liquid:untreated,melatonin (10^-8 mol·L^-1 MT),melatonin receptor antagonist (10^-8 mol·L^-1 LZU),melatonin receptor agonist (10^-8 mol·L^-1 ⅡK7),melatonin+ melatonin receptor antagonist (10^-8 mol·L^-1 MT+10^-8 mol·L^-1 LZU).The first polar body excretion rate of oocyte was calculated and the first polar body oocytes were detected by ELISA Kit after 24 h,and then mature oocytes were fertilized in vitro and their rates of division and blastocyst were calculated.The results showed that:1)The both of the MT1 and MT2 receptors were presented in the buffalo cumulus cells and oocytes; 2)The first polar body exhaust rates of oocytes cultured in the maturation medium added with different concentrations of melatonin (10^-9,10^-8,10^-7 mol·L^-1) were significantly higher than the 0 mol·L^-1MT group and the embryonic cleavage rate in each treatment group was also significantly higher than in 0 mol·L^-1 MT group (P<0.05),moreover,the blastocyst rate of 10^-8 and 10^-7 mol·L^-1 MT groups was significantly higher than that of control group (P<0.05); 3)The oocyte maturation rate, division rate and blastocyst rate of embryo in the melatonin receptor antagonist group (10^-8 mol·L^-1 LZU) were not significantly different from those in the untreated group (P>0.05).The oocyte maturation rate, embryo division rate and blastocyst rate after in vitro fertilization were significantly higher in the group of melatonin receptor agonist group(10^-8 mol·L^-1 ⅡK7) than these in the untreated group(P<0.05),however, the difference was not significant compared with the melatonin group (10^-8 mol·L^-1 MT) (P>0.05); The oocyte maturation rate,division rate and blastocyst rate of embryo in melatonin+melatonin receptor antagonist group (10^-8 mol·L^-1 MT+10^-8 mol·L^-1 LZU) were not significantly different compared with that in untreated group (P>0.05). 4) The concentration of cAMP in the oocyte of melatonin group was significantly reduced compared with that of the untreated group, but the concentration of cGMP was significantly increased than the untreated group (P<0.05). It can be seen from this study, by binding to the cell membrane G protein coupled receptor MT1 and MT2, melatonin inhibits cAMP synthesis and increases cGMP content, thereby promoting the mature and early embryonic development of oocytes.

The purpose of this study was to investigate the effects of exogenous melatonin on in vitro maturation of buffalo oocytes and their receptor mediated mechanisms. The following tests:1)The expression of melatonin receptors MT1 and MT2 in cumulus cell and oocytes of buffalo were detected by immunofluorescence.2)The effects of different concentrations of melatonin (10^-9, 10^-8, 10^-7 mol·L^-1) added in the maturation medium on the efficiency of buffalo oocytes IVM and the development of the embryos after in vitro fertilization (IVF) were explored.3)According to the optimal concentration of melatonin, different treatment combinations were added into the mature liquid:untreated,melatonin (10^-8 mol·L^-1 MT),melatonin receptor antagonist (10^-8 mol·L^-1 LZU),melatonin receptor agonist (10^-8 mol·L^-1 ⅡK7),melatonin+ melatonin receptor antagonist (10^-8 mol·L^-1 MT+10^-8 mol·L^-1 LZU).The first polar body excretion rate of oocyte was calculated and the first polar body oocytes were detected by ELISA Kit after 24 h,and then mature oocytes were fertilized in vitro and their rates of division and blastocyst were calculated.The results showed that:1)The both of the MT1 and MT2 receptors were presented in the buffalo cumulus cells and oocytes; 2)The first polar body exhaust rates of oocytes cultured in the maturation medium added with different concentrations of melatonin (10^-9,10^-8,10^-7 mol·L^-1) were significantly higher than the 0 mol·L^-1MT group and the embryonic cleavage rate in each treatment group was also significantly higher than in 0 mol·L^-1 MT group (P<0.05),moreover,the blastocyst rate of 10^-8 and 10^-7 mol·L^-1 MT groups was significantly higher than that of control group (P<0.05); 3)The oocyte maturation rate, division rate and blastocyst rate of embryo in the melatonin receptor antagonist group (10^-8 mol·L^-1 LZU) were not significantly different from those in the untreated group (P>0.05).The oocyte maturation rate, embryo division rate and blastocyst rate after in vitro fertilization were significantly higher in the group of melatonin receptor agonist group(10^-8 mol·L^-1 ⅡK7) than these in the untreated group(P<0.05),however, the difference was not significant compared with the melatonin group (10^-8 mol·L^-1 MT) (P>0.05); The oocyte maturation rate,division rate and blastocyst rate of embryo in melatonin+melatonin receptor antagonist group (10^-8 mol·L^-1 MT+10^-8 mol·L^-1 LZU) were not significantly different compared with that in untreated group (P>0.05). 4) The concentration of cAMP in the oocyte of melatonin group was significantly reduced compared with that of the untreated group, but the concentration of cGMP was significantly increased than the untreated group (P<0.05). It can be seen from this study, by binding to the cell membrane G protein coupled receptor MT1 and MT2, melatonin inhibits cAMP synthesis and increases cGMP content, thereby promoting the mature and early embryonic development of oocytes.

The aim of this study was to investigate the effects of dietary methionine on the regeneration performance of goose down, serum indexes and the expression of genes related to cysteine and methionine metabolic pathways in Hortobagy goose (Goosebag goose). One hundred and twenty eight 150-day-old healthy Hortobagy geese were selected and randomly divided into 4 groups after epilation. Four replicates for each group, and 8 geese for each replicate. Geese in the 4 groups were fed diets with additional methionine levels of 0, 0.28%, 0.56% and 0.84%, respectively. The formal experimental period was 42 days. The data of goose down such as the thousand plush weight,velvet length, serum biochemical indicators, growth factors and antioxidant factors levels were measured, and the expression levels of genes related to cysteine and methionine metabolism pathways were determined by RT-qPCR. The results showed as the following:1) There was no significant difference in the TP,ALB,BUN,TG,HDL and LDL levels in serum among different groups (P>0.05). At the same time, with the increase of dietary methionine concentration, the growth factor (IGF-1), prolactin(PRL) and the glutathione (GSH-Px) levels in serum were also significantly increased(P<0.05), but when the methionine level added to 0.84%, the concentration of the above factors reduced. 2) Supplementation of methionine in diet could significantly increase the thousand down weight and velvet length of back(P<0.05), and promote the regeneration of down, but there was no significant difference in the yield of down among the different methionine supplementation groups(P>0.05). 3) The results of RT-qPCR showed that, with the methionine addition, the mRNA expression of MAT1A gene in cysteine and methionine metabolism pathway decreased significantly (P<0.05), and the expression of ACHY mRNA extremely significantly decreased in the 0.28% methionine supplementation group(P<0.01). The results indicate that suitable concentration of methionine in diet can promote the secretion of IGF-1 and PRL growth factors and antioxidant factors in goose. It can also significantly increase the thousand plush weight and velvet length of back, reduce the mRNA expression of the MAT1A and ACHY genes in cysteine and methionine metabolism pathways and promote the regeneration of the down feather, and the suitable total addition level is 0.55%-0.83%.

The aim of this study was to investigate the effects of dietary methionine on the regeneration performance of goose down, serum indexes and the expression of genes related to cysteine and methionine metabolic pathways in Hortobagy goose (Goosebag goose). One hundred and twenty eight 150-day-old healthy Hortobagy geese were selected and randomly divided into 4 groups after epilation. Four replicates for each group, and 8 geese for each replicate. Geese in the 4 groups were fed diets with additional methionine levels of 0, 0.28%, 0.56% and 0.84%, respectively. The formal experimental period was 42 days. The data of goose down such as the thousand plush weight,velvet length, serum biochemical indicators, growth factors and antioxidant factors levels were measured, and the expression levels of genes related to cysteine and methionine metabolism pathways were determined by RT-qPCR. The results showed as the following:1) There was no significant difference in the TP,ALB,BUN,TG,HDL and LDL levels in serum among different groups (P>0.05). At the same time, with the increase of dietary methionine concentration, the growth factor (IGF-1), prolactin(PRL) and the glutathione (GSH-Px) levels in serum were also significantly increased(P<0.05), but when the methionine level added to 0.84%, the concentration of the above factors reduced. 2) Supplementation of methionine in diet could significantly increase the thousand down weight and velvet length of back(P<0.05), and promote the regeneration of down, but there was no significant difference in the yield of down among the different methionine supplementation groups(P>0.05). 3) The results of RT-qPCR showed that, with the methionine addition, the mRNA expression of MAT1A gene in cysteine and methionine metabolism pathway decreased significantly (P<0.05), and the expression of ACHY mRNA extremely significantly decreased in the 0.28% methionine supplementation group(P<0.01). The results indicate that suitable concentration of methionine in diet can promote the secretion of IGF-1 and PRL growth factors and antioxidant factors in goose. It can also significantly increase the thousand plush weight and velvet length of back, reduce the mRNA expression of the MAT1A and ACHY genes in cysteine and methionine metabolism pathways and promote the regeneration of the down feather, and the suitable total addition level is 0.55%-0.83%.

The objective of this study was to investigate the effect of dietary energy levels on nutrient apparent digestibility, growth performance and rumen fermentation of Chinese Holstein heifers. Forty-five Chinese Holstein heifers averaged at aged 8 months were selected and randomly allocated into 3 groups with 15 heifers in each group. Animals in group I were fed a low-energy diet (NE_L 5.64 MJ·kg^-1), those in group Ⅱ were fed a medium energy diet (NE_L 5.94 MJ·kg^-1), and those in group Ⅲ were fed a high-energy diet (NE_L 6.21 MJ·kg^-1). The experiment lasted for 110 d. Protein levels of the 3 diets were similar(CP14.04%). The growth performance indicators of heifer, including body weight, withers height, body length, heart girth, circumference of cannon bone, teat length and teat spacing, were determined, and fecal samples were collected for the analysis of apparent digestibility of nutrient, and the rumen fluid samples were collected for the determination of rumen pH, microbial protein(MCP), NH₃-N and volatile fatty acid(VFA). The results showed that:1) Compared with group I and Ⅱ, the apparent digestibility of neutral detergent fiber (NDF) and acid detergent fiber (ADF) in group Ⅲ were significantly decreased (P<0.05). Compared with group I, the apparent digestibility of NDF in group Ⅱ and Ⅲ were decreased by 4.84% and 7.03%, and the apparent digestibility of ADF were decreased by 2.99% and 7.50%, respectively. There was no significant difference in the dry matter intake(DMI),the apparent digestibility of crude protein(CP), crude fat(EE), calcium(Ca) and phosphorus(P) among group I, Ⅱ and Ⅲ (P>0.05). 2) The average daily gain (ADG) in group I, Ⅱ and Ⅲ were 0.70, 0.80 and 0.91 kg·d^-1, respectively. Compared with group I, ADG in group Ⅱ and Ⅲ were increased by 14.28% and 30.00%(P<0.05), respectively. There was no significant differences in body measurements (withers height, body length, heart girth, circumference of cannon bone) among group I, Ⅱ and Ⅲ (P>0.05). 3) Compared with group I and Ⅲ, the average length of anterior teats in group Ⅱ were increased 4.15% (P>0.05) and 9.06% (P<0.05), and the average length of posterior teats were increased 9.02%(P<0.05) and 11.54%(P<0.05), respectively. 4) There was no significant differences in rumen pH, MCP content, the concentration of acetic acid, butyric acid, total volatile fatty acids (T-VFA) and the ration of acetic acid/propionic acid (A/P) among group I, Ⅱ and Ⅲ (P>0.05). The content of ammonia nitrogen (NH₃-N) was the lowest in group Ⅱ, which was significantly lower than that of group I by 3.56% (P<0.05), but there was no significant difference between group Ⅱ and Ⅲ (P>0.05). Propionic acid content in group Ⅲ was higher than that in groupⅠand Ⅱ by 4.99% (P<0.05) and 4.83%(P>0.05), respectively. In conclusion, a dietary NE_L level of 5.94 MJ·kg^-1 and CP level of 14% are recommended for Chinese Holstein heifers averaged at 8-11 months old.

The objective of this study was to investigate the effect of dietary energy levels on nutrient apparent digestibility, growth performance and rumen fermentation of Chinese Holstein heifers. Forty-five Chinese Holstein heifers averaged at aged 8 months were selected and randomly allocated into 3 groups with 15 heifers in each group. Animals in group I were fed a low-energy diet (NE_L 5.64 MJ·kg^-1), those in group Ⅱ were fed a medium energy diet (NE_L 5.94 MJ·kg^-1), and those in group Ⅲ were fed a high-energy diet (NE_L 6.21 MJ·kg^-1). The experiment lasted for 110 d. Protein levels of the 3 diets were similar(CP14.04%). The growth performance indicators of heifer, including body weight, withers height, body length, heart girth, circumference of cannon bone, teat length and teat spacing, were determined, and fecal samples were collected for the analysis of apparent digestibility of nutrient, and the rumen fluid samples were collected for the determination of rumen pH, microbial protein(MCP), NH₃-N and volatile fatty acid(VFA). The results showed that:1) Compared with group I and Ⅱ, the apparent digestibility of neutral detergent fiber (NDF) and acid detergent fiber (ADF) in group Ⅲ were significantly decreased (P<0.05). Compared with group I, the apparent digestibility of NDF in group Ⅱ and Ⅲ were decreased by 4.84% and 7.03%, and the apparent digestibility of ADF were decreased by 2.99% and 7.50%, respectively. There was no significant difference in the dry matter intake(DMI),the apparent digestibility of crude protein(CP), crude fat(EE), calcium(Ca) and phosphorus(P) among group I, Ⅱ and Ⅲ (P>0.05). 2) The average daily gain (ADG) in group I, Ⅱ and Ⅲ were 0.70, 0.80 and 0.91 kg·d^-1, respectively. Compared with group I, ADG in group Ⅱ and Ⅲ were increased by 14.28% and 30.00%(P<0.05), respectively. There was no significant differences in body measurements (withers height, body length, heart girth, circumference of cannon bone) among group I, Ⅱ and Ⅲ (P>0.05). 3) Compared with group I and Ⅲ, the average length of anterior teats in group Ⅱ were increased 4.15% (P>0.05) and 9.06% (P<0.05), and the average length of posterior teats were increased 9.02%(P<0.05) and 11.54%(P<0.05), respectively. 4) There was no significant differences in rumen pH, MCP content, the concentration of acetic acid, butyric acid, total volatile fatty acids (T-VFA) and the ration of acetic acid/propionic acid (A/P) among group I, Ⅱ and Ⅲ (P>0.05). The content of ammonia nitrogen (NH₃-N) was the lowest in group Ⅱ, which was significantly lower than that of group I by 3.56% (P<0.05), but there was no significant difference between group Ⅱ and Ⅲ (P>0.05). Propionic acid content in group Ⅲ was higher than that in groupⅠand Ⅱ by 4.99% (P<0.05) and 4.83%(P>0.05), respectively. In conclusion, a dietary NE_L level of 5.94 MJ·kg^-1 and CP level of 14% are recommended for Chinese Holstein heifers averaged at 8-11 months old.

The aim of this study was to investigate molecular prevalence of bovine coronavirus (BCoV) in yak in Qinghai-Tibetan Plateau and to isolate BCoV from clinical samples. RT-PCR assay was used to detect BCoV from 336 diarrhea feces samples of yak from Tibet, Qinghai, Sichuan and Yunnan, and the S, HE, and N gene fragments were further amplified, moreover, positive samples were used to isolate BCoV strain. Results were as follows:Approximate 69.05% (232/336) diarrheic samples were detected BCoV positive by RT-PCR. Sequence and phylogenetic analysis showed that BCoV S1 subunit sequence, HE gene fragment and N gene fragment from 32 clinical samples in this study had unique evolutionary features. Notably, a BCoV strain of yak was successfully isolated firstly, the TCID₅₀ of this strain was 10^-7.17·0.1 mL^-1,and a recombinant event was predicted in S gene of this strain. These results indicate that BCoV widely circulates in yak in the Qinghai-Tibetan Plateau of China and exhibit unique evolution.

The aim of this study was to investigate molecular prevalence of bovine coronavirus (BCoV) in yak in Qinghai-Tibetan Plateau and to isolate BCoV from clinical samples. RT-PCR assay was used to detect BCoV from 336 diarrhea feces samples of yak from Tibet, Qinghai, Sichuan and Yunnan, and the S, HE, and N gene fragments were further amplified, moreover, positive samples were used to isolate BCoV strain. Results were as follows:Approximate 69.05% (232/336) diarrheic samples were detected BCoV positive by RT-PCR. Sequence and phylogenetic analysis showed that BCoV S1 subunit sequence, HE gene fragment and N gene fragment from 32 clinical samples in this study had unique evolutionary features. Notably, a BCoV strain of yak was successfully isolated firstly, the TCID₅₀ of this strain was 10^-7.17·0.1 mL^-1,and a recombinant event was predicted in S gene of this strain. These results indicate that BCoV widely circulates in yak in the Qinghai-Tibetan Plateau of China and exhibit unique evolution.

The study aims to isolate bluetongue virus (BTV) endemic in Yunnan Province of China and to understand the genetic and infection characteristics of the isolated BTV. Virus isolation was performed by inoculation method of "Embryos-C6/36 cells-BHK-21 cells" with BTV nucleic acid positive blood samples collected from sentinel cattle. Serotype of the isolated virus was decided by micro-neutralization test and sequencing the ORF regions of Segment 2 and Segment 6. The proliferation characteristics of isolated virus in BHK-21 cells were determined by the tests of growth curve and plaque formation. The viral load and neutralizing antibody of infected animal were analyzed by qRT-PCR and neutralization test. Results were as follows:In August 2013, BTV strain of V013/YN/2013 was isolated form sentinel cattle in Mangshi, Yunnan Province. Micro-neutralization test showed that V013/YN/2013 belonging to serotype of BTV-9. Further sequence analysis confirmed the identity of isolated virus by correlation with other isolated BTV-9 strains belonging to Eastern topotype and showed closest relationship with BTV-9 strains isolated from Japan and Australia. The results of growth curve and plaque formation showed that the proliferation capacity of V013/YN/2013 is significantly higher than BTV-9 reference strain. Although specific neutralizing antibodies are produced in infected animals, the presence of the virus in the blood can be continuously detected, suggesting that the virus can cause a long-lasting infection in cattle. This study reported for the first time the isolation of an BTV-9 strain V013/YN/2013 belonging to Eastern topotype in China, these results will provide a basis for further whole-genome sequencing, establishment of diagnostic methods, epidemiological investigation and pathogenicity study of BTV-9 in China.

The study aims to isolate bluetongue virus (BTV) endemic in Yunnan Province of China and to understand the genetic and infection characteristics of the isolated BTV. Virus isolation was performed by inoculation method of "Embryos-C6/36 cells-BHK-21 cells" with BTV nucleic acid positive blood samples collected from sentinel cattle. Serotype of the isolated virus was decided by micro-neutralization test and sequencing the ORF regions of Segment 2 and Segment 6. The proliferation characteristics of isolated virus in BHK-21 cells were determined by the tests of growth curve and plaque formation. The viral load and neutralizing antibody of infected animal were analyzed by qRT-PCR and neutralization test. Results were as follows:In August 2013, BTV strain of V013/YN/2013 was isolated form sentinel cattle in Mangshi, Yunnan Province. Micro-neutralization test showed that V013/YN/2013 belonging to serotype of BTV-9. Further sequence analysis confirmed the identity of isolated virus by correlation with other isolated BTV-9 strains belonging to Eastern topotype and showed closest relationship with BTV-9 strains isolated from Japan and Australia. The results of growth curve and plaque formation showed that the proliferation capacity of V013/YN/2013 is significantly higher than BTV-9 reference strain. Although specific neutralizing antibodies are produced in infected animals, the presence of the virus in the blood can be continuously detected, suggesting that the virus can cause a long-lasting infection in cattle. This study reported for the first time the isolation of an BTV-9 strain V013/YN/2013 belonging to Eastern topotype in China, these results will provide a basis for further whole-genome sequencing, establishment of diagnostic methods, epidemiological investigation and pathogenicity study of BTV-9 in China.

To explore the cellular transcriptional gene differentially expressed in MDBK cells infected with caprine parainfluenza virus type 3 (CPIV3), we analyzed the mRNA expression profiles in MDBK cells infected with 1 MOI CPIV3 JSHA2014-1 strain at 24 hpi using high-throughput sequencing. The results indicated that a total of 261 genes were obviously differentially expressed in the infected MDBK cells including 140 up-regulated genes and 121 down-regulated genes. The eight of these genes were verified by RT-qPCR assay, and the results were consistent with those of high-throughput sequencing. GO analysis classification showed that differentially expressed genes were mainly involved in biological processes, cellular components and molecular functions. KEGG analysis shows that the signaling pathways involved in these genes included metabolism-related signaling pathways, biological systems, cellular processes, gene information processes and environmental information processes. This study laid the foundation for further exploring the pathogenesis of CPIV3.

To explore the cellular transcriptional gene differentially expressed in MDBK cells infected with caprine parainfluenza virus type 3 (CPIV3), we analyzed the mRNA expression profiles in MDBK cells infected with 1 MOI CPIV3 JSHA2014-1 strain at 24 hpi using high-throughput sequencing. The results indicated that a total of 261 genes were obviously differentially expressed in the infected MDBK cells including 140 up-regulated genes and 121 down-regulated genes. The eight of these genes were verified by RT-qPCR assay, and the results were consistent with those of high-throughput sequencing. GO analysis classification showed that differentially expressed genes were mainly involved in biological processes, cellular components and molecular functions. KEGG analysis shows that the signaling pathways involved in these genes included metabolism-related signaling pathways, biological systems, cellular processes, gene information processes and environmental information processes. This study laid the foundation for further exploring the pathogenesis of CPIV3.

The study was conducted to understand the difference of viremia, anti-PRRSV antibody and anti-PRRSV neutralizing antibody in piglets vaccinated against porcine reproductive and respiratory syndrome virus (PRRSV) VR2332 attenuated strain which was given by nasal drip and injection. Nine 20-day-old health piglets were randomly selected and separated into 3 groups which designated as nose dropping group, injection group and control group respectively. Blood samples were collected by the anterior vena cava at day 0, 3, 7, 11, 15, 19, 23, 27, 31, 35, 39, 43 after infection, spleen, submandibular lymph nodes, inguinal lymph nodes and mesenteric lymph nodes were obtained at day 43, PAMs were collected in a sterile room meanwhile. Viral load in piglets was detected with the real-time fluorescent quantitative PCR. The anti-PRRSV antibody titer was tested by ELISA, and the anti-PRRSV neutralizing antibody titer was detected by the method based on PAMs. The results showed that, ①PRRSV started to replicate in only 2 piglet's sera at day 3 after infection, viremia peaked at day 15-19, the virus copy number was greater than 10⁴ copies·μL^-1 and viremia disappeared after day 27 in nose dropping group. PRRSV started to replicate in all 3 piglet's serum at day 3 after infection, viremia peaked at day 15-23, the virus copy number was up to 10⁵ copies·μL^-1 at day 19 and viremia disappeared after day 31 in injection group. ②PRRRSV can be detected in these lymph nodes and PAMs, and the virus copy number of injection group was greater than nose dropping group. ③Anti-PRRSV antibody was produced in piglets at day 27 after infection from serum, and anti-PRRSV antibody levels of injection group was always higher than nose dropping group. ④Anti-PRRSV neutralizing antibody titers was 1:2 at day 43 and lower than 1:2 before day 35 in nose dropping group. Anti-PRRSV neutralizing antibody titers was 1:4 at day 27, 1:8 at day 35, 1:4 at day 43 and lower than 1:2 at day 3, 11 and 19 in injection group. The results showed that the virus copy number in injection group was always ten to one hundred times higher than that of nose dropping group, and duration of viremia was longer. Anti-PRRSV antibody producing time in injection group was earlier than nose dropping group, and antibody levels was higher. Anti-PRRSV neutralizing antibody levels in nose dropping group was very low, anti-PRRSV neutralizing antibody producing time in injection group was earlier than nose dropping group, and antibody levels were higher. In conclusion, higher level neutralizing antibody can be stimulated in piglets after inoculating PRRSV VR2332 attenuated strain by injection than by nasal drip, but higher level viremia also can be induced meanwhile.

The study was conducted to understand the difference of viremia, anti-PRRSV antibody and anti-PRRSV neutralizing antibody in piglets vaccinated against porcine reproductive and respiratory syndrome virus (PRRSV) VR2332 attenuated strain which was given by nasal drip and injection. Nine 20-day-old health piglets were randomly selected and separated into 3 groups which designated as nose dropping group, injection group and control group respectively. Blood samples were collected by the anterior vena cava at day 0, 3, 7, 11, 15, 19, 23, 27, 31, 35, 39, 43 after infection, spleen, submandibular lymph nodes, inguinal lymph nodes and mesenteric lymph nodes were obtained at day 43, PAMs were collected in a sterile room meanwhile. Viral load in piglets was detected with the real-time fluorescent quantitative PCR. The anti-PRRSV antibody titer was tested by ELISA, and the anti-PRRSV neutralizing antibody titer was detected by the method based on PAMs. The results showed that, ①PRRSV started to replicate in only 2 piglet's sera at day 3 after infection, viremia peaked at day 15-19, the virus copy number was greater than 10⁴ copies·μL^-1 and viremia disappeared after day 27 in nose dropping group. PRRSV started to replicate in all 3 piglet's serum at day 3 after infection, viremia peaked at day 15-23, the virus copy number was up to 10⁵ copies·μL^-1 at day 19 and viremia disappeared after day 31 in injection group. ②PRRRSV can be detected in these lymph nodes and PAMs, and the virus copy number of injection group was greater than nose dropping group. ③Anti-PRRSV antibody was produced in piglets at day 27 after infection from serum, and anti-PRRSV antibody levels of injection group was always higher than nose dropping group. ④Anti-PRRSV neutralizing antibody titers was 1:2 at day 43 and lower than 1:2 before day 35 in nose dropping group. Anti-PRRSV neutralizing antibody titers was 1:4 at day 27, 1:8 at day 35, 1:4 at day 43 and lower than 1:2 at day 3, 11 and 19 in injection group. The results showed that the virus copy number in injection group was always ten to one hundred times higher than that of nose dropping group, and duration of viremia was longer. Anti-PRRSV antibody producing time in injection group was earlier than nose dropping group, and antibody levels was higher. Anti-PRRSV neutralizing antibody levels in nose dropping group was very low, anti-PRRSV neutralizing antibody producing time in injection group was earlier than nose dropping group, and antibody levels were higher. In conclusion, higher level neutralizing antibody can be stimulated in piglets after inoculating PRRSV VR2332 attenuated strain by injection than by nasal drip, but higher level viremia also can be induced meanwhile.

To better understand the situation and evolution of poultry infected with H3 subtype avian influenza viruses around Dongting Lake, active monitoring at live-poultry markets and Free-range duck farms at this region were implemented in 2011-2015, and a total of 31 strains of H3 subtype AIV were isolated. Gene sequencing and evolutionary analysis of these strains were conducted in our study. Sequencing results showed that all the viruses were located in the Eurasian lineage except for two H3N8s in North America lineage, all isolates were low pathogenic avian influenza virus. Phylogenetic tree showed that PA and PB2 genes of some viruses were clustered with H5, while NP, PA and PB1 were clustered with H7, suggesting that complicated gene exchange were occurred. Internal genes of some viruses isolated from free-range poultry show very high homology with wild birds origin H3, H7, H11 from neighboring countries such as Vietnam and South Korea, may share the same evolutionary origin. Further genome sequence alignment can divided all viruses into 21 different genotypes. The source of H3 subtype avian influenza virus in the Dongting Lake area is complicated, showing a clear genetic diversity.

To better understand the situation and evolution of poultry infected with H3 subtype avian influenza viruses around Dongting Lake, active monitoring at live-poultry markets and Free-range duck farms at this region were implemented in 2011-2015, and a total of 31 strains of H3 subtype AIV were isolated. Gene sequencing and evolutionary analysis of these strains were conducted in our study. Sequencing results showed that all the viruses were located in the Eurasian lineage except for two H3N8s in North America lineage, all isolates were low pathogenic avian influenza virus. Phylogenetic tree showed that PA and PB2 genes of some viruses were clustered with H5, while NP, PA and PB1 were clustered with H7, suggesting that complicated gene exchange were occurred. Internal genes of some viruses isolated from free-range poultry show very high homology with wild birds origin H3, H7, H11 from neighboring countries such as Vietnam and South Korea, may share the same evolutionary origin. Further genome sequence alignment can divided all viruses into 21 different genotypes. The source of H3 subtype avian influenza virus in the Dongting Lake area is complicated, showing a clear genetic diversity.

This experiment was conducted to study the effect of ATP-binding cassette G5 gene of Toxocara canis. Specific siRNAs of the Tc-abcg-5 gene fragment were synthesized in vitro, and adult T. canis and eggs were interfered by using RNAi techniques. Results were as follows:Compared with siRNA-1020 group and siRNA-432 group, the transcriptional level of Tc-abcg-5 in siRNA-744 group was significantly reduced after 24 and 48 h's treatments, indicating that siRNA-744 had higher silencing efficiency. The delivery efficiency of siRNA-744 to eggs was compared by electroporation and immersion methods. The results showed that immersion method was more efficient. Interference of T. canis infective eggs were performed using siRNA-744 by soaking. Pathology study was used to analysis of mouse liver, brain and lung tissues on the 7th day after oral administration of interfered eggs, which showed that RNAi reduced the infectivity of T. canis infective eggs, thereby reducing the lung and liver lesions. It is suggested that the Tc-abcg-5 gene may participate in the growth and development of T. canis by affecting the development of eggs.

This experiment was conducted to study the effect of ATP-binding cassette G5 gene of Toxocara canis. Specific siRNAs of the Tc-abcg-5 gene fragment were synthesized in vitro, and adult T. canis and eggs were interfered by using RNAi techniques. Results were as follows:Compared with siRNA-1020 group and siRNA-432 group, the transcriptional level of Tc-abcg-5 in siRNA-744 group was significantly reduced after 24 and 48 h's treatments, indicating that siRNA-744 had higher silencing efficiency. The delivery efficiency of siRNA-744 to eggs was compared by electroporation and immersion methods. The results showed that immersion method was more efficient. Interference of T. canis infective eggs were performed using siRNA-744 by soaking. Pathology study was used to analysis of mouse liver, brain and lung tissues on the 7th day after oral administration of interfered eggs, which showed that RNAi reduced the infectivity of T. canis infective eggs, thereby reducing the lung and liver lesions. It is suggested that the Tc-abcg-5 gene may participate in the growth and development of T. canis by affecting the development of eggs.

This experiment was conducted to clear if chicken invariant chain (Ii) cytoplasmic/transmembrane domains-Ii_(Cyt/Tra), as immune carrier-is able to carry antigen peptide to associate with MHC classⅡ molecules and enter the endosomes in cell transport pathway. In this study, 4 gene segments, such as Ii, Ii_(Cyt/Tra), F2 (Newcastle disease virus F protein fragment) and Ii_(Cyt/Tra)/F2, were amplified and constructed by PCR. They were inserted into prokaryotic expression plasmid pET-32a and eukaryotic expression plasmid pmCherry-C1/N1 respectively, and 8 recombinant plasmids were constructed. Then they were transferred into the engineering bacteria (E. coli) and human renal cell (293 T) respectively. The binding of the target protein to the MHCⅡβ was detected by pull-down assay, and the co-localization in the eukaryotic cells with the MHCⅡβ and the localization in the endosomes were determined by laser confocal. The results showed that all the recombinant target protein molecules were expressed in prokaryotic or eukaryotes; Ii, Ii_(Cyt/Tra) and Ii_(Cyt/Tra)/F2 were able to bind to MHCⅡβ as well as to enter endosomes except F2. To sum up, the active domains, Ii_(Cyt/Tra), not only itself had the effect of associating MHCⅡβ, but also could bring antigenic peptides to bind MHCⅡβ and transport the endosome together, and finally enter the antigen presentation pathway. This provides a theoretical basis for further study of Ii as immune carrier.

This experiment was conducted to clear if chicken invariant chain (Ii) cytoplasmic/transmembrane domains-Ii_(Cyt/Tra), as immune carrier-is able to carry antigen peptide to associate with MHC classⅡ molecules and enter the endosomes in cell transport pathway. In this study, 4 gene segments, such as Ii, Ii_(Cyt/Tra), F2 (Newcastle disease virus F protein fragment) and Ii_(Cyt/Tra)/F2, were amplified and constructed by PCR. They were inserted into prokaryotic expression plasmid pET-32a and eukaryotic expression plasmid pmCherry-C1/N1 respectively, and 8 recombinant plasmids were constructed. Then they were transferred into the engineering bacteria (E. coli) and human renal cell (293 T) respectively. The binding of the target protein to the MHCⅡβ was detected by pull-down assay, and the co-localization in the eukaryotic cells with the MHCⅡβ and the localization in the endosomes were determined by laser confocal. The results showed that all the recombinant target protein molecules were expressed in prokaryotic or eukaryotes; Ii, Ii_(Cyt/Tra) and Ii_(Cyt/Tra)/F2 were able to bind to MHCⅡβ as well as to enter endosomes except F2. To sum up, the active domains, Ii_(Cyt/Tra), not only itself had the effect of associating MHCⅡβ, but also could bring antigenic peptides to bind MHCⅡβ and transport the endosome together, and finally enter the antigen presentation pathway. This provides a theoretical basis for further study of Ii as immune carrier.

The purpose of this study was to evaluate the immunomodulatory effects of antimicrobial peptide sublancin and astragalus polysaccharides (APS) on immunosuppressive mice induced by cyclophosphamide (CTX). Sixty healthy female BALB/c mice aged 4-6 weeks were randomly divided into 6 groups of 10 each. Normal control group:d 1 to d 3, intraperitoneally injected with 0.9% physiological saline, d 4 to d 10, gavage 0.9% physiological saline. The other 5 groups:d 1 to d 3, injected with CTX (80 mg·kg^-1). On the d 4 to d 10, the negative control group:gavage 0.9% physiological saline; low or high concentration sublancin treatment group:sublancin solution was administered at a dose of 4.0 and 8.0 mg·kg^-1; the APS-treated group:gavage APS (200 mg·kg^-1); positive control group:gavage levamisole (10 mg·kg^-1, BW). All treatments were performed once a day and 0.2 mL each time. The mice were weighed before the mice were intraperitoneally injected with CTX on the first day of the experiment, on the d 4 before gavage and on the d 11. After weighing the body weight on the d 11, peripheral blood and spleen were collected. Compared with the normal control group, the peripheral blood red blood cell, hemoglobin, and white blood cell counts in the negative control group were significantly reduced (P<0.05); the gene expression of IL-2, IL-4, and IL-6 in the spleen was significantly reduced (P<0.05). Compared with the negative control group, there was no difference in body weight between the APS group and the high concentration Sublancin group after intragastric administration but there is an upward trend, indicating that high concentration Sublancin and APS had a positive effect on body weight of mice; the leukocyte content in peripheral blood of low and high concentration Sublancin, APS, and positive control groups were significantly increased (P<0.05); in the high concentration Sublancin-treated group, red blood cells and hemoglobin were significantly increased (P<0.05); CD4⁺ levels in peripheral blood of mice treated with low or high concentration Sublancin and APS were significantly increased (P<0.05); in both low and high concentration Sublancin, APS and positive control groups, CD8⁺ was significantly decreased (P<0.05); in low and high concentration sublancin groups, CD4⁺/CD8⁺ was significantly increased (P<0.05); in low and high concentration sublancin, APS and positive control groups, mouse spleen IL-4 were significantly increased (P<0.05); in high concentration sublancin, APS and positive control groups, the expressions of IL-2 and IL-6 in the spleen of mice were significantly increased (P<0.05). In summary, it can be concluded that appropriate doses of the antimicrobial peptides sublancin and APS can all alleviate the immunosuppression caused by cyclophosphamide. Compared with APS, the role of antimicrobial peptide sublancin in the effect on alleviating the immunosuppression of CTX is more comprehensive.

The purpose of this study was to evaluate the immunomodulatory effects of antimicrobial peptide sublancin and astragalus polysaccharides (APS) on immunosuppressive mice induced by cyclophosphamide (CTX). Sixty healthy female BALB/c mice aged 4-6 weeks were randomly divided into 6 groups of 10 each. Normal control group:d 1 to d 3, intraperitoneally injected with 0.9% physiological saline, d 4 to d 10, gavage 0.9% physiological saline. The other 5 groups:d 1 to d 3, injected with CTX (80 mg·kg^-1). On the d 4 to d 10, the negative control group:gavage 0.9% physiological saline; low or high concentration sublancin treatment group:sublancin solution was administered at a dose of 4.0 and 8.0 mg·kg^-1; the APS-treated group:gavage APS (200 mg·kg^-1); positive control group:gavage levamisole (10 mg·kg^-1, BW). All treatments were performed once a day and 0.2 mL each time. The mice were weighed before the mice were intraperitoneally injected with CTX on the first day of the experiment, on the d 4 before gavage and on the d 11. After weighing the body weight on the d 11, peripheral blood and spleen were collected. Compared with the normal control group, the peripheral blood red blood cell, hemoglobin, and white blood cell counts in the negative control group were significantly reduced (P<0.05); the gene expression of IL-2, IL-4, and IL-6 in the spleen was significantly reduced (P<0.05). Compared with the negative control group, there was no difference in body weight between the APS group and the high concentration Sublancin group after intragastric administration but there is an upward trend, indicating that high concentration Sublancin and APS had a positive effect on body weight of mice; the leukocyte content in peripheral blood of low and high concentration Sublancin, APS, and positive control groups were significantly increased (P<0.05); in the high concentration Sublancin-treated group, red blood cells and hemoglobin were significantly increased (P<0.05); CD4⁺ levels in peripheral blood of mice treated with low or high concentration Sublancin and APS were significantly increased (P<0.05); in both low and high concentration Sublancin, APS and positive control groups, CD8⁺ was significantly decreased (P<0.05); in low and high concentration sublancin groups, CD4⁺/CD8⁺ was significantly increased (P<0.05); in low and high concentration sublancin, APS and positive control groups, mouse spleen IL-4 were significantly increased (P<0.05); in high concentration sublancin, APS and positive control groups, the expressions of IL-2 and IL-6 in the spleen of mice were significantly increased (P<0.05). In summary, it can be concluded that appropriate doses of the antimicrobial peptides sublancin and APS can all alleviate the immunosuppression caused by cyclophosphamide. Compared with APS, the role of antimicrobial peptide sublancin in the effect on alleviating the immunosuppression of CTX is more comprehensive.

The experiment was conducted to discuss the biological function of HSPs during the progress of tumor induced by the Marek's disease, the relationship between pathological lesion of liver and distribution, expression level of HSPs were studied. The tumor animal model was established using MD virus. Chickens were timely killed post artificial infection and the livers were sampled. Pathological lesion of liver and distribution, transcription and expression level of HSPs were studied during the progress of tumor by the methods of histopathology, immunohistochemistry, FQ RT-PCR and ELISA. Results were as follows:At 14 days, obvious pathological damage appeared in the livers of chickens attracted by MDV, and unequal-sized lymphoid cells infiltration was found among liver cells. HSP60 and HSP90 were mainly located in the cytoplasm of tumor cell, while HSP27 and HSP70 were mainly located in the hepatocyte around the tumor tissues. Within the course of MD, the transcription level of HSP70 and HSP90 mRNA of challenge group were always significantly higher than those of blank control and vaccine control groups. The expression level of HSP90 of challenge group was always significantly higher than those of blank control group in the test process, while the expression level of HSP70 of challenge group was significantly higher than those of blank controrl group at 14 and 21 d, was significantly higher at 28 and 35 d, and no significant difference after 42 d. The distribution among HSP60, HSP90, HSP27, HSP70 and the trend of expression level between HSP90 and HSP70 are completely different during the progress of tumor induced by MDV, which indicates that different HSPs have different biological functions.

The experiment was conducted to discuss the biological function of HSPs during the progress of tumor induced by the Marek's disease, the relationship between pathological lesion of liver and distribution, expression level of HSPs were studied. The tumor animal model was established using MD virus. Chickens were timely killed post artificial infection and the livers were sampled. Pathological lesion of liver and distribution, transcription and expression level of HSPs were studied during the progress of tumor by the methods of histopathology, immunohistochemistry, FQ RT-PCR and ELISA. Results were as follows:At 14 days, obvious pathological damage appeared in the livers of chickens attracted by MDV, and unequal-sized lymphoid cells infiltration was found among liver cells. HSP60 and HSP90 were mainly located in the cytoplasm of tumor cell, while HSP27 and HSP70 were mainly located in the hepatocyte around the tumor tissues. Within the course of MD, the transcription level of HSP70 and HSP90 mRNA of challenge group were always significantly higher than those of blank control and vaccine control groups. The expression level of HSP90 of challenge group was always significantly higher than those of blank control group in the test process, while the expression level of HSP70 of challenge group was significantly higher than those of blank controrl group at 14 and 21 d, was significantly higher at 28 and 35 d, and no significant difference after 42 d. The distribution among HSP60, HSP90, HSP27, HSP70 and the trend of expression level between HSP90 and HSP70 are completely different during the progress of tumor induced by MDV, which indicates that different HSPs have different biological functions.

The study was aimed to evaluate the probable protective effect of Shugan Yiyang Capsule (SGYY, Chinese herbal extract with highly efficient antioxidant activities) against testicular toxicity and oxidative stress in adult male rats exposed to quinestrol (QES) and study the possible mechanisms underlying these effects. Twenty 8-week-old male SD rats were randomly divided into 4 groups. After 1 week of adaptation, the rats were treated with gavage once daily for 2 weeks. Control group:0.1 mL physiological saline+0.1 mL olive oil; SGYY group:100 mg·kg^-1 SGYY; QES group:0.1 mg·kg^-1 QES; QES+SGYY group:0.1 mg·kg^-1 QES+100 mg·kg^-1 SGYY. QES was dissolved in olive oil; SGYY was dissolved in physiological saline. After the treatments, the testis, epididymis, seminal vesicle and prostate were weighed, and the long and short diameters were measured. The changes in the histological structure of seminiferous tubules and the proportion of spermatogenic cells were observed in testicular tissue sections by HE staining. The expression of PCNA in spermatogenic cells of testis was detected by immunohistochemistry, and the apoptosis was detected by Tunel. The blood plasma was separated and the content of testosterone was detected. The antioxidant enzyme activity was detected in the testicular homogenate. The results showed that exposure to QES decreased the weights of reproductive organs and the sperms counts in cauda epididymis of rats. A significant decrease in the seminiferous tubular area, tubular diameter, height of germinal epithelium, number of spermatogenic cell (per seminiferous tubules) and expression of PCNA level with a significant increase in the Tunel levels were observed in the testis of experimental rats over the controls. These events were accompanied by a significant reduction in the plasma testosterone levels in QES exposed rats, indicating reduced steroidogenesis. Significant reduction in the activities of SOD, GSH-Px and T-AOC and a significant increase in MDA were also observed in the testis of QES exposed rats over the controls. Conversely, supplementation of SGYY (100 mg·kg^-1) ameliorated the male reproductive health in rats exposed to QES as evidenced by increased reproductive organ weights, sperm counts, testicular steroidogenesis, spermatogenic cell proliferation and testicular antioxidants whereas MDA and Tunel levels decreased. To sum up, the results show that SGYY increased the number of spermatogenic cells and improved spermatogenesis in the testis by improving antioxidant function, inhibiting oxidative stress and promoting testosterone secretion.

The study was aimed to evaluate the probable protective effect of Shugan Yiyang Capsule (SGYY, Chinese herbal extract with highly efficient antioxidant activities) against testicular toxicity and oxidative stress in adult male rats exposed to quinestrol (QES) and study the possible mechanisms underlying these effects. Twenty 8-week-old male SD rats were randomly divided into 4 groups. After 1 week of adaptation, the rats were treated with gavage once daily for 2 weeks. Control group:0.1 mL physiological saline+0.1 mL olive oil; SGYY group:100 mg·kg^-1 SGYY; QES group:0.1 mg·kg^-1 QES; QES+SGYY group:0.1 mg·kg^-1 QES+100 mg·kg^-1 SGYY. QES was dissolved in olive oil; SGYY was dissolved in physiological saline. After the treatments, the testis, epididymis, seminal vesicle and prostate were weighed, and the long and short diameters were measured. The changes in the histological structure of seminiferous tubules and the proportion of spermatogenic cells were observed in testicular tissue sections by HE staining. The expression of PCNA in spermatogenic cells of testis was detected by immunohistochemistry, and the apoptosis was detected by Tunel. The blood plasma was separated and the content of testosterone was detected. The antioxidant enzyme activity was detected in the testicular homogenate. The results showed that exposure to QES decreased the weights of reproductive organs and the sperms counts in cauda epididymis of rats. A significant decrease in the seminiferous tubular area, tubular diameter, height of germinal epithelium, number of spermatogenic cell (per seminiferous tubules) and expression of PCNA level with a significant increase in the Tunel levels were observed in the testis of experimental rats over the controls. These events were accompanied by a significant reduction in the plasma testosterone levels in QES exposed rats, indicating reduced steroidogenesis. Significant reduction in the activities of SOD, GSH-Px and T-AOC and a significant increase in MDA were also observed in the testis of QES exposed rats over the controls. Conversely, supplementation of SGYY (100 mg·kg^-1) ameliorated the male reproductive health in rats exposed to QES as evidenced by increased reproductive organ weights, sperm counts, testicular steroidogenesis, spermatogenic cell proliferation and testicular antioxidants whereas MDA and Tunel levels decreased. To sum up, the results show that SGYY increased the number of spermatogenic cells and improved spermatogenesis in the testis by improving antioxidant function, inhibiting oxidative stress and promoting testosterone secretion.

The aim of this study was to prove that α-mangostin can alleviate the lipopolysaccharide (LPS)-induced inflammation of IEC-6 cells and reveal its mechanism. In this study, we constructed an LPS-induced inflammation model in IEC-6 cells in vitro, and examined whether LPS-induced inflammation was reduced by α-mangostin by means of the methods of rheumatology, ELISA, Q-PCR, and Western blot. The results showed that pretreatment with 5 μmol·L^-1 α-mangostin significantly reduced the secretion of prostaglandin (PG) E2, interleukin (IL)-1β, IL-6, tumor necrosis factor (TNF)-α and the expression of COX-2, IL-6, TNF-α, IL-1β mRNA induced by LPS in IEC-6 cells (P<0.05). By investigating the inflammatory-related signaling pathway, NF-κB, we found that α-mangostin significantly inhibited the activation of TLR4 mRNA and NF-κB-related protein phosphorylated IκBα (pIκBα) and phosphorylated p65 (pp65) (P<0.05). In summary, α-mangostin can inhibit LPS-induced inflammation of IEC-6 cells through the TLR4/NF-κB signaling pathway and may be a potential treatment for inflammatory diseases.

The aim of this study was to prove that α-mangostin can alleviate the lipopolysaccharide (LPS)-induced inflammation of IEC-6 cells and reveal its mechanism. In this study, we constructed an LPS-induced inflammation model in IEC-6 cells in vitro, and examined whether LPS-induced inflammation was reduced by α-mangostin by means of the methods of rheumatology, ELISA, Q-PCR, and Western blot. The results showed that pretreatment with 5 μmol·L^-1 α-mangostin significantly reduced the secretion of prostaglandin (PG) E2, interleukin (IL)-1β, IL-6, tumor necrosis factor (TNF)-α and the expression of COX-2, IL-6, TNF-α, IL-1β mRNA induced by LPS in IEC-6 cells (P<0.05). By investigating the inflammatory-related signaling pathway, NF-κB, we found that α-mangostin significantly inhibited the activation of TLR4 mRNA and NF-κB-related protein phosphorylated IκBα (pIκBα) and phosphorylated p65 (pp65) (P<0.05). In summary, α-mangostin can inhibit LPS-induced inflammation of IEC-6 cells through the TLR4/NF-κB signaling pathway and may be a potential treatment for inflammatory diseases.

This study aimed to systematically compare the accuracy of genomic selection for important economic traits in pigs by using 8 models including GBLUP, SSGBLUP, BayesA, BayesB, BayesC, BayesLASSO, BSLMM and BayesR. The data of age at 100 kg, backfat thickness at 100 kg and teat numbers were collected from 2 585 Yorkshire sows and all the individuals were genotyped by using PorcineSNP50K Beadchip. The accuracy of genomic selection for the 8 models were compared by a 5-fold cross-validation procedure based on additive model. The results demonstrated that the accuracy of genomic selection were positively correlated with the calculated heritabilities of different traits. The cross-validation analysis indicated that the prediction accuracy of age at 100 kg was the highest among the 3 different traits, but different models performed dissimilarly in different traits. The prediction accuracy of SSGBLUP was the highest for both age at 100 kg and backfat thickness at 100 kg, and the prediction accuracy of BayesA was the highest for teat numbers. In conclusion, SSGBLUP model can be used for the traits with moderate and high heritabilities when conducting genomic prediction for small sample size and BayesA is suitable to the traits with low heritability. How to optimize and select a model that is applicable to all traits may be a research direction in the future.

This study aimed to systematically compare the accuracy of genomic selection for important economic traits in pigs by using 8 models including GBLUP, SSGBLUP, BayesA, BayesB, BayesC, BayesLASSO, BSLMM and BayesR. The data of age at 100 kg, backfat thickness at 100 kg and teat numbers were collected from 2 585 Yorkshire sows and all the individuals were genotyped by using PorcineSNP50K Beadchip. The accuracy of genomic selection for the 8 models were compared by a 5-fold cross-validation procedure based on additive model. The results demonstrated that the accuracy of genomic selection were positively correlated with the calculated heritabilities of different traits. The cross-validation analysis indicated that the prediction accuracy of age at 100 kg was the highest among the 3 different traits, but different models performed dissimilarly in different traits. The prediction accuracy of SSGBLUP was the highest for both age at 100 kg and backfat thickness at 100 kg, and the prediction accuracy of BayesA was the highest for teat numbers. In conclusion, SSGBLUP model can be used for the traits with moderate and high heritabilities when conducting genomic prediction for small sample size and BayesA is suitable to the traits with low heritability. How to optimize and select a model that is applicable to all traits may be a research direction in the future.

The aim of this study was to screen the suitable reference gene in mouse ovaries, which provided important foundation for mRNA expression analysis during ovary development. Mouse ovaries tissues at 4 different development stages (0 day, 3 weeks, 5 weeks, 8 weeks old) were selected as experimental materials. The Trizol method was used to extract the total RNA and synthesize cDNA. The primers of 8 commonly used internal reference genes (Gapdh, β-actin, β-tubulin, 18S rRNA, 16S rRNA, H2afz, Ubc, Rpl13a) were designed based on the sequence of Mus musculus. Ovaries cDNA were diluted gradiently (1:10) to construct standard curves by qRT-PCR. GeNorm was used to calculate gene expression stability measure (M) of the 8 candidated reference genes. NormFinder analysis was performed to compare the stability between selected reference genes, and BestKeeper ranked the standard deviation (SD) and coefficient of variance (CV) of candidate gene expression to determine the optimal reference gene. The result indicated that the primers of 8 internal reference genes were highly specific and had good linear relationship. According to the results of geNorm, the stabilities of selected reference genes were Gapdh=β-actin > 18S rRNA > Ubc > 16S rRNA > H2afz > Rpl13a > β-tubulin. Based on the results of NormFinder, Gapdh had the best stability in the ovaries of mouse as the standard deviation and coefficient of variation were the smallest (SD:0.32, CV:1.95), while H2afz was considered as unstable with SD>1.0, CV=5.76. In conclusion, we have successfully identified Gapdh, β-actin as the most suitable reference genes during ovary development after birth, which could be used as reference genes for normalizing genes analysis.

The aim of this study was to screen the suitable reference gene in mouse ovaries, which provided important foundation for mRNA expression analysis during ovary development. Mouse ovaries tissues at 4 different development stages (0 day, 3 weeks, 5 weeks, 8 weeks old) were selected as experimental materials. The Trizol method was used to extract the total RNA and synthesize cDNA. The primers of 8 commonly used internal reference genes (Gapdh, β-actin, β-tubulin, 18S rRNA, 16S rRNA, H2afz, Ubc, Rpl13a) were designed based on the sequence of Mus musculus. Ovaries cDNA were diluted gradiently (1:10) to construct standard curves by qRT-PCR. GeNorm was used to calculate gene expression stability measure (M) of the 8 candidated reference genes. NormFinder analysis was performed to compare the stability between selected reference genes, and BestKeeper ranked the standard deviation (SD) and coefficient of variance (CV) of candidate gene expression to determine the optimal reference gene. The result indicated that the primers of 8 internal reference genes were highly specific and had good linear relationship. According to the results of geNorm, the stabilities of selected reference genes were Gapdh=β-actin > 18S rRNA > Ubc > 16S rRNA > H2afz > Rpl13a > β-tubulin. Based on the results of NormFinder, Gapdh had the best stability in the ovaries of mouse as the standard deviation and coefficient of variation were the smallest (SD:0.32, CV:1.95), while H2afz was considered as unstable with SD>1.0, CV=5.76. In conclusion, we have successfully identified Gapdh, β-actin as the most suitable reference genes during ovary development after birth, which could be used as reference genes for normalizing genes analysis.

The aim of this study was to establish a sensitive and specific method for detection of antibody against porcine circovirus type 3 (PCV3). The bioinformatical prediction shows that most antigen epitopes of PCV3 Cap protein are clustered at the C terminal (carboxyl terminal) of PCV3-Cap protein, while first 33 amino acids in the N terminal of Cap protein are the nuclear localization sequences. Primers were designed to amplify a part of ORF2 gene which does not contain first 120 amino acids of N terminal and PCV3 positive samples were used as templates for PCR amplification. The recombinant plasmid was constructed by cloning the PCR product into pET-30a vector. The recombinant plasmid was transfected into E. coli BL21 competent cells, and the best induction conditions were determined. The expression protein was purified by Ni-NTA affinity chromatography. Finally, using the Cap protein as a coating protein, an indirect ELISA for detection of antibody against PCV3 was established and then primary applied to clinical detection. As a result:PCV3 ORF2 gene fragment with size of 285 bp was amplified from positive sample. The recombinant plasmid was constructed by inserting the amplicon into pET-30a, followed by enzymatical digestion and sequencing. Expression of the recombinant protein was induced by adding 1 mmol·L^-1 IPTG, incubating at 37℃ for 6 h. Western blot result showed that the recombinant protein possessed good reactivity with positive serum against PCV3. The optimal antigen concentration, dilution of testing serum, working dilution of enzyme conjugated antibody of ELISA were 1 μg·mL^-1, 1:20 and 1:5 000, respectively. The positive value was 0.273 and the coefficients of inter-and intra-batches were less than 10%, indicating the good repeatability of the method. PCV2 positive serum was negative in this method, indicating its high specificity. Finally, 439 clinical serum samples were detected through this ELISA method and 60.59% (266/439) of them were identified as positive. In summary, a rapid, easy to use, sensitive and specific method for detection of antibody against porcine circovirus type 3 (PCV3) was established.

The aim of this study was to establish a sensitive and specific method for detection of antibody against porcine circovirus type 3 (PCV3). The bioinformatical prediction shows that most antigen epitopes of PCV3 Cap protein are clustered at the C terminal (carboxyl terminal) of PCV3-Cap protein, while first 33 amino acids in the N terminal of Cap protein are the nuclear localization sequences. Primers were designed to amplify a part of ORF2 gene which does not contain first 120 amino acids of N terminal and PCV3 positive samples were used as templates for PCR amplification. The recombinant plasmid was constructed by cloning the PCR product into pET-30a vector. The recombinant plasmid was transfected into E. coli BL21 competent cells, and the best induction conditions were determined. The expression protein was purified by Ni-NTA affinity chromatography. Finally, using the Cap protein as a coating protein, an indirect ELISA for detection of antibody against PCV3 was established and then primary applied to clinical detection. As a result:PCV3 ORF2 gene fragment with size of 285 bp was amplified from positive sample. The recombinant plasmid was constructed by inserting the amplicon into pET-30a, followed by enzymatical digestion and sequencing. Expression of the recombinant protein was induced by adding 1 mmol·L^-1 IPTG, incubating at 37℃ for 6 h. Western blot result showed that the recombinant protein possessed good reactivity with positive serum against PCV3. The optimal antigen concentration, dilution of testing serum, working dilution of enzyme conjugated antibody of ELISA were 1 μg·mL^-1, 1:20 and 1:5 000, respectively. The positive value was 0.273 and the coefficients of inter-and intra-batches were less than 10%, indicating the good repeatability of the method. PCV2 positive serum was negative in this method, indicating its high specificity. Finally, 439 clinical serum samples were detected through this ELISA method and 60.59% (266/439) of them were identified as positive. In summary, a rapid, easy to use, sensitive and specific method for detection of antibody against porcine circovirus type 3 (PCV3) was established.