In this study, we evaluated cuticle quality and other eggshell quality traits in the Rhode Island Red hens, and we also calculated the genetic and phenotypic correlations between cuticle quality and other eggshells quality traits, which could provide theoretical support for cuticle quality optimization and breeding in the domestic brown/pink egg layer, as well as enhancement of antibacterial properties of egg. We measured cuticle qualities of 3 283 Rhode Island Red hens (120 pedigrees) in Beijing Huadu Yukou Poultry Industry Co. LTD.. Cuticle quality heritability, genetic and phenotypic correlation between cuticle quality and other eggshell quality traits were evaluated by DMU software. The results showed that the value of cuticle quality was (24.24±9.13)%, and its heritability was 0.40 (medium heritability). The values of eggshell color (L^*, a^*, b^*), eggshell strength and eggshell thickness were (61.45±4.91), (17.79±2.34), (30.59±2.50), (3.22±0.75) kg·cm^-2 and (324.85±42.37)μm, respectively. Their heritability were from 0.39 to 0.17, which represented heritability from medium to low. Furthermore, the genetic correlations between cuticle quality and eggshell color (L^*, a^*, b^*) were —0.49, 0.43 and —0.27, respectively. Both eggshell strength and eggshell thickness showed weak correlation with cuticle quality, which were 0.02 and 0.13, respectively. The genetic correlation between eggshell strength and eggshell thickness was 0.72. In conclusion, direct selection of cuticle quality (high heritability) could accelerate genetic progress and improve breeding efficiency. Selection of cuticle quality might deepen eggshell color, since it was related to the L^*, a^* and b^* values. Besides, selection of cuticle quality would not significantly impact eggshell strength and thickness owing to low genetic correlations between them.

In this study, we evaluated cuticle quality and other eggshell quality traits in the Rhode Island Red hens, and we also calculated the genetic and phenotypic correlations between cuticle quality and other eggshells quality traits, which could provide theoretical support for cuticle quality optimization and breeding in the domestic brown/pink egg layer, as well as enhancement of antibacterial properties of egg. We measured cuticle qualities of 3 283 Rhode Island Red hens (120 pedigrees) in Beijing Huadu Yukou Poultry Industry Co. LTD.. Cuticle quality heritability, genetic and phenotypic correlation between cuticle quality and other eggshell quality traits were evaluated by DMU software. The results showed that the value of cuticle quality was (24.24±9.13)%, and its heritability was 0.40 (medium heritability). The values of eggshell color (L^*, a^*, b^*), eggshell strength and eggshell thickness were (61.45±4.91), (17.79±2.34), (30.59±2.50), (3.22±0.75) kg·cm^-2 and (324.85±42.37)μm, respectively. Their heritability were from 0.39 to 0.17, which represented heritability from medium to low. Furthermore, the genetic correlations between cuticle quality and eggshell color (L^*, a^*, b^*) were —0.49, 0.43 and —0.27, respectively. Both eggshell strength and eggshell thickness showed weak correlation with cuticle quality, which were 0.02 and 0.13, respectively. The genetic correlation between eggshell strength and eggshell thickness was 0.72. In conclusion, direct selection of cuticle quality (high heritability) could accelerate genetic progress and improve breeding efficiency. Selection of cuticle quality might deepen eggshell color, since it was related to the L^*, a^* and b^* values. Besides, selection of cuticle quality would not significantly impact eggshell strength and thickness owing to low genetic correlations between them.

The present study aimed to explore the promoter activity of Nhlh2 gene and the relationship between the promoter activity and transcription factors in cells, and further provide a theoretical basis for the regulating mechanism of Nhlh2 gene in pigs. Based on the template of porcine ovarian genomic DNA, PCR was used to amplify different length deletion fragments of Nhlh2 gene promoter. These fragments were cloned into pGL3-basic to build the promoter recombinant plasmid, and then were transfected into the porcine ovarian granulosa cells. The relative luciferase activities of these plasmids were further measured by using the dual luciferase assay system. Chromatin immunoprecipitation assay (ChIP) was used to confirm the interaction between YY1, C/EBPβ and the promoter of Nhlh2. Finally, the overexpression and mutant vectors, small interfering RNA (siRNA) of YY1 and C/EBPβ were constructed, the luciferase activity was detected by dual luciferase reporter assay system. Results showed that transcription activity of P7 (—238-+129) region was the highest, —654-—238 was the potential region containing the negative regulatory element, and —238-—20 was the potential region containing the positive regulatory element; After the bioinformatic analysis and ChIP test, we found the binding sites of YY1 and C/EBPβ at Nhlh2 gene promoter were —101-—85 and —153-—140, respectively. After mutating the binding sites of YY1 and C/EBPβ, the transcription activity of P7 was significantly up-regulated; The overexpression of YY1 and C/EBPβ significantly reduced the transcription activity of P7, and the transcription activity was significantly up-regulated by YY1-siRNA and C/EBPβ-siRNA. These results indicate that the core promoter region of Nhlh2 is —238-+129. Furthermore, YY1 and C/EBPβ respectively bound at —101-—85 and —153-—140 of Nhlh2 gene promoter to inhibit the transcription of Nhlh2 gene in pigs.

The present study aimed to explore the promoter activity of Nhlh2 gene and the relationship between the promoter activity and transcription factors in cells, and further provide a theoretical basis for the regulating mechanism of Nhlh2 gene in pigs. Based on the template of porcine ovarian genomic DNA, PCR was used to amplify different length deletion fragments of Nhlh2 gene promoter. These fragments were cloned into pGL3-basic to build the promoter recombinant plasmid, and then were transfected into the porcine ovarian granulosa cells. The relative luciferase activities of these plasmids were further measured by using the dual luciferase assay system. Chromatin immunoprecipitation assay (ChIP) was used to confirm the interaction between YY1, C/EBPβ and the promoter of Nhlh2. Finally, the overexpression and mutant vectors, small interfering RNA (siRNA) of YY1 and C/EBPβ were constructed, the luciferase activity was detected by dual luciferase reporter assay system. Results showed that transcription activity of P7 (—238-+129) region was the highest, —654-—238 was the potential region containing the negative regulatory element, and —238-—20 was the potential region containing the positive regulatory element; After the bioinformatic analysis and ChIP test, we found the binding sites of YY1 and C/EBPβ at Nhlh2 gene promoter were —101-—85 and —153-—140, respectively. After mutating the binding sites of YY1 and C/EBPβ, the transcription activity of P7 was significantly up-regulated; The overexpression of YY1 and C/EBPβ significantly reduced the transcription activity of P7, and the transcription activity was significantly up-regulated by YY1-siRNA and C/EBPβ-siRNA. These results indicate that the core promoter region of Nhlh2 is —238-+129. Furthermore, YY1 and C/EBPβ respectively bound at —101-—85 and —153-—140 of Nhlh2 gene promoter to inhibit the transcription of Nhlh2 gene in pigs.

This study aimed to predict and validate the crucial miRNAs that regulate FoxO1 gene expression, and to elucidate the functional mechanism of these miRNAs in ovine adipose differentiation.Four online softwares were used to predict the miRNAs that might target FoxO1. A dual-luciferase reporter assay system was employed to detect luciferase activity. qPCR, Western blotting and Oil red O staining were used to detect the effect of these miRNAs on the expression of FoxO1 both at mRNA and protein levels, and on the regulation of ovine preadipocyte differentiation. The results showed that FoxO1 was targeted by both miR-142 and miR-144.Overexpression of miR-142 or miR-144 significantly inhibited the luciferase activity of the dual-luciferase reporter vector, and reduced the expression of FoxO1 mRNA (P<0.01) and protein (P<0.05). Both miR-142 and miR-144 promoted the differentiation of ovine preadipocytes and accelerated the formation of lipid droplets. In conclusion, miR-142 and miR-144 can down-regulate the expression of FoxO1, and further down-regulate PPARγ expression,thus promote the differentiation of ovine preadipocytes.

This study aimed to predict and validate the crucial miRNAs that regulate FoxO1 gene expression, and to elucidate the functional mechanism of these miRNAs in ovine adipose differentiation.Four online softwares were used to predict the miRNAs that might target FoxO1. A dual-luciferase reporter assay system was employed to detect luciferase activity. qPCR, Western blotting and Oil red O staining were used to detect the effect of these miRNAs on the expression of FoxO1 both at mRNA and protein levels, and on the regulation of ovine preadipocyte differentiation. The results showed that FoxO1 was targeted by both miR-142 and miR-144.Overexpression of miR-142 or miR-144 significantly inhibited the luciferase activity of the dual-luciferase reporter vector, and reduced the expression of FoxO1 mRNA (P<0.01) and protein (P<0.05). Both miR-142 and miR-144 promoted the differentiation of ovine preadipocytes and accelerated the formation of lipid droplets. In conclusion, miR-142 and miR-144 can down-regulate the expression of FoxO1, and further down-regulate PPARγ expression,thus promote the differentiation of ovine preadipocytes.

The aim of this study was to investigate the effect of Wnt10b gene on the precursor adipocytes differentiation of goat by RNA interference. The goat Wnt10b siRNA sequence was designed and compounded,and the goat subcutaneous and intramuscular precursor adipocytes were obtained by collagenase digestion. The effect of Wnt10b gene expression on the cell differentiation and adipocyte differentiation marker genes expression were detected by using the effective siRNA sequence to interfere the expression of Wnt10b gene in goat subcutaneous and intramuscular precursor adipocytes.The results showed that an effective goat Wnt10b siRNA sequence was obtained,and the expression of Wnt10b was down-regulated by 63% (P<0.01) and 67% (P<0.01), respectively after transfection in goat subcutaneous and intramuscular precursor adipocytes. Results of Oil red O staining showed that the accumulation of lipid droplets were inhibited significantly by Wnt10b RNAi in goat subcutaneous and intramuscular adipocytes. The C/EBPβ, PPARγ,SREBP1 and Pref1 genes were extremely significantly down-regulated in the subcutaneous precursor adipocyte differentiation after Wnt10b RNAi (P<0.01), LPL was significantly down-regulated (P<0.05). The AP2 and LPL were extremely significantly down-regulated (P<0.01), C/EBPβ was significantly down-regulated (P<0.05), and Pref1 was extremely significantly up-regulated (P<0.01) in intramuscular precursor adipocytes differentiation. In this study, it was found that Wnt10b gene could promote the differentiation of goat subcutaneous precursor adipocytes by regulating the expression of C/EBPβ, PPARγ, SREBP1 and LPL, and promote the differentiation of goat intramuscular precursor adipocytes by regulating the expression of AP2, LPL, C/EBPβ and Pref1.

The aim of this study was to investigate the effect of Wnt10b gene on the precursor adipocytes differentiation of goat by RNA interference. The goat Wnt10b siRNA sequence was designed and compounded,and the goat subcutaneous and intramuscular precursor adipocytes were obtained by collagenase digestion. The effect of Wnt10b gene expression on the cell differentiation and adipocyte differentiation marker genes expression were detected by using the effective siRNA sequence to interfere the expression of Wnt10b gene in goat subcutaneous and intramuscular precursor adipocytes.The results showed that an effective goat Wnt10b siRNA sequence was obtained,and the expression of Wnt10b was down-regulated by 63% (P<0.01) and 67% (P<0.01), respectively after transfection in goat subcutaneous and intramuscular precursor adipocytes. Results of Oil red O staining showed that the accumulation of lipid droplets were inhibited significantly by Wnt10b RNAi in goat subcutaneous and intramuscular adipocytes. The C/EBPβ, PPARγ,SREBP1 and Pref1 genes were extremely significantly down-regulated in the subcutaneous precursor adipocyte differentiation after Wnt10b RNAi (P<0.01), LPL was significantly down-regulated (P<0.05). The AP2 and LPL were extremely significantly down-regulated (P<0.01), C/EBPβ was significantly down-regulated (P<0.05), and Pref1 was extremely significantly up-regulated (P<0.01) in intramuscular precursor adipocytes differentiation. In this study, it was found that Wnt10b gene could promote the differentiation of goat subcutaneous precursor adipocytes by regulating the expression of C/EBPβ, PPARγ, SREBP1 and LPL, and promote the differentiation of goat intramuscular precursor adipocytes by regulating the expression of AP2, LPL, C/EBPβ and Pref1.

This study has investigated the effect of ambient temperature on pig energy metabolism, aiming to provide a theoretical foundation of molecular regulation mechanism for pig adaptive evolution. The brain, heart, liver, lung, kidney, eye muscle and psoas major muscle of 12 Rongchang pigs (6-month-old) were collected during the seasonal high and low temperature period,respectively, the mtDNA copy number and the transcriptional levels of energy metabolism-related genes were tested. The results showed that there was a significant correlation between body weight growth rate and ambient temperature (|r| ≥ 0.77, P<0.01), the mtDNA copy number in different tissues were highly consistent between low and high temperature groups (r=0.92, P<0.01); The mtDNA copy number was significantly higher in low temperature group than in high temperature group (P<0.05 or P<0.01) of all tissues, except for the heart (P>0.05), and the transcriptional level of TWINKLE gene regulating mtDNA biosynthesis significantly decreased in different tissues in the high temperature group (P<0.05 or P<0.01); The transcriptional level of genes (ND1, COX1, ATP6 and CYTB) related to energy metabolism in low temperature group basically were significantly (P<0.05) or very significantly (P<0.01) higher than that in high temperature group. However, the transcriptional levels of ND1 and COX1 genes in eye muscle, CYTB gene in liver and heart were no significantly different between low and high temperature groups (P>0.05), and the transcriptional level of CYTB gene in brain in high temperature group was significantly higher than in low temperature group (P<0.05). Furthermore, except for the lung tissue (P<0.01), the transcriptional level of HSP70 gene in other tissues had no significant difference between low and high temperature groups (P>0.05). These results suggest that pig could reshape the metabolism through regulating the gene transcription level and mtDNA copy number, and adapt to ambient temperature change.

This study has investigated the effect of ambient temperature on pig energy metabolism, aiming to provide a theoretical foundation of molecular regulation mechanism for pig adaptive evolution. The brain, heart, liver, lung, kidney, eye muscle and psoas major muscle of 12 Rongchang pigs (6-month-old) were collected during the seasonal high and low temperature period,respectively, the mtDNA copy number and the transcriptional levels of energy metabolism-related genes were tested. The results showed that there was a significant correlation between body weight growth rate and ambient temperature (|r| ≥ 0.77, P<0.01), the mtDNA copy number in different tissues were highly consistent between low and high temperature groups (r=0.92, P<0.01); The mtDNA copy number was significantly higher in low temperature group than in high temperature group (P<0.05 or P<0.01) of all tissues, except for the heart (P>0.05), and the transcriptional level of TWINKLE gene regulating mtDNA biosynthesis significantly decreased in different tissues in the high temperature group (P<0.05 or P<0.01); The transcriptional level of genes (ND1, COX1, ATP6 and CYTB) related to energy metabolism in low temperature group basically were significantly (P<0.05) or very significantly (P<0.01) higher than that in high temperature group. However, the transcriptional levels of ND1 and COX1 genes in eye muscle, CYTB gene in liver and heart were no significantly different between low and high temperature groups (P>0.05), and the transcriptional level of CYTB gene in brain in high temperature group was significantly higher than in low temperature group (P<0.05). Furthermore, except for the lung tissue (P<0.01), the transcriptional level of HSP70 gene in other tissues had no significant difference between low and high temperature groups (P>0.05). These results suggest that pig could reshape the metabolism through regulating the gene transcription level and mtDNA copy number, and adapt to ambient temperature change.

The objective of the study was to investigate the effect of TGF-β on the expression of proliferation-related proteins and genes of boar Sertoli cells, and reveal the mechanism of TGF-β regulating proliferation of boar Sertoli cells. In this study, TGF-β and TGF-β/Smad pathway inhibitor LY2109761 was used to stimulate cultured immature boar Sertoli cells. Cell viability and cell cycle were detected by CCK-8 kit and flow cytometry, respectively. The phosphorylation levels of Smad3 were detected by Western blotting. The expressions of c-Myc mRNA, Skp2 mRNA and ssc-miRNA-24 were detected by real-time quantitative PCR (RT-qPCR). Furthermore, ssc-miRNA-24 mimics and inhibitor were transfected into Sertoli cells, and then Sertoli cells were treated with TGF-β, following detected the expression of related genes and proteins. The results showed that TGF-β influenced cell viability in a dose-dependent manner. 180 pg·mL^-1 TGF-β increased cell proliferation index (PI)and S-phase cell fraction (SPF), inhibited the phosphorylation of Smad3 in a time-dependent (0-24 h) manner, increased the abundance of c-Myc mRNA, Skp2 mRNA, ssc-miRNA-24. 10 μmol·L^-1 LY2109761 promoted TGF-β-induced cell viability, increased cell proliferation index and S-phase cell fraction. In addition, ssc-miRNA-24 mimics decreased phosphorylation level of Smad3, promoted the expression of c-Myc and Skp2 mRNA. However, ssc-miRNA-24 inhibitor had an opposite effect on these parameters. The above results revealed that TGF-β decreased phosphorylation level of Smad3 by increasing the expression of ssc-miRNA-24, and enhanced the expression of c-Myc and Skp2 mRNA to promote the proliferation of boar Sertoli cells.

The objective of the study was to investigate the effect of TGF-β on the expression of proliferation-related proteins and genes of boar Sertoli cells, and reveal the mechanism of TGF-β regulating proliferation of boar Sertoli cells. In this study, TGF-β and TGF-β/Smad pathway inhibitor LY2109761 was used to stimulate cultured immature boar Sertoli cells. Cell viability and cell cycle were detected by CCK-8 kit and flow cytometry, respectively. The phosphorylation levels of Smad3 were detected by Western blotting. The expressions of c-Myc mRNA, Skp2 mRNA and ssc-miRNA-24 were detected by real-time quantitative PCR (RT-qPCR). Furthermore, ssc-miRNA-24 mimics and inhibitor were transfected into Sertoli cells, and then Sertoli cells were treated with TGF-β, following detected the expression of related genes and proteins. The results showed that TGF-β influenced cell viability in a dose-dependent manner. 180 pg·mL^-1 TGF-β increased cell proliferation index (PI)and S-phase cell fraction (SPF), inhibited the phosphorylation of Smad3 in a time-dependent (0-24 h) manner, increased the abundance of c-Myc mRNA, Skp2 mRNA, ssc-miRNA-24. 10 μmol·L^-1 LY2109761 promoted TGF-β-induced cell viability, increased cell proliferation index and S-phase cell fraction. In addition, ssc-miRNA-24 mimics decreased phosphorylation level of Smad3, promoted the expression of c-Myc and Skp2 mRNA. However, ssc-miRNA-24 inhibitor had an opposite effect on these parameters. The above results revealed that TGF-β decreased phosphorylation level of Smad3 by increasing the expression of ssc-miRNA-24, and enhanced the expression of c-Myc and Skp2 mRNA to promote the proliferation of boar Sertoli cells.

The study aimed to investigate the transcriptome differential expression between pre-and post-vitrified yak blastocysts in order to understand the effect of vitrification on the gene expression profile of yak blastocysts. Total RNAs were extracted respectively from fresh blastocysts (FRB) and vitrified-thawed blastocysts (VTB) that were in vitro-produced from yaks, and then the Smart-seq2 method was used to amplify and construct RNA libraries, and finally transcriptome was sequenced using RNA-seq. The|log₂ (VTB/FRB)| ≥ 1 and Q-value<0.05 were set as thresholds for identifying differentially expressed gene (DEG). The DEGs were then searched against the GO and KEGG database for enrichment analysis. The results showed that 9 827 and 13 567 transcripts were detected in FRB and VTB, respectively. A total of 11 174 DEGs were identified between the 2 libraries, in which 7 037 were up-regulated, and 4 137 were down-regulated in VTB. 10 538 DEGs were significantly enriched in 479 GO terms (P<0.05), and there were 318 pathways involved, of which 8 pathways including ribosome biogenesis in eukaryotes, spliceosome and neuroactive ligand-receptor interaction were significantly enriched (P<0.05). In conclusion, this report analyzed the transcriptome change pre-and post-vitrification of yak blastocysts using RNA-seq, which provides a theoretical basis to explore the mechanism of vitrification damage and improve vitrification technology of yak blastocysts.

The study aimed to investigate the transcriptome differential expression between pre-and post-vitrified yak blastocysts in order to understand the effect of vitrification on the gene expression profile of yak blastocysts. Total RNAs were extracted respectively from fresh blastocysts (FRB) and vitrified-thawed blastocysts (VTB) that were in vitro-produced from yaks, and then the Smart-seq2 method was used to amplify and construct RNA libraries, and finally transcriptome was sequenced using RNA-seq. The|log₂ (VTB/FRB)| ≥ 1 and Q-value<0.05 were set as thresholds for identifying differentially expressed gene (DEG). The DEGs were then searched against the GO and KEGG database for enrichment analysis. The results showed that 9 827 and 13 567 transcripts were detected in FRB and VTB, respectively. A total of 11 174 DEGs were identified between the 2 libraries, in which 7 037 were up-regulated, and 4 137 were down-regulated in VTB. 10 538 DEGs were significantly enriched in 479 GO terms (P<0.05), and there were 318 pathways involved, of which 8 pathways including ribosome biogenesis in eukaryotes, spliceosome and neuroactive ligand-receptor interaction were significantly enriched (P<0.05). In conclusion, this report analyzed the transcriptome change pre-and post-vitrification of yak blastocysts using RNA-seq, which provides a theoretical basis to explore the mechanism of vitrification damage and improve vitrification technology of yak blastocysts.

The aim of the present study was to investigate the expression of SOX5 in the testis of chickens and identify its possible roles in testis development and sperm motility regulation. The mRNA and protein expression of SOX5 in the testis of roosters at 0,5,15,40 and 60 weeks of age were determined by RT-qPCR and Western blot, respectively. Its cellular localization in the testis was determined by immunohistochemistry. The expression of SOX5 in the testis of normal and asthenospermia roosters at 40 weeks of age was also compared. The results showed that SOX5 mRNA and protein were differentially expressed in the testis at different ages (P<0.05). The expression pattern of SOX5 protein was in accordance with the mRNA. The immunohistochemical staining revealed that SOX5 was expressed in the Sertoli cells and spermatogonia at 0 and 5 weeks of age. SOX5 was high-expressed in Sertoli cells, spermatocytes, secondary spermatocytes, round and elongated spermatids, and mature sperm in the testis at 15 weeks of age. The expression started to decrease at 40 weeks. The expression pattern of SOX5 was in accordance with the sexual development and recession of the roosters, i.e. high-expressed around the sexual maturation and reproduction peak and low-expressed before the puberty and sexual recession. In addition, the expression of SOX5 in the asthenospermia roosters was lower than the normal ones at 40 weeks of age (P<0.05). In conclusion, this study indicated that the expression of SOX5 was associated with testis development and sperm motility in chickens, suggesting its regulatory roles in these processes.

The aim of the present study was to investigate the expression of SOX5 in the testis of chickens and identify its possible roles in testis development and sperm motility regulation. The mRNA and protein expression of SOX5 in the testis of roosters at 0,5,15,40 and 60 weeks of age were determined by RT-qPCR and Western blot, respectively. Its cellular localization in the testis was determined by immunohistochemistry. The expression of SOX5 in the testis of normal and asthenospermia roosters at 40 weeks of age was also compared. The results showed that SOX5 mRNA and protein were differentially expressed in the testis at different ages (P<0.05). The expression pattern of SOX5 protein was in accordance with the mRNA. The immunohistochemical staining revealed that SOX5 was expressed in the Sertoli cells and spermatogonia at 0 and 5 weeks of age. SOX5 was high-expressed in Sertoli cells, spermatocytes, secondary spermatocytes, round and elongated spermatids, and mature sperm in the testis at 15 weeks of age. The expression started to decrease at 40 weeks. The expression pattern of SOX5 was in accordance with the sexual development and recession of the roosters, i.e. high-expressed around the sexual maturation and reproduction peak and low-expressed before the puberty and sexual recession. In addition, the expression of SOX5 in the asthenospermia roosters was lower than the normal ones at 40 weeks of age (P<0.05). In conclusion, this study indicated that the expression of SOX5 was associated with testis development and sperm motility in chickens, suggesting its regulatory roles in these processes.

The experiment was conducted to study the effects of wheat grinding particle size in pellet diets on growth performance and intestinal barrier function of broilers. A total of 385 1-day-old AA male broilers were assigned to 5 groups with 7 replicates per group and 11 broilers per replicate. The 5 groups differed only in aperture size of screen surface (2, 4, 6, 8, 10 mm). The diet was wheat-soybean meal diet. The experiment lasted for 42 days, 1 to 21 days and 22 to 42 days stages. On 22 and 43 days of age, one bird per replicate was slaughtered to determine jejunum morphology, the gene expression of ZO-1, occludin, claudin-1 and inflammatory factors in jejunum mucosa, and microbial count in caecum digesta. The results showed that:1) Wheat grinding particle size had significant effect on body weight gain,feed intake and F/G from 1 to 21 days of age (P<0.05),and had a significant quadratic effect on body weight gain and F/G (P<0.05). Wheat grinding particle size had no significant effect on body weight gain and feed intake from 22 to 42 days and 1 to 42 days (P>0.05), but significantly increased F/G and had linearly effect on F/G (P<0.05).2) With the wheat grinding particle size increased, the villus height and villus height/crypt depth significantly increased in jejunum of broilers (P<0.05), and the wheat grinding particle size had a significant quadratic effect on the crypt depth (P<0.05). 3) The relative expression of occludin and IL-4 mRNA were significantly affected by wheat grinding particle size in broilers at 21 d (P<0.05). Wheat grinding particle size had significant linear effect on microbial count in caecum (P<0.05). The count of Lactobacillus increased significantly and the count of Salmonella, Escherichia coli and C.perfringens decreased significantly (P<0.05) with the wheat grinding particle size increasing. In conclusion, the wheat grinding particle size of 580-760 μm (aperture size of screen surface is 4-6 mm)in pellet diet (contain xylanase) is advantageous to growth performance and intestinal barrier function of broilers.

The experiment was conducted to study the effects of wheat grinding particle size in pellet diets on growth performance and intestinal barrier function of broilers. A total of 385 1-day-old AA male broilers were assigned to 5 groups with 7 replicates per group and 11 broilers per replicate. The 5 groups differed only in aperture size of screen surface (2, 4, 6, 8, 10 mm). The diet was wheat-soybean meal diet. The experiment lasted for 42 days, 1 to 21 days and 22 to 42 days stages. On 22 and 43 days of age, one bird per replicate was slaughtered to determine jejunum morphology, the gene expression of ZO-1, occludin, claudin-1 and inflammatory factors in jejunum mucosa, and microbial count in caecum digesta. The results showed that:1) Wheat grinding particle size had significant effect on body weight gain,feed intake and F/G from 1 to 21 days of age (P<0.05),and had a significant quadratic effect on body weight gain and F/G (P<0.05). Wheat grinding particle size had no significant effect on body weight gain and feed intake from 22 to 42 days and 1 to 42 days (P>0.05), but significantly increased F/G and had linearly effect on F/G (P<0.05).2) With the wheat grinding particle size increased, the villus height and villus height/crypt depth significantly increased in jejunum of broilers (P<0.05), and the wheat grinding particle size had a significant quadratic effect on the crypt depth (P<0.05). 3) The relative expression of occludin and IL-4 mRNA were significantly affected by wheat grinding particle size in broilers at 21 d (P<0.05). Wheat grinding particle size had significant linear effect on microbial count in caecum (P<0.05). The count of Lactobacillus increased significantly and the count of Salmonella, Escherichia coli and C.perfringens decreased significantly (P<0.05) with the wheat grinding particle size increasing. In conclusion, the wheat grinding particle size of 580-760 μm (aperture size of screen surface is 4-6 mm)in pellet diet (contain xylanase) is advantageous to growth performance and intestinal barrier function of broilers.

The present study was conducted to investigate the effects of fermented corn stalk on rumen bacterial diversity of sheep via 16S rDNA high-throughput sequencing. Twelve F1 generation mature rams of South African Merino sheep♂×Northeast China Fine-fleece sheep♀ with rumen fistula were divided into two groups randomly and fed with corn stalk silage and fermented corn stalk, respectively. Rumen fluid was collected at different time points as following:1 day before feeding, 6 h after the 7th and 14th day morning feeding. Samples collected at the same time point in the same group were mixed well and analyzed for bacterial diversity by high-throughput sequencing, which were marked as:CS group (YD0, YD7, YD21) from the corn stalk silage group and FCS group (YS0, YS7, YS21) from the fermented corn stalk group.The results showed that:1)The bacterial community in collected rumen fluid was consisted of 29 phyla, 74 classes, 135 orders, 215 families and 428 genera.2) The dominant bacteria were identified as Bacteroidetes followed by Lentisphaerae, Firmicutes and Fibrobacteres with successively decreased amount in the FCS group, while successively Bacteroidetes, Firmicutes, Lentisphaerae and Fibrobacteres in the CS group. In addition, the abundance of Bacteroidetes and Firmicutes in CS group were lower compared with FCS group. The abundance of Lentisphaerae and Fibrobacteres increased in both groups, while a larger increase in CS group after 21 days feeding. 3)Prevotella was found to be the dominant genera in YS7 (15.56%), however its abundance reduced dramatically in CS group (from 10.48% to 3.17%) after 7 days feeding; the increase in abundance of Fibrobacter was higher in the CS group than that in FCS group after 21 days feeding. 4)Furthermore, with 97 similarity, the Shannon and Simpson indexes of YS21 in the FCS group were higher than that of YD21 in the CS group, indicating the higher bacterial community diversity. In conclusion, the fermented corn stalk with complex bacteria increased the rumen bacteria diversity.The ratio of Bacteroidetes and Firmicutes in sheep rumen reduced,while the populations of Lentisphaerae and Fibrobacteres increased in the FCS diet.

The present study was conducted to investigate the effects of fermented corn stalk on rumen bacterial diversity of sheep via 16S rDNA high-throughput sequencing. Twelve F1 generation mature rams of South African Merino sheep♂×Northeast China Fine-fleece sheep♀ with rumen fistula were divided into two groups randomly and fed with corn stalk silage and fermented corn stalk, respectively. Rumen fluid was collected at different time points as following:1 day before feeding, 6 h after the 7th and 14th day morning feeding. Samples collected at the same time point in the same group were mixed well and analyzed for bacterial diversity by high-throughput sequencing, which were marked as:CS group (YD0, YD7, YD21) from the corn stalk silage group and FCS group (YS0, YS7, YS21) from the fermented corn stalk group.The results showed that:1)The bacterial community in collected rumen fluid was consisted of 29 phyla, 74 classes, 135 orders, 215 families and 428 genera.2) The dominant bacteria were identified as Bacteroidetes followed by Lentisphaerae, Firmicutes and Fibrobacteres with successively decreased amount in the FCS group, while successively Bacteroidetes, Firmicutes, Lentisphaerae and Fibrobacteres in the CS group. In addition, the abundance of Bacteroidetes and Firmicutes in CS group were lower compared with FCS group. The abundance of Lentisphaerae and Fibrobacteres increased in both groups, while a larger increase in CS group after 21 days feeding. 3)Prevotella was found to be the dominant genera in YS7 (15.56%), however its abundance reduced dramatically in CS group (from 10.48% to 3.17%) after 7 days feeding; the increase in abundance of Fibrobacter was higher in the CS group than that in FCS group after 21 days feeding. 4)Furthermore, with 97 similarity, the Shannon and Simpson indexes of YS21 in the FCS group were higher than that of YD21 in the CS group, indicating the higher bacterial community diversity. In conclusion, the fermented corn stalk with complex bacteria increased the rumen bacteria diversity.The ratio of Bacteroidetes and Firmicutes in sheep rumen reduced,while the populations of Lentisphaerae and Fibrobacteres increased in the FCS diet.

This experiment was conducted to study the effects of dietary copper levels on copper balance, serum trace element contents, serum lipid metabolism parameters and serum antioxidant indices in male mink. Eighty healthy (60±3) day-old male minks were randomly divided into 8 groups with 10 minks in each group. 0 (Control), 4 (Cu4), 8 (Cu8), 16 (Cu16), 32 (Cu32), 64 (Cu64), 128 (Cu128)and 256 mg·kg^-1 (Cu256)of copper levels were added in basic diet, respectively. The pre-test period lasted for 7 days, and the trial lasted for 45 days. On day 30 of the trail, eight animals from each treatment group were selected randomly to determine copper balance. Blood samples were collected from mink before sacrifice to measure the serum biochemical indices. The results showed that:1) Intake of copper, fecal copper, urinary copper, retention copper had a linear and quadratic (P<0.01) increase with the increasing dietary copper level. 2) There was a linear and quadratic (P<0.01) effect of dietary copper level on plasma Cu concentrations. 3) Serum TC (linear, quadratic, P<0.01), TG (linear, P<0.05;quadratic, P<0.01) contents were decreased with the increasing dietary copper level. 4)There was a quadratic (P<0.01) effect of dietary copper level on plasma ceruloplasmin concentration and Cu-Zn superoxide dismutase activity. These results indicate that supplementing Cu in the diet can increase Cu retention and plasma Cu concentrations, but reduce serum TC and TG levels, and improve antioxidant enzyme activity.

This experiment was conducted to study the effects of dietary copper levels on copper balance, serum trace element contents, serum lipid metabolism parameters and serum antioxidant indices in male mink. Eighty healthy (60±3) day-old male minks were randomly divided into 8 groups with 10 minks in each group. 0 (Control), 4 (Cu4), 8 (Cu8), 16 (Cu16), 32 (Cu32), 64 (Cu64), 128 (Cu128)and 256 mg·kg^-1 (Cu256)of copper levels were added in basic diet, respectively. The pre-test period lasted for 7 days, and the trial lasted for 45 days. On day 30 of the trail, eight animals from each treatment group were selected randomly to determine copper balance. Blood samples were collected from mink before sacrifice to measure the serum biochemical indices. The results showed that:1) Intake of copper, fecal copper, urinary copper, retention copper had a linear and quadratic (P<0.01) increase with the increasing dietary copper level. 2) There was a linear and quadratic (P<0.01) effect of dietary copper level on plasma Cu concentrations. 3) Serum TC (linear, quadratic, P<0.01), TG (linear, P<0.05;quadratic, P<0.01) contents were decreased with the increasing dietary copper level. 4)There was a quadratic (P<0.01) effect of dietary copper level on plasma ceruloplasmin concentration and Cu-Zn superoxide dismutase activity. These results indicate that supplementing Cu in the diet can increase Cu retention and plasma Cu concentrations, but reduce serum TC and TG levels, and improve antioxidant enzyme activity.

In this study, we investigated the proliferation of avian leukosis virus subgroup K (ALV-K) and the activity of LTR. An ALV-K strain was used to infect DF-1 cells and the LTR was cloned into pGL3-Basic vector. The replication ability of the virus on ALV susceptible cell DF-1 was evaluated by ALV p27 antigen ELISA, and the promoter activities of the LTR of ALV-K, ALV-E and ALV-J strains were measured using a Dual-Glo Luciferase Assay System. The results showed that promoter activity of ALV-K LTR was significantly lower than those of other exogenous ALV in DF-1, CEF and 293T cells. The relatively poorer replication ability of ALV-K may be related to its weak promoter activity of the LTR.

In this study, we investigated the proliferation of avian leukosis virus subgroup K (ALV-K) and the activity of LTR. An ALV-K strain was used to infect DF-1 cells and the LTR was cloned into pGL3-Basic vector. The replication ability of the virus on ALV susceptible cell DF-1 was evaluated by ALV p27 antigen ELISA, and the promoter activities of the LTR of ALV-K, ALV-E and ALV-J strains were measured using a Dual-Glo Luciferase Assay System. The results showed that promoter activity of ALV-K LTR was significantly lower than those of other exogenous ALV in DF-1, CEF and 293T cells. The relatively poorer replication ability of ALV-K may be related to its weak promoter activity of the LTR.

The aim of this study was to isolate and identify the Palyam serogroup virus (PALV) prevalent in China from the sentinel herds and to clarify the relationship between the native strains and the foreign strains. For virus isolation, PALV-positive blood samples were collected from sentinel herds and blind passed on BHK-21 cells. Specific primers targeting Seg-2 and Seg-7 of PALV were designed for identification and sequence analysis of the isolated virus by RT-PCR amplification. Genomic RNA of the isolated virus was extracted from the infected cells and determined by high resolution agarose gel electrophoresis. Cross-reactivity and cross-neutralizing activity of positive sera from different serotypes of PALV were analyzed by immunofluorescence and serum neutralization test. The results were as follows:During 2012 to 2016, a total of 19 strains of PALV were isolated form Yunnan, Guangxi and Guangdong provinces. Seg-2 and Seg-7 sequencing and phylogenetic analysis showed that the Chinese strains of PALV belonged to Chuzan virus (CHUV), D'Aguilar virus (DAV) and Bunyip Creek virus (BCV) and showed closest genetic relationship with the Japanese strains, but showed relative far relationship with the Australian and African strains. High resolution agarose gel electrophoresis showed that the genomic dsRNA of different serotypes of representative PALV strains presenting "3-3-4" banding characteristics. Immunofluorescence results showed that BHK-21 cells infected with CHUV, DAV or BCV presented specific fluorescence when using polyclonal antibody against CHUV as detection antibodies. Serological neutralization tests showed that there was no cross-neutralization protection between the positive sera of CHUV, DAV and BCV viruses. This study reported the molecular characteristics of PALV isolated in China from 2012 to 2016. It's the first time, we reported the DAV and BCV isolated in China. These results will be helpful to better understand the distribution and genetic characteristics of Chinese PALV and provide basis for development of diagnosis reagent and carried out epidemiological and pathogenic research on PALV.

The aim of this study was to isolate and identify the Palyam serogroup virus (PALV) prevalent in China from the sentinel herds and to clarify the relationship between the native strains and the foreign strains. For virus isolation, PALV-positive blood samples were collected from sentinel herds and blind passed on BHK-21 cells. Specific primers targeting Seg-2 and Seg-7 of PALV were designed for identification and sequence analysis of the isolated virus by RT-PCR amplification. Genomic RNA of the isolated virus was extracted from the infected cells and determined by high resolution agarose gel electrophoresis. Cross-reactivity and cross-neutralizing activity of positive sera from different serotypes of PALV were analyzed by immunofluorescence and serum neutralization test. The results were as follows:During 2012 to 2016, a total of 19 strains of PALV were isolated form Yunnan, Guangxi and Guangdong provinces. Seg-2 and Seg-7 sequencing and phylogenetic analysis showed that the Chinese strains of PALV belonged to Chuzan virus (CHUV), D'Aguilar virus (DAV) and Bunyip Creek virus (BCV) and showed closest genetic relationship with the Japanese strains, but showed relative far relationship with the Australian and African strains. High resolution agarose gel electrophoresis showed that the genomic dsRNA of different serotypes of representative PALV strains presenting "3-3-4" banding characteristics. Immunofluorescence results showed that BHK-21 cells infected with CHUV, DAV or BCV presented specific fluorescence when using polyclonal antibody against CHUV as detection antibodies. Serological neutralization tests showed that there was no cross-neutralization protection between the positive sera of CHUV, DAV and BCV viruses. This study reported the molecular characteristics of PALV isolated in China from 2012 to 2016. It's the first time, we reported the DAV and BCV isolated in China. These results will be helpful to better understand the distribution and genetic characteristics of Chinese PALV and provide basis for development of diagnosis reagent and carried out epidemiological and pathogenic research on PALV.

To analyze the characterization of the mink calicivirus in China, the genome of the mink calicivirus detected in the farm of Qingdao was sequenced by high-throughput sequencing. The genome of the isolate was analyzed and compared with other caliciviruses. The results showed that the length of the isolate was 8 427 bp, which possessed the similar genomic organization, structure and gene order with the other calicivirus. It possessed 5'-untranslated region and 3'-poly (A) tail. The comparing analysis of the genome and capsid protein amino acid sequence of the virus and the 19 representative viruses showed that the identity between this virus and the Chinese virus detected in 2011 was high. The BLAST results showed that this virus was not belong to mink enteric calicivirus. The analysis was also useful to supply reference to the classification of the caliciviruses which haven't been defied into genera.

To analyze the characterization of the mink calicivirus in China, the genome of the mink calicivirus detected in the farm of Qingdao was sequenced by high-throughput sequencing. The genome of the isolate was analyzed and compared with other caliciviruses. The results showed that the length of the isolate was 8 427 bp, which possessed the similar genomic organization, structure and gene order with the other calicivirus. It possessed 5'-untranslated region and 3'-poly (A) tail. The comparing analysis of the genome and capsid protein amino acid sequence of the virus and the 19 representative viruses showed that the identity between this virus and the Chinese virus detected in 2011 was high. The BLAST results showed that this virus was not belong to mink enteric calicivirus. The analysis was also useful to supply reference to the classification of the caliciviruses which haven't been defied into genera.

This experiment was conducted to obtain recombinant mutant of Clostridium perfringens ε toxin and subsequently evaluate the virulence and immunogenicity of the recombinant toxin. The ε toxin gene of C. perfringens type D strain using optimized codons was synthesized based on the sequence reported. At the same time, three amino acid mutations:Y30 and Y196 substituted with alanine, H106 substituted with proline, were introduced into this sequence synthesized. Then,this ε toxin gene was cloned into prokaryotic expression vector pET3a- (+) to construct a recombinant E. coli, followed by induction with IPTG to yield the recombinant protein,following with purification. The reactivity of the purified protein with antiserum of C. perfringens type D was determined by Western blot and the mouse was used to evaluate the virulence of purified protein. The rabbit antiserum against the recombinant proteins was prepared and the neutralizing titer was measured according to the method prescribed in Chinese Veterinary Pharmacopoeia (2015). The results showed that recombinant protein was expressed at a high level in a soluble form with a ratio of about 40% by gray scale scanning, And the protein could react with the antiserum of C. perfringens type D. The protein with the injection volume of 6.25×10⁶ ng·kg^-1 still shows no virulence to mouse. The titer of rabbit antiserum against Clostridium perfringens type D could reach 50-60 and 400-450 Minimum Lethal Dose (MLD) after the first and second immunization and respectively.After challenge with 1MLD of C. perfringens type D toxin, all the rabbits immunized with recombinant protein were protected. The results suggest that the recombinant mutant of C. perfringens ε toxin without virulence retains the good immunogenic antigen, which provides important experimental data for the development of novel C. perfringens vaccine.

This experiment was conducted to obtain recombinant mutant of Clostridium perfringens ε toxin and subsequently evaluate the virulence and immunogenicity of the recombinant toxin. The ε toxin gene of C. perfringens type D strain using optimized codons was synthesized based on the sequence reported. At the same time, three amino acid mutations:Y30 and Y196 substituted with alanine, H106 substituted with proline, were introduced into this sequence synthesized. Then,this ε toxin gene was cloned into prokaryotic expression vector pET3a- (+) to construct a recombinant E. coli, followed by induction with IPTG to yield the recombinant protein,following with purification. The reactivity of the purified protein with antiserum of C. perfringens type D was determined by Western blot and the mouse was used to evaluate the virulence of purified protein. The rabbit antiserum against the recombinant proteins was prepared and the neutralizing titer was measured according to the method prescribed in Chinese Veterinary Pharmacopoeia (2015). The results showed that recombinant protein was expressed at a high level in a soluble form with a ratio of about 40% by gray scale scanning, And the protein could react with the antiserum of C. perfringens type D. The protein with the injection volume of 6.25×10⁶ ng·kg^-1 still shows no virulence to mouse. The titer of rabbit antiserum against Clostridium perfringens type D could reach 50-60 and 400-450 Minimum Lethal Dose (MLD) after the first and second immunization and respectively.After challenge with 1MLD of C. perfringens type D toxin, all the rabbits immunized with recombinant protein were protected. The results suggest that the recombinant mutant of C. perfringens ε toxin without virulence retains the good immunogenic antigen, which provides important experimental data for the development of novel C. perfringens vaccine.

In order to establish a method for detecting specific antibody against Lawsonia intracellularis, the codon-optimized LsaA gene encoding surface protein LsaA of L. intracellularis was cloned into pET32a (+) vector, and the expression of LsaA protein was induced in E. coli host BL21 (DE3). After optimization of a series of conditions, an indirect ELISA for detecting antibodies against L. intracellularis was established by using purified LsaA protein as coating antigen. The results showed that the anti- L. intracellularis antibodies were detected specifically by the established ELISA, and there were no cross-reactions with the antibodies against common diarrhea-causing pathogens such as E. coli, Salmonella choleraesuis, porcine epidemic diarrhea virus and transmissible gastroenteritis virus. Serum samples from fecal L. intracellularis-positive pigs which were identified by PCR, were also positive for anti- L. intracellularis antibodies detected by the established ELISA. The ELISA has good sensitivity and repeatability. Anti- L. intracellularis positive serum with 1:800 dilution was still ELISA positive. The intra-and inter-assay coefficient of variation was 0.352%-2.752% and 0.877%-3.000%, respectively. Seroepidemiological prevalence of L. intracellularis infection in partial pig farms in Henan province was investigated by using the established ELISA. The results indicated that all the tested pig farms were anti- L. intracellularis antibody positive with positive rate of 13.3% to 57.1%, with average positive rate of 30.6%. The results showed that the established ELISA in this study can be used for the detection of antibodies against L. intracellularis, and provides a method for the monitoring and epidemiological investigation of L. intracellularis.

In order to establish a method for detecting specific antibody against Lawsonia intracellularis, the codon-optimized LsaA gene encoding surface protein LsaA of L. intracellularis was cloned into pET32a (+) vector, and the expression of LsaA protein was induced in E. coli host BL21 (DE3). After optimization of a series of conditions, an indirect ELISA for detecting antibodies against L. intracellularis was established by using purified LsaA protein as coating antigen. The results showed that the anti- L. intracellularis antibodies were detected specifically by the established ELISA, and there were no cross-reactions with the antibodies against common diarrhea-causing pathogens such as E. coli, Salmonella choleraesuis, porcine epidemic diarrhea virus and transmissible gastroenteritis virus. Serum samples from fecal L. intracellularis-positive pigs which were identified by PCR, were also positive for anti- L. intracellularis antibodies detected by the established ELISA. The ELISA has good sensitivity and repeatability. Anti- L. intracellularis positive serum with 1:800 dilution was still ELISA positive. The intra-and inter-assay coefficient of variation was 0.352%-2.752% and 0.877%-3.000%, respectively. Seroepidemiological prevalence of L. intracellularis infection in partial pig farms in Henan province was investigated by using the established ELISA. The results indicated that all the tested pig farms were anti- L. intracellularis antibody positive with positive rate of 13.3% to 57.1%, with average positive rate of 30.6%. The results showed that the established ELISA in this study can be used for the detection of antibodies against L. intracellularis, and provides a method for the monitoring and epidemiological investigation of L. intracellularis.

The present study was designed to analyze the immune genes against Salmonella infection and their regulatory network in yak at the genomic transcriptional level. Using yak Salmonella as the model pathogen and yak as the object, transcriptome sequencing was performed on the central immune organ of spleen in infected yak by RNA-Seq sequencing technology at three different periods of 12, 24, and 48 hours, respectively. Transcriptome data from the three different periods were compared between the infected and the control healthy yaks and differentially expressed genes were screened out. The GO and KEGG functional analysis of these differentially expressed genes were conducted and the co-expression network was further analyzed. Results showed that 413 differentially expressed genes were selected, of which 185 were up-regulated and 228 were down regulated. GO analysis showed that genes related to biological process accounted for the largest percentage, among which the most enriched ones were those involved in cellular process, metabolic process, biological regulation, regulation of biological process and single organism process. KEGG analysis revealed that infection-and immune-related pathways presented a larger portion in the top 20 pathways enriched in all three periods, and the major genes involved in these pathways were chemokines. WGCNA analysis showed 66 genes in network hubs, and those in the network center were SCOC and NOCT. The present study explored the molecular interaction mechanism of spleen cells against Salmonella in yak at the omics level.

The present study was designed to analyze the immune genes against Salmonella infection and their regulatory network in yak at the genomic transcriptional level. Using yak Salmonella as the model pathogen and yak as the object, transcriptome sequencing was performed on the central immune organ of spleen in infected yak by RNA-Seq sequencing technology at three different periods of 12, 24, and 48 hours, respectively. Transcriptome data from the three different periods were compared between the infected and the control healthy yaks and differentially expressed genes were screened out. The GO and KEGG functional analysis of these differentially expressed genes were conducted and the co-expression network was further analyzed. Results showed that 413 differentially expressed genes were selected, of which 185 were up-regulated and 228 were down regulated. GO analysis showed that genes related to biological process accounted for the largest percentage, among which the most enriched ones were those involved in cellular process, metabolic process, biological regulation, regulation of biological process and single organism process. KEGG analysis revealed that infection-and immune-related pathways presented a larger portion in the top 20 pathways enriched in all three periods, and the major genes involved in these pathways were chemokines. WGCNA analysis showed 66 genes in network hubs, and those in the network center were SCOC and NOCT. The present study explored the molecular interaction mechanism of spleen cells against Salmonella in yak at the omics level.

The present study was designed to investigate the effects of Haemonchus contortus Cysteine protease on immune functions of peripheral blood mononuclear cells (PBMCs) of goat. PBMCs were separated from goat blood sample, monocytes were isolated from PBMCs because of their adherent growth. Recombinant protein of different concentrations (0, 10, 20, 40 μg·mL^-1) was cultured with PBMCs or monocytes in vitro. Nitric oxide (NO) secretion was measured by nitrate reductase assay. Cell migration was tested by Cell Culture Inserts. The mRNA transcriptional levels of IL-2, IL-4, IL-10, IL-17, IFN-γ and TGF-β were detected by real-time fluorescent quantitative PCR. Then cell phagocytosis and apoptosis were calculated by flow cytometry. The result revealed that Hc58 was able to dramatically enhance NO release and phagocytosis of monocytes. Hc58 might significantly up-regulate the production of IL-2, IL-4, IL-17 and IFN-γ. Furthermore, it also could notably increase apoptosis ratio and migration of PBMCs. There were signs that Haemonchus contortus Cysteine protease impacted on the immune functions of goat PBMCs via different pathways.

The present study was designed to investigate the effects of Haemonchus contortus Cysteine protease on immune functions of peripheral blood mononuclear cells (PBMCs) of goat. PBMCs were separated from goat blood sample, monocytes were isolated from PBMCs because of their adherent growth. Recombinant protein of different concentrations (0, 10, 20, 40 μg·mL^-1) was cultured with PBMCs or monocytes in vitro. Nitric oxide (NO) secretion was measured by nitrate reductase assay. Cell migration was tested by Cell Culture Inserts. The mRNA transcriptional levels of IL-2, IL-4, IL-10, IL-17, IFN-γ and TGF-β were detected by real-time fluorescent quantitative PCR. Then cell phagocytosis and apoptosis were calculated by flow cytometry. The result revealed that Hc58 was able to dramatically enhance NO release and phagocytosis of monocytes. Hc58 might significantly up-regulate the production of IL-2, IL-4, IL-17 and IFN-γ. Furthermore, it also could notably increase apoptosis ratio and migration of PBMCs. There were signs that Haemonchus contortus Cysteine protease impacted on the immune functions of goat PBMCs via different pathways.

The objective of this study was to evaluate the transcriptional changes of immune related genes and the effection of recombinant chicken glutathione S-transferase A3 (chGSTA3) protein on the transcription of immune related genes in erythrocytes of Tibial Dyschondroplasia (TD) broiler chickens. Therefore, the model of TD broiler chickens was established, in which 120 broiler chickens were randomly broken up into 6 groups; group A, B and C (fed with basal diet), group D, E and F (fed with basal diet containing 100 mg·kg^-1 Thiram) after one week normal feeding. The broiler chickens of group B and E were treated with chGSTA3 protein by intramuscular injection (low dose:20 μg·kg^-1) and the broilers of group C and F were treated intra-muscularly with chGSTA3 protein (high dose:50 μg·kg^-1), while groups A and D received the same dosage of phosphate buffered solution (PBS). We used Real-time PCR to investigate the mRNA expression of the immune-related genes in chicken erythrocytes. The transcripts for TLR2, TLR3, TLR4, TLR5, TLR7, TLR15, myeloid differentiation factor 88 (MyD88), major histocompatibility complex (MHC) class Ⅱ, Nucleotidebinding oligomerization domain receptor caspase recruitment domain 5 (NLRC5), TNF receptor-associated factor 6 (TRAF6) and Interleukin (IL-7) were constitutively expressed in erythrocytes of healthy broiler chickens. Contrasted with group A with phosphate buffered solution (PBS) treatment, the mRNA transcription level of TLR2, TLR3, TLR4, TLR5, TLR7, TLR15, MyD88, MHCⅡ, NLRC5, TRAF6 of Thiram-treated chicken erythrocytes in group D on the 4^th day were significantly upregulated (P<0.05), whereas IL-7 was significantly downregulated. We observed that the transcripts of immune-related genes:TLR2, TLR4, MyD88 and NLRC5 in the chicken erythrocytes of group D were significantly downregulated than that in group A (P<0.05), but IL-7, TLR3, TLR5, TLR7, TLR15, MHCⅡ and TRAF6 were insignificant (P>0.05) on the 15^th day. The changes of above mentioned genes mRNA expression in group E and F were significant except TLR4, TLR5, MyD88 in group E as compared with group D on the 4^th day. The changes of TLR4, TLR5, MyD88, NLRC5 genes in group E and TLR3, TLR5, MyD88, TRAF6, IL-7 genes in group F were insignificant, whereas the others were significant compared with group D on the 15^th day. Broiler chicken erythrocytes could induce the occurrence of TD in the early stage and alleviate TD symptoms in the late stage by changing the mRNA expression of IL-7, TLR2, TLR3, TLR4, TLR5, TLR7, TLR15, MyD88, MHCⅡ, NLRC5, TRAF6. The recombinant chicken glutathione S-transferase A3 protein could influence TD development caused by Thiram.

The objective of this study was to evaluate the transcriptional changes of immune related genes and the effection of recombinant chicken glutathione S-transferase A3 (chGSTA3) protein on the transcription of immune related genes in erythrocytes of Tibial Dyschondroplasia (TD) broiler chickens. Therefore, the model of TD broiler chickens was established, in which 120 broiler chickens were randomly broken up into 6 groups; group A, B and C (fed with basal diet), group D, E and F (fed with basal diet containing 100 mg·kg^-1 Thiram) after one week normal feeding. The broiler chickens of group B and E were treated with chGSTA3 protein by intramuscular injection (low dose:20 μg·kg^-1) and the broilers of group C and F were treated intra-muscularly with chGSTA3 protein (high dose:50 μg·kg^-1), while groups A and D received the same dosage of phosphate buffered solution (PBS). We used Real-time PCR to investigate the mRNA expression of the immune-related genes in chicken erythrocytes. The transcripts for TLR2, TLR3, TLR4, TLR5, TLR7, TLR15, myeloid differentiation factor 88 (MyD88), major histocompatibility complex (MHC) class Ⅱ, Nucleotidebinding oligomerization domain receptor caspase recruitment domain 5 (NLRC5), TNF receptor-associated factor 6 (TRAF6) and Interleukin (IL-7) were constitutively expressed in erythrocytes of healthy broiler chickens. Contrasted with group A with phosphate buffered solution (PBS) treatment, the mRNA transcription level of TLR2, TLR3, TLR4, TLR5, TLR7, TLR15, MyD88, MHCⅡ, NLRC5, TRAF6 of Thiram-treated chicken erythrocytes in group D on the 4^th day were significantly upregulated (P<0.05), whereas IL-7 was significantly downregulated. We observed that the transcripts of immune-related genes:TLR2, TLR4, MyD88 and NLRC5 in the chicken erythrocytes of group D were significantly downregulated than that in group A (P<0.05), but IL-7, TLR3, TLR5, TLR7, TLR15, MHCⅡ and TRAF6 were insignificant (P>0.05) on the 15^th day. The changes of above mentioned genes mRNA expression in group E and F were significant except TLR4, TLR5, MyD88 in group E as compared with group D on the 4^th day. The changes of TLR4, TLR5, MyD88, NLRC5 genes in group E and TLR3, TLR5, MyD88, TRAF6, IL-7 genes in group F were insignificant, whereas the others were significant compared with group D on the 15^th day. Broiler chicken erythrocytes could induce the occurrence of TD in the early stage and alleviate TD symptoms in the late stage by changing the mRNA expression of IL-7, TLR2, TLR3, TLR4, TLR5, TLR7, TLR15, MyD88, MHCⅡ, NLRC5, TRAF6. The recombinant chicken glutathione S-transferase A3 protein could influence TD development caused by Thiram.

The experiment was conducted to evaluate the effect of betulinic acid (BA) on intestinal oxidative damage induced by cyclophosphamide (Cy) in mice. Male Kunming mice were randomly divided into 5 groups:control group (NC), Cy group, 0.5 mg·kg^-1 BA group, 5.0 mg·kg^-1 BA group and 50.0 mg·kg^-1 BA group. The control and Cy group were administered orally with 1% starch and the other groups were administered orally with different doses of BA (0.5, 5.0, and 50.0 mg·kg^-1) daily for 14 days. The model of intestinal oxidative damage was set up in mice induced by Cy at the dosage of 50 mg·kg^-1 bw for 2 days after last administration of BA. The liver, spleen and thymus index of mice were measured, the activity of secretory immunoglobulin a (SIgA) in intestinal mucosa and the levels of the superoxide dismutase (SOD), malondialdehyde (MDA) and glutathione (GSH) in intestine and serum were determined. BA pretreatment relieved atrophy of thymus, spleen and liver, inhibited the decline of SIgA activity in intestinal mucosa induced by Cy. Pretreatment with BA reduced the contents of GSH and MDA in small intestine and serum induced by Cy, but had no effect on SOD activity. BA could improve intestinal lipid peroxidation and increase the secretion of SIgA in the intestine mucosa and shows preventive protection of Intestinal oxidative damage induced by Cy.

The experiment was conducted to evaluate the effect of betulinic acid (BA) on intestinal oxidative damage induced by cyclophosphamide (Cy) in mice. Male Kunming mice were randomly divided into 5 groups:control group (NC), Cy group, 0.5 mg·kg^-1 BA group, 5.0 mg·kg^-1 BA group and 50.0 mg·kg^-1 BA group. The control and Cy group were administered orally with 1% starch and the other groups were administered orally with different doses of BA (0.5, 5.0, and 50.0 mg·kg^-1) daily for 14 days. The model of intestinal oxidative damage was set up in mice induced by Cy at the dosage of 50 mg·kg^-1 bw for 2 days after last administration of BA. The liver, spleen and thymus index of mice were measured, the activity of secretory immunoglobulin a (SIgA) in intestinal mucosa and the levels of the superoxide dismutase (SOD), malondialdehyde (MDA) and glutathione (GSH) in intestine and serum were determined. BA pretreatment relieved atrophy of thymus, spleen and liver, inhibited the decline of SIgA activity in intestinal mucosa induced by Cy. Pretreatment with BA reduced the contents of GSH and MDA in small intestine and serum induced by Cy, but had no effect on SOD activity. BA could improve intestinal lipid peroxidation and increase the secretion of SIgA in the intestine mucosa and shows preventive protection of Intestinal oxidative damage induced by Cy.

The aims of this study were to analyze the effect of haplotypes of ADIPOQ gene on growth traits in male and female lambs, to filter haplotypes of ADIPOQ gene which had different effects on growth traits of male and female lambs, and to provide basis for improving the efficiency of production and breeding improvement. The mutations and haplotypes of ADIPOQ gene were detected in total 1 185 New Zealand Romney lambs (556 male and 629 female) by using PCR-SSCP method. The relationship between haplotypes and growth traits of male and female lambs was investigated using GLMs model. The effects of haplotypes of ADIPOQ on ovine growth traits were estimated and the haplotypes which had differents effects on growth traits of male and female lambs were identified. The results showed that ovine ADIPOQ gene had rich nucleotide mutations and haplotypes, and the frequencies of haplotypes were different in male and female lambs (haplotypes D₁-A₂ and D₁-C₂ were only detected in female lambs in this study). The associations analysis results showed that haplotypes of ovine ADIPOQ gene had different effects on growth traits of male and female lambs. In male lambs, the presence of A₁-A₂ was associated with decreased tailing weight (P=0.033), weaning weight (P=0.020) and pre-weaning growth rate (P=0.026). No associations were detected between the other haplotypes and growth traits of male lambs. No associations were found between the ADIPOQ haplotypes and growth traits of female lambs. Frequencies of ovine ADIPOQ haplotypes are different, and haplotypes may have different effects on growth traits of male and female lambs. The presence of A₁-A₂ is associated with decreased growth production, and may be used as a theoretical guidance for improving the growth performance of male lambs.

The aims of this study were to analyze the effect of haplotypes of ADIPOQ gene on growth traits in male and female lambs, to filter haplotypes of ADIPOQ gene which had different effects on growth traits of male and female lambs, and to provide basis for improving the efficiency of production and breeding improvement. The mutations and haplotypes of ADIPOQ gene were detected in total 1 185 New Zealand Romney lambs (556 male and 629 female) by using PCR-SSCP method. The relationship between haplotypes and growth traits of male and female lambs was investigated using GLMs model. The effects of haplotypes of ADIPOQ on ovine growth traits were estimated and the haplotypes which had differents effects on growth traits of male and female lambs were identified. The results showed that ovine ADIPOQ gene had rich nucleotide mutations and haplotypes, and the frequencies of haplotypes were different in male and female lambs (haplotypes D₁-A₂ and D₁-C₂ were only detected in female lambs in this study). The associations analysis results showed that haplotypes of ovine ADIPOQ gene had different effects on growth traits of male and female lambs. In male lambs, the presence of A₁-A₂ was associated with decreased tailing weight (P=0.033), weaning weight (P=0.020) and pre-weaning growth rate (P=0.026). No associations were detected between the other haplotypes and growth traits of male lambs. No associations were found between the ADIPOQ haplotypes and growth traits of female lambs. Frequencies of ovine ADIPOQ haplotypes are different, and haplotypes may have different effects on growth traits of male and female lambs. The presence of A₁-A₂ is associated with decreased growth production, and may be used as a theoretical guidance for improving the growth performance of male lambs.

The objective of this study was to conduct a genome-wide association study (GWAS) for carcass weight and bone weight by using two statistic models in 1 301 Chinese Simmental beef cattle and promote the research of GWAS method by comparing the results of the two models. The samples and phenotype data were obtained by field collection and measurement, and the genotype data were obtained by Illumina Bovine HDBeadChip (770K) after genomic DNA extracted from samples. Two statistic models were used to conduct the GWAS, which including linear mixed model (LMM) and composite interval mapping-linear mixed model (CIM-LMM), and the significant single nucleotide polymorphisms (SNPs) and candidate genes associated with target traits were identified. In addition, the performance of the models was assessed according to GWAS results. The results showed that the more significant SNPs were detected by CIM-LMM (P<0.05), which overlapped the SNPs detected by LMM and performed a higher detection power than LMM.There were 8 and 7 SNPs associated with carcass weight and bone weight, respectively and 2 SNPs associated with both traits. These SNPs were mapped on chromosome 3, 5, 6, 10, 14, 16 and 17, while mainly focused on chromosome 6 and 14. Finally, 11 candidate genes were identified and the functions of GCNT4, ALDH1A2, LCORL and WDFY3 genes were discussed. This research further explore the potential genetic mechanism of carcass traits in Chinese Simmental beef cattle and offer new ideas for GWAS methods.

The objective of this study was to conduct a genome-wide association study (GWAS) for carcass weight and bone weight by using two statistic models in 1 301 Chinese Simmental beef cattle and promote the research of GWAS method by comparing the results of the two models. The samples and phenotype data were obtained by field collection and measurement, and the genotype data were obtained by Illumina Bovine HDBeadChip (770K) after genomic DNA extracted from samples. Two statistic models were used to conduct the GWAS, which including linear mixed model (LMM) and composite interval mapping-linear mixed model (CIM-LMM), and the significant single nucleotide polymorphisms (SNPs) and candidate genes associated with target traits were identified. In addition, the performance of the models was assessed according to GWAS results. The results showed that the more significant SNPs were detected by CIM-LMM (P<0.05), which overlapped the SNPs detected by LMM and performed a higher detection power than LMM.There were 8 and 7 SNPs associated with carcass weight and bone weight, respectively and 2 SNPs associated with both traits. These SNPs were mapped on chromosome 3, 5, 6, 10, 14, 16 and 17, while mainly focused on chromosome 6 and 14. Finally, 11 candidate genes were identified and the functions of GCNT4, ALDH1A2, LCORL and WDFY3 genes were discussed. This research further explore the potential genetic mechanism of carcass traits in Chinese Simmental beef cattle and offer new ideas for GWAS methods.

The main objective of this study was focused on expression of low abundance protein and details of the experimental procedure detected by Western blot. Brown adipose tissue (BAT) from 6-8 week old C57/BL6 mice were chosen as experimental materials and detected for the expression of Melanocortin-4-receptor (MC4R) using different parameters including pH and content of Sodium Dodecyl Sulfate (SDS) in electrotransfer buffers. The effects of protein processing methods, components and pH of electrotransfer buffers on transfer efficiency were investigated. The results showed that initial voltage and power were significantly related to the content of sodium SDS and pH of the electrotransfer buffers. The concentration of SDS in the transmembrane buffer was inverse to the transfer voltage and power under the constant circulating membrane. The transmembrane voltage and power of buffer in pH 8.0 were significantly higher than those of buffers in pH 7.6 (P<0.01) and pH 8.6 (P<0.001). The expression of relating proteins, especially MC4R, in group of alcohols mixed lysate (20% Methanol+1% Triton+1% SDS+RIPA) was significantly higher than that in the single RIPA group (P<0.05) and Trizol group (P<0.01). The result indicate that adding enhancers (Triton and SDS) to the lysate buffer can promote the efficiency of protein extraction and improve the sensitivity of antibody for proteins. Those procedure may help to understand the details and improve the sensitivity and repeatability of the Western blot.

The main objective of this study was focused on expression of low abundance protein and details of the experimental procedure detected by Western blot. Brown adipose tissue (BAT) from 6-8 week old C57/BL6 mice were chosen as experimental materials and detected for the expression of Melanocortin-4-receptor (MC4R) using different parameters including pH and content of Sodium Dodecyl Sulfate (SDS) in electrotransfer buffers. The effects of protein processing methods, components and pH of electrotransfer buffers on transfer efficiency were investigated. The results showed that initial voltage and power were significantly related to the content of sodium SDS and pH of the electrotransfer buffers. The concentration of SDS in the transmembrane buffer was inverse to the transfer voltage and power under the constant circulating membrane. The transmembrane voltage and power of buffer in pH 8.0 were significantly higher than those of buffers in pH 7.6 (P<0.01) and pH 8.6 (P<0.001). The expression of relating proteins, especially MC4R, in group of alcohols mixed lysate (20% Methanol+1% Triton+1% SDS+RIPA) was significantly higher than that in the single RIPA group (P<0.05) and Trizol group (P<0.01). The result indicate that adding enhancers (Triton and SDS) to the lysate buffer can promote the efficiency of protein extraction and improve the sensitivity of antibody for proteins. Those procedure may help to understand the details and improve the sensitivity and repeatability of the Western blot.

A case of canine ovarian teratoma complicated with Leydig cell tumor of ovary is reported in this article. It is rare in veterinary clinics and has not been reported in literature. A 2-year-old female border collie represented the clitoris hypertrophy, no signs of oestrus and occasional mating behavior. Abdominal palpation showed softness and no pain, but an egg sized mass that was tough and removable was palpated in right lower abdomen. In the exploratory laparotomy, a cyst-like mass was seen in the right side. The dog was sterilized and its ovarian mass was examined histopathologically. Resultant diagnosis was ovarian teratoma complicated with leydig cell tumor of ovary, the cystic region of tumor was filled with the fat, skin and their appendages. No recurrence was noted in our patient after one year of follow-up. Ovarian mature cystic teratoma complicated with Leydig cell tumor has not been previously reported in dog. This report will contribute to a better understanding of the pathology of this rare tumor in dog and has certain reference value for clinical diagnosis and treatment of related diseases.

A case of canine ovarian teratoma complicated with Leydig cell tumor of ovary is reported in this article. It is rare in veterinary clinics and has not been reported in literature. A 2-year-old female border collie represented the clitoris hypertrophy, no signs of oestrus and occasional mating behavior. Abdominal palpation showed softness and no pain, but an egg sized mass that was tough and removable was palpated in right lower abdomen. In the exploratory laparotomy, a cyst-like mass was seen in the right side. The dog was sterilized and its ovarian mass was examined histopathologically. Resultant diagnosis was ovarian teratoma complicated with leydig cell tumor of ovary, the cystic region of tumor was filled with the fat, skin and their appendages. No recurrence was noted in our patient after one year of follow-up. Ovarian mature cystic teratoma complicated with Leydig cell tumor has not been previously reported in dog. This report will contribute to a better understanding of the pathology of this rare tumor in dog and has certain reference value for clinical diagnosis and treatment of related diseases.

Porcine deltacoronavirus (PDCoV) and porcine epidemic diarrhea virus (PEDV) are enterpathogenic coronavirus which cause acute diarrhea, vomiting,dehydration and mortality in neonatal piglets. In order to establish a real-time reverse transcription quantitative PCR (RT-qPCR) assay to detect PDCoV and PEDV, two pairs of specific primers were designed according to the conservative sequence of N gene (PDCoV) and M gene (PEDV) registered in GenBank. The genes of the conservative region were amplified and cloned into pMD19-T vector. Taking Mix 10 times the gradient dilution of recombinant plasmid as a standard template, a RT-qPCR to PDCoV and PEDV was established. The sensitivity, specificity and repeatability were tested. Results showed that the sensitivity of this RT-qPCR assay was 51 and 32 copies·μL^-1 for PDCoV and PEDV, respectively. No cross-reaction was detected to PBoV, TGEV, PRV and PCV2. The RT-qPCR assay was applied to the detection of 130 clinical samples collected from Hebei province during 2016-2017. PDCoV positive rate was 16.9%, PEDV positive rate was 66.2%, and co-infection was 2.3%. The sensitivity of fluorescence quantitative PCR was significantly higher than that of ordinary RT-PCR. These results indicated that the detection assay could be applied for rapid and high through-put diagnosis of PDCoV and PEDV.

Porcine deltacoronavirus (PDCoV) and porcine epidemic diarrhea virus (PEDV) are enterpathogenic coronavirus which cause acute diarrhea, vomiting,dehydration and mortality in neonatal piglets. In order to establish a real-time reverse transcription quantitative PCR (RT-qPCR) assay to detect PDCoV and PEDV, two pairs of specific primers were designed according to the conservative sequence of N gene (PDCoV) and M gene (PEDV) registered in GenBank. The genes of the conservative region were amplified and cloned into pMD19-T vector. Taking Mix 10 times the gradient dilution of recombinant plasmid as a standard template, a RT-qPCR to PDCoV and PEDV was established. The sensitivity, specificity and repeatability were tested. Results showed that the sensitivity of this RT-qPCR assay was 51 and 32 copies·μL^-1 for PDCoV and PEDV, respectively. No cross-reaction was detected to PBoV, TGEV, PRV and PCV2. The RT-qPCR assay was applied to the detection of 130 clinical samples collected from Hebei province during 2016-2017. PDCoV positive rate was 16.9%, PEDV positive rate was 66.2%, and co-infection was 2.3%. The sensitivity of fluorescence quantitative PCR was significantly higher than that of ordinary RT-PCR. These results indicated that the detection assay could be applied for rapid and high through-put diagnosis of PDCoV and PEDV.

To investigate the variation of PEDV S gene in Henan province, specific primers were designed and synthesized, S genes of 38 PEDV strains from different pig farms in Henan provinces from 2014-2015 were amplified by RT-PCR, then cloned and sequenced. The phylogenetic relationships and protein characterization of the full-length PEDV S protein in Henan of China were analyzed with other PEDV reference strains. Phylogenetic analysis indicated that all the amplified S genes had a close relationship with some variant strains isolated from China and USA in recent years. Sequence alignment analysis showed that extensive amino acid (AA) insertion and deletion were found in the S protein. Furthermore, there were 11 AA deletion at the C terminal of S protein encoded by amplicated S gene. Many strains had wide AA substitution in the neutralizing epitopes COE and 2C10. These results will provide valuable research basis for understanding the variant characterization of PEDV and controlling the outbreak of PED in this area.

To investigate the variation of PEDV S gene in Henan province, specific primers were designed and synthesized, S genes of 38 PEDV strains from different pig farms in Henan provinces from 2014-2015 were amplified by RT-PCR, then cloned and sequenced. The phylogenetic relationships and protein characterization of the full-length PEDV S protein in Henan of China were analyzed with other PEDV reference strains. Phylogenetic analysis indicated that all the amplified S genes had a close relationship with some variant strains isolated from China and USA in recent years. Sequence alignment analysis showed that extensive amino acid (AA) insertion and deletion were found in the S protein. Furthermore, there were 11 AA deletion at the C terminal of S protein encoded by amplicated S gene. Many strains had wide AA substitution in the neutralizing epitopes COE and 2C10. These results will provide valuable research basis for understanding the variant characterization of PEDV and controlling the outbreak of PED in this area.

The experiment was conducted to set up the immunohistochemitry assay to detect chicken TRIM25 expression in the chicken's tissues. Chicken TRIM25 gene was amplified from chicken's spleen and the gene was cloned into a prokaryotic expression vector pEASY-Blunt E1 to construct recombinant plasmid named as pEASY-Blunt E1-TRIM25. The recombinant plasmid was expressed in E. coli and then identified by SDS-PAGE and Western blot. After being purified, the fusion protein was inoculated to rabbits and BALB/c mice as antigen for preparing polyclonal antibody. The titer of the antibody was detected by ELISA, and the sera antibodies were used to setup immunohistochemistry assay; the tissue sections of chicken's lung and spleen and liver were assayed. The results showed that TRIM25 was expressed as fusion protein with 48 kD. The titers of the antibody in rabbits' and mice's sera were above 1:10⁶. The results of immunohistochemistry assay indicated that TRIM25 mainly exists in the cytoplasm of lymphocytes, hepatocyte, and epithelial cells in chicken's lung, spleen and liver. In conclusion, the immunohistochemitry assay mediated with polyclonal antibody against chicken TRIM25 were successfully set up in this study and TRIM25 distribution in some tissues of chickens was firstly reported.

The experiment was conducted to set up the immunohistochemitry assay to detect chicken TRIM25 expression in the chicken's tissues. Chicken TRIM25 gene was amplified from chicken's spleen and the gene was cloned into a prokaryotic expression vector pEASY-Blunt E1 to construct recombinant plasmid named as pEASY-Blunt E1-TRIM25. The recombinant plasmid was expressed in E. coli and then identified by SDS-PAGE and Western blot. After being purified, the fusion protein was inoculated to rabbits and BALB/c mice as antigen for preparing polyclonal antibody. The titer of the antibody was detected by ELISA, and the sera antibodies were used to setup immunohistochemistry assay; the tissue sections of chicken's lung and spleen and liver were assayed. The results showed that TRIM25 was expressed as fusion protein with 48 kD. The titers of the antibody in rabbits' and mice's sera were above 1:10⁶. The results of immunohistochemistry assay indicated that TRIM25 mainly exists in the cytoplasm of lymphocytes, hepatocyte, and epithelial cells in chicken's lung, spleen and liver. In conclusion, the immunohistochemitry assay mediated with polyclonal antibody against chicken TRIM25 were successfully set up in this study and TRIM25 distribution in some tissues of chickens was firstly reported.