The purpose of this study was to explore the effect of pig LYRM1 gene on the fat deposition. The expression levels of LYRM1 gene in different tissues of Baixi pigs aged 10 months were first detected by qRT-PCR. CDS of LYRM1 gene was amplified by PCR. The recombinant eukaryotic expression vector pEGFP-N3-LYRM1 was constructed and then transfected into progenitor cells of subcutaneous adipose tissue of Baixi pig. The cell apoptosis of transfected cells was analyzed by triacylglycerol concentration detection, serum starvation method and MTT assay. The mRNA expression levels of LYRM1, PPARγ, ATGL and FAS genes were detected by qRT-PCR. The results showed that LYRM1 gene had the highest expression level in adipose tissues of Baixi pigs. The concentration of triacylglycerol in progenitor cells of subcutaneous adipose tissue transfected with pEGFP-N3-LYRM1 was higher than that in the control group. The apoptosis rates of the experimental group cells and the control group cells were similar and no significant difference was found. The mRNA expression levels of LYRM1, PPARγ, ATGL and FAS genes in the experimental group were significantly higher than that in the control group (P<0.01). In conclusion, overexpression of LYRM1 gene can increase the concentration of triacylglycerol, promote fat deposition, enhance the mRNA expression level of genes related to fat deposition, and indirectly increase the level of fat deposition. Therefore, LYRM1 gene can be regarded as a candidate gene to study the fat deposition, which provide a theoretical basis for investigating the mechanism of fat deposition.

The purpose of this study was to explore the effect of pig LYRM1 gene on the fat deposition. The expression levels of LYRM1 gene in different tissues of Baixi pigs aged 10 months were first detected by qRT-PCR. CDS of LYRM1 gene was amplified by PCR. The recombinant eukaryotic expression vector pEGFP-N3-LYRM1 was constructed and then transfected into progenitor cells of subcutaneous adipose tissue of Baixi pig. The cell apoptosis of transfected cells was analyzed by triacylglycerol concentration detection, serum starvation method and MTT assay. The mRNA expression levels of LYRM1, PPARγ, ATGL and FAS genes were detected by qRT-PCR. The results showed that LYRM1 gene had the highest expression level in adipose tissues of Baixi pigs. The concentration of triacylglycerol in progenitor cells of subcutaneous adipose tissue transfected with pEGFP-N3-LYRM1 was higher than that in the control group. The apoptosis rates of the experimental group cells and the control group cells were similar and no significant difference was found. The mRNA expression levels of LYRM1, PPARγ, ATGL and FAS genes in the experimental group were significantly higher than that in the control group (P<0.01). In conclusion, overexpression of LYRM1 gene can increase the concentration of triacylglycerol, promote fat deposition, enhance the mRNA expression level of genes related to fat deposition, and indirectly increase the level of fat deposition. Therefore, LYRM1 gene can be regarded as a candidate gene to study the fat deposition, which provide a theoretical basis for investigating the mechanism of fat deposition.

We studied the possibility of the PDGF-D gene to be a novel candidate gene affecting the sheep tail traits, in order to verify the previous results, as well as to find new molecular markers related to the sheep tail traits. In the 533 sheep experiment group (Hu sheep, Tibetan sheep, Dorper sheep×Hu sheep), 30 DNA samples with a fixed concentration from Hu sheep and Tibetan sheep respectively were selected to construct the DNA pool, primers were designed to amplify exons and 1 000 bp of upstream and downstream regulatory regions. PCR products were tested and the target segments were sequenced. Sequenom Mass-array technology was used to genotype the detected SNPs. Haploview 4.1 was used to construct haplotypes and analyze linkage disequilibrium of the polymorphic loci. Association analysis between the SNPs of PDGF-D gene and the tail traits was performed using SPSS 19.0 software. The results showed that 6 SNPs were detected. rs5, rs27 and rs28 were significantly related to tail length, tail width and tail perimeter(P<0.05). rs6 and rs19 were only significantly involved in tail length(P<0.05). rs13 was significantly related to tail width and tail perimeter(P<0.05). The result of linkage disequilibrium analysis showed that there was close linkage from rs6 to rs13. Meanwhile, the association analysis of combined genotypes of rs6-rs13 with tail traits revealed that individuals with CCGG combined genotype were significantly lower than the individuals with other combined genotypes in tail length, tail width and tail perimeter (P<0.05 or P<0.01), but the number of individuals with CCGG was less in population. The results indicate that PDGF-D gene has significant effect on sheep tail traits, and the SNPs of PDGF-D gene are potentially valuable as genetic markers for breeding. The CCGG genotype can be considered as the potential molecular marker to breed small-tailed sheep. The amount of individuals with CCGG combined genotype should be expanded further.

We studied the possibility of the PDGF-D gene to be a novel candidate gene affecting the sheep tail traits, in order to verify the previous results, as well as to find new molecular markers related to the sheep tail traits. In the 533 sheep experiment group (Hu sheep, Tibetan sheep, Dorper sheep×Hu sheep), 30 DNA samples with a fixed concentration from Hu sheep and Tibetan sheep respectively were selected to construct the DNA pool, primers were designed to amplify exons and 1 000 bp of upstream and downstream regulatory regions. PCR products were tested and the target segments were sequenced. Sequenom Mass-array technology was used to genotype the detected SNPs. Haploview 4.1 was used to construct haplotypes and analyze linkage disequilibrium of the polymorphic loci. Association analysis between the SNPs of PDGF-D gene and the tail traits was performed using SPSS 19.0 software. The results showed that 6 SNPs were detected. rs5, rs27 and rs28 were significantly related to tail length, tail width and tail perimeter(P<0.05). rs6 and rs19 were only significantly involved in tail length(P<0.05). rs13 was significantly related to tail width and tail perimeter(P<0.05). The result of linkage disequilibrium analysis showed that there was close linkage from rs6 to rs13. Meanwhile, the association analysis of combined genotypes of rs6-rs13 with tail traits revealed that individuals with CCGG combined genotype were significantly lower than the individuals with other combined genotypes in tail length, tail width and tail perimeter (P<0.05 or P<0.01), but the number of individuals with CCGG was less in population. The results indicate that PDGF-D gene has significant effect on sheep tail traits, and the SNPs of PDGF-D gene are potentially valuable as genetic markers for breeding. The CCGG genotype can be considered as the potential molecular marker to breed small-tailed sheep. The amount of individuals with CCGG combined genotype should be expanded further.

To investigate the mRNA expression patterns of IGF1R and IGF2R genes in different tissues and muscle cells of goats, real-time quantitative PCR was used to detect the mRNA levels of IGF1R and IGF2R in longissimus dorsi muscle at 3 prenatal stages (E45, E60 and E105), in muscle tissues (longissimus dorsi muscle, triceps brachii muscle) and visceral tissues (heart, liver and lung) at 5 postnatal stages (B3, B30, B60, B90 and B120) of Nanjiang Brown goats. Moreover, the expression patterns of IGF1R and IGF2R during skeletal muscle satellite cells (SMSCs) proliferation and differentiation was determined. The results showed that, in longissimus dorsi muscle of prenatal goats, the expression of IGF1R and IGF2R increased, then decreased. And the IGF1R expression level was higher than IGF2R in E45 and E60 (P<0.05, P>0.05), but lower than IGF2R in E105 (P<0.01). At the postnatal stages, the expression level of IGF1R was the highest in lung, and the lowest in liver; In contrast, the expression level of IGF2R was the lowest in lung but the highest in longissimus dorsi muscle. With the development of lambs, IGF1R and IGF2R exhibited ascending expression in heart, fluctuated (down-up-down) in liver, while the trends of IGF1R and IGF2R expression was reverse in lung (before B90). Furthermore, in longissimus dorsi and triceps brachii muscle, IGF1R showed increasing expression tendency(before B90), while IGF2R showed a down-up-down trend. Furthermore, IGF1R and IGF2R showed a up-down expression pattern during the proliferation stages (24, 48 and 72 h) of SMSCs, but was low at the early differentiation stages from 0 to 3 day, followed by significantly increased at 5 day (P<0.05), and then decreased. Expression patterns of IGF1R and IGF2R genes in different tissues and SMSCs of Nanjiang Brown goats were presented in this study. It is preliminarily reveal that high expression of IGF1R in lung and low expression in liver play a key role in organ growth and function maintenance, IGF1R synergizes with IGF2R to promote muscle tissue development and muscle cell proliferation and differentiation.

To investigate the mRNA expression patterns of IGF1R and IGF2R genes in different tissues and muscle cells of goats, real-time quantitative PCR was used to detect the mRNA levels of IGF1R and IGF2R in longissimus dorsi muscle at 3 prenatal stages (E45, E60 and E105), in muscle tissues (longissimus dorsi muscle, triceps brachii muscle) and visceral tissues (heart, liver and lung) at 5 postnatal stages (B3, B30, B60, B90 and B120) of Nanjiang Brown goats. Moreover, the expression patterns of IGF1R and IGF2R during skeletal muscle satellite cells (SMSCs) proliferation and differentiation was determined. The results showed that, in longissimus dorsi muscle of prenatal goats, the expression of IGF1R and IGF2R increased, then decreased. And the IGF1R expression level was higher than IGF2R in E45 and E60 (P<0.05, P>0.05), but lower than IGF2R in E105 (P<0.01). At the postnatal stages, the expression level of IGF1R was the highest in lung, and the lowest in liver; In contrast, the expression level of IGF2R was the lowest in lung but the highest in longissimus dorsi muscle. With the development of lambs, IGF1R and IGF2R exhibited ascending expression in heart, fluctuated (down-up-down) in liver, while the trends of IGF1R and IGF2R expression was reverse in lung (before B90). Furthermore, in longissimus dorsi and triceps brachii muscle, IGF1R showed increasing expression tendency(before B90), while IGF2R showed a down-up-down trend. Furthermore, IGF1R and IGF2R showed a up-down expression pattern during the proliferation stages (24, 48 and 72 h) of SMSCs, but was low at the early differentiation stages from 0 to 3 day, followed by significantly increased at 5 day (P<0.05), and then decreased. Expression patterns of IGF1R and IGF2R genes in different tissues and SMSCs of Nanjiang Brown goats were presented in this study. It is preliminarily reveal that high expression of IGF1R in lung and low expression in liver play a key role in organ growth and function maintenance, IGF1R synergizes with IGF2R to promote muscle tissue development and muscle cell proliferation and differentiation.

The aim of this study was to explore the factors affecting temperament of Chinese Holstein, estimate the variance component and genetic parameters, and reveal its genetic trend. The temperament score (TS) of 9 016 healthy lactating cows from 13 herds were collected from 2015 to 2017 in Beijing. Logistic model was used to analyze the factors affecting temperament, the animal model and DMU software were employed to estimate heritability of TS, rectal temperature(RT), daily milk yield(DMY) and reproductive traits(interval from calving to first service(ICF) and number of services(NS)) and genetic correlation between these traits. Genetic trends were analyzed according to the estimated breeding value. The results showed that the proportion of TS as 1, 2 and 3 in Chinese Holstein herds in Beijing area were 75.00%, 19.25% and 5.75%, respectively; The parity and farm-scorer had significant impact on TS (P<0.05); Estimated heritability of TS was 0.03, which defining TS as a low inheritable trait. The genetic correlation of TS with NS was moderate(r=0.56), with ICF and RT were negative and moderate (r=-0.30, r=-0.17, respectively), however, there was a low genetic correlation between TS and DMY (r=0.07); In general, no obvious change was evidenced for the genetic trend of temperament from 2001 to 2017. It is the first report to evaluate genetic parameters of temperament in Chinese Holstein. The results could provide theoretical basis for understanding the behavior characteristics of Chinese Holstein and facilitate future balanced breeding.

The aim of this study was to explore the factors affecting temperament of Chinese Holstein, estimate the variance component and genetic parameters, and reveal its genetic trend. The temperament score (TS) of 9 016 healthy lactating cows from 13 herds were collected from 2015 to 2017 in Beijing. Logistic model was used to analyze the factors affecting temperament, the animal model and DMU software were employed to estimate heritability of TS, rectal temperature(RT), daily milk yield(DMY) and reproductive traits(interval from calving to first service(ICF) and number of services(NS)) and genetic correlation between these traits. Genetic trends were analyzed according to the estimated breeding value. The results showed that the proportion of TS as 1, 2 and 3 in Chinese Holstein herds in Beijing area were 75.00%, 19.25% and 5.75%, respectively; The parity and farm-scorer had significant impact on TS (P<0.05); Estimated heritability of TS was 0.03, which defining TS as a low inheritable trait. The genetic correlation of TS with NS was moderate(r=0.56), with ICF and RT were negative and moderate (r=-0.30, r=-0.17, respectively), however, there was a low genetic correlation between TS and DMY (r=0.07); In general, no obvious change was evidenced for the genetic trend of temperament from 2001 to 2017. It is the first report to evaluate genetic parameters of temperament in Chinese Holstein. The results could provide theoretical basis for understanding the behavior characteristics of Chinese Holstein and facilitate future balanced breeding.

To investigate the association of genetic resistance loci to subgroup A avian leukosis virus(ALV) with important economic traits, the genetic variations within resistance loci of tva gene were screened in an F₂ resource population of broilers, which contained 788 healthy chicken. Production-related traits such as body weight, breast muscle weight, leg muscle weight on 6, 8, 10, 12 weeks and slaughter weight were measured, separately, and their association with genetic resistance loci to subgroup A avian leukosis virus was analyzed. Three variants (tva^r3, tva^r5 and tva^r6, named tva^mut502-516) were discovered in the F₂ population. Association analysis revealed that the tva^mut502-516 was significantly associated with abdominal fat weight (AFW) and abdominal fat rate (AFR) (P<0.05), but not significantly associated with growth, feed efficiency and other carcass traits (P>0.05). These results indicate that the mutation of genetic resistance loci to subgroup A avian leukosis virus can significantly decrease the AFW and AFR of chicken, and had no unfavorable effect on growth, feed efficiency and other carcass traits, therefore, these loci can balance the ALV-A resistance and growth development, can be applied in subgroup A ALV resistant breeding of broilers.

To investigate the association of genetic resistance loci to subgroup A avian leukosis virus(ALV) with important economic traits, the genetic variations within resistance loci of tva gene were screened in an F₂ resource population of broilers, which contained 788 healthy chicken. Production-related traits such as body weight, breast muscle weight, leg muscle weight on 6, 8, 10, 12 weeks and slaughter weight were measured, separately, and their association with genetic resistance loci to subgroup A avian leukosis virus was analyzed. Three variants (tva^r3, tva^r5 and tva^r6, named tva^mut502-516) were discovered in the F₂ population. Association analysis revealed that the tva^mut502-516 was significantly associated with abdominal fat weight (AFW) and abdominal fat rate (AFR) (P<0.05), but not significantly associated with growth, feed efficiency and other carcass traits (P>0.05). These results indicate that the mutation of genetic resistance loci to subgroup A avian leukosis virus can significantly decrease the AFW and AFR of chicken, and had no unfavorable effect on growth, feed efficiency and other carcass traits, therefore, these loci can balance the ALV-A resistance and growth development, can be applied in subgroup A ALV resistant breeding of broilers.

The aim of this study was to investigate whether endoplasmic reticulum stress (ERS) marker gene Grp78 was involved in regulation on the expression of inflammation/immunity-related genes in goose fatty liver. Thirty healthy 70-day-old Landaise geese (male or female) with similar body weight were randomly divided into the control group (n=15) and the overfeeding group (n=15). The relative expression of Grp78 in different tissues at different overfeeding periods (7, 14, 19 d) was determined by fluorescent quantitative PCR to clarify the relationship between Grp78 and geese fatty liver formation; Grp78 was overexpressed in goose primary hepatocytes, and the signaling pathways and genes affected by Grp78 were analyzed by RNA-seq (n=3); The expression of genes related to inflammation/immunity was determined in goose fatty liver by fluorescent quantitative PCR (n=4) to clarify the association of these genes with Grp78. The results showed that the expression of Grp78 in the liver and abdominal fat was affected by overfeeding (decreased in liver and increased in abdominal fat). The result of overexpression of Grp78 gene suggested that ERS not only participated in the known metabolic diseases and cell apoptosis pathways, but also had an effect on signal transduction and immune pathways. The Toll-like receptor signaling pathway, TNFα signaling pathway and NF-kappa B signaling pathway were the most prominent ones in regard to immunity/inflammation. In addition, the expression of immune/inflammation related genes, Tnfsf10 and Map3k7cl, was affected by Grp78 overexpression in the goose primary hepatocytes and was affected by overfeeding in goose fatty liver. In summary, this study revealed a new biological function of Grp78/ERS, that is, to regulate cellular immune/inflammatory state and participate in the formation of goose fatty liver.

The aim of this study was to investigate whether endoplasmic reticulum stress (ERS) marker gene Grp78 was involved in regulation on the expression of inflammation/immunity-related genes in goose fatty liver. Thirty healthy 70-day-old Landaise geese (male or female) with similar body weight were randomly divided into the control group (n=15) and the overfeeding group (n=15). The relative expression of Grp78 in different tissues at different overfeeding periods (7, 14, 19 d) was determined by fluorescent quantitative PCR to clarify the relationship between Grp78 and geese fatty liver formation; Grp78 was overexpressed in goose primary hepatocytes, and the signaling pathways and genes affected by Grp78 were analyzed by RNA-seq (n=3); The expression of genes related to inflammation/immunity was determined in goose fatty liver by fluorescent quantitative PCR (n=4) to clarify the association of these genes with Grp78. The results showed that the expression of Grp78 in the liver and abdominal fat was affected by overfeeding (decreased in liver and increased in abdominal fat). The result of overexpression of Grp78 gene suggested that ERS not only participated in the known metabolic diseases and cell apoptosis pathways, but also had an effect on signal transduction and immune pathways. The Toll-like receptor signaling pathway, TNFα signaling pathway and NF-kappa B signaling pathway were the most prominent ones in regard to immunity/inflammation. In addition, the expression of immune/inflammation related genes, Tnfsf10 and Map3k7cl, was affected by Grp78 overexpression in the goose primary hepatocytes and was affected by overfeeding in goose fatty liver. In summary, this study revealed a new biological function of Grp78/ERS, that is, to regulate cellular immune/inflammatory state and participate in the formation of goose fatty liver.

The study was aimed to study the immunological and biological efficacy of 3-different types of GnRH-like constructs in male Sprague Dawley (SD) rats. The designing strategies for the new GnRH-like peptide constructs are based on:1) increasing the number of GnRH copy; 2) increasing the molecular weight of GnRH antigen. The new GnRH-like peptide constructs were designed based on a GnRH-tandem peptide, which was modified by relacing Gly at position 6 of the decapeptide with D-Lys (G6KT) to allow the conjugation of the GnRH peptide to ovalbumin (OVA). The first new GnRH-like construct was prepared by the dimerization of G6KT (G6KTD); the second and third were prepared by conjugating G6KT and G6KTD to OVA, respectively (G6KT-OVA and G6KTD-OVA). Sixty male SD rats at the age of 6 weeks, were randomly allocated to 5 groups (n=12):intact control (no treatment), surgically castrated or vaccinated against 100 μg peptide equivalent of G6KTD, G6KT-OVA and G6KTD-OVA in Specol adjuvant, respectively, at 6 weeks of age (with a booster 8 weeks later). Serum anti-GnRH antibody titers and reproductive hormone concentrations were assayed by RIA and ELISA, respectively. mRNA expressions of reproduction-related genes in pituitary-testicular axis were quantified using qPCR.The results shown that:1) active immunization against all of the 3 different new GnRH-like constructs could intrigue a good antibody responses in male SD rats; 2) Only with the first injection, active immunization against both G6KT-OVA and G6KTD-OVA intrigued a high antibody generation(P<0.05) and reduced serum concentrations of LH, FSH, testosterone and inhibin B(P<0.05), and further caused antrophy of testes with completely disrupted spermatogenesis and downregulated mRNA expressions of pituitary GnRHR, LHβ, FSHβ and testicular LHR, FSHR, INHα, INβA and INHβB (P<0.05) with the booster injection; 3) in contrast, the significant decrease of serum concentrations of LH, FSH, testosterone and inhibin B in rats actively immunized against G6KTD only occurred after the booster injection(P<0.05), but which still caused antrophy of testes with obviously disrupted spermatogenesis at decapitation (P<0.05) in 9 of 12 (75%) rats. In conclusion,based on those results, we concluded that both G6KT-OVA and G6KTD-OVA were highly efficacious GnRH antigen, which could be used as one-shot immunocastration vaccine in the furthure, whereas G6KTD itself was also effective, which might be used as GnRH antigen so as to avoid the drawbacks derived from the conjugation and purification processes of GnRH-carrier conjugates. Our findings promoted the further development and large scale commercial applications of GnRH vaccine.

The study was aimed to study the immunological and biological efficacy of 3-different types of GnRH-like constructs in male Sprague Dawley (SD) rats. The designing strategies for the new GnRH-like peptide constructs are based on:1) increasing the number of GnRH copy; 2) increasing the molecular weight of GnRH antigen. The new GnRH-like peptide constructs were designed based on a GnRH-tandem peptide, which was modified by relacing Gly at position 6 of the decapeptide with D-Lys (G6KT) to allow the conjugation of the GnRH peptide to ovalbumin (OVA). The first new GnRH-like construct was prepared by the dimerization of G6KT (G6KTD); the second and third were prepared by conjugating G6KT and G6KTD to OVA, respectively (G6KT-OVA and G6KTD-OVA). Sixty male SD rats at the age of 6 weeks, were randomly allocated to 5 groups (n=12):intact control (no treatment), surgically castrated or vaccinated against 100 μg peptide equivalent of G6KTD, G6KT-OVA and G6KTD-OVA in Specol adjuvant, respectively, at 6 weeks of age (with a booster 8 weeks later). Serum anti-GnRH antibody titers and reproductive hormone concentrations were assayed by RIA and ELISA, respectively. mRNA expressions of reproduction-related genes in pituitary-testicular axis were quantified using qPCR.The results shown that:1) active immunization against all of the 3 different new GnRH-like constructs could intrigue a good antibody responses in male SD rats; 2) Only with the first injection, active immunization against both G6KT-OVA and G6KTD-OVA intrigued a high antibody generation(P<0.05) and reduced serum concentrations of LH, FSH, testosterone and inhibin B(P<0.05), and further caused antrophy of testes with completely disrupted spermatogenesis and downregulated mRNA expressions of pituitary GnRHR, LHβ, FSHβ and testicular LHR, FSHR, INHα, INβA and INHβB (P<0.05) with the booster injection; 3) in contrast, the significant decrease of serum concentrations of LH, FSH, testosterone and inhibin B in rats actively immunized against G6KTD only occurred after the booster injection(P<0.05), but which still caused antrophy of testes with obviously disrupted spermatogenesis at decapitation (P<0.05) in 9 of 12 (75%) rats. In conclusion,based on those results, we concluded that both G6KT-OVA and G6KTD-OVA were highly efficacious GnRH antigen, which could be used as one-shot immunocastration vaccine in the furthure, whereas G6KTD itself was also effective, which might be used as GnRH antigen so as to avoid the drawbacks derived from the conjugation and purification processes of GnRH-carrier conjugates. Our findings promoted the further development and large scale commercial applications of GnRH vaccine.

This experiment was conducted to compare the distribution characteristics of TGF-β1 and its receptors(TGF-βRⅠ) in Tibetan sheep testis before and after sexual mature and attempt to explore their relationships with testicular spermatogenesis in sexual mature. The testis of 1.0-, 1.5-, 2.0-year-old Tibetan sheep were prepared by testicular removal surgery for describe the distribution density and the location of TGF-β1 and TGF-βRⅠ in the testis tissues using the Western blot, immunohistochemical SP combined with IPP (Image-Pro Plus) statistics methods. The results suggested that:1) The reactions of TGF-β1 antibody were weakly and there were no significant difference in Tibetan sheep testis before and after sexual mature(P>0.05);The expression of TGF-βR Ⅰ in the 1.5-year-old sheep testis tissue were significantly higher than 1.0-and 2.0-year-old(P<0.05). 2)The TGF-β1 was mainly present in Leydig cells and endothelial cells of small vascular at different ages, there were medium positive expression of TGF-β1 in Sertoli cells and spermatocytes at 1.5-year-old Tibetan sheep but weakly expressed in seminiferous epithelium at other 2 groups. The TGF-βRⅠwere expressed in seminiferous epithelium at 3 ages. In the 1.0-year-old sheep testis, the expression of TGF-βRⅠwas weakly expressed in Leydig cells, and at the age of 1.5, its expression in the round spermatid, Leydig cells and lymphatic endothelial cells were strongly positive, also there were immunolocalized in Leydig cells and capillary lymph vessel at 2-year-old, basically the expression of TGF-βRⅠin the 1.5-year-old sheep testis was more stronger than that of other 2 groups.3)The positive signal statistics indicated that the average absorbance of TGF-βRⅠ expression was significantly higher in the testis than TGF-β1,and the expression of TGF-β1 were not different in the testis of the Tibetan sheep before and after sexual mature(P> 0.05),the TGF-βRⅠwas expressed significantly higher in testis tissue of 1.5-year-old Tibetan sheep than that of 1.0-and 2.0-year-old sheep(P<0.05). In conclusion, these results demonstrate that the expression of TGF-β1 and TGF-βRⅠin Tibetan sheep testis appered with rise trend first and then fall with age in sexual mature, their expression in Sertoli cells had correlaton with the regulation of spermatogenesis; and the mainly changes in Leydig cells serve as a meaningful index for the further research of TGF-β1/TGF-βRⅠpathways involved in its steroid biosynthesis and basic insights into testicular development.

This experiment was conducted to compare the distribution characteristics of TGF-β1 and its receptors(TGF-βRⅠ) in Tibetan sheep testis before and after sexual mature and attempt to explore their relationships with testicular spermatogenesis in sexual mature. The testis of 1.0-, 1.5-, 2.0-year-old Tibetan sheep were prepared by testicular removal surgery for describe the distribution density and the location of TGF-β1 and TGF-βRⅠ in the testis tissues using the Western blot, immunohistochemical SP combined with IPP (Image-Pro Plus) statistics methods. The results suggested that:1) The reactions of TGF-β1 antibody were weakly and there were no significant difference in Tibetan sheep testis before and after sexual mature(P>0.05);The expression of TGF-βR Ⅰ in the 1.5-year-old sheep testis tissue were significantly higher than 1.0-and 2.0-year-old(P<0.05). 2)The TGF-β1 was mainly present in Leydig cells and endothelial cells of small vascular at different ages, there were medium positive expression of TGF-β1 in Sertoli cells and spermatocytes at 1.5-year-old Tibetan sheep but weakly expressed in seminiferous epithelium at other 2 groups. The TGF-βRⅠwere expressed in seminiferous epithelium at 3 ages. In the 1.0-year-old sheep testis, the expression of TGF-βRⅠwas weakly expressed in Leydig cells, and at the age of 1.5, its expression in the round spermatid, Leydig cells and lymphatic endothelial cells were strongly positive, also there were immunolocalized in Leydig cells and capillary lymph vessel at 2-year-old, basically the expression of TGF-βRⅠin the 1.5-year-old sheep testis was more stronger than that of other 2 groups.3)The positive signal statistics indicated that the average absorbance of TGF-βRⅠ expression was significantly higher in the testis than TGF-β1,and the expression of TGF-β1 were not different in the testis of the Tibetan sheep before and after sexual mature(P> 0.05),the TGF-βRⅠwas expressed significantly higher in testis tissue of 1.5-year-old Tibetan sheep than that of 1.0-and 2.0-year-old sheep(P<0.05). In conclusion, these results demonstrate that the expression of TGF-β1 and TGF-βRⅠin Tibetan sheep testis appered with rise trend first and then fall with age in sexual mature, their expression in Sertoli cells had correlaton with the regulation of spermatogenesis; and the mainly changes in Leydig cells serve as a meaningful index for the further research of TGF-β1/TGF-βRⅠpathways involved in its steroid biosynthesis and basic insights into testicular development.

The aim of this study was to investigate the effects of defatted rice bran substituting corn levels on growth performance, intestinal development and nutrient apparent digestibility of Suhuai pigs, and select the optimal level of defatted rice bran substituting corn for Suhuai pig.Thirty-five healthy Suhuai castrated boars with similar body weight at (62.90±0.78) kg were selected and randomly divided into 5 treatments (control group, group Ⅰ-Ⅳ) with 7 duplicates for 1 per duplicate. The pre-feeding period of the trial was 10 days and all pigs were fed with the same control diet ad libitum. The pigs in control were provided control diet without defatted rice bran, and the pigs in group Ⅰ-Ⅳ were provided diets that using 7%, 14%, 21%, 28% of defatted rice bran replaced the equivalent corn respectively for 28 days during the experiment period. The neutral detergent fiber (NDF) levels in the 5 groups were 8.89%, 11.80%, 12.93%, 14.35% and 17.94%, respectively. All pigs were provided diets and water ad libitum. The feed intake and body weight of all pigs were recorded daily during the experiment period. The fecal samples were collected on the 0th and 14th day of the experiment period, and the rectal contents were collected on the 28th day of the experiment period, which were used for the determination of apparent digestibility of nutrients. All pigs were slaughtered on the 28th day of the experiment period, and the weight and length of the gastrointestinal tract were measured. The results showed that:1) No significant difference was observed in the initial body weight, body weight of 14th of the experiment period, terminal body weight, average daily feed intake (ADFI), average daily gain (ADG), average daily gain from 0th to 14th day of the experiment period, average daily gain from 14th to 28th day of the experiment period and feed to gain ratio (F/G) among the 5 treatments(P>0.05). However, the average daily gain from 14th to 28th day were significantly higher than that from 0th to 14th day in control group and group Ⅰ-Ⅳ (P<0.05). The growth curve showed that the inflection points of the growth of the pigs in each group were 18.51th day(75.87 kg), 15.92th day(73.61 kg), 17.39th day(75.91 kg), 17.58th day(75.45 kg), 16.29th day(76.15 kg), respectively. Compared with the control group, pigs in the experimental groups reached the inflection point of growth earlier but there was no obvious difference in the growth trend among different groups. 2) Diets with different fiber levels had no significant effect on the relative length and weight of gastrointestinal tract, ratio of large intestine's weight and length to intestinal tract (P>0.05). 3) On the 14th and 28th day of the experiment period, with the dietary fiber level increased, no significant difference was observed in the apparent digestibility of NDF among the 5 treatments(P>0.05). The apparent digestibility of acidic detergent fiber (ADF) and crude protein (CP) decreased (linear, quadratic and cubic, P<0.01). On the 14th day, the apparent digestibility of ether extract (EE) had a decreased trend (quadratic, P<0.10). 4) According to the prediction of cubic curve regression model, the appropriate levels of defatted rice bran substituting corn were 12.77% and 14.78% respectively, when the apparent digestibility of ADF and CP were considered as dependent variance. When the digestive energy and CP of 5 diets were the same and enough, increasing dietary fiber level had no siginficant effect on growth performance, relative intestinal weight and length, apparent digestibility of NDF, but decreased the apparent digestibility of ADF and CP. The defatted rice bran substituting part of the corn was feasible, the optimal ratio was 12.77%.

The aim of this study was to investigate the effects of defatted rice bran substituting corn levels on growth performance, intestinal development and nutrient apparent digestibility of Suhuai pigs, and select the optimal level of defatted rice bran substituting corn for Suhuai pig.Thirty-five healthy Suhuai castrated boars with similar body weight at (62.90±0.78) kg were selected and randomly divided into 5 treatments (control group, group Ⅰ-Ⅳ) with 7 duplicates for 1 per duplicate. The pre-feeding period of the trial was 10 days and all pigs were fed with the same control diet ad libitum. The pigs in control were provided control diet without defatted rice bran, and the pigs in group Ⅰ-Ⅳ were provided diets that using 7%, 14%, 21%, 28% of defatted rice bran replaced the equivalent corn respectively for 28 days during the experiment period. The neutral detergent fiber (NDF) levels in the 5 groups were 8.89%, 11.80%, 12.93%, 14.35% and 17.94%, respectively. All pigs were provided diets and water ad libitum. The feed intake and body weight of all pigs were recorded daily during the experiment period. The fecal samples were collected on the 0th and 14th day of the experiment period, and the rectal contents were collected on the 28th day of the experiment period, which were used for the determination of apparent digestibility of nutrients. All pigs were slaughtered on the 28th day of the experiment period, and the weight and length of the gastrointestinal tract were measured. The results showed that:1) No significant difference was observed in the initial body weight, body weight of 14th of the experiment period, terminal body weight, average daily feed intake (ADFI), average daily gain (ADG), average daily gain from 0th to 14th day of the experiment period, average daily gain from 14th to 28th day of the experiment period and feed to gain ratio (F/G) among the 5 treatments(P>0.05). However, the average daily gain from 14th to 28th day were significantly higher than that from 0th to 14th day in control group and group Ⅰ-Ⅳ (P<0.05). The growth curve showed that the inflection points of the growth of the pigs in each group were 18.51th day(75.87 kg), 15.92th day(73.61 kg), 17.39th day(75.91 kg), 17.58th day(75.45 kg), 16.29th day(76.15 kg), respectively. Compared with the control group, pigs in the experimental groups reached the inflection point of growth earlier but there was no obvious difference in the growth trend among different groups. 2) Diets with different fiber levels had no significant effect on the relative length and weight of gastrointestinal tract, ratio of large intestine's weight and length to intestinal tract (P>0.05). 3) On the 14th and 28th day of the experiment period, with the dietary fiber level increased, no significant difference was observed in the apparent digestibility of NDF among the 5 treatments(P>0.05). The apparent digestibility of acidic detergent fiber (ADF) and crude protein (CP) decreased (linear, quadratic and cubic, P<0.01). On the 14th day, the apparent digestibility of ether extract (EE) had a decreased trend (quadratic, P<0.10). 4) According to the prediction of cubic curve regression model, the appropriate levels of defatted rice bran substituting corn were 12.77% and 14.78% respectively, when the apparent digestibility of ADF and CP were considered as dependent variance. When the digestive energy and CP of 5 diets were the same and enough, increasing dietary fiber level had no siginficant effect on growth performance, relative intestinal weight and length, apparent digestibility of NDF, but decreased the apparent digestibility of ADF and CP. The defatted rice bran substituting part of the corn was feasible, the optimal ratio was 12.77%.

This study aimed to investigate the effects of dietary soybean oil supplementation and different birth types on collagen characteristics and related genes expression in muscle of lamb. Sixty-four male Hu lambs with twin born (TW, 32 lambs) and triplet born (TR, 32 lambs) types weaned at 2 months old were selected and fed basal diet (CON) or diet with 2% soybean oil supplemented (SBO). Lambs were randomly divided into 4 treatments (TW-CON, TW-SBO, TR-CON, TR-SBO) according to 2×2 factorial experimental design, each treatment had 16 biological replication, and 1 lamb in each replication. Lambs were slaughtered after 90 days of feeding, and longissimus dorsi muscle samples were collected for detecting the content of collagen, enzymes activity and muscle development-related genes expression level. The results indicated that:1) The collagen type Ⅰ content in TW groups was significantly higher than that in TR groups (P<0.05). Dietary soybean oil supplementation increased collagen content (0.05 < P < 0.10), while had no significant effect on collagen solubility (P>0.05). 2) Matrix metalloproteinases-13 (MMP-13) content decreased significantly with diet soybean oil supplementation (P<0.05), while pyridinoline (PYD) and tissue inhibitor of metalloproteinases (TIMP-1) were affected significantly by the interaction effects between dietary soybean oil supplementation and birth types (P<0.05). 3) MYOD1 expression level was significantly higher in TW groups than that in TR groups (P<0.05). The expression level of FN1 decreased significantly with soybean oil supplemented in diet (P<0.05), whereas FGF2 and COL3A1 expression levels were affected by the interaction effects between dietary soybean oil supplementation and birth types (0.05 < P < 0.10). In addition, the expression levels of FGFR1, TGFβ1 and COL1A1 were not significantly affected by the soybean oil supplementation or birth types (P>0.05). In conclusion, twin born lambs contain more collagen in muscle than triplet born lambs, and soybean oil supplementation show a trend to increase the collagen content in muscle. Moreover, dietary soybean oil supplementation and birth types are combined together to regulate the content of collagen metabolism enzymes and related genes expression level, and then modulate the collagen content in muscle.

This study aimed to investigate the effects of dietary soybean oil supplementation and different birth types on collagen characteristics and related genes expression in muscle of lamb. Sixty-four male Hu lambs with twin born (TW, 32 lambs) and triplet born (TR, 32 lambs) types weaned at 2 months old were selected and fed basal diet (CON) or diet with 2% soybean oil supplemented (SBO). Lambs were randomly divided into 4 treatments (TW-CON, TW-SBO, TR-CON, TR-SBO) according to 2×2 factorial experimental design, each treatment had 16 biological replication, and 1 lamb in each replication. Lambs were slaughtered after 90 days of feeding, and longissimus dorsi muscle samples were collected for detecting the content of collagen, enzymes activity and muscle development-related genes expression level. The results indicated that:1) The collagen type Ⅰ content in TW groups was significantly higher than that in TR groups (P<0.05). Dietary soybean oil supplementation increased collagen content (0.05 < P < 0.10), while had no significant effect on collagen solubility (P>0.05). 2) Matrix metalloproteinases-13 (MMP-13) content decreased significantly with diet soybean oil supplementation (P<0.05), while pyridinoline (PYD) and tissue inhibitor of metalloproteinases (TIMP-1) were affected significantly by the interaction effects between dietary soybean oil supplementation and birth types (P<0.05). 3) MYOD1 expression level was significantly higher in TW groups than that in TR groups (P<0.05). The expression level of FN1 decreased significantly with soybean oil supplemented in diet (P<0.05), whereas FGF2 and COL3A1 expression levels were affected by the interaction effects between dietary soybean oil supplementation and birth types (0.05 < P < 0.10). In addition, the expression levels of FGFR1, TGFβ1 and COL1A1 were not significantly affected by the soybean oil supplementation or birth types (P>0.05). In conclusion, twin born lambs contain more collagen in muscle than triplet born lambs, and soybean oil supplementation show a trend to increase the collagen content in muscle. Moreover, dietary soybean oil supplementation and birth types are combined together to regulate the content of collagen metabolism enzymes and related genes expression level, and then modulate the collagen content in muscle.

The aim of this study was to explore the possible role of TLR-2 and Dectin-1 in the expression of β-defensin-1 (SBD-1) in ovine ruminal epithelial cells (ORECs) induced by Saccharomyces cerevisiae β-glucan. The immunohistochemistry, RT-PCR, immunofluorescence and Western blot were used to detect the expression of Dectin-1 in ORECs, and qPCR and Western blot were used to detected the expression changes of Dectin-1 and TLR-2 after β-glucan stimulated ORECs. Then, ORECs were pretreated using Dectin-1 blocker laminarin or TLR-2 blocking antibody with different concentrations, and the expression of SBD-1 were detected by qPCR and ELISA to determine the involvement of Dectin-1 and TLR-2 in the induction expression of SBD-1 induced by β-glucan. The results showed that:1) Dectin-1 was expressed in ovine ruminal tissues and ORECs, and the expression levels of Dectin-1 and TLR-2 were significantly increased after β-glucan stimulating ORECs (P<0.05); 2)The laminarin or TLR-2 blocking antibody with different concentrations significantly decreased the expression of SBD-1 induced by β-glucan (P<0.01), and this effect became stronger as the laminarin and blocking antibodies concentration increased. The results indicate that Dectin-1 is expressed in both ovine ruminal tissues and ORECs, and the Saccharomyces cerevisiae β-glucan inducing SBD-1 expression in ORECs is mediated by Dectin-1 and TLR-2.

The aim of this study was to explore the possible role of TLR-2 and Dectin-1 in the expression of β-defensin-1 (SBD-1) in ovine ruminal epithelial cells (ORECs) induced by Saccharomyces cerevisiae β-glucan. The immunohistochemistry, RT-PCR, immunofluorescence and Western blot were used to detect the expression of Dectin-1 in ORECs, and qPCR and Western blot were used to detected the expression changes of Dectin-1 and TLR-2 after β-glucan stimulated ORECs. Then, ORECs were pretreated using Dectin-1 blocker laminarin or TLR-2 blocking antibody with different concentrations, and the expression of SBD-1 were detected by qPCR and ELISA to determine the involvement of Dectin-1 and TLR-2 in the induction expression of SBD-1 induced by β-glucan. The results showed that:1) Dectin-1 was expressed in ovine ruminal tissues and ORECs, and the expression levels of Dectin-1 and TLR-2 were significantly increased after β-glucan stimulating ORECs (P<0.05); 2)The laminarin or TLR-2 blocking antibody with different concentrations significantly decreased the expression of SBD-1 induced by β-glucan (P<0.01), and this effect became stronger as the laminarin and blocking antibodies concentration increased. The results indicate that Dectin-1 is expressed in both ovine ruminal tissues and ORECs, and the Saccharomyces cerevisiae β-glucan inducing SBD-1 expression in ORECs is mediated by Dectin-1 and TLR-2.

Natural antimicrobial peptides have a strong bactericidal ability, however, low cell selectivity of natural antimicrobial peptides due to their strong cytotoxicity. In order to improve the specificity of antibacterial peptides, the effects of different hydrophobic amino acids on the biological activity of α-helix antimicrobial peptides and bacteriostatic mechanisms were investigated. We employed hydrophobic amino acid tryptophan (Try, T), phenylalanine (Phe, P), valine (Val, V), alanine (Ala, A), leucine (Leu, L) and isoleucine (Ile, I) to replace X position of GRX₂RX₃RX₂RG template. Our study evaluated the secondary structure, hemolytic activity, bacteriostatic activity, salt resistance and studied bacteriostatic mechanism of peptides. The results of CD spectroscopy showed that GF, GI, GA and GL displayed typical α helix structure in the simulated environment of cell membrane, while only GV exhibited α helix structure in TFE. Hemolysis test showed that GV and GA did not exhibit hemolytic activity in the concentration of 128 μmol·L^-1, while other peptides showed high hemolytic activity. The geometric average value of minimal inhibitory concentration (MIC) of GV which has the highest therapeutic index is 3.7 μmol·L^-1. The stability test showed that GV had the stable antibacterial activity against E. coli ATCC 25922 in the presence of NH₄⁺, Zn²⁺ and Fe³⁺. The antimicrobial mechanism of antimicrobial peptide GV was further analyzed by scanning electron microscopy and inner membrane permeability test. The results showed that GV induced outflow of bacterial content via penetrating membrane of E. coli ATCC 25922 and Staphylococcus aureus ATCC 29213 and penetrated inner membrane of E. coli ATCC 25922 by time-and dose-dependence. Collectivity, the Val-rich antimicrobial peptide GV had the highest cell selectivity, and is potential for development of highly effective antibacterial drugs.

Natural antimicrobial peptides have a strong bactericidal ability, however, low cell selectivity of natural antimicrobial peptides due to their strong cytotoxicity. In order to improve the specificity of antibacterial peptides, the effects of different hydrophobic amino acids on the biological activity of α-helix antimicrobial peptides and bacteriostatic mechanisms were investigated. We employed hydrophobic amino acid tryptophan (Try, T), phenylalanine (Phe, P), valine (Val, V), alanine (Ala, A), leucine (Leu, L) and isoleucine (Ile, I) to replace X position of GRX₂RX₃RX₂RG template. Our study evaluated the secondary structure, hemolytic activity, bacteriostatic activity, salt resistance and studied bacteriostatic mechanism of peptides. The results of CD spectroscopy showed that GF, GI, GA and GL displayed typical α helix structure in the simulated environment of cell membrane, while only GV exhibited α helix structure in TFE. Hemolysis test showed that GV and GA did not exhibit hemolytic activity in the concentration of 128 μmol·L^-1, while other peptides showed high hemolytic activity. The geometric average value of minimal inhibitory concentration (MIC) of GV which has the highest therapeutic index is 3.7 μmol·L^-1. The stability test showed that GV had the stable antibacterial activity against E. coli ATCC 25922 in the presence of NH₄⁺, Zn²⁺ and Fe³⁺. The antimicrobial mechanism of antimicrobial peptide GV was further analyzed by scanning electron microscopy and inner membrane permeability test. The results showed that GV induced outflow of bacterial content via penetrating membrane of E. coli ATCC 25922 and Staphylococcus aureus ATCC 29213 and penetrated inner membrane of E. coli ATCC 25922 by time-and dose-dependence. Collectivity, the Val-rich antimicrobial peptide GV had the highest cell selectivity, and is potential for development of highly effective antibacterial drugs.

The objective of this study was to investigate the epidemiological situation of swine influenza and viral molecule characteristics in North-eastern China. One H1N2 subtype swine influenza virus, designated as A/swine/Liaoning/FX575/2016 (H1N2) (FX575), was isolated from a slaughterhouse in Liaoning province during an active surveillance in 2016. The complete genomic sequence of the strain was sequenced and subjected to phylogenetic analysis, molecular characteristic analysis and genotypic analysis. Then, the viral pathogenesis was carried out by intranasal infecting the six-week-old female BALB/c mice and evaluated by measuring body weight loss of mice and virus replication titer. FX575 was a triple reassortant H1N2 virus, which had the nearest genetic relationship with the H1N2 influenza viruses previously isolated from China. Phylogenetic analysis results demonstrated that HA gene of the isolate belonged to the Eurasian avian-like swine H1N1 lineage, the NA gene was clustered into the North American swine H1N2 lineage, and the six internal genes fell into the pandemic 2009/H1N1 virus lineage, respectively. The virus FX575 could cause significant weight losses of mice within one week after infection, and one mouse with its weight loss exceeding 25 percent was considered dead. The viral replications occurred both in lungs and nasal turbinates, with the titers of 4.82 and 4.20 log₁₀EID₅₀·mL^-1, respectively. The results indicated that continuous reassortments of different genotypes of influenza viruses occurred in the swine population, and the novel reassortant virus showed moderate pathogenicity to mice, which further suggested that the active monitoring of SIV should be strengthened in the future.

The objective of this study was to investigate the epidemiological situation of swine influenza and viral molecule characteristics in North-eastern China. One H1N2 subtype swine influenza virus, designated as A/swine/Liaoning/FX575/2016 (H1N2) (FX575), was isolated from a slaughterhouse in Liaoning province during an active surveillance in 2016. The complete genomic sequence of the strain was sequenced and subjected to phylogenetic analysis, molecular characteristic analysis and genotypic analysis. Then, the viral pathogenesis was carried out by intranasal infecting the six-week-old female BALB/c mice and evaluated by measuring body weight loss of mice and virus replication titer. FX575 was a triple reassortant H1N2 virus, which had the nearest genetic relationship with the H1N2 influenza viruses previously isolated from China. Phylogenetic analysis results demonstrated that HA gene of the isolate belonged to the Eurasian avian-like swine H1N1 lineage, the NA gene was clustered into the North American swine H1N2 lineage, and the six internal genes fell into the pandemic 2009/H1N1 virus lineage, respectively. The virus FX575 could cause significant weight losses of mice within one week after infection, and one mouse with its weight loss exceeding 25 percent was considered dead. The viral replications occurred both in lungs and nasal turbinates, with the titers of 4.82 and 4.20 log₁₀EID₅₀·mL^-1, respectively. The results indicated that continuous reassortments of different genotypes of influenza viruses occurred in the swine population, and the novel reassortant virus showed moderate pathogenicity to mice, which further suggested that the active monitoring of SIV should be strengthened in the future.

As a new pathogen, the pathogenesis of Senecavirus A (SVA) remains unclear. Through microscopic observation, it was found that SVA can infect PK-15 cells and causes significant cytopathic (CPE). Concomitant with CPE, apoptosis is commonly observed in infected cells. In this study, the function of each individual SVA protein in the induction of apoptosis was studied. Firstly, using Western blotting confirmed the successful expression of SVA proteins. Subsequently, the SVA proteins were screened for their apoptotic function using flow cytometry with Annexin V-FITC/PI double staining. Then, the extent of externalized phospholipid phosphatidylserine and nuclear condensation induced by VP1 was analyzed by fluorescence microscope using Annexin V-FITC/PI and Hoechst staining. Finally, Western blotting and RT-PCR technique were used to analyze the effects of VP1 on the protein and mRNA levels of the major apoptosis regulatory molecules Caspase3, Caspase8 and Bax. SVA-VP1 was found to significantly induce early and late apoptosis in cells. And it also could promote the up-regulation of pro-apoptotic protein Caspase3, Caspase8 and Bax protein and mRNA levels. This study provides a theoretical basis for further research in molecular mechanism and the pathogenesis of SVA-regulated apoptosis.

As a new pathogen, the pathogenesis of Senecavirus A (SVA) remains unclear. Through microscopic observation, it was found that SVA can infect PK-15 cells and causes significant cytopathic (CPE). Concomitant with CPE, apoptosis is commonly observed in infected cells. In this study, the function of each individual SVA protein in the induction of apoptosis was studied. Firstly, using Western blotting confirmed the successful expression of SVA proteins. Subsequently, the SVA proteins were screened for their apoptotic function using flow cytometry with Annexin V-FITC/PI double staining. Then, the extent of externalized phospholipid phosphatidylserine and nuclear condensation induced by VP1 was analyzed by fluorescence microscope using Annexin V-FITC/PI and Hoechst staining. Finally, Western blotting and RT-PCR technique were used to analyze the effects of VP1 on the protein and mRNA levels of the major apoptosis regulatory molecules Caspase3, Caspase8 and Bax. SVA-VP1 was found to significantly induce early and late apoptosis in cells. And it also could promote the up-regulation of pro-apoptotic protein Caspase3, Caspase8 and Bax protein and mRNA levels. This study provides a theoretical basis for further research in molecular mechanism and the pathogenesis of SVA-regulated apoptosis.

The purpose of this study was to investigate the relationship between the two-component system phoP/Q and flagella type Ⅲ secretory system (flagellar T3SS) proteins in Avian Pathogenic Escherichia coli (APEC). We used software to analyze the function and to predict the relationship between phoP/Q and flagella T3SS, and used gene chip technology to compare the transcriptome differences between APEC wild strain and ΔphoP/Q deletion mutant, and screening for variable genes in flagellum T3SS pedestal. The changes of gene expression were detected by real-time PCR. The results predicted by STRING showed that phoP/Q may regulate flagellar T3SS indirectly by regulating the two-component system near the flagellar T3SS locus. Gene chip screening and real-time PCR revealed that the phoP/Q deletion could significantly differ the expression of most genes in the core component of flagellar T3SS, and also affect the expression of motA, motB and cheY in the two components. In this study, the relationship between phoP/Q two-component system and flagellar T3SS was predicted and explored, which provided a new breakthrough point and research direction for further research on bacterial activity and pathogenic mechanism.

The purpose of this study was to investigate the relationship between the two-component system phoP/Q and flagella type Ⅲ secretory system (flagellar T3SS) proteins in Avian Pathogenic Escherichia coli (APEC). We used software to analyze the function and to predict the relationship between phoP/Q and flagella T3SS, and used gene chip technology to compare the transcriptome differences between APEC wild strain and ΔphoP/Q deletion mutant, and screening for variable genes in flagellum T3SS pedestal. The changes of gene expression were detected by real-time PCR. The results predicted by STRING showed that phoP/Q may regulate flagellar T3SS indirectly by regulating the two-component system near the flagellar T3SS locus. Gene chip screening and real-time PCR revealed that the phoP/Q deletion could significantly differ the expression of most genes in the core component of flagellar T3SS, and also affect the expression of motA, motB and cheY in the two components. In this study, the relationship between phoP/Q two-component system and flagellar T3SS was predicted and explored, which provided a new breakthrough point and research direction for further research on bacterial activity and pathogenic mechanism.

Enterotoxigenic E. coli (ETEC) is one of the most important cause of neonatal and post-weaning diarrhea (PWD) in piglets. The interaction between ETEC and intestinal epithelial cells is the prerequisite of bacterial pathogenesis, as well as the first step of triggering host immune response. The aim of this study was to determine the expression profile and host defense mechanisms of IL-17 cytokine in porcine intestinal epithelial cells against ETEC infection. Firstly, we determined the expression of IL-17 cytokines and several mucosal defense genes by the application of RT-PCR, ELISA, immunohistochemistry (IHC) assays and Western Blotting in the porcine intestinal epithelial cell line IPEC-J2 infected with a F4⁺ETEC reference strain C83901. Later on, the regulating effect of IL-17 cytokine on the expression of above mucosal defense genes in IPEC-J2 cells was explored. The results showed that C83901 induced the mRNA expression of all IL-17 cytokines (IL-17D was not detected), particularly upregulated the expression of IL-17A and IL-17C both at the transcriptional and protein level. The upregulation of IL-17A and IL-17C in the protein level were confirmed by IHC and ELISA. The existence of IL-17A and/or IL-17C either induced by ETEC infection or supplemented artificially in IPEC-J2 cells can elicit the expression of host defense gene expression of mucin-2, claudin-1, claudin-2 and beta defensins-2. These results indicated that IL-17 induction in intestinal epithelial cells upon ETEC infection are beneficial for the mucosal host defense by promoting the intestinal barrier integrity.

Enterotoxigenic E. coli (ETEC) is one of the most important cause of neonatal and post-weaning diarrhea (PWD) in piglets. The interaction between ETEC and intestinal epithelial cells is the prerequisite of bacterial pathogenesis, as well as the first step of triggering host immune response. The aim of this study was to determine the expression profile and host defense mechanisms of IL-17 cytokine in porcine intestinal epithelial cells against ETEC infection. Firstly, we determined the expression of IL-17 cytokines and several mucosal defense genes by the application of RT-PCR, ELISA, immunohistochemistry (IHC) assays and Western Blotting in the porcine intestinal epithelial cell line IPEC-J2 infected with a F4⁺ETEC reference strain C83901. Later on, the regulating effect of IL-17 cytokine on the expression of above mucosal defense genes in IPEC-J2 cells was explored. The results showed that C83901 induced the mRNA expression of all IL-17 cytokines (IL-17D was not detected), particularly upregulated the expression of IL-17A and IL-17C both at the transcriptional and protein level. The upregulation of IL-17A and IL-17C in the protein level were confirmed by IHC and ELISA. The existence of IL-17A and/or IL-17C either induced by ETEC infection or supplemented artificially in IPEC-J2 cells can elicit the expression of host defense gene expression of mucin-2, claudin-1, claudin-2 and beta defensins-2. These results indicated that IL-17 induction in intestinal epithelial cells upon ETEC infection are beneficial for the mucosal host defense by promoting the intestinal barrier integrity.

In this article, high-throughput transcriptome sequencing (RNA-seq) technology was used to screen potential regulatory target genes of small RNA (sRNA) gcvB in Salmonella. Comparative analysis of mRNA in wild-type strain and knockout small RNA gcvB strain has been used. The number and type of GcvB target genes were comprehensively predicted, the main functions and the biological processes of the selected genes were compared by the GO and KEGG databases for further analysis. Some differentially expressed genes were verified by real-time PCR. The results showed that there were 1 244 differential expression genes have been screened out, including 678 up-regulated genes and 566 down-regulated genes. GO enrichment analysis showed that the differentially expressed genes mainly involved molecular functions such as redox activity, iron binding, locomotor activity, cellular components of bacterial flagella, cellular respiration, cobalamin metabolism, and cobalamin biosynthesis. KEGG database analysis showed that differential expression genes mainly participated in 14 signaling pathways, including bacterial chemotaxis, porphyrin and chlorophyll metabolism, microbial metabolism in different environments, two-component systems and methane metabolism. Ten differentially expressed genes in WT strain and ΔGcvB strain were selected from transcriptome analysis and carried out the further Real-time PCR test, and the results show that the trend of gene expression is consistent in both transcriptome analysis and in Real-time PCR test. The study lays the foundation for further exploration of the interaction between sRNA GcvB and target genes, the regulation of sRNA and the pathogenic mechanism in Salmonella.

In this article, high-throughput transcriptome sequencing (RNA-seq) technology was used to screen potential regulatory target genes of small RNA (sRNA) gcvB in Salmonella. Comparative analysis of mRNA in wild-type strain and knockout small RNA gcvB strain has been used. The number and type of GcvB target genes were comprehensively predicted, the main functions and the biological processes of the selected genes were compared by the GO and KEGG databases for further analysis. Some differentially expressed genes were verified by real-time PCR. The results showed that there were 1 244 differential expression genes have been screened out, including 678 up-regulated genes and 566 down-regulated genes. GO enrichment analysis showed that the differentially expressed genes mainly involved molecular functions such as redox activity, iron binding, locomotor activity, cellular components of bacterial flagella, cellular respiration, cobalamin metabolism, and cobalamin biosynthesis. KEGG database analysis showed that differential expression genes mainly participated in 14 signaling pathways, including bacterial chemotaxis, porphyrin and chlorophyll metabolism, microbial metabolism in different environments, two-component systems and methane metabolism. Ten differentially expressed genes in WT strain and ΔGcvB strain were selected from transcriptome analysis and carried out the further Real-time PCR test, and the results show that the trend of gene expression is consistent in both transcriptome analysis and in Real-time PCR test. The study lays the foundation for further exploration of the interaction between sRNA GcvB and target genes, the regulation of sRNA and the pathogenic mechanism in Salmonella.

The present study aimed to develop a new differential chromogenic medium for fast identification of pathogens associated with mastitis in dairy cows, and check its identification consistency with two traditional approaches including normal isolation and identification media combined with biochemical identifications and 16S rRNA sequencing. Specific enzymes and the only carbon source that could be used to produce acid in bacteria of each genus were screened via bioinformatics tools. Synthesized chromogenic substrate of specific enzyme, carbon and acid-base indicator were mixed into basal enrichment medium to prepare differential chromogenic media. Standard strains and wild-type strains of related pathogens were inoculated on differential chromogenic media, and morphology of colonies and color changes of colony and medium that could be considered as identification standards were observed. Milk samples from mastitic quarters (samples diagnosed as subclinical mastitis by California Mastitis Test or samples of clinical mastitis with presence of flakes, clots, or serous milk; n=482) were cultured aerobically at 37℃ and identified using differential chromogenic media and traditional culture methods. Colonies from differential chromogenic media were purified and DNA was extracted for 16S rRNA sequencing (n=194). The reliability of identification by differential chromogenic media was diagnosed referring to results of two traditional approaches, based on which consistency inspection parameter, namely, simple Cohen's kappa coefficient was calculated via FREQ program in SAS Version 9.4. The colony and color of medium matrix from common bovine mastitis-associated pathogens of different genus grown on differential chromogenic media, regardless of standard strains or wild-type strains, were morphologically distinct and visible to the naked eyes. Consistency inspection parameter of differential chromogenic media referred to normal isolation and identification media combined with biochemical identifications and 16S rRNA sequencing was seperately κ=0.70 and κ=0.96. In conclusion, differential chromogenic media showed great potential in identifying common pathogens of bovine mastitis.

The present study aimed to develop a new differential chromogenic medium for fast identification of pathogens associated with mastitis in dairy cows, and check its identification consistency with two traditional approaches including normal isolation and identification media combined with biochemical identifications and 16S rRNA sequencing. Specific enzymes and the only carbon source that could be used to produce acid in bacteria of each genus were screened via bioinformatics tools. Synthesized chromogenic substrate of specific enzyme, carbon and acid-base indicator were mixed into basal enrichment medium to prepare differential chromogenic media. Standard strains and wild-type strains of related pathogens were inoculated on differential chromogenic media, and morphology of colonies and color changes of colony and medium that could be considered as identification standards were observed. Milk samples from mastitic quarters (samples diagnosed as subclinical mastitis by California Mastitis Test or samples of clinical mastitis with presence of flakes, clots, or serous milk; n=482) were cultured aerobically at 37℃ and identified using differential chromogenic media and traditional culture methods. Colonies from differential chromogenic media were purified and DNA was extracted for 16S rRNA sequencing (n=194). The reliability of identification by differential chromogenic media was diagnosed referring to results of two traditional approaches, based on which consistency inspection parameter, namely, simple Cohen's kappa coefficient was calculated via FREQ program in SAS Version 9.4. The colony and color of medium matrix from common bovine mastitis-associated pathogens of different genus grown on differential chromogenic media, regardless of standard strains or wild-type strains, were morphologically distinct and visible to the naked eyes. Consistency inspection parameter of differential chromogenic media referred to normal isolation and identification media combined with biochemical identifications and 16S rRNA sequencing was seperately κ=0.70 and κ=0.96. In conclusion, differential chromogenic media showed great potential in identifying common pathogens of bovine mastitis.

This research was conducted to establish a nude mice model of canine mammary cancer cell,and study its biological characteristics as well as dynamic changes. The green fluorescent protein (GFP) was introduced into the CHMm and CHMp cells of canine breast cancer cell. The nude mice model was established by implantation of tumor cell suspension in right second breast of the nude mice. The dynamic changes of tumor in nude mice at different time periods were studied by the technique of living fluorescence imaging and pathological examination. Results were as follows:GFP labeled CHMm-GFP and CHMp-GFP cells were observed to emit stable green fluorescence signals under inverted fluorescence microscope, and macroscopic tumor appeared on the 6th day after implantation. The tumor growth curve and weight change curve were drawn by fluorescence signal detection and pathological examination on the 25th day and the 60th day after implantation. On the 25th day after implantation, the fluorescence signal of CHMm-GFP was the strongest in nude mice, while that of CHMp-GFP was strongest on 35th day. On the 25th day after implantation, it was found that one lymph node of each group of nude mice showed fluorescence signal. On the 35th day, fluorescent signals were displayed in lymph nodes of 3 nude mice, but no fluorescence signals were found in other organs. On the 25th day, one nude mouse tumor metastasized to the lymph node, and at the 35th day and the 60th day, the tumor was found to have metastasized to the lymph node. Canine breast tumor models of two cell lines were established in this study. The two cell lines showed different growth characteristics in nude mice at different periods and could be metastasized, which provided a reference for the further study of the occurrence and development of mammary tumors.

This research was conducted to establish a nude mice model of canine mammary cancer cell,and study its biological characteristics as well as dynamic changes. The green fluorescent protein (GFP) was introduced into the CHMm and CHMp cells of canine breast cancer cell. The nude mice model was established by implantation of tumor cell suspension in right second breast of the nude mice. The dynamic changes of tumor in nude mice at different time periods were studied by the technique of living fluorescence imaging and pathological examination. Results were as follows:GFP labeled CHMm-GFP and CHMp-GFP cells were observed to emit stable green fluorescence signals under inverted fluorescence microscope, and macroscopic tumor appeared on the 6th day after implantation. The tumor growth curve and weight change curve were drawn by fluorescence signal detection and pathological examination on the 25th day and the 60th day after implantation. On the 25th day after implantation, the fluorescence signal of CHMm-GFP was the strongest in nude mice, while that of CHMp-GFP was strongest on 35th day. On the 25th day after implantation, it was found that one lymph node of each group of nude mice showed fluorescence signal. On the 35th day, fluorescent signals were displayed in lymph nodes of 3 nude mice, but no fluorescence signals were found in other organs. On the 25th day, one nude mouse tumor metastasized to the lymph node, and at the 35th day and the 60th day, the tumor was found to have metastasized to the lymph node. Canine breast tumor models of two cell lines were established in this study. The two cell lines showed different growth characteristics in nude mice at different periods and could be metastasized, which provided a reference for the further study of the occurrence and development of mammary tumors.

In this study, IPEC-J2 cells (porcine intestinal epithelial cells) were cultured in vitro to study the effect of different concentrations of 7S β-conglycinin on IPEC-J2. The experiment was designed as six groups:A, B, C, D, E, and F, A was the control group, and the other groups were added 1, 5, 10, 5 and 5 mg·mL^-1β-conglycinin, respectively, and 1 μmol·L^-1 NF-κB(PDTC) and iNOS (L-NAME) inhibitors were added to groups E and F, respectively. Cell viability was measured by CCK-8 method, the contents of NO, DAO, 5-HT, IL-6 and IL-10 were detected by ELISA, p-NF-κB, p65, iNOS and COX-2 protein expression levels were detected by Western blot, the relative transcription of NF-κB p65, IKKβ, iNOS, IKKα and COX-2 mRNA were determined by qPCR. The results showed that β-conglycinin reduced the viability of IPEC-J2 cells, promoted the secretion of NO, 5-HT and IL-6, decreased the secretion of IL-10, and increased the expression of p-NF-κB p65, iNOS and COX-2 proteins, transcription of NF-κB p65, IKKβ, iNOS, IKKα and COX-2 mRNA, PDTC and L-NAME can inhibit the effects of β-conglycinin. The results showed that β-conglycinin caused damage to IPEC-J2 and increased the damage with increasing concentration, PDTC and L-NAME can reduce the damage of β-conglycinin to cells.

In this study, IPEC-J2 cells (porcine intestinal epithelial cells) were cultured in vitro to study the effect of different concentrations of 7S β-conglycinin on IPEC-J2. The experiment was designed as six groups:A, B, C, D, E, and F, A was the control group, and the other groups were added 1, 5, 10, 5 and 5 mg·mL^-1β-conglycinin, respectively, and 1 μmol·L^-1 NF-κB(PDTC) and iNOS (L-NAME) inhibitors were added to groups E and F, respectively. Cell viability was measured by CCK-8 method, the contents of NO, DAO, 5-HT, IL-6 and IL-10 were detected by ELISA, p-NF-κB, p65, iNOS and COX-2 protein expression levels were detected by Western blot, the relative transcription of NF-κB p65, IKKβ, iNOS, IKKα and COX-2 mRNA were determined by qPCR. The results showed that β-conglycinin reduced the viability of IPEC-J2 cells, promoted the secretion of NO, 5-HT and IL-6, decreased the secretion of IL-10, and increased the expression of p-NF-κB p65, iNOS and COX-2 proteins, transcription of NF-κB p65, IKKβ, iNOS, IKKα and COX-2 mRNA, PDTC and L-NAME can inhibit the effects of β-conglycinin. The results showed that β-conglycinin caused damage to IPEC-J2 and increased the damage with increasing concentration, PDTC and L-NAME can reduce the damage of β-conglycinin to cells.

The study was conducted to investigate the effects of astragaloside (Ast) on microvascular vasomotion at Hegu acupoint, nitric oxide and endothelin-1 released by rat myocardial microvascular endothelial cells (RMMECs). Laser Doppler flowmetry combined with iontophoresis was used to detect the amplitude of microvascular vasomotion. ELISA method was used to detect the levels of NO and ET-1 released by RMMECs incubated with different doses of Ast for 3, 6, 9 and 12 hours. Real-Time PCR was used to detect the gene transcription of NO and ET-1 in RMMECs treated with 10 μg·mL^-1Ast. The results were as follows:After introduction of Ast, the amplitude of microvascular vasomotion at Hegu acupoint was improved. Compared with control group (RMMECs), the levels of NO and ET-1 were improved, NO/ET-1 value was increased by Ast in Ast incubated RMMECs. When RMMECs were incubated with 10 μg·mL^-1 Ast for 3 hours, the NO/ET-1 value was the highest, and the gene transcription levels of NO and ET-1 were improved, which were consistent with the results of secretion level. When 25 μg·mL^-1 Ast incubated RMMECs for 3 hours, its NO/ET-1 value was slightly lower than that of 10 μg·mL^-1 Ast group, but its ratio could be maintained at a relatively high level. The results showed that Ast could increase the amplitude of microvascular vasomotion, and its mechanism might be through regulating the dynamic balance of NO and ET-1 released by RMMECs in circulatory system.

The study was conducted to investigate the effects of astragaloside (Ast) on microvascular vasomotion at Hegu acupoint, nitric oxide and endothelin-1 released by rat myocardial microvascular endothelial cells (RMMECs). Laser Doppler flowmetry combined with iontophoresis was used to detect the amplitude of microvascular vasomotion. ELISA method was used to detect the levels of NO and ET-1 released by RMMECs incubated with different doses of Ast for 3, 6, 9 and 12 hours. Real-Time PCR was used to detect the gene transcription of NO and ET-1 in RMMECs treated with 10 μg·mL^-1Ast. The results were as follows:After introduction of Ast, the amplitude of microvascular vasomotion at Hegu acupoint was improved. Compared with control group (RMMECs), the levels of NO and ET-1 were improved, NO/ET-1 value was increased by Ast in Ast incubated RMMECs. When RMMECs were incubated with 10 μg·mL^-1 Ast for 3 hours, the NO/ET-1 value was the highest, and the gene transcription levels of NO and ET-1 were improved, which were consistent with the results of secretion level. When 25 μg·mL^-1 Ast incubated RMMECs for 3 hours, its NO/ET-1 value was slightly lower than that of 10 μg·mL^-1 Ast group, but its ratio could be maintained at a relatively high level. The results showed that Ast could increase the amplitude of microvascular vasomotion, and its mechanism might be through regulating the dynamic balance of NO and ET-1 released by RMMECs in circulatory system.

The objective of this study was to investigate the phenotypic and genotypic basis of bovine Shiga toxin-producing Escherichia coli (STEC) from Xinjiang and obtain the evolution and spread of STECs resistance. Susceptibility testing to 18 antimicrobials was performed on bovine (non-O157:H7)STEC isolates from 6 regions of Xinjiang. Meanwhile, the extended spectrum β-lactamases(ESBL) genes were amplified. In this study 4.31% STEC isolates were multidrug resistant to antimicrobials (MDR), and 1.91% were ESBLs-producing strains. The predominant ESBL genes detected were bla_TEM and bla_CTX-M. This is the first report of bla_TEM and bla_CTX-M in STEC isolates in Xinjiang. Most resistant STECs (91.67%) isolated in this study belong to phylogenetic groups A. These findings suggest that MDR STECs are emerging as a result of nonpathogenic E. coli acquiring virulence and resistance genes. This may convey a certain competitive advantage for the colonization of these STECs in cattle when antimicrobial selective pressures are present, potentially leading to an increase in contamination of food with resistant STECs.

The objective of this study was to investigate the phenotypic and genotypic basis of bovine Shiga toxin-producing Escherichia coli (STEC) from Xinjiang and obtain the evolution and spread of STECs resistance. Susceptibility testing to 18 antimicrobials was performed on bovine (non-O157:H7)STEC isolates from 6 regions of Xinjiang. Meanwhile, the extended spectrum β-lactamases(ESBL) genes were amplified. In this study 4.31% STEC isolates were multidrug resistant to antimicrobials (MDR), and 1.91% were ESBLs-producing strains. The predominant ESBL genes detected were bla_TEM and bla_CTX-M. This is the first report of bla_TEM and bla_CTX-M in STEC isolates in Xinjiang. Most resistant STECs (91.67%) isolated in this study belong to phylogenetic groups A. These findings suggest that MDR STECs are emerging as a result of nonpathogenic E. coli acquiring virulence and resistance genes. This may convey a certain competitive advantage for the colonization of these STECs in cattle when antimicrobial selective pressures are present, potentially leading to an increase in contamination of food with resistant STECs.

Nebovirus (NeV) is an emerging causative agent of calf diarrhea in China, the aim of the study was to establish a real-time RT-PCR assay for detecting NeV. The TB Green real-time RT-PCR assay was successfully established through designing primers targeted to RdRp fragment of NeV strains in China and optimizing the reaction conditions and system. The Real-time RT-PCR assay showed a good linear relationship between 2.9×10¹ and 2.9×10⁹ copies·μL^-1, linear correlation coefficient R²=0.999 4, and amplification efficiency was 98%. This method only specifically detected NeV, and did not detect other unrelated pathogens; the minimum detection limit was 29 copies·μL^-1; the intra-and inter-coefficients of variation were 0.89%-1.89% and 0.83%-1.15%, respectively; comparing to other three reported RT-PCR assays, the assay in this study has a significant high detection rate for NeV in clinical samples (P<0.05). In 135 diarrhea samples of dairy cows from Xinjiang and Liaoing regions from August to November 2018, the NeV detection rate was 67.41%, and the farms positive rate was 100% (15/15). The assay for detecting NeV established in this study has a good specificity and stability, which provide an effective means for detection and epidemiological investigation of NeV.

Nebovirus (NeV) is an emerging causative agent of calf diarrhea in China, the aim of the study was to establish a real-time RT-PCR assay for detecting NeV. The TB Green real-time RT-PCR assay was successfully established through designing primers targeted to RdRp fragment of NeV strains in China and optimizing the reaction conditions and system. The Real-time RT-PCR assay showed a good linear relationship between 2.9×10¹ and 2.9×10⁹ copies·μL^-1, linear correlation coefficient R²=0.999 4, and amplification efficiency was 98%. This method only specifically detected NeV, and did not detect other unrelated pathogens; the minimum detection limit was 29 copies·μL^-1; the intra-and inter-coefficients of variation were 0.89%-1.89% and 0.83%-1.15%, respectively; comparing to other three reported RT-PCR assays, the assay in this study has a significant high detection rate for NeV in clinical samples (P<0.05). In 135 diarrhea samples of dairy cows from Xinjiang and Liaoing regions from August to November 2018, the NeV detection rate was 67.41%, and the farms positive rate was 100% (15/15). The assay for detecting NeV established in this study has a good specificity and stability, which provide an effective means for detection and epidemiological investigation of NeV.