Mycoplasma is one of the smallest prokaryotic microorganisms without cell wall, and it relies on the cell membrane to maintain individual morphology and physiological functions. Mycoplasma is a pathogenic microorganism, which could cause variety of diseases for both human and animals. It is seriously affected the development of animal husbandry and threatened human health. Diseases caused by Mycoplasma are very difficult to prevent and control due to lack of the sensitive diagnostic tools, the clinical efficacy of antibiotics and vaccine. For further development, it is urgently necessary to undertake an in-depth study of its unclear pathogenesis. As much attention were paid on Mycoplasma infection and some progress were achieved, this article summarizes the mycoplasma-induced host cell apoptosis, autophagy and carcinogenic in order to contribute to Mycoplasma pathogenesis research.

Mycoplasma is one of the smallest prokaryotic microorganisms without cell wall, and it relies on the cell membrane to maintain individual morphology and physiological functions. Mycoplasma is a pathogenic microorganism, which could cause variety of diseases for both human and animals. It is seriously affected the development of animal husbandry and threatened human health. Diseases caused by Mycoplasma are very difficult to prevent and control due to lack of the sensitive diagnostic tools, the clinical efficacy of antibiotics and vaccine. For further development, it is urgently necessary to undertake an in-depth study of its unclear pathogenesis. As much attention were paid on Mycoplasma infection and some progress were achieved, this article summarizes the mycoplasma-induced host cell apoptosis, autophagy and carcinogenic in order to contribute to Mycoplasma pathogenesis research.

With the Human genome project's(HGP)implementation and propulsion, life science research has entered a post-genome era. In this era, proteomics has become a hot study of life science. Echinococcosis is a significant health burden for humans in developing world and also lead to substantial economic losses in livestock production around the world. Therefore, the prevention, diagnosis and treatment had been a major issue in parasite research. This paper reviews present advances in the proteomics of Echinococcus granulosus (E.granulosus), highlights some means and methods. This will highlight some of the advances that have been made in understanding the host-parasite in E.granulosus infection, provide the base-data for development of diagnostic methods, new medicine and vaccines for E.granulosus.

With the Human genome project's(HGP)implementation and propulsion, life science research has entered a post-genome era. In this era, proteomics has become a hot study of life science. Echinococcosis is a significant health burden for humans in developing world and also lead to substantial economic losses in livestock production around the world. Therefore, the prevention, diagnosis and treatment had been a major issue in parasite research. This paper reviews present advances in the proteomics of Echinococcus granulosus (E.granulosus), highlights some means and methods. This will highlight some of the advances that have been made in understanding the host-parasite in E.granulosus infection, provide the base-data for development of diagnostic methods, new medicine and vaccines for E.granulosus.

The aim of this study were to clone alternative splicing isoforms of pig MYNN gene, predict the structures and functions of their coding proteins, and investigate the temporal-spatial expression characteristics of each transcript. The full-length CDS of MYNN was cloned by RT-PCR and the biological characteristics of MYNN protein was analyzed by bioinformatics in Mashen pig. Quantitative real-time PCR was employed to detect the expression patterns of MYNN gene in heart, liver, spleen, lung, kidney, cerebellum, small intestine, stomach, pancreas, longissimus dorsi and fat tissues of Mashen pig, and to study the developmental expression patterns in stomach and longissimus dorsi tissues. Two transcripts of MYNN gene were successfully cloned in present study, and named MYNN-1 (GenBank accession number:KY470829) and MYNN-2 (GenBank accession number:KY670835), respectively. MYNN-1 CDS was composed of 1 830 bp encoding 609 amino acids, which belonged to the stable alkaline soluble protein. Whereas MYNN-2 CDS was composed of 1 746 bp encoding 581 amino acids, which belonged to the unstable alkaline soluble protein. MYNN-2 was 84 bp less than MYNN-1, and lacked the exon6. MYNN-2 was found a C₂H₂ type zinc finger protein domain less than MYNN-1 by the prediction of function domain. The analysis of homology and phylogenetic tree showed that the two transcripts amino acid sequences of pig MYNN gene had high homology and close genetic distance with polar bear, goat, horse, dog, and so on, which proved that the pig MYNN gene was very conservative during evolutionary process. MYNN-1 and MYNN-2 were universally expressed in all pig tissues detected, and there were significant differences in expression among different tissues (P<0.05). The two variants had higher expression levels in stomach, small intestine and pancreas of Mashen pig, and their expression were the lowest in fat tissue. The expression level of MYNN-1 was significantly or extremely significantly higher than that of MYNN-2 in all tissues except kidney (P<0.05, P<0.01), which testified that MYNN-1 was the main variant in pig. The expression of MYNN-1 and MYNN-2 decreased gradually with the increase of age in the stomach of Mashen pig. In longissimus dorsi, the expression of MYNN-1 and MYNN-2 increased firstly and then decreased with the increase of age. In this study, two transcripts of pig MYNN gene were successfully cloned. And it was speculated that MYNN play important role in the process of digestion and absorption, as well as growth and development of skeletal muscle in pig, whereas the specific mechanisms were still remaining to be further elucidated.

The aim of this study were to clone alternative splicing isoforms of pig MYNN gene, predict the structures and functions of their coding proteins, and investigate the temporal-spatial expression characteristics of each transcript. The full-length CDS of MYNN was cloned by RT-PCR and the biological characteristics of MYNN protein was analyzed by bioinformatics in Mashen pig. Quantitative real-time PCR was employed to detect the expression patterns of MYNN gene in heart, liver, spleen, lung, kidney, cerebellum, small intestine, stomach, pancreas, longissimus dorsi and fat tissues of Mashen pig, and to study the developmental expression patterns in stomach and longissimus dorsi tissues. Two transcripts of MYNN gene were successfully cloned in present study, and named MYNN-1 (GenBank accession number:KY470829) and MYNN-2 (GenBank accession number:KY670835), respectively. MYNN-1 CDS was composed of 1 830 bp encoding 609 amino acids, which belonged to the stable alkaline soluble protein. Whereas MYNN-2 CDS was composed of 1 746 bp encoding 581 amino acids, which belonged to the unstable alkaline soluble protein. MYNN-2 was 84 bp less than MYNN-1, and lacked the exon6. MYNN-2 was found a C₂H₂ type zinc finger protein domain less than MYNN-1 by the prediction of function domain. The analysis of homology and phylogenetic tree showed that the two transcripts amino acid sequences of pig MYNN gene had high homology and close genetic distance with polar bear, goat, horse, dog, and so on, which proved that the pig MYNN gene was very conservative during evolutionary process. MYNN-1 and MYNN-2 were universally expressed in all pig tissues detected, and there were significant differences in expression among different tissues (P<0.05). The two variants had higher expression levels in stomach, small intestine and pancreas of Mashen pig, and their expression were the lowest in fat tissue. The expression level of MYNN-1 was significantly or extremely significantly higher than that of MYNN-2 in all tissues except kidney (P<0.05, P<0.01), which testified that MYNN-1 was the main variant in pig. The expression of MYNN-1 and MYNN-2 decreased gradually with the increase of age in the stomach of Mashen pig. In longissimus dorsi, the expression of MYNN-1 and MYNN-2 increased firstly and then decreased with the increase of age. In this study, two transcripts of pig MYNN gene were successfully cloned. And it was speculated that MYNN play important role in the process of digestion and absorption, as well as growth and development of skeletal muscle in pig, whereas the specific mechanisms were still remaining to be further elucidated.

To establish simple and feasible methods to evaluate the temperament of dairy cow and study the importance of temperament in dairy production, totally 3 988 milking Holstein cows from 7 large-scale farms in Beijing were assessed to derive their temperament scores based on two easy and quick scoring methods of temperament in dairy cows during July and August in 2016. The influence factors of temperament score and the effects of temperament on milk yield and body condition score (BCS) were analyzed using fixed effect model. The results showed that both of the two methods could reveal the variation of the temperament among dairy cows, and there was no significant differences between the ranking of the two scoring methods based on rank correlation test, there was a trivial difference of 0.04 between the mean values of two scoring methods. Farm, parity and lactation stage had significant effects on temperament score (P<0.05). The proportion of bad temperament cows tended to be higher in the first parity, and probability of dairy cows with bad temperament during lactation peak period (45-99 d) was higher than that in other lactation stages, especially when comparing to cows in the stage of later than 300 days in milk. The effects of temperament score on corrected milk yield and BCS were not significant (P>0.05), however cows with different temperament scores differed in their milk production. The two temperament scoring methods for dairy cow proposed in this study are simple and easy to apply, and can be used to assess the temperament of cows in large-scale farms efficiently, which can provide convenient tools to improve the cow welfare and management.

To establish simple and feasible methods to evaluate the temperament of dairy cow and study the importance of temperament in dairy production, totally 3 988 milking Holstein cows from 7 large-scale farms in Beijing were assessed to derive their temperament scores based on two easy and quick scoring methods of temperament in dairy cows during July and August in 2016. The influence factors of temperament score and the effects of temperament on milk yield and body condition score (BCS) were analyzed using fixed effect model. The results showed that both of the two methods could reveal the variation of the temperament among dairy cows, and there was no significant differences between the ranking of the two scoring methods based on rank correlation test, there was a trivial difference of 0.04 between the mean values of two scoring methods. Farm, parity and lactation stage had significant effects on temperament score (P<0.05). The proportion of bad temperament cows tended to be higher in the first parity, and probability of dairy cows with bad temperament during lactation peak period (45-99 d) was higher than that in other lactation stages, especially when comparing to cows in the stage of later than 300 days in milk. The effects of temperament score on corrected milk yield and BCS were not significant (P>0.05), however cows with different temperament scores differed in their milk production. The two temperament scoring methods for dairy cow proposed in this study are simple and easy to apply, and can be used to assess the temperament of cows in large-scale farms efficiently, which can provide convenient tools to improve the cow welfare and management.

This experiment was conducted to investigate the growth rule of the body weight and body size traits for Chinese Simmental beef cattle population in Ulgai of Inner Mongolia, and to construct the regression equation of body weight by body size. We measured the growth traits of 2 162 Simmental cattle including body weight, withers height, hip height, body length, chest circumference and abdominal girth from birth to 20 month old. Using Logistic, Brody, Gompertz and Bertallanffy models, we fitted the growth curves of these traits, and calculated the related parameters in the 4 curve equations, respectively. We also conducted correlation and regression analysis between body measurements and body weight. The results showed that all of the 4 kinds of growth curve fitting models in Chinese Simmental beef cattle had good fitting degree(R² >0.98) for the growth of body measurements and body weight, but for different traits, the optimal fitting model was different. The correlation analysis between body weight and body measurements showed that correlations between body weight and body length, chest circumference, abdominal girth were higher, respectively. The correlation coefficient between body weight and chest circumference was the highest (0.958 55). The extremely significant correlations between body measurements and body weight were observed. The regression equation predicting body weight was Y=-366.485 70+1.307 37 X₃+3.896 12 X₄-0.417 50 X₅. The results indicated that the fitting for body weights of Gompertz and Bertallanffy were better than the other two models, and the Brody model was the best for fitting the body height, body length, chest circumference, abdominal girth and hip height. The regression equation predicting body weight was proved to be of statistical significance, which will provide the data basis for the further breeding of Chinese Simmental beef cattle.

This experiment was conducted to investigate the growth rule of the body weight and body size traits for Chinese Simmental beef cattle population in Ulgai of Inner Mongolia, and to construct the regression equation of body weight by body size. We measured the growth traits of 2 162 Simmental cattle including body weight, withers height, hip height, body length, chest circumference and abdominal girth from birth to 20 month old. Using Logistic, Brody, Gompertz and Bertallanffy models, we fitted the growth curves of these traits, and calculated the related parameters in the 4 curve equations, respectively. We also conducted correlation and regression analysis between body measurements and body weight. The results showed that all of the 4 kinds of growth curve fitting models in Chinese Simmental beef cattle had good fitting degree(R² >0.98) for the growth of body measurements and body weight, but for different traits, the optimal fitting model was different. The correlation analysis between body weight and body measurements showed that correlations between body weight and body length, chest circumference, abdominal girth were higher, respectively. The correlation coefficient between body weight and chest circumference was the highest (0.958 55). The extremely significant correlations between body measurements and body weight were observed. The regression equation predicting body weight was Y=-366.485 70+1.307 37 X₃+3.896 12 X₄-0.417 50 X₅. The results indicated that the fitting for body weights of Gompertz and Bertallanffy were better than the other two models, and the Brody model was the best for fitting the body height, body length, chest circumference, abdominal girth and hip height. The regression equation predicting body weight was proved to be of statistical significance, which will provide the data basis for the further breeding of Chinese Simmental beef cattle.

The purpose of this study was to explore the genetic rules of polydactyly traits and the expression of its candidate genes during embryogenesis in order to provide a basis for the selection and breeding of polydactyly traits lines in Royal chicken. Royal chicken with different toe types (four-four toe, four-five toe, five-five toe) were selected, and hybridization and crossbreeding test were carried out to analyze the proportions of various toe types in offsprings, then it was compared with the theoretical values by Chi square test. The limb bud tissue samples from 5th to 10th day during embryonic development were collected and the expression of the polydactyly traits candidate genes(Lmbr1, SHH, RNF32, NOM1, MNX1 and Gli3) were analyzed. The results showed that the ratio of polydactyly to normal dactyly in four-five toe♂×four-five toe♀ group was 3.67:1, which was in accordance with the theoretical ratio of 3:1 (P>0.05). The other two groups did not fit the theoretical ratio (P<0.05). Secondly, the result of crossbreeding test showed that there were 3 toe types in offsprings from each cross male. Finally, the expression of SHH gene was very low from the 5th to 10th day during the development of limb bud tissue in Royal chicken. Lmbr1, RNF32, NOM1, MNX1, Gli3 gene expression peaks appeared on the 7th and 9th day during the development of limb bud tissue. The polydactyly trait of Royal chicken is in form of autosomal dominant inheritance, but its penetrance is different. The results indicate that the polydactyly traits may be regulated by multiple genes such as Lmbr1 and RNF32, and the critical period may be from the 7th to 9th day during embryo development.

The purpose of this study was to explore the genetic rules of polydactyly traits and the expression of its candidate genes during embryogenesis in order to provide a basis for the selection and breeding of polydactyly traits lines in Royal chicken. Royal chicken with different toe types (four-four toe, four-five toe, five-five toe) were selected, and hybridization and crossbreeding test were carried out to analyze the proportions of various toe types in offsprings, then it was compared with the theoretical values by Chi square test. The limb bud tissue samples from 5th to 10th day during embryonic development were collected and the expression of the polydactyly traits candidate genes(Lmbr1, SHH, RNF32, NOM1, MNX1 and Gli3) were analyzed. The results showed that the ratio of polydactyly to normal dactyly in four-five toe♂×four-five toe♀ group was 3.67:1, which was in accordance with the theoretical ratio of 3:1 (P>0.05). The other two groups did not fit the theoretical ratio (P<0.05). Secondly, the result of crossbreeding test showed that there were 3 toe types in offsprings from each cross male. Finally, the expression of SHH gene was very low from the 5th to 10th day during the development of limb bud tissue in Royal chicken. Lmbr1, RNF32, NOM1, MNX1, Gli3 gene expression peaks appeared on the 7th and 9th day during the development of limb bud tissue. The polydactyly trait of Royal chicken is in form of autosomal dominant inheritance, but its penetrance is different. The results indicate that the polydactyly traits may be regulated by multiple genes such as Lmbr1 and RNF32, and the critical period may be from the 7th to 9th day during embryo development.

The aim of this study was to investigate the expression patterns of fibroblast growth factor (FGF) 7 subfamily (FGF7, FGF10 and FGF22) in the first hair follicle growth cycle of mice. The Real-time PCR, Western blot and immunohistochemistry were adopted to study the expression of FGF7/FGF10/FGF22 mRNA and protein in mice back skin on 1,3,5,8,16,18,20,23 postnatal days. The results of immunohistochemistry showed that FGF7 protein was mainly located in the inner root sheath and epidermal; FGF10 and FGF22 were widely located in various structure of hair follicle. The results of real-time PCR, Western blot as well as immunohistochemistry showed that the FGF7 level was the lowest in catagen(16 d).And relative level of FGF7 was significantly increased in telogen (18 d) and anagen (1-8, 20-23 d), compared to its expression in catagen (16 d) (P<0.05, P<0.01). The FGF10 level was the lowest in the end of anagen (8 d). And the relative level of FGF10 was significantly increased in prophase and metaphase of anagen (1-5, 20-23 d), catagen (16 d) and telogen (18 d) compared to its expression in the end of anagen (8 d) (P<0.05, P<0.01). The FGF22 level was the highest in catagen (16 d). And relative level of FGF22 was significantly decreased in telogen (18 d) and anagen (1-8, 20-23 d) compared to its expression in the catagen (16 d) (P<0.05;P<0.01). These results suggested that FGF7 and FGF10 might play an important role in inducing hair follicle into the new cycle during the development of hair follicle, and FGF22 might have an important effect on the induction of hair follicles into catagen.

The aim of this study was to investigate the expression patterns of fibroblast growth factor (FGF) 7 subfamily (FGF7, FGF10 and FGF22) in the first hair follicle growth cycle of mice. The Real-time PCR, Western blot and immunohistochemistry were adopted to study the expression of FGF7/FGF10/FGF22 mRNA and protein in mice back skin on 1,3,5,8,16,18,20,23 postnatal days. The results of immunohistochemistry showed that FGF7 protein was mainly located in the inner root sheath and epidermal; FGF10 and FGF22 were widely located in various structure of hair follicle. The results of real-time PCR, Western blot as well as immunohistochemistry showed that the FGF7 level was the lowest in catagen(16 d).And relative level of FGF7 was significantly increased in telogen (18 d) and anagen (1-8, 20-23 d), compared to its expression in catagen (16 d) (P<0.05, P<0.01). The FGF10 level was the lowest in the end of anagen (8 d). And the relative level of FGF10 was significantly increased in prophase and metaphase of anagen (1-5, 20-23 d), catagen (16 d) and telogen (18 d) compared to its expression in the end of anagen (8 d) (P<0.05, P<0.01). The FGF22 level was the highest in catagen (16 d). And relative level of FGF22 was significantly decreased in telogen (18 d) and anagen (1-8, 20-23 d) compared to its expression in the catagen (16 d) (P<0.05;P<0.01). These results suggested that FGF7 and FGF10 might play an important role in inducing hair follicle into the new cycle during the development of hair follicle, and FGF22 might have an important effect on the induction of hair follicles into catagen.

To elucidate the association between tissue expression level and polymorphism of FGF 7 and litter size in sheep, semi-quantitative reverse transcription polymerase chain reaction (sqRT-PCR) and quantitative real-time PCR (qPCR) were performed to investigate the expression of FGF 7 gene in multiparous Small Tail Han sheep and uniparous Sunite sheep and different genotypes of FecB gene in Small Tail Han sheep. Multiparous (380 Small Tail Han sheep) and uniparous (total 380 for Tan, Sunite, Suffolk, Dorper and Prairie Tibetan sheep) sheep breeds were selected, and the locus of g.57842893C>T in FGF 7 gene was genotyped using Sequenom MassARRAY^® SNP assay. Then the association was analyzed between FGF 7 gene and litter size in Small Tail Han sheep. The results showed that the expression of FGF 7 was at high level in heart and lung in both uniparous and multiparous sheep breeds and at moderate or low level in other tissues. The expression of FGF 7 was higher in most tissues in multiparous Small Tail Han sheep than in uniparous Sunite sheep, but the difference was not significant (P>0.05), while the expression of FGF 7 had no obvious differences among 3 genotypes of FecB gene in Small Tail Han sheep. Three genotypes (CC, CT and TT) were found at g.57842893C>T locus in both uniparous and multiparous sheep, both genotype frequency and allele frequency reached significant difference between uniparous and multiparous sheep (P ≤ 0.05). Population genetic analysis indicated that the g.57842893C>T locus was at moderate polymorphism (0.25 < PIC < 0.50) in Sunite, Suffolk and Dorper and at low polymorphism (PIC<0.25) in other sheep breeds; χ² test revealed that the g.57842893C>T locus was under Hardy-Weinberg equilibrium in Sunite, Suffolk and Dorper sheep (P>0.05). Association analysis indicated that the g.57842893C>T locus didn't have significant correlation with the litter size of the first, second or third parity in Small Tail Han sheep (P>0.05), while the litter size of individuals with CC was higher than those with TT for each parity. Therefore, we concluded that there might be a certain positive correlation between the expression level of FGF 7and the litter size in sheep, although it may not be the key gene for litter size, the g.57842893C>T locus might provide a basis for litter size trait selection in sheep.

To elucidate the association between tissue expression level and polymorphism of FGF 7 and litter size in sheep, semi-quantitative reverse transcription polymerase chain reaction (sqRT-PCR) and quantitative real-time PCR (qPCR) were performed to investigate the expression of FGF 7 gene in multiparous Small Tail Han sheep and uniparous Sunite sheep and different genotypes of FecB gene in Small Tail Han sheep. Multiparous (380 Small Tail Han sheep) and uniparous (total 380 for Tan, Sunite, Suffolk, Dorper and Prairie Tibetan sheep) sheep breeds were selected, and the locus of g.57842893C>T in FGF 7 gene was genotyped using Sequenom MassARRAY^® SNP assay. Then the association was analyzed between FGF 7 gene and litter size in Small Tail Han sheep. The results showed that the expression of FGF 7 was at high level in heart and lung in both uniparous and multiparous sheep breeds and at moderate or low level in other tissues. The expression of FGF 7 was higher in most tissues in multiparous Small Tail Han sheep than in uniparous Sunite sheep, but the difference was not significant (P>0.05), while the expression of FGF 7 had no obvious differences among 3 genotypes of FecB gene in Small Tail Han sheep. Three genotypes (CC, CT and TT) were found at g.57842893C>T locus in both uniparous and multiparous sheep, both genotype frequency and allele frequency reached significant difference between uniparous and multiparous sheep (P ≤ 0.05). Population genetic analysis indicated that the g.57842893C>T locus was at moderate polymorphism (0.25 < PIC < 0.50) in Sunite, Suffolk and Dorper and at low polymorphism (PIC<0.25) in other sheep breeds; χ² test revealed that the g.57842893C>T locus was under Hardy-Weinberg equilibrium in Sunite, Suffolk and Dorper sheep (P>0.05). Association analysis indicated that the g.57842893C>T locus didn't have significant correlation with the litter size of the first, second or third parity in Small Tail Han sheep (P>0.05), while the litter size of individuals with CC was higher than those with TT for each parity. Therefore, we concluded that there might be a certain positive correlation between the expression level of FGF 7and the litter size in sheep, although it may not be the key gene for litter size, the g.57842893C>T locus might provide a basis for litter size trait selection in sheep.

This research was conducted to clone the Lysine-specific histone demethylase 2B (KDM2B) gene in yak, identify its expression pattern in various tissues and meiosis process of oocytes, respectively, which might provide reliable basis for studying the mechanism of KDM2B in oocyte meiosis of yak. The samples of yak heart, liver, spleen, lung, kidney, brain, small intestine, stomach, muscle, ovary, testicle and uterus were collected after slaughtering. Total RNAs in different samples were extracted.Ovaries from healthy yaks of 3 to 5 years old were selected for collecting the cumulus-oocyte complex (COCs), and then oocytes and cumulus cells were collected by hyaluronidase. The coding sequence of KDM2B gene was cloned by RT-PCR. The mRNA expression of KDM2B in different tissues was determined by quantitative real-time PCR (qRT-PCR). The COCs in 3 stages of GV, MI and MⅡ were cultured in vitro, and the expression of KDM2B in the process of oocyte meiosis was detected by qRT-PCR. The results showed that the CDS region of KDM2B was 3 930 bp, encoding 1 309 amino acids. Compared with the existing predicted yak sequence, it belonged to long-form transcript and had high homology with cattle, sheep and goat, but had lower homology with zabra, fish and chicken. KDM2B gene of yak was widely expressed in various tissues, and its expression was relatively higher in spleen, uterus, testicle and ovary. The expression level of KDM2B in MI stage was significantly higher than that in GV and MⅡ stage in oocytes (P<0.01). In cumulus granulosa cells, the expression level of KDM2B was significantly increased by the process of oocyte meiosis (P<0.01). This research provided basic data for further research of oocyte meiosis mechanism and improving breeding efficiency in yak.

This research was conducted to clone the Lysine-specific histone demethylase 2B (KDM2B) gene in yak, identify its expression pattern in various tissues and meiosis process of oocytes, respectively, which might provide reliable basis for studying the mechanism of KDM2B in oocyte meiosis of yak. The samples of yak heart, liver, spleen, lung, kidney, brain, small intestine, stomach, muscle, ovary, testicle and uterus were collected after slaughtering. Total RNAs in different samples were extracted.Ovaries from healthy yaks of 3 to 5 years old were selected for collecting the cumulus-oocyte complex (COCs), and then oocytes and cumulus cells were collected by hyaluronidase. The coding sequence of KDM2B gene was cloned by RT-PCR. The mRNA expression of KDM2B in different tissues was determined by quantitative real-time PCR (qRT-PCR). The COCs in 3 stages of GV, MI and MⅡ were cultured in vitro, and the expression of KDM2B in the process of oocyte meiosis was detected by qRT-PCR. The results showed that the CDS region of KDM2B was 3 930 bp, encoding 1 309 amino acids. Compared with the existing predicted yak sequence, it belonged to long-form transcript and had high homology with cattle, sheep and goat, but had lower homology with zabra, fish and chicken. KDM2B gene of yak was widely expressed in various tissues, and its expression was relatively higher in spleen, uterus, testicle and ovary. The expression level of KDM2B in MI stage was significantly higher than that in GV and MⅡ stage in oocytes (P<0.01). In cumulus granulosa cells, the expression level of KDM2B was significantly increased by the process of oocyte meiosis (P<0.01). This research provided basic data for further research of oocyte meiosis mechanism and improving breeding efficiency in yak.

This study investigated the multi-directional differentiation potential of mammary stem cells in Holstein cows and the effect of prolactin on its proliferative activity. The Nissl staining was performed to identify the nerve cells induced from the mammary stem cells in Holstein cows, and the marker genes of neuronal cells were analyzed by RT-PCR. The effect of prolactin on the proliferation of mammary stem cells was detected by MTT.The results indicated that the neuron-like cells and microtubule-like structure were found during induction of mammary stem cells, which was positive by Nissl staining. The marker gene (β-Tubulin Ⅲ and NSE) of nerve cells were positive expression after induction. Prolactin could improve the proliferation of mammary stem cells in Holstein cows, and the optimal proliferation concentration of prolactin to mammary stem cells was at 100 ng·mL^-1.

This study investigated the multi-directional differentiation potential of mammary stem cells in Holstein cows and the effect of prolactin on its proliferative activity. The Nissl staining was performed to identify the nerve cells induced from the mammary stem cells in Holstein cows, and the marker genes of neuronal cells were analyzed by RT-PCR. The effect of prolactin on the proliferation of mammary stem cells was detected by MTT.The results indicated that the neuron-like cells and microtubule-like structure were found during induction of mammary stem cells, which was positive by Nissl staining. The marker gene (β-Tubulin Ⅲ and NSE) of nerve cells were positive expression after induction. Prolactin could improve the proliferation of mammary stem cells in Holstein cows, and the optimal proliferation concentration of prolactin to mammary stem cells was at 100 ng·mL^-1.

The objective of this study was to evaluate the effect of glucagon-like peptide-2 (GLP-2) on gastrointestinal weight, rumen fermentation and expression of small intestinal epithelial development-related genes in lambs. In the present study, 10 lambs were randomly assigned into 2 groups:control group (0.5% bovine serum albumin(BSA) in saline, n=5) and GLP-2 group (50 μg·kg^-1 BW GLP-2 in 0.5% bovine serum albumin, n=5). At 28 days-old, lambs were subcutaneously injected with BSA or GLP-2 once every 12 hours in the following 14 days. At 42 days-old, all lambs were slaughtered at 2 h after the first injection. Rumen fluid was collected for the analysis of rumen pH and VFA concentration, and the small intestine epithelium was used for quantitative analysis. The results showed that:1) The body weight and gastrointestinal weight of lambs in GLP-2 group were not significantly affected(P>0.05). GLP-2 treatment had a tendency to increase the weight of jejunum(P=0.083) and ileum(P=0.060). 2) GLP-2 injection significantly increased plasma GLP-2 concentration(P=0.025), while there was no significant change in plasma glucose, rumen pH and VFA concentration (P > 0.05) compared with control group. 3) Compared with the control group, GLP-2 injection significantly increased the mRNA expression of Cyclin B1 (P=0.001), Cyclin D1 (P=0.044), CDK1 (P=0.028), CDK6 (P=0.016) in duodenum epithelium and Cyclin A (P=0.028), Cyclin B1 (P=0.016), CDK1 (P=0.013), CDK4 (P=0.023), CDK6 (P=0.028) in jejunum epithelium, and Cyclin A (P=0.025), Cyclin D1 (P=0.001), CDK2 (P=0.013), CDK4 (P=0.020) in ileum epithelium. 4) The mRNA expression of GCG (P=0.036), IGF-1R (P=0.031) in duodenum epithelium and the mRNA expression of GCG (P=0.049), IGF-1 (P=0.027), IGF-1R (P=0.036) and GLP-2R (P=0.011) in jejunum epithelium, and the mRNA expression of GCG (P=0.025), IGF-1 (P=0.029), IGF-1R (P=0.029) and GLP-2R (P=0.032) in ileum epithelium in GLP-2 group were significantly higher than that in the control group.These results suggest that subcutaneous injection of GLP-2 have no significant effect on body weight, gastrointestinal weight and rumen environment in lambs. However, GLP-2 have a tendency to increase jejunum weight and ileum weight, elevate plasma GLP-2 concentration, significantly affect the expression of small intestinal epithelial development-related genes, and promote the development of the small intestine in lambs. These findings will provide new insight into nutritional strategies for promoting the development of small intestine in lambs.

The objective of this study was to evaluate the effect of glucagon-like peptide-2 (GLP-2) on gastrointestinal weight, rumen fermentation and expression of small intestinal epithelial development-related genes in lambs. In the present study, 10 lambs were randomly assigned into 2 groups:control group (0.5% bovine serum albumin(BSA) in saline, n=5) and GLP-2 group (50 μg·kg^-1 BW GLP-2 in 0.5% bovine serum albumin, n=5). At 28 days-old, lambs were subcutaneously injected with BSA or GLP-2 once every 12 hours in the following 14 days. At 42 days-old, all lambs were slaughtered at 2 h after the first injection. Rumen fluid was collected for the analysis of rumen pH and VFA concentration, and the small intestine epithelium was used for quantitative analysis. The results showed that:1) The body weight and gastrointestinal weight of lambs in GLP-2 group were not significantly affected(P>0.05). GLP-2 treatment had a tendency to increase the weight of jejunum(P=0.083) and ileum(P=0.060). 2) GLP-2 injection significantly increased plasma GLP-2 concentration(P=0.025), while there was no significant change in plasma glucose, rumen pH and VFA concentration (P > 0.05) compared with control group. 3) Compared with the control group, GLP-2 injection significantly increased the mRNA expression of Cyclin B1 (P=0.001), Cyclin D1 (P=0.044), CDK1 (P=0.028), CDK6 (P=0.016) in duodenum epithelium and Cyclin A (P=0.028), Cyclin B1 (P=0.016), CDK1 (P=0.013), CDK4 (P=0.023), CDK6 (P=0.028) in jejunum epithelium, and Cyclin A (P=0.025), Cyclin D1 (P=0.001), CDK2 (P=0.013), CDK4 (P=0.020) in ileum epithelium. 4) The mRNA expression of GCG (P=0.036), IGF-1R (P=0.031) in duodenum epithelium and the mRNA expression of GCG (P=0.049), IGF-1 (P=0.027), IGF-1R (P=0.036) and GLP-2R (P=0.011) in jejunum epithelium, and the mRNA expression of GCG (P=0.025), IGF-1 (P=0.029), IGF-1R (P=0.029) and GLP-2R (P=0.032) in ileum epithelium in GLP-2 group were significantly higher than that in the control group.These results suggest that subcutaneous injection of GLP-2 have no significant effect on body weight, gastrointestinal weight and rumen environment in lambs. However, GLP-2 have a tendency to increase jejunum weight and ileum weight, elevate plasma GLP-2 concentration, significantly affect the expression of small intestinal epithelial development-related genes, and promote the development of the small intestine in lambs. These findings will provide new insight into nutritional strategies for promoting the development of small intestine in lambs.

This experiment aimed to study the effect of FOS on metabolism of phenylalanine(Phe) and tryptophan (Trp) in pig hindgut bacteria fermentation broths in vitro. Ileum, cecum and colon chyme in pigs were used as inoculum. Phe and Trp concentrations were kept at 10 mmol·L^-1 in fermentation broth. The dose of FOS was 0, 0.5 and 0.75 g, respectively. Fermentation samples collected at 24 h and 37℃ were used to measure concentrations of amino aicds, ammonia nitrogen (NH₃-N), microbial crude protein (MCP), indole and skatole. Real-time PCR was used to quantify the bacterial numbers. The results showed that the fermentation characteristic of Phe and Trp differed in different gut locations. The degradation rate of Phe was significantly affected by FOS(P<0.05). The concentrations of NH₃-N, MCP, skatole and number of total bacterial were significantly affected by FOS (P<0.05), while indole was not affected by FOS (P>0.05). In 0.75 g FOS group, Trp degradation rate decreased significantly in the hindgut intestine (P<0.05). Trp was the major factor affecting indole and skatole production in fermentation broths. Collectively, FOS could alter the metabolic patterns of phenylalanine and tryptophan by gut bacteria, and decreased the conversion of tryptophan to skatole.

This experiment aimed to study the effect of FOS on metabolism of phenylalanine(Phe) and tryptophan (Trp) in pig hindgut bacteria fermentation broths in vitro. Ileum, cecum and colon chyme in pigs were used as inoculum. Phe and Trp concentrations were kept at 10 mmol·L^-1 in fermentation broth. The dose of FOS was 0, 0.5 and 0.75 g, respectively. Fermentation samples collected at 24 h and 37℃ were used to measure concentrations of amino aicds, ammonia nitrogen (NH₃-N), microbial crude protein (MCP), indole and skatole. Real-time PCR was used to quantify the bacterial numbers. The results showed that the fermentation characteristic of Phe and Trp differed in different gut locations. The degradation rate of Phe was significantly affected by FOS(P<0.05). The concentrations of NH₃-N, MCP, skatole and number of total bacterial were significantly affected by FOS (P<0.05), while indole was not affected by FOS (P>0.05). In 0.75 g FOS group, Trp degradation rate decreased significantly in the hindgut intestine (P<0.05). Trp was the major factor affecting indole and skatole production in fermentation broths. Collectively, FOS could alter the metabolic patterns of phenylalanine and tryptophan by gut bacteria, and decreased the conversion of tryptophan to skatole.

The objective of this study was to investigate the effects of different dietary fiber levels on cecum microflora diversity of Carlos geese. Ninety healthy Carlos male geese with 35 days old were divided into 2 groups (n=45) with 3 duplicates for 15 per duplicate, and then fed with diets containing 5% (L group) or 8%(H group) crude fiber using alfalfa as the crude fiber sources, respectively. After feeding 42 days, caecum microflora was determined using MiSeq PE300 sequencing platform. The results showed that caecum microflora of goose in H group included 17 phylums and 199 genera, while caecum microflora of goose in L group included 19 phylums and 226 genera. The bacteria diversity of each sample between the two groups was similar (P>0.05). The relative abundance of Verrucomicrobia, Deferribacteres, Desulfovibrio, Prevotella, Helicobacter, Mucispirillum and Akkermansia of H group significantly increased compared to L group (P<0.05); The relative abundance of Synergistetes, Euryarchaeota, Bacteroides and Methanobrevibacter of L group significantly increased compared to H group (P<0.05). These results suggest that there is no significant difference in the bacteria diversity of cecum of goose fed with 5% or 8% crude fiber, however, the relative abundance of Desulfovibrio, Prevotella, Helicobacter, Mucispirillum, Akkermansia, Bacteroides, Methanobrevibacter are significantly different between the two groups.

The objective of this study was to investigate the effects of different dietary fiber levels on cecum microflora diversity of Carlos geese. Ninety healthy Carlos male geese with 35 days old were divided into 2 groups (n=45) with 3 duplicates for 15 per duplicate, and then fed with diets containing 5% (L group) or 8%(H group) crude fiber using alfalfa as the crude fiber sources, respectively. After feeding 42 days, caecum microflora was determined using MiSeq PE300 sequencing platform. The results showed that caecum microflora of goose in H group included 17 phylums and 199 genera, while caecum microflora of goose in L group included 19 phylums and 226 genera. The bacteria diversity of each sample between the two groups was similar (P>0.05). The relative abundance of Verrucomicrobia, Deferribacteres, Desulfovibrio, Prevotella, Helicobacter, Mucispirillum and Akkermansia of H group significantly increased compared to L group (P<0.05); The relative abundance of Synergistetes, Euryarchaeota, Bacteroides and Methanobrevibacter of L group significantly increased compared to H group (P<0.05). These results suggest that there is no significant difference in the bacteria diversity of cecum of goose fed with 5% or 8% crude fiber, however, the relative abundance of Desulfovibrio, Prevotella, Helicobacter, Mucispirillum, Akkermansia, Bacteroides, Methanobrevibacter are significantly different between the two groups.

This experiment was conducted to study the characterization and diagnostic value of cystatin of Echinococcus granulosus and provide basis for prevention and control of Echinococcus granulosus. We cloned and expressed Eg-cystatin, and presented a bioinformatic characterization, Western blotting. Immunofluorescence localization was performed to determine the distribution of cystatin from E. granulosus, and explore its potential for diagnosis of cystic echinococcosis (CE) in sheep based on indirect ELISA. The results showed that Eg-cystatin is belong to a typical type Ⅱ cystatin with an N-terminal signal peptide and the cystatin-like domain (G, Q-X-V-X-G, P-W). Phylogenetic analysis indicated that Eg-cystatin belongs to the cestode cystatin clade, and shares 72.20%-80.66% identity with cystatins from other cestodes. Native Eg-cystatin was located on the tegument and hooks of protoscoleces (PSCs), the whole germinal layer, and the inner body and eggs of adult worms. Indirect ELISA exhibited low specificity (79.1%) and sensitivity (83.3%), and the diagnostic accordance rate was 81.25% compared with the results of necropsy. Eg-cystatin was widely distributed in the larva and adult worm of E. granulosus, suggesting that it is closely related to the life activity of this parasite. However, rEg-cystatin is not a suitable antigen for the detection of sheep CE.

This experiment was conducted to study the characterization and diagnostic value of cystatin of Echinococcus granulosus and provide basis for prevention and control of Echinococcus granulosus. We cloned and expressed Eg-cystatin, and presented a bioinformatic characterization, Western blotting. Immunofluorescence localization was performed to determine the distribution of cystatin from E. granulosus, and explore its potential for diagnosis of cystic echinococcosis (CE) in sheep based on indirect ELISA. The results showed that Eg-cystatin is belong to a typical type Ⅱ cystatin with an N-terminal signal peptide and the cystatin-like domain (G, Q-X-V-X-G, P-W). Phylogenetic analysis indicated that Eg-cystatin belongs to the cestode cystatin clade, and shares 72.20%-80.66% identity with cystatins from other cestodes. Native Eg-cystatin was located on the tegument and hooks of protoscoleces (PSCs), the whole germinal layer, and the inner body and eggs of adult worms. Indirect ELISA exhibited low specificity (79.1%) and sensitivity (83.3%), and the diagnostic accordance rate was 81.25% compared with the results of necropsy. Eg-cystatin was widely distributed in the larva and adult worm of E. granulosus, suggesting that it is closely related to the life activity of this parasite. However, rEg-cystatin is not a suitable antigen for the detection of sheep CE.

To study the biological character of Lipase from Haemonchus contortus, the lipase gene of H. contortus preserved in our laboratory was cloned by PCR, and then expressed in procaryotic expression system. The expression vector was constructed to obtain the fusion protein. The antigenicity of the protein was analyzed by Western blot. The Lipase activity of the fusion protein was detected by esterase reaction. The location of the protein was tested by the indirect immunofluorescence. The transcription of lipase in different stages of H. contortus was detected by real-time quantification PCR. The results showed that the gene fragment encoding Lipase (without signal peptide region) sized as 864 bp. The recombinant protein weighted as 50 kD was fusion expressed. The fusion protein had highest lipase enzyme activity under 37℃ and pH7. Hc-Lipase was localized at outer and inter membrane as well as the intestinal tracts of adult worms.The expression of Hc-Lipase was higher in the male than other stages. It is indicated that Lipase from H. contortus could play important roles in worm development and modulation of the host immunity. Therefore, it is expected that Hc-Lipase could be a new target in prevention and therapy of Haemonchosis.

To study the biological character of Lipase from Haemonchus contortus, the lipase gene of H. contortus preserved in our laboratory was cloned by PCR, and then expressed in procaryotic expression system. The expression vector was constructed to obtain the fusion protein. The antigenicity of the protein was analyzed by Western blot. The Lipase activity of the fusion protein was detected by esterase reaction. The location of the protein was tested by the indirect immunofluorescence. The transcription of lipase in different stages of H. contortus was detected by real-time quantification PCR. The results showed that the gene fragment encoding Lipase (without signal peptide region) sized as 864 bp. The recombinant protein weighted as 50 kD was fusion expressed. The fusion protein had highest lipase enzyme activity under 37℃ and pH7. Hc-Lipase was localized at outer and inter membrane as well as the intestinal tracts of adult worms.The expression of Hc-Lipase was higher in the male than other stages. It is indicated that Lipase from H. contortus could play important roles in worm development and modulation of the host immunity. Therefore, it is expected that Hc-Lipase could be a new target in prevention and therapy of Haemonchosis.

The aim of this study was to investigate the immune efficacy of the transferrin-binding protein A of HPS and to lay a foundation for research and development of new vaccine. The piglets were immunized with the inactivated whole cell vaccine from HPS serotype 13 and recombinant protein TbpA(0.5 and 0.25 mg·4 mL^-1). The immunization was conducted at week 0, 2, 4 and then challenged with HPSLJ3 (7 LD₅₀). Serum antibody (IgG) and cytokines (IL-5, IL-10, IFN-γ, TNF-α) levels were measured by ELISA and pathological examination was performed. Results were as follows:Immunization and challenge assay demonstrated the recombinant protein TbpA could induce high levels of antibody (IgG) and cytokines. The protective efficacy of the inactivated whole cell and TbpA (0.5 mg·4mL^-1) vaccines was 100%. There was a significant difference between the pathological changes and the control group. TbpA can stimulate the humoral and cellular immunity, and produce powerful immune protection. These results suggested that TbpA is a potential candidate for developing a novel HPS vaccine.

The aim of this study was to investigate the immune efficacy of the transferrin-binding protein A of HPS and to lay a foundation for research and development of new vaccine. The piglets were immunized with the inactivated whole cell vaccine from HPS serotype 13 and recombinant protein TbpA(0.5 and 0.25 mg·4 mL^-1). The immunization was conducted at week 0, 2, 4 and then challenged with HPSLJ3 (7 LD₅₀). Serum antibody (IgG) and cytokines (IL-5, IL-10, IFN-γ, TNF-α) levels were measured by ELISA and pathological examination was performed. Results were as follows:Immunization and challenge assay demonstrated the recombinant protein TbpA could induce high levels of antibody (IgG) and cytokines. The protective efficacy of the inactivated whole cell and TbpA (0.5 mg·4mL^-1) vaccines was 100%. There was a significant difference between the pathological changes and the control group. TbpA can stimulate the humoral and cellular immunity, and produce powerful immune protection. These results suggested that TbpA is a potential candidate for developing a novel HPS vaccine.

To understand the epidemiology of the pigs diarrhea virus in Hebei province, a total of 1 855 clinical samples were collected from April, 2016 to February, 2017 and subjected to detection of porcine epidemic diarrhea virus (PEDV), transmissible gastroenteritis virus (TGEV) and porcine rotavirus (PoRV) by real-time PCR. Spike (S) genes of nine amplified PEDV were sequenced and analyzed. The results showed that positive rate of diarrhea was 9.70% and highest in winter and spring seasons. The positive rates of PEDV of total diarrhea samples, piglets diarrhea samples and fattening pigs diarrhea samples were 13.89%, 19.28% and 17.24%, respectively. The phylogenetic analysis of PEDV S genes amino acid sequence showed that the detected 9 S genes from Hebei were distributed in two branches of group Ⅱ. The detail analysis showed several unique amino acid mutations including deletions and insertions in the 9 S genes. These results indicate PEDV infection has fell on piglets but rise on fattening pigs, and the genetic diversity and mutations of S genes were potentially cause alternations of pathogenesis and antigenicity of pandemic PEDV in Hebei province.

To understand the epidemiology of the pigs diarrhea virus in Hebei province, a total of 1 855 clinical samples were collected from April, 2016 to February, 2017 and subjected to detection of porcine epidemic diarrhea virus (PEDV), transmissible gastroenteritis virus (TGEV) and porcine rotavirus (PoRV) by real-time PCR. Spike (S) genes of nine amplified PEDV were sequenced and analyzed. The results showed that positive rate of diarrhea was 9.70% and highest in winter and spring seasons. The positive rates of PEDV of total diarrhea samples, piglets diarrhea samples and fattening pigs diarrhea samples were 13.89%, 19.28% and 17.24%, respectively. The phylogenetic analysis of PEDV S genes amino acid sequence showed that the detected 9 S genes from Hebei were distributed in two branches of group Ⅱ. The detail analysis showed several unique amino acid mutations including deletions and insertions in the 9 S genes. These results indicate PEDV infection has fell on piglets but rise on fattening pigs, and the genetic diversity and mutations of S genes were potentially cause alternations of pathogenesis and antigenicity of pandemic PEDV in Hebei province.

This study was conducted to investigate the infection status of Bovine viral diarrhea virus (BVDV) and the main epidemic subtypes and genetic variation of BVDV in the Sichuan-Tibet Plateau region. A total of 149 clinical diarrhea yak fecal samples were collected from Sichuan province and Tibet region in 2016, and the genetic variation of BVDV in yak was studied according to the variation of 5'-UTR, Npro and E2. Meanwhile, the BVDV strain of yak was isolated and identified by RT-PCR, cell isolation and indirect immunofluorescence. The results showed that the positive rate of BVDV was 19.46% (95% confidence interval (CI)=13.4% -26.7%) in 149 samples of clinical diarrhea yak feces. The positive rate of BVDV in yak in Tibet region was 27.14% (95% CI=17.2% -39.1%) and 12.66% (95% CI=6.2%-22.0%) in Sichuan province. The phylogenetic tree was established based on the sequences of 5'-UTR, Npro and E2. The results showed that 10 samples belongs to BVDV-1, BVDV-1a subtype (n=4) and 1d (n=6). In addition, two strains of cytopathic (cp) BVDV were identified and identified as subgroups of BVDV-1a and 1d, named SWU-Z10 and SWU-L8, respectively. The results showed that BVDV infection is present in yak in the Sichuan-Tibet Plateau region, and BVDV-1a and 1d are the dominant subtype of yak infection in Sichuan-Tibet Plateau region. The results of the study provide basic data for the comprehensive prevention and control of yak diarrhea in Sichuan-Tibet Plateau region. The data obtained enrich the molecular epidemiological investigation data of BVDV, and provide the theoretical basis for the development of BVDV vaccine for yak.

This study was conducted to investigate the infection status of Bovine viral diarrhea virus (BVDV) and the main epidemic subtypes and genetic variation of BVDV in the Sichuan-Tibet Plateau region. A total of 149 clinical diarrhea yak fecal samples were collected from Sichuan province and Tibet region in 2016, and the genetic variation of BVDV in yak was studied according to the variation of 5'-UTR, Npro and E2. Meanwhile, the BVDV strain of yak was isolated and identified by RT-PCR, cell isolation and indirect immunofluorescence. The results showed that the positive rate of BVDV was 19.46% (95% confidence interval (CI)=13.4% -26.7%) in 149 samples of clinical diarrhea yak feces. The positive rate of BVDV in yak in Tibet region was 27.14% (95% CI=17.2% -39.1%) and 12.66% (95% CI=6.2%-22.0%) in Sichuan province. The phylogenetic tree was established based on the sequences of 5'-UTR, Npro and E2. The results showed that 10 samples belongs to BVDV-1, BVDV-1a subtype (n=4) and 1d (n=6). In addition, two strains of cytopathic (cp) BVDV were identified and identified as subgroups of BVDV-1a and 1d, named SWU-Z10 and SWU-L8, respectively. The results showed that BVDV infection is present in yak in the Sichuan-Tibet Plateau region, and BVDV-1a and 1d are the dominant subtype of yak infection in Sichuan-Tibet Plateau region. The results of the study provide basic data for the comprehensive prevention and control of yak diarrhea in Sichuan-Tibet Plateau region. The data obtained enrich the molecular epidemiological investigation data of BVDV, and provide the theoretical basis for the development of BVDV vaccine for yak.

A colloidal gold test strip for rapid detection of RHDV antibody was developed based on colloidal gold immunochromatographic assay. The colloidal gold particles were labeled with recombinant RHDV VP60 protein. The anti-VP60 monoclonal antibody A3C and Staphylococcus aureus protein A (SPA) were respectively coated on the control line (C line) and test line (T line). The strip was optimized by detecting strong positive, positive, weak positive and negative serum, and its qualities and coincidence rate of HI test were verified. The results indicated that the T and C lines were the same color when the positive serum was detected. The T line was darker than the C line when the strong positive serum was detected. The T line was lighter than the C line when the weak positive serum was detected. Compared with HI test, the coincidence rates of strong positive, positive, weak positive, negative serum were 93.75%, 88.75%, 91.25%, 95.00%, respectively, while the total coincidence rate was 91.54%. The colloidal gold strip has specificity, sensitivity, repeatability and stability, and provide a tool for epidemiological survey and antibody level monitoring.

A colloidal gold test strip for rapid detection of RHDV antibody was developed based on colloidal gold immunochromatographic assay. The colloidal gold particles were labeled with recombinant RHDV VP60 protein. The anti-VP60 monoclonal antibody A3C and Staphylococcus aureus protein A (SPA) were respectively coated on the control line (C line) and test line (T line). The strip was optimized by detecting strong positive, positive, weak positive and negative serum, and its qualities and coincidence rate of HI test were verified. The results indicated that the T and C lines were the same color when the positive serum was detected. The T line was darker than the C line when the strong positive serum was detected. The T line was lighter than the C line when the weak positive serum was detected. Compared with HI test, the coincidence rates of strong positive, positive, weak positive, negative serum were 93.75%, 88.75%, 91.25%, 95.00%, respectively, while the total coincidence rate was 91.54%. The colloidal gold strip has specificity, sensitivity, repeatability and stability, and provide a tool for epidemiological survey and antibody level monitoring.

The study aimed to investigate the effects of Chinese medicine prescriptions on production performance, physiological and biochemical indexes of beef cattle under heat stress. Eighteen healthy uncastrated beef cattle with 22 month old and initial body weight of (433.41±15.10) kg were randomly allotted into 3 groups.One group was the control group and the other two groups were the treatment groups. There were 6 cattle in each group. The experiment was designed by single factor random arrangement. The cattle in the control group were fed the basal diet. And the cattle in the two treatment groups were fed the basal diets with Chinese medicine prescription Ⅰ (strengthening the spleen group, SS) and prescription Ⅱ (clearing heat group, CH) supplied with 100 g per day per cattle, respectively. All cattle were fed under high temperature and humidity in summer of south in China. The duration of preliminary experiment was 10 days and the duration of the formal experiment was 60 days. Feed intake was recorded everyday, the body temperature and respiratory rate were determined at 1, 20, 40 and 60 d, and serum samples were collected for detecting biochemical indexes at the end of the experiment. The results showed that:1) The body temperature of individuals in SS group significantly decreased on day 40 and 60 (P<0.05), and body temperature of individuals in CH group significantly decreased on day 20 and 60 (P<0.05) compared with control group. The respiratory rate of individuals in SS and CH groups significantly reduced on day 20 and 40 compared with control group (P<0.05). 2) On day 60, the content of serum total protein, albumin, triglyceride of individuals in SS group significantly increased, but their total cholesterol levels significantly decreased compared with control group (P<0.05). Moreover, serum triglyceride levels of individuals in the CH group were lower but their total cholesterol levels were higher than those in the SS group (P<0.05). 3) Compared with control group, the levels of serum insulin and adiponectin of individuals in the SS group significantly increased (P<0.05), but serum insulin and growth hormone levels of individuals in the CH group significantly decreased (P<0.05), and the thyroid hormone (T3 and T4) levels of individuals in CH group significantly increased(P<0.05). The levels of serum insulin, growth hormone and adiponectin of individuals in the CH group were lower but the thyroid hormone (T3 and T4) were higher than those in the SS group (P<0.05). 4) The average daily feed intake (ADFI) and the average daily gain (ADG) of beef cattle in the SS group significantly decreased (P<0.05), but the F:G ratio significantly increased compared with control group (P<0.05). The present results suggested that dietary supplemented with two Chinese medicine prescriptions could alleviate the heat stress response of beef cattle, and dietary supplemented with the SS promoted the synthesis and metabolism of fat, but had a negative impact on the beef cattle production performance.

The study aimed to investigate the effects of Chinese medicine prescriptions on production performance, physiological and biochemical indexes of beef cattle under heat stress. Eighteen healthy uncastrated beef cattle with 22 month old and initial body weight of (433.41±15.10) kg were randomly allotted into 3 groups.One group was the control group and the other two groups were the treatment groups. There were 6 cattle in each group. The experiment was designed by single factor random arrangement. The cattle in the control group were fed the basal diet. And the cattle in the two treatment groups were fed the basal diets with Chinese medicine prescription Ⅰ (strengthening the spleen group, SS) and prescription Ⅱ (clearing heat group, CH) supplied with 100 g per day per cattle, respectively. All cattle were fed under high temperature and humidity in summer of south in China. The duration of preliminary experiment was 10 days and the duration of the formal experiment was 60 days. Feed intake was recorded everyday, the body temperature and respiratory rate were determined at 1, 20, 40 and 60 d, and serum samples were collected for detecting biochemical indexes at the end of the experiment. The results showed that:1) The body temperature of individuals in SS group significantly decreased on day 40 and 60 (P<0.05), and body temperature of individuals in CH group significantly decreased on day 20 and 60 (P<0.05) compared with control group. The respiratory rate of individuals in SS and CH groups significantly reduced on day 20 and 40 compared with control group (P<0.05). 2) On day 60, the content of serum total protein, albumin, triglyceride of individuals in SS group significantly increased, but their total cholesterol levels significantly decreased compared with control group (P<0.05). Moreover, serum triglyceride levels of individuals in the CH group were lower but their total cholesterol levels were higher than those in the SS group (P<0.05). 3) Compared with control group, the levels of serum insulin and adiponectin of individuals in the SS group significantly increased (P<0.05), but serum insulin and growth hormone levels of individuals in the CH group significantly decreased (P<0.05), and the thyroid hormone (T3 and T4) levels of individuals in CH group significantly increased(P<0.05). The levels of serum insulin, growth hormone and adiponectin of individuals in the CH group were lower but the thyroid hormone (T3 and T4) were higher than those in the SS group (P<0.05). 4) The average daily feed intake (ADFI) and the average daily gain (ADG) of beef cattle in the SS group significantly decreased (P<0.05), but the F:G ratio significantly increased compared with control group (P<0.05). The present results suggested that dietary supplemented with two Chinese medicine prescriptions could alleviate the heat stress response of beef cattle, and dietary supplemented with the SS promoted the synthesis and metabolism of fat, but had a negative impact on the beef cattle production performance.

The aim of this study was to evaluate the inhibitory effect of Andrographolide on the Streptococcus suis. The dilution method was conducted to determine the minimum inhibitory biofilm formation of S. suis and to provide an alternative therapy for the drug resistance of concentration (MIC) and minimum bactericidal concentration (MBC) of Andrographolide on S. suis; The effects of Andrographolide on the biofilm formation of S. suis were detected by crystal violet staining (CV) and scanning electron microscopy (SEM). Result were as follows:1)The MIC and MBC of Andrographolide on S. suis were 0.25 and 0.5 mg·mL^-1, respectively. 2)Scanning electron microscopy showed that the bacteria were embedded in the biofilm in the blank control group. Andrographolide(1/2MIC, 1/4MIC, 1/8MIC)can make the number of bacteria in the bacterial biofilm and the morphology of the biofilm changed greatly. According to the dose of the trend changed, bacterial biofilm gradually reduced. The results showed that Andrographolide had inhibitory effect on the formation of S. suis biofilm in a dose-dependent manner. 3)Through quantitatively detected the expression of luxS gene and virulence gene in S. suis by RT-PCR, we can see the sub-MICs (1/2MIC, 1/4MIC) of Andrographolide can respectively reduce the expression of gapdh, sly, fbps, ef and luxS genes and significantly increase the expression of cps2J, mrp, and gdh genes. 4)Detected the bacterial culture medium by Bioluminescence of Vibrio harveyi showed that Andrographolide (1/2MIC, 1/4MIC, 1/8MIC) had inhibitory effect on the production of AI-2 signal molecule in S. suis, in a dose-dependent manner. Intervention of Andrographolide can inhibit the formation of biofilm in S. suis, and can cause the difference of virulence gene expression levels.

The aim of this study was to evaluate the inhibitory effect of Andrographolide on the Streptococcus suis. The dilution method was conducted to determine the minimum inhibitory biofilm formation of S. suis and to provide an alternative therapy for the drug resistance of concentration (MIC) and minimum bactericidal concentration (MBC) of Andrographolide on S. suis; The effects of Andrographolide on the biofilm formation of S. suis were detected by crystal violet staining (CV) and scanning electron microscopy (SEM). Result were as follows:1)The MIC and MBC of Andrographolide on S. suis were 0.25 and 0.5 mg·mL^-1, respectively. 2)Scanning electron microscopy showed that the bacteria were embedded in the biofilm in the blank control group. Andrographolide(1/2MIC, 1/4MIC, 1/8MIC)can make the number of bacteria in the bacterial biofilm and the morphology of the biofilm changed greatly. According to the dose of the trend changed, bacterial biofilm gradually reduced. The results showed that Andrographolide had inhibitory effect on the formation of S. suis biofilm in a dose-dependent manner. 3)Through quantitatively detected the expression of luxS gene and virulence gene in S. suis by RT-PCR, we can see the sub-MICs (1/2MIC, 1/4MIC) of Andrographolide can respectively reduce the expression of gapdh, sly, fbps, ef and luxS genes and significantly increase the expression of cps2J, mrp, and gdh genes. 4)Detected the bacterial culture medium by Bioluminescence of Vibrio harveyi showed that Andrographolide (1/2MIC, 1/4MIC, 1/8MIC) had inhibitory effect on the production of AI-2 signal molecule in S. suis, in a dose-dependent manner. Intervention of Andrographolide can inhibit the formation of biofilm in S. suis, and can cause the difference of virulence gene expression levels.

Porcine reproductive and respiratory syndrome virus (PRRSV) remains the most economically important pathogen impacting swine industry worldwide. Recently, the novel NADC30-like strains of PRRSV resulting in the re-emerging of clinically PRRS outbreaks has been characterized in China. In order to provide a basis for research on the mechanisms in relation to the genetic variation and evolution of PRRSV NADC30-like strain, CHsx1401, as well as the differential pathogenesis between this virus and other PRRSV strains, a full-length cDNA clone of NADC30-like CHsx1401 was generated. Four fragments of the whole genome of CHsx1401 were amplified by RT-PCR, and each was cloned into pJET1.2 blunt, and each clone was then ligated into the modified low-copy-number vector pWSK29 to generate the full-length cDNA clone, plasmid pWSK-CHsx1401. To distinguish the rescued virus from its parental virus, a restriction enzyme site AscⅠ was introduced between fragment C and D through synonymous mutation (T11582G) as a genetic marker. Subsequently, the full-length cDNA clone was transfected into MARC-145 cells using Lipofectamine LTX and plus reagents to rescue the virus. As the results showed, the full-length cDNA clone (plasmid pWSK-CHsx1401) was constructed successfully, cytopathic effect (CPE) typical of PRRSV in MARC-145 cells could be observed at the second passage. The virus could be rescued from the plasmid pWSK-CHsx1401 by IFA, RT-PCR and sequencing. The rescued virus could be passaged stably in MARC-145 cells, and showed a similar growth curve to its parental isolate in vitro, with slightly lower titers at some time points than CHsx1401 in MARC-145 cells. Our results indicate that the full-length cDNA clone of the PRRSV NADC30-like CHsx1401 is infectious and viable virus can be rescued. The rescued virus (RvCHsx1401) exhibited similar growth ability to its parental virus in vitro. Our present study provides a platform for further research on the molecular mechanisms in relation to the variation and evolution of PRRSV NADC30-like and its differential pathogenesis from other strains of PRRSV.

Porcine reproductive and respiratory syndrome virus (PRRSV) remains the most economically important pathogen impacting swine industry worldwide. Recently, the novel NADC30-like strains of PRRSV resulting in the re-emerging of clinically PRRS outbreaks has been characterized in China. In order to provide a basis for research on the mechanisms in relation to the genetic variation and evolution of PRRSV NADC30-like strain, CHsx1401, as well as the differential pathogenesis between this virus and other PRRSV strains, a full-length cDNA clone of NADC30-like CHsx1401 was generated. Four fragments of the whole genome of CHsx1401 were amplified by RT-PCR, and each was cloned into pJET1.2 blunt, and each clone was then ligated into the modified low-copy-number vector pWSK29 to generate the full-length cDNA clone, plasmid pWSK-CHsx1401. To distinguish the rescued virus from its parental virus, a restriction enzyme site AscⅠ was introduced between fragment C and D through synonymous mutation (T11582G) as a genetic marker. Subsequently, the full-length cDNA clone was transfected into MARC-145 cells using Lipofectamine LTX and plus reagents to rescue the virus. As the results showed, the full-length cDNA clone (plasmid pWSK-CHsx1401) was constructed successfully, cytopathic effect (CPE) typical of PRRSV in MARC-145 cells could be observed at the second passage. The virus could be rescued from the plasmid pWSK-CHsx1401 by IFA, RT-PCR and sequencing. The rescued virus could be passaged stably in MARC-145 cells, and showed a similar growth curve to its parental isolate in vitro, with slightly lower titers at some time points than CHsx1401 in MARC-145 cells. Our results indicate that the full-length cDNA clone of the PRRSV NADC30-like CHsx1401 is infectious and viable virus can be rescued. The rescued virus (RvCHsx1401) exhibited similar growth ability to its parental virus in vitro. Our present study provides a platform for further research on the molecular mechanisms in relation to the variation and evolution of PRRSV NADC30-like and its differential pathogenesis from other strains of PRRSV.

Our study was conducted to explore protein structure, function and the possibility as a vaccine candidate antigen of immune-related gene, CAMK/TSSK protein kinase (CTPK) gene of Parabronema skrjabini. RNA was extracted from Parabronema skrjabini and cDNA was obtained by reverse transcription. A pair of specific primers was designed to amplify the coding region of CTPK gene. The protein structure and antigenic epitopes of CTPK were analyzed by using bioinformatics softwares. The bioinformatic analysis results showed that the length of CTPK gene was 519 bp, contained an open reading frame of 519 bp which encoded a polypeptide of 173 amino acids with a predicted molecular mass of 20.264 kDa and PI of 9.53. Analyses of domain and structure indicated that the protein was dominantly composed of 60% hydrophillic amino acid residues, with numerous curls and no transmembrane structure. The antigenic epitopes prediction results indicated that CTPK protein is a hydrophilic protein with high antigenicity. It contained more potential B cell antigen epitopes and more T cell antigen epitopes. These results possibly suggested that the CTPK antigen has been promised as a candidate for immune diagnostics and could be used as a vaccine against Parabronema skrjabini. This research provide the theoretical basis in order to establish iELISA corresponding diagnostic kits and development DNA vaccine.

Our study was conducted to explore protein structure, function and the possibility as a vaccine candidate antigen of immune-related gene, CAMK/TSSK protein kinase (CTPK) gene of Parabronema skrjabini. RNA was extracted from Parabronema skrjabini and cDNA was obtained by reverse transcription. A pair of specific primers was designed to amplify the coding region of CTPK gene. The protein structure and antigenic epitopes of CTPK were analyzed by using bioinformatics softwares. The bioinformatic analysis results showed that the length of CTPK gene was 519 bp, contained an open reading frame of 519 bp which encoded a polypeptide of 173 amino acids with a predicted molecular mass of 20.264 kDa and PI of 9.53. Analyses of domain and structure indicated that the protein was dominantly composed of 60% hydrophillic amino acid residues, with numerous curls and no transmembrane structure. The antigenic epitopes prediction results indicated that CTPK protein is a hydrophilic protein with high antigenicity. It contained more potential B cell antigen epitopes and more T cell antigen epitopes. These results possibly suggested that the CTPK antigen has been promised as a candidate for immune diagnostics and could be used as a vaccine against Parabronema skrjabini. This research provide the theoretical basis in order to establish iELISA corresponding diagnostic kits and development DNA vaccine.

The aim of this study was to investigate the effect of sterol regulatory element-binding protein cleavage activating protein (SCAP) on the expression of stearoyl-CoA desaturase(SCD) gene regulated by steroid regulatory element binding protein (SREBP1) in dairy mammary epithelial cells. The dairy mammary epithelial cells were cultured and transfected with SCD gene promoter vectors, then SREBP1 and SCAP eukaryotic expression vectors were transfected into cells as treatment factor.The SCD promoter activity was detected by dual luciferase reporter gene system.The expression of SREBP1 in nucleus was observed by immunofluorescence. qRT-PCR was used to measure the mRNA expression of SCD gene. The results showed that, compared with pcDNA3.1 control group, transfection of SCAP had no significant effect on SCD promoter activity. Transfection of SREBP1 and co-transfection of SCAP/SREBP1 significantly increased the SCD promoter activity (P<0.01). There was a significant dose-response relationship between the transfection dose of SCAP and the promoter activity of SCD (P<0.01). The expression of SREBP1 in nucleus could be enhanced by SCAP transfection in mammary epithelial cells. The mRNA expression of SCD was up-regulated by 1.23-fold and 1.54-fold (P <0.05) after transfected SREBP1 or co-transfected SCAP/SREBP1, respectively. In conclusion, SCAP can increase the expression of SREBP1 protein in nucleus which enhance the transcriptional activation of SCD gene.

The aim of this study was to investigate the effect of sterol regulatory element-binding protein cleavage activating protein (SCAP) on the expression of stearoyl-CoA desaturase(SCD) gene regulated by steroid regulatory element binding protein (SREBP1) in dairy mammary epithelial cells. The dairy mammary epithelial cells were cultured and transfected with SCD gene promoter vectors, then SREBP1 and SCAP eukaryotic expression vectors were transfected into cells as treatment factor.The SCD promoter activity was detected by dual luciferase reporter gene system.The expression of SREBP1 in nucleus was observed by immunofluorescence. qRT-PCR was used to measure the mRNA expression of SCD gene. The results showed that, compared with pcDNA3.1 control group, transfection of SCAP had no significant effect on SCD promoter activity. Transfection of SREBP1 and co-transfection of SCAP/SREBP1 significantly increased the SCD promoter activity (P<0.01). There was a significant dose-response relationship between the transfection dose of SCAP and the promoter activity of SCD (P<0.01). The expression of SREBP1 in nucleus could be enhanced by SCAP transfection in mammary epithelial cells. The mRNA expression of SCD was up-regulated by 1.23-fold and 1.54-fold (P <0.05) after transfected SREBP1 or co-transfected SCAP/SREBP1, respectively. In conclusion, SCAP can increase the expression of SREBP1 protein in nucleus which enhance the transcriptional activation of SCD gene.