Copy number variation (CNV), as an important component of genomic structural variation (SV), plays important roles in phenotypic variability, disease susceptibility estimates and species evolution. Currently, a large number of studies have been widely conducted to analyze genome-wide CNV of domestic animals using high throughput and density technologies, which enable CNV as an important molecular marker for complex traits in genome-wide association analysis study (GWAS). In addition, CNV provides a new insight into the analysis of population genetic structure. Therefore, this review comprehensively summarized definition, formation mechanism, distribution characteristics, analyzing methods, genetic effect of CNV in cattle and the CNV on bovine Y chromosome based on previous studies. Meanwhile, we also raised some problems in current CNV studies, and provide a brief outlook of the future for CNV studies, which would supply guidance for cattle genome CNV studies in the future.
Haemophilus parasuis (Hps) is a major bacterial pathogen in pigs that is prevalent in many countries worldwide. It can easily parasitize the upper respiratory tract of newborn piglets through contact transmission. Under certain conditions, Hps infection leads to systematic inflammations, in which the most typical clinical symptom is the so-called Glässer's disease. Co-infections of Hps with other bacteria and/or viruses have been reported, which can impair the host protective immunity and affect the production efficiency seriously. In the last decade, Hps infection has continuously attracted scientists and great progress in the basic research fields of prevention, control, and in vivo/in vitro challenges have been made. Whereas the molecular mechanism of Hps and host interaction is still fuzzy that warrants further investigation. Based on our studies and results from other teams, we speculate that the "Macrophage (MΦ)-γδT cell" immune axis may play a crucial role in the pathogenesis of Glässer's disease. In order to discuss better with our academic faculties, we herein present a deep review on this hypotheses. Objectives of this article are to discuss key issues of host immune response and to discover the underlying main cellular players. We hope to provide insight for the signaling pathway studies and Hps resistant genetic marker discoveries in pigs.
This study intended to understand the genetic status of Shanghai White pig population through detection and annotation analysis of genome-wide genetic variation in order to better purify, rejuvenate and exploit the resource. In this study, genome sequencing was performed on 99 Shanghai White pigs using the genotype by genome simplifying and sequencing platform-GGRS. A total of 447 sequencing data including Taihu local pig breeds and western introduced breeds were collected for SNP and InDel genetic variation detection and functional annotation analysis. The results showed that the average coverage of Shanghai White pig genome was 2.87% and the average sequencing depth was 3.9 times. A total of 328 586 SNPs and 693 220 InDels were detected. Further analysis showed that the distribution of SNP and Indel on chromosomes were relatively uniform, but the distributions of number and density on different chromosomes were different. Structural annotation showed that the corresponding gene number of SNP and InDel were 11 496 and 13 216, respectively. According to the structural region classification of the genes, the distribution characteristics of SNP and InDel were consistent in all kinds of functional gene regions. The most of SNP and InDel variation distributed in the intergenic region, with the ratio of 74.61% and 83.38%, respectively, followed by intron, with 22.76% and 14.98%, respectively. Gene enrichment analysis showed that SNPs were mainly related to protein metabolism and macromolecule metabolism. InDel mostly enriched in tissues, organ development and disease-related pathways. In addition, we also found that the QTLs containing high-density SNP and InDel mainly focused on meat quality, carcass and healthy traits. The whole genome genetic variation of Shanghai White pig was investigated by advanced simplified genomic sequencing. The comparative analysis between Shanghai White pig, Taihu pig breeds and western pig breeds explained the chromosomal distribution characteristics,gene distribution and functional annotation information of these genetic variations, which laid a solid foundation for subsequent exploitation and utilization.
The aim of this study was to investigate the regulation function of methylation in promoter region of TLR5 gene on the resistance to E. coli F18 of weaned piglets. The differential expressions of TLR5 gene and protein in small intestine (duodenum and jejunum) between E. coli F18-resistant and E. coli F18-sensitive Sutai weaned piglets were detected by qPCR and Western blot, respectively. The core promoter region, CpG islands and their acting elements of TLR5 were determined by bioinformatics analysis and dual luciferase reporter system, and the correlation between the methylation degree in promoter region and the expression of TLR5 gene in E. coli F18-resistant and E. coli F18-sensitive Sutai weaned piglets was detected and analyzed. The results showed that expression levels of TLR5 gene in duodenum and jejunum of sensitive individuals were significantly and very significantly higher than that in resistant individuals (P<0.05 and P<0.01), respectively. And the protein expressions of TLR5 in duodenum and jejunum of sensitive individuals were significantly higher than that of resistant individuals (P<0.05). The core promoter region of TLR5 gene included 2 CpG islands and 16 acting elements, the methylation level of the mC-6 site in the second CpG island of the promoter region had a certain regulation effect on the expression of TLR5 gene. The site located in the core promoter bound by transcription factor Sp1. The above results indicated that TLR5 gene played an important regulating role in the process of E. coli invasion, whose low expression was conducive to the piglets' resistance to E. coli. The methylation of the mC-6 CG site in the second CpG island of the TLR5 gene promoter could inhibit the binding of the transcription factor Sp1, which inhibited the expression of the TLR5 gene and ultimately affected the resistance of weaned piglets to E. coli.
The aim of this study was to determine the most suitable reference genes in different periods of goat intramuscular preadipocytes differentiation in goat by comparing the expression levels of 9 candidate reference genes. The intramuscular preadipocytes of goat were obtained by collagenase digestion technique. The adipocytes induced for 0, 1, 2, 3, 5 and 6 d were used in this study. The expression levels of the 9 candidate reference genes (GAPDH, PPIA, 18S rRNA, PPIB, UXT, RPLP0, ACTB, EIF3K and TBP) were detected by real-time quantitative PCR (qPCR), and the expression stability was evaluated by geNorm, NormFinder and BestKeeper programs, individually. Meanwhile, the suitability of the selected reference genes for normalization was further assessed by the expression of KLF3 and KLF12. The results showed that UXT was identified as the most suitable reference gene by both geNorm and NormFinder analysis, while the PPIB was identified as the most suitable reference gene by BestKeeper analysis. The combined use of UXT and PPIB as the reference genes resulted in the optimal selection for normalization of analyzed genes in different periods of intramuscular preadipocytes differentiation in goat based on the analysis of geNorm, NormFinder and BestKeeper; Furthermore, the expression levels of KLF3 and KLF12 normalized by the most stable reference genes (UXT and PPIB, UXT) were significantly different from that normalized by the least stable ones (EIF3K, 18S rRNA). In conclusion, the expression of UXT was the most stable and it was the most suitable reference genes during the differentiation of goat intramuscular preadipocyte in goat, meanwhile the combined use of UXT and PPIB was found to lead to a more accurate result for target genes normalization. These results provided a foundation for functional research of genes related to preadipocytes differentiation in goats.
The present study aimed to understand the sequence characterization and transcriptional regulation of the 5'-UTR of Hu sheep FSHR gene, thus provided the basis for further exploring of transcription regulatory mechanism of FSHR gene. Adult female Hu sheep (n=3) were slaughtered for 11 tissues sampling of heart, liver, spleen, lung, kidney, stomach, muscle, rectum, intestinal, uterus and ovary. PCR amplification and clone sequencing were performed to determine the 5'-UTR sequence, and then the sequence characterization of Hu sheep FSHR gene was analyzed by bioinformatic method. Tissue expression patterns of transcription factor PAX4 of Hu sheep were detected by RT-PCR. Overexpression vector (pcDNA3.1-PAX4) of Hu sheep PAX4 gene was synthesized, which was then transfected into pig ovarian granulosa cells (GCs), and the apoptosis rate of GCs was measured by flow cytometry. The dul-luciferase reporter assay system was used to evaluate the effect of PAX4 on transcriptional activity of Hu sheep FSHR gene. The results showed that the full length of the 5'-UTR of Hu sheep FSHR gene was 161 bp, which contained several typical transcriptional regulatory elements such as E-box, CAAT-box and GC-box. A few of binding sites for transcription factor were also found, such as GATA-2, Sp1 and PAX4. RT-PCR showed that transcription factor PAX4 was expressed in 11 tissues of Hu sheep, and moderately expressed in ovarian tissue. After transfecting with pcDNA3.1-PAX4, the mRNA levels of PAX4 was increased (P<0.01) in GCs. Overexpression of PAX4 significantly increased the apoptosis rate of GCs (P<0.05), and decreased the luciferase activity of luciferase reporter vectors containing FSHR 5'-UTR in both GCs (P<0.05) and COS-7 cells (P<0.01). Together, the findings demonstrated that transcription factor PAX4 could enhance GC apoptosis by inhibiting transcriptional activity of FSHR gene in Hu sheep.
In present study, we aimed to explore the effect of the cell wall of Saccharomyces cerevisiae (S. cerevisiae) on the expression of sheep beta defensin-1 (SBD-1) in primary culture of sheep's ruminal epithelial cells (SRECs). Firstly, the cell wall of S.cerevisiae was extracted and subjected to quality test. Then, SRECs were cultured and stimulated with cell wall of S. cerevisiae (0, 25, 50, 100, 200, 400 μg·mL-1) for different times (0, 2, 4, 8, 12, 24 h). RT-PCR and ELISA were performed to grope the optimal concentration and time for SBD-1 expression. The results showed that:1) The cell wall of S. cerevisiae extracted was pure and suitable for the subsequent experiments; 2) SRECs were in vitro cultured successfully (cobblestone pavement), that meet the experimental requirement; 3) Compared with other concentration groups, the expression of SBD-1 mRNA and protein reached the highest when the cell wall of S. cerevisiae of 200 μg·mL-1 was used to stimulate SRECs for 12 h (P<0.001). The results indicated that the cell wall of S. cerevisiae could improve the expression of SBD-1 in SRECs, and optimal condition was 200 μg·mL-1for 12 h.
The purpose of this study was to investigate the heritability of somatic cell score (SCS) difference and analyze its changing pattern in Chinese Holstein population in Beijing. Dairy herd improvement (DHI) data of 78 386 Chinese Holstein dairy cows (from 1998 to 2016) obtained from 121 different dairy farms in Beijing were analyzed by the least square method and the SCS difference was calculated using DHI data between the SCSs of the current test-month and the previous test-month. The variance components and heritability of SCS difference were estimated by the linear mixed model and DMU software. The results revealed significant effect (P<0.01) of herd scale, test year, test season, parity, calving season and current lactation month on SCS difference. The estimated heritability of SCS difference in the current study was (0.054±0.003) for Chinese Holstein cows in Beijing. Among them, the heritability of SCS difference in the first, the second and the third parities were (0.070±0.005),(0.045±0.004), (0.052±0.007), respectively. Since the SCS difference is regarded as a timely simply indicator to detect the occurrence of subclinical mastitis, the estimated heritability of SCS difference might be helpful in the selection practice of resistance to subclinical mastitis in Chinese Holstein cattle.
This research was designed to investigate the effect of nutrient treatments during luteal phase on follicle development, apoptotic rate of ovarian cells, protein expression of glucose transporter-4 (GLUT4), and expression levels of genes involved in glucose metabolic pathway, to explore the possible mechanism involved in follicle development response to nutrients. After estrous synchronization with intravaginal progestogen sponges, estrous behavior was detected by vasectomized rams from the second day of pessary removal. The end of estrous behavior was considered to be day 0 of the estrous cycle. All 30 multiparous Hu sheep received a total mixed ration diet based on the feeding standards for maintenance requirement (M) from day 0 to day 6 of estrous cycle, and were randomly divided to 0.5M, 1.5M and 1.0M groups from day 7 to day 14 of estrous cycle. Six ewes from each group were randomly selected to be slaughtered on day 15 of estrous cycle. The remaining ewes from all groups were fed the maintenance diet, and the estrous behavior was examined individually. The results indicated that the feed restriction of 0.5M significantly delayed the estrous cycle (P<0.05), which accompanied with significantly increased and decreased ratio of small follicle (<2.5 mm in diameter) and large follicle (≥ 2.5 mm in diameter) to total follicle (≥ 1.0 mm in diameter), respectively (P<0.05); likewise, the apoptotic rate of ovarian tissue in 0.5M group was greater than those in 1.5M and 1.0M groups (P<0.05; TUNEL method results). GLUT4 were immunolocalization detected in all stages of follicle development of ovarian tissue within each group, meanwhile, within <2.5 mm follicle, the protein expression level of GLUT4 of 1.5M group was significantly greater than that of 0.5M and 1.0M groups (P<0.05). Analysis of expression levels of key genes involved in glucose metabolism pathway(polyol pathway, glycolysis pathway, hexosamine biosynthesis pathway (HBP), and pentose phosphate pathway (PPP) by qRT-PCR showed that, the gene expression level of glutamine:fructose-6-phosphate amidotransferase-2 (GFPT2), the enzyme which catalyzes the rate-limiting enzyme of HBP, were significantly decreased by increasing of feed intake in <2.5 mm follicles (P<0.05), however, the expression levels of GFPT2 in ≥ 2.5 mm follicles were significantly increased by increasing of feed intake (P<0.05). Additionally, the gene expression level of Glucose-6-phosphate dehydrogenase (G6PDH), the enzyme which catalyzes the rate determining step of the PPP, was significantly lower in ≥ 2.5 mm follicles of the 0.5M group than that of 1.0M and 1.5M groups (P<0.05). Overall, the feed restriction during luteal phase prolonged estrous cycle of ewes, this response might be related to the increased of ovarian cell apoptosis rate and decrease of GLUT4 expression, but decreased HBP intensity within <2.5 mm follicles, and also be related to the (decreased HBP and enhanced PPP) intensity within ≥ 2.5 mm follicles, respectively. The current research will enrich the mechanism in follicle development regulated by different nutritional levels, by exploring the changing pattern of estrous cycle, ovarian cell apoptosis, intra-follicular glucose metabolic pathway, to provide a theoretical basis for reproductive improvement for non-pregnant ewes by feeding and management.
The aim of this study was to determine if the expression of leptin receptor (LEPR) and leptin (LEP) genes was associated with litter size of Small Tail Han ewes. Twenty one healthy Small Tail Han ewes, composed of 9 FecB wild type (++) and 12 FecB mutation homozygous type (BB), about 2-3 years old, were used to investigate the ovulation rate by laparoscopy after spontaneous estrus with CIDR. Real-time PCR was used to detect the expression of LEPR and LEP genes in 11 tissues (heart,liver,spleen,lung,kidney,adrenal gland, fallopian tube,uterus,hypothalamus,pituitary and ovary)of Small Tail Han ewes during follicular phase and in hypothalamus-pituitary-ovary gonadal axis of Small Tail Han ewes (++ and BB genotypes) during luteal phase and follicular phase. The results showed that both ovulation number (P=0.003 5) and litter size (P=0.004 0) of individuals with BB genotype were significantly higher than that of individuals with ++ genotype. These 2 genes were all expressed in 11 tissues during follicular phase, the expression of LEPR gene was high in ovary, uterus and adrenal gland, and the expression of LEP gene was high in hypothalamus, pituitary, ovary and kidney. In ovary, the expression of LEPR gene of individuals with ++ genotype during follicular phase was higher (P>0.05) than that during luteal phase. The LEPR expression of individuals with BB genotype during follicular phase was significantly higher than that of individuals with ++ genotype during follicular phase (P=0.007), BB genotype during luteal phase (P=0.003) and ++ genotype during luteal phase (P=0.003). Therefore, we concluded that there was a positive correlation between the high expression of LEPR gene during follicular phase and high litter size in Small Tail Han ewes, which provided a new clue for improving litter size in Small Tail Han sheep.
The present study was conducted to investigate the effects of Enterococcus faecium administration on the growth performance, serum biochemical parameters and cecal microflora's structure in E. coli-challenged broilers. A total of 360 1-day-old White Feather chickens with the approximative body weight were randomly divided into 4 groups, six replicates (15 birds per replicate) in each group:negative control (NC), positive control (basal diet, PC), E. faecium-fed (E. f) and antibiotic-fed birds (Anti). The birds in PC, E. f and Anti groups were challenged with E. coli K88 on d 7, the whole test lasted for 28 d. And the serum levels of complement components and immunoglobulins were measured by the specific kits, while the change of cecal microbiota was determined by PCR-DGGE. Data showed that:1) The birds in E. f group had significant higher body weight than that of birds in NC and PC groups on d 14, 21 and 28(P<0.05); The birds in E. f group had significant higher ADG than that of birds in NC and PC groups during d 10-14, 15-21 and 10-28 (P<0.05). 2) The birds in E. f and Anti groups had significant higher level of serum IgA than that of birds in NC and PC groups on d 14, 21 (P<0.05); Birds in E. f and Anti groups had significant higher level of serum IgG than that of birds in NC group in d 10-28(P<0.05). 3) Compared with birds in PC group, birds in E. f group had significant higher C3 concentration on d 21(P<0.05). The birds in PC, E. f and Anti groups had higher serum lysozyme than that of birds in NC group on d 10, 14 and 21 (P<0.05). 4) Compared with the birds in PC group, birds in E. f group had significant higher Shannon index of cecal microflora (P<0.05) on d 28. In conclusion, the dietary Enterococcus faecium improved the growth performance, serum immune parameters and changed the diversity of cecal intestinal microflora in the E. coli K88-challenged chicken.
The objective of this study was to investigate the effect of early weaned on morphological development of stomach for housed lambs. Fifty-five male lambs (Gansu modern breeding sheep group, singleton) were divided into 3 treatments, 15 animals were used in group A (weaned at 28 d of age) and B (weaned at 42 d of age), respectively, 25 suckling lambs were used in group C (control). All lambs were raised with the supplementary feed from 7 d of age. Lambs were killed and sampled from the group A and B at 0, 7, 14 days after weaned, respectively, and lambs in the control(5 lambs) were killed and sampled at the same time. The morphological indexes of rumen ventral sac and dorsal sac, fundus reticuli, omasum, and cardiac, fundus, pyloric of abomasum were measured, respectively. The results showed that, group A was weaned for 7 days, group A and B were weaned for 14 days, the papilla height of the rumen ventral sac were significantly higher than that of group C(P<0.05). The growth rate of the papilla height in rumen dorsal sac in group A was higher than that in group B(83.4% vs. 46.9%) at weaned for 14 days. The average papilla height of fundus reticuli in group A and B were higher than that in group C(1 194.0 μm vs. 632.2 μm;953.1 μm vs. 661.3 μm). The growth rate of the epithelial thickness in omasum in group A was higher than that in group B (40.6% vs. 31.9%) at weaned for 14 days. And the mucosa thickness of cardiac gland in abomasums in group B was significantly higher than that in group C at weaned for 14 days (P<0.05). The average submucosa thickness of fundus gland abomasum in group A and B were higher than that in group C(326.8 μm vs. 245.2 μm;303.4 μm vs. 172.7 μm). And the average muscular thickness of pyloric gland abomasum in group A and B were higher than that in group C(1 218.4 μm vs. 1 047.4 μm;1 466.3 μm vs. 1 253.7 μm). The result indicated that, with feasible raised and supplied the suited supplementary diet from 7 d of age, there were promotion for morphplogical development of stomach in lambs. And it had better promotion for the lambs weaned at 28 d of age than at 42 d of age for the morphplogical development of stomach.
The aim of this study was to investigate the effects of dietary vitamin D and calcium levels on production performance, serum biochemical parameters and organ indexes of male mink under the fixed ratio of calcium to phosphorus. One hundred and seventeen healthy male minks with (135±5) day-old with similar body weight were randomly divided into 9 groups with 13 replicates per group and 1 mink per replicate. The experiment was conducted with a 3×3 factorial design using a basal diet containing 2 300 IU·kg-1 vitamin D and 2.0% Ca. The dietary ratio of calcium to phosphorus was fixed to 1.7. Nine kinds of diets were prepared in the experiment. The supplementary vitamin D levels were 0, 2 000 and 4 000 IU·kg-1 of the diets, respectively, while supplementary Ca levels were 0%, 0.4%, 0.8%, respectively. Calcium and vitamin D levels were 2.0% versus 2 300 IU·kg-1, 2.0% versus 4 300 IU·kg-1, 2.0% versus 6 300 IU·kg-1, 2.4% versus 2 300 IU·kg-1, 2.4% versus 4 300 IU·kg-1, 2.4% versus 6 300 IU·kg-1, 2.8% versus 2 300 IU·kg-1, 2.8% versus 4 300 IU·kg-1, 2.8% versus 6 300 IU·kg-1. The test lasted for 60 days. Production performance, serum nitrogen metabolism indexes and fat metabolism indexes were measured, and organ index was calculated. The results showed that:1) When the calcium supplementation level was 0%, the skin length of mink significantly increased (P<0.05). The vitamin D and calcium levels had no significant effect on the body weight, body length and fur quality of mink (P>0.05). 2)The serum TP concentration significantly increased(P<0.05) and serum TG concentration significantly decreased (P<0.05) when the addtion level of vitamin D was 2 000 IU·kg-1. When the supplementary level of calcium was 0.8%, the serum HDL concentration of mink significantly increased (P<0.05), and the concentration of LDL significantly decreased (P<0.01).3) 0.8% calcium supplementation significantly increased mink heart index, liver index and spleen index(P<0.05), vitamin D supplementation in 0-2 000 IU·kg-1 significantly increased mink kidney index(P<0.01). In conclusion,under the conditions of this experiment, when the dietary supplementation is 0-2 000 IU·kg-1 vitamin D (total 2 300-4 300 IU·kg-1 vitamin D in diet), protein metabolism and kidney index of mink can be promoted;when the dietary supplementation is 0.8% Ca(total 2.8% Ca in diet), heart index, liver index and spleen index of mink can be promoted.
Foot-and-mouth disease and vesicular stomatitis are serious cute disease common in cattle, and prevalence in large parts of the word. The aim of this study was to establish a duplex fluorescence-based reverse transcript loop-mediated isothermal amplification assay for detection of foot-and-mouth disease virus (FMDV) and vesicular stomatitis virus (VSV). Two sets of specific primers were designed according to the conserved sequences of FMDV 3D gene and VSV N gene, and the inner primers were synthesized with two different fluorophores at its 5'ends. A duplex fluorescence-based reverse transcript loop-mediated isothermal amplification assay was developed and used for detection of FMDV and VSV. The results showed that this assay could detect at least 100 copies of premixed plasmid containing the targets gene of FMDV and VSV per tube. The specificity of this assay was high, and be able to detect FMDV and VSV without any cross-reactions with other known non-targeted bovine pathogens. Different concentration template of FMDV and VSV could be identified when mixed together without any interference. These findings indicate that this duplex RT-LAMP assay is a simple, rapid, specific, and sensitive method for detection of FMDV and VSV, and can be applied in clinical detection and epidemiological investigation of FMDV and VSV.
Specific pathogen free Leghorn chickens were infected with MDV, and lymphoma from spleens and livers were collected post infection. The qRT-PCR assay showed that microRNA gga-miR-140-3p was downregulated in MDV-induced tumorous spleen and lymphoma from liver. To elucidate the functions of this miRNA in Marek's disease tumor transformation, we transfected MDV-transformed lymphoid cell line, MDCC-MSB1 with gga-miR-140-3p agomir or agomir NC, and inspected their effects on cell proliferation and migration as well as MMP2 and MMP9 genes expression levels whose expression levels were closely correlated with invasion. The proliferation of MSB1 cells decreased at 24, 36, 48, 60 and 72 h post gga-miR-140-3p agomir transfection. The cell number of migration also decreased after transfection with gga-miR-140-3p agomir. MMP2 expression was significantly downregulated at 24, 48 and 72 h; in addition, MMP9 transcription was remarkably downregulated at 48 h post gga-miR-140-3p agomir transfection. In conclusion, gga-miR-140-3p which was downregulated in MDV-induced tumorous tissues had an effect on features of tumor cells such as proliferation, migration and invasion, which indicates that this miRNA may be involved in Marek's disease tumor transformation.
In order to evaluate the genetic variation and population structure of Psoroptes ovis var. cuniculi, the full-length of the mitochondrial 16S rRNA of 83 isolates of Psoroptes ovis var. cuniculi from 5 geographic regions in China (including North, East, central, southwest, northwest zones) were amplified using the PCR technology, sequenced and then genetically analyzed. The results showed that the full 16S rRNA gene from 83 Psoroptes ovis var.cuniculi isolates were 1 025 bp in length, and that there were a total of 83 polymorphic sites and 52 haplotypes; the values of Fst and the gene flow between 5 geographical populations were -0.037 59-0.587 63 and -6.900 7-31.117 62, respectively; NJ tree and haplotype network analysis further showed these 52 haplotypes formed two branches with a messy distribution, and no population cluster patterns according to the geographical area, temperature zone and rabbit breeds were observed. These results indicate that Psoroptes ovis var.cuniculi population in China appeared a high genetic diversity and differentiation, but the geographical population structure has not formed.
The aim of this study was to investigate the effects of the flagellin protein FliC of Salmonella abortus equi on immune response of Streptococcus equi subsp. equi SeM and provide basis for the development of a new effective strangle vaccine. The recombinant protein FliC was constructed, expressed and purified using affinity chromatography, and its immunogenicity was analyzed by Western blot. The purified FliC and SeM were used in-combination to immunize C57BL/6 mice and the serum antibody level, antibody subtype of immunized mice and challenge protection rate were investigated. SDS-PAGE result indicated that the expressed recombinant FliC protein was 52 kDa. The results of antibody typing showed that the main antibodies were mainly IgG1, and the challenge protection rate of FliC+SeM immunization group (87%) was higher than that of SeM immunization group (67%). The flagellin FliC of Salmonella abortus equi could enhance the immune efficacy of SeM and this result laid experimental foundation for the further utilization of SeM.
The purpose of this study was to evaluate the therapeutic effect of traditional Chinese herbal compound of Qinhuo oral liquid on ducklings that infected with duck hepatitis virus. Three hundred feathers healthy 1-day-old ducklings were randomly divided into 5 groups, 60 feathers in each group. The test groups were Qinhuo high dose group, middle dose group, low dose group, virus group and blank control group, respectively. At the age of 6 days old, except for the blank control group, the other groups were intramuscular injected with DHAV-1 to establish artificial infection model; 1 h later, Qinhuo high dose group, middle dose group and low dose group were given high dose (0.6 mL per feather), middle dose (0.4 mL per feather) and low dose (0.2 mL per feather) of Qinhuo oral liquid in the water for treatment, once a day for 3 days, the virus group and blank control group treated without medicine. The samples were collected at the 4th, 8th and 48th hour during the experiment for measuring related indexes. During the experiment, the mortality rate of each group was compared; the liver pathological examination and HE staining section were observed; relative blood DHAV-1 VP1 gene copies number was analyzed; the contents of hepatic function biochemical indexes (AST, ALT, TP, ALB, GLO) and serum cytokines (IL-2, IL-6, IL-8, IFN-β) were detected. All indexes were severed to evaluate the therapeutic effect of Qinhuo oral liquid on duck virus hepatitis. The results showed that Qinhuo oral liquid can effectively reduce the mortality rate of infected ducklings; compared with virus group, Qinhuo oral liquid can significantly reduce the copies number of DHAV-1 VP1 in the blood (P<0.05); repair the liver damage casused by DHAV-1; reduce the contents of AST and ALT; enhance the contents of IL-2, IL-6, IL-8, IFN-β in serum. Considering all factors, the high dose of the Qinhuo oral liquid has a better therapeutic effect on ducklings infected with duck hepatitis virus.
The aim of this study was to investigate the effect of Quercetin (QE) on the reproductive system damage induced by cadmium (Cd) in male rats and the role of Nrf2-Keap1 signaling pathway in this process. A total 32 3-month-old SPF Wistar male rats were randomly divided into four groups with eight rats in each group:control group[0.5 mL·(kg·d)-1 physiological saline], Cd group[5 mg·(kg·d)-1 CdCl2], QE group[15 mg·(kg·d)-1 QE], Cd+ QE group[5 mg·(kg·d)-1 CdCl2+15 mg·(kg·d)-1 QE], the study lasted for 30 days. At the end of the trial, the testis and epididymis organs coefficients were calculated, then the sperm quality and the levels of anti-oxidation of testis were measured. RT-qPCR and Western blot were used to detect the changes of mRNA and protein expressions of Nrf2 and its related genes, respectively. The results showed that the reproductive function of rats in Cd group was seriously damaged compared with the control group. Compared with Cd group, the coefficients of testis and epididymis (P<0.01) and the secretion testosterone of rats in Cd+QE group were significantly increased (P<0.05). The semen quality was improved, furthermore, the levels of MDA, H2O2 were significantly decreased (P<0.01) and the levels of GSH (P<0.05), CAT, T-SOD, GSH-Px (P<0.01) were significantly increased in Cd+QE group. The analysis showed that the mRNA and protein expression levels of Nrf2, HO-1, NQO1, GSH-Px, γ-GCS in testis were significantly increased (P<0.05 or P <0.01) and Keap1 mRNA and protein expression levels were significantly decreased (P <0.05) in Cd+QE group. In conclusion, QE can effectively alleviate the reproductive system damage of male rats induced by Cd through activating Nrf2-Keap1 signaling pathway.
OPG/RANKL/RANK signal pathway plays critical roles in the formation of osteoclasts. Inhibitive roles of calcium chelated sheep bone collagen peptide (SBCP-Ca) in the development of osteoporosis (OP) and its mechanism based on the OPG/RANKL/RANK signal pathway were investigated in this study. OP models of SD rats were established by bilateral ovariectomy. SBCP-Ca (100 mg·mL-1) was intragastrically administered at 10 mL·(kg·d)-1 for 8 weeks. After treatment, scanning electron microscopy for metaphysis of distal femur showed that OP models were successfully established. The serum levels of PINP and β-CTx, which reflected bone formation and bone resorption respectively, were significantly higher in model group (P<0.01), while the calcium and proline contents in femur bone were significantly lower than the sham group (P<0.01).The levels of PINP and β-CTx in SBCP-Ca group showed no statistical differences compared with the sham group (P>0.05), both Ca and proline content were higher than that of the model group (P<0.01). The results of real-time PCR suggested that the relative mRNA levels of RANK and RANKL in model group were significantly higher than that of the sham group (P<0.01), while the expression of OPG showed no significant changes. The results showed that the transcriptional levels of RANK and RANKL were significantly lower in SBCP-Ca group than that of the model group(P<0.01), and the OPG level was significantly higher than that of the other groups(P<0.01). It can be concluded that SBCP-Ca could inhibit the occurrence of osteoporosis effectively by inhibiting the expression of RANK and RANKL, and promoting the expression of OPG in bone tissue. The triple actions of SBCP-Ca inhibited the formation and the resorption activity of osteoclasts.
Studies have shown that isoflurane could cause autotrophy and apoptosis in the developing rat brain. Based on the preliminary experiment, forty 7 day Sprague-Dawley rat pups were selected and divided into control group and groups of 1.5% isoflurane anesthesia for 2, 3, 4 and 6 h randomly. After anesthesia, the rats were euthanized, then hippocampus were collected for TUNEL-staining, LC3-Ⅱ immunohistochemistry and Western blotting, to investigate the level of autophagy and apoptosis. The results showed that with the increasing time exposed to isoflurane, the percent of positive cells were significantly increased at 3 h, the results of LC3-Ⅱ immunohistochemistry showed that the percent of positive cells were increased at 3 and 4 h, but decreased at 6 h; and the expression of Caspase-3 was increased gradually compared with control group, although the expression of Bcl-2 was decreased. However, the expression of Beclin-1 and LC3-Ⅱ/I were increased at the first 4 h with the most expressions at 3 h (P<0.05) and then decreased; In addition, compared with isoflurane anesthesia at 3 and 4 h, the expression amount of Beclin-1 was significantly decreased at 6 h while Caspase-3 significantly increased (P<0.05). These results showed that isoflurane can not only induce extensive apoptosis of hippocampal neurons in young SD rats, but also induce autophagy in hippocampal neurons. And the level of apoptosis gradually increased with the prolongation of anesthetic time, while the level of autophagy increased first and then decreased. Autophagy may play a protective role in the apoptosis of hippocampal neurons induced by isoflurane.
The present study was conducted to study the effects of phosphorus (P) forms (CaHPO4(DCP), Ca(H2PO4)2·H2O+CaHPO4·2H2O (MDCP), Ca(H2PO4)2(MCP) and plant derived dicalcium phosphate (PDCP)) and P supplemented levels (0.1% and 0.2%) on the production performance, contents of serum calcium(Ca) and P and the metabolism of Ca, P and nitrogen (N) in laying hens. The factorial arrangement of treatments (4×2) was used involving four P sources(DCP, MDCP, MCP, PDCP) and two P levels (low P (0.1%) addition group and high P (0.2%) addition group), with a total of 8 treatment groups. A total of 480 53-week-old Hyline Brown laying hens were randomly allocated to 8 dietary treatments (6 replications of 10 hens per replication) and were fed corn-soybean meal basic diet(non-phytic acid phosphorus 0.14%), the nutrient contents were same except P. The experiment lasted for 20 weeks. Daily records of egg production, egg weight and weekly records of feed consumption were maintained throughout the experimental period and average egg production rate, average egg yield and feed/egg ratio were calculated. The blood samples were collected from the winged veins at the tenth and 20th weekends to determine the contents of Ca and P in the serum. In addition, the feces of each group were collected every 5 weeks to determine the contents of Ca, P and N in the feces. The results showed as follows:1) P source, P level and their interactions had no significant influence on average egg production rate, average egg yield and feed/egg ratio of laying hens, however, all of the above indexes in groups of PDCP, MDCP and MCP were better than those in DCP (P>0.05), and the above indexes in groups of the P level of 0.2% were also better than those in the level of 0.1% (P>0.05). 2) P source, P level and their interactions had no significant effects on serum Ca (P>0.05), meanwhile, P source and the interaction between P source and P level had no significant effects on serum P (P>0.05). 3) Compared with the DCP group, the excretion of Ca (P<0.05) and P (P<0.01) in the PDCP group decreased significantly, P excretion in the MCP group decreased significantly (P<0.05). P source had no significant effect on N excretion (P>0.05). Compared with the P level of 0.1% supplemented group, the Ca excretion in the P level of 0.2% group decreased significantly (P<0.01), and P excretion increased significantly (P<0.01). The interaction between P source and P level had significant effects on P excretion (P<0.01), while had no significant effect on excretion of N (P>0.05) in laying hens. The results suggested that adding 0.1%-0.2% P level to the basal diet (the level of NPP was 0.14%) could satisfy the production performance of the laying hens, and P forms of PDCP and MDCP were better for laying hens.
This paper aims at understanding the information about the antimicrobial resistance and the existence of extended-spectrum β-lactamases (ESBL) genes in livestock-associated Staphylococcus aureus (S. aureus) at some animal farms of Chongqing. One thousand three hundred and seventy-one samples were collected from animal farms. After pretreating, enriching and culturing. The S. aureus specific gene nuc was amplified from the suspected colonies, and the antimicrobial susceptibility of the identified S. aureus was tested. Meanwhile, the ESBL genes were amplified. Results were as follows:Eighty-nine S. aureus clinical strains were isolated and identified from 1 371 samples, including 25 pig-originated strains, 32 chicken-originated strains, 6 dairy cattle-originated strains, 10 goat-originated strains, and 16 rabbit-originated strains. The isolates were most resistant to penicillins, and also more resistant to tetracyclines, macrolides, and quinolones. Except for 2 rabbit-originated strains, other isolates exhibited varying degrees of multi-drug resistance, defined as resistance to 2 to 21 antimicrobials. The most prevalent multi-drug resistant strains were pig-originated. The ESBL genes detection result showed that 86 (96.6%) organisms contained blaTEM-1a. The S. aureus isolates were resistant to tested antimicrobials seriously, and most of them carried TEM-1a type ESBL.
To investigate current infection statuses of Salmonella in dead chicken embryos, Salmonella strains was isolated and identified from dead embryo samples collected from 4 hatcheries attached to their parental flocks of Sanhuang Chickens, and the drug resistance and resistance genes were detected by Kirby-Bauer Test and PCR method, respectively. A total of 173 Salmonella isolates were obtained from 356 dead embryos,including 5 serotypes:S. pullorum(162),S. blegdam (5),S. Cerro (4),S. enteritidis(1), S. Fillmore(1). Results showed that the isolation rate and drug resistance of Salmonella in dead embryos from different sources were significantly different. Of total 173 Salmonella isolates, the drug resistance rate to Sulfisoxazole (SF), Ampicillin (AMP), Tetracycline (TE) and Oxytetracycline (OT) were 82.1%, 74.0%, 49.1% and 48.6%. And 43.4% (75/173) isolates showed multiple drug resistance. The resistance to other drugs was mild, and the resistance rate was below 11%. The isolates were sensitive to Ceftazidime (CAZ), Meropenem (MEM), Imipenem (IPM), Polymyxin E (CT), Chloramphenicol (C), Enrofloxacin (ENR) and Ciprofloxacin (CIP). In addition, 13 drug-resistant genes were detected by PCR, and the coincidence rate between drug-resistant phenotype and drug-resistant genes was more than 72%, especially the rate were 100% to extended-spectrum beta-lactamases (ESBLs), aminoglycosides, cephalosporins. The results indicate that serotypes of Salmonella infected in Sanhuang chicken embryos are complex, the multi-drug resistance is serious and the resistance genes are widely existed in these resistant strains.
In order to clarify the tumorigenicity of Jaagsiekte sheep retrovirus (JSRV) envelope protein (Env), the recombinant eukaryotic expression plasmids pEGFP-C1/exJSRV-env and pEGFP-C1/enJSRV-env were transfected into immortalized sheep chorionic trophoblast cells (STCs) respectively. The optimal transfection conditions were screened and the soft agar colony forming assay was used to detect the malignant transformation of the cells, and the effect of the expression of the envelope protein on cell proliferation was detected by plate cloning assay. Results were as follows:Sheep trophoblast cells transfected with pEGFP-C1/exJSRV-env acquired colony forming ability in soft agar and lost contact inhibition, while cells transfected with pEGFP-C1/enJSRV-env can't form colony. The results of plate cloning experiments were analyzed by SPSS software, the clone formation rate of sheep trophoblast cells transfected with pEGFP-C1/exJSRV-env was significantly higher than that of transfected with pEGFP-C1/enJSRV-env, pEGFP-C1 empty carrier and non-transfected (P<0.01). The expression of exJSRV-Env can induce malignant transformation and the proliferation of STCs. This study may provide a theoretical basis for further study on the structure and function of Jaagsiekte sheep retrovirus envelope protein, and provide a new idea for the study of the mechanism of sheep pulmonary adenomatosis.
China is the largest swine industry country as well as the biggest pork consumption country in the world. Pork yield and quality have been being great significance to public health, living standard and food safety. China has the most rich indigenous pig resources which are crucial in pig breeding. In order to establish a sound theoretical foundation for promoting the genetic improvement and utilization of resources of Chinese domestic pig breeds, The National Natural Science Foundation of China (NSFC) proposed the major program entitled "Genetic Mechanisms of Myogenesis and Intramuscular Adipogenesis in Chinese Indigenous Pigs". After public application, peer review, panel meeting and NSFC committee, the application by Professor Lusheng Huang from Jiangxi Agricultural University was funded.
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