Exploiting the method of liposome transfection to obtain modified Toll-like receptors 4(TLR4) gene fibroblast cells as competent donor, build reconstructed embryos, which finally produced transfected TLR4 gene and gender controllable Large-tailed Han sheep by somatic cell nuclear transfer technology.In this study, through primary culturing the fetus dermal fibroblasts from Large-tailed Han sheep, to identify the SRY gene gender.Using the method of liposome transfection transferred TLR4 gene into fibroblast cells, then detected whether TLR4 gene expression stably or not in morphology level and molecular level respectively, transplanting fibroblast cells which stably expressed TLR4 gene into oocytes without nucleus from Large-tailed Han sheep for building reconstructed embryos.To harvest 5 positive clones by fibroblast cells with stable expression level of TLR4.According to the identification of the SRY gene gender, the cell lines were female.The analysis of Real-time fluorescent quantitative PCR, the fourth generation of transfected cells had the highest expression level of TLR4, which were higher than control group in 7.4816 times (P<0.01).The cleavage reconstructed embryos had stable expression level of EGFP.We succeeded in building reconstructed embryos with TLR4 gene and lay the foundation for producing the disease-resistant and gender controllable transgenic sheep in the future.

The study was aimed to obtain carp iNOS cDNA full-length sequence, and investigate the changes in peripheral blood leukocyte expression of iNOS in the stimulation of different mitogens.Based on EST sequences of iNOS that obtained from carp normal peripheral blood leukocytes cDNA library, full-length cDNA sequence of carp iNOS was successfully amplified by using gene library screening and rapid-amplification of cDNA 5'ends (5'-RACE) method.Carp peripheral blood leukocytes were divided into control and experimental groups and cultured.The experimental groups were stimulated by LPS (1.0 μg/mL) and ConA (1.0 μg/mL) for 4 and 12 h, respectively.And control groups were in the same cultured conditon time without mitogen stimulated.Real-time PCR was used to detect the expression of iNOS in peripheral blood leukocytes of each group.The results showed that the cDNA fragment was total 3 704 bp containing 74 bp 5'untranslated region (UTR), 246 bp 3'UTR and a 3 384 bp ORF encoding 1 127 amino acids.Sequence homology analysis showed that the amino acid sequence of carp iNOS shared 100% identity with Carassius auratus.After LPS and ConA stimulation, the iNOS expression of peripheral blood leukocytes in the experimental groups were increased, and the expression in the short time stimulation condition (4 h) was higher than the long time (LPS 12 h; ConA 24 h).In conclusion, iNOS expression of peripheral blood leukocytes was raised after LPS and ConA stimulation, suggesting that there were dynamic changes in the inflammation.

The study was conducted to reveal the genetic basis of abdominal fat weight trait and scan molecular markers which could be used to improve the abdominal fat weight.The abdominal fat weight of female Jinghai Yellow chicken in core group were measured after being slaughtered.Genome-wide association study was carried out using specific-locus amplified fragment sequencing technology to detect SNPs associated with abdominal fat weight.Finally, five SNPs that associated with abdominal fat weight and reached suggestive genome-wide significance (P<1.1×10^-5) were found, and four of them (rs18144674, rs18144680, rs12246236 and rs12257795) reached chromosome significance level.Three SNPs (rs18144674, rs18144680 and rs19745739) located in a 1.6 Mb area of chromosome 2, and two SNPs (rs12246236 and rs12257795) located in a 12 kb area of chromosome 14.Genes in the candidate regions with 0.1 Mb windows were screened.Finally, eleven candidate genes of VIM, TRDMT1, AXIN1, PDIA2, ARHGDIG, gga-mir-1763, gga-mir-1564, CLCN7, ECL1, DNASE1 and SDOS were obtained.The molecular function and biological processes were analyzed using GO database.The results showed that those genes might be candidate genes affecting abdominal fat weight in Jinghai Yellow chicken.

In order to study the epidemiology and genetic evolution characteristics of H9N2 subtype avain influenza virus (AIV) isolated from some areas of China, the hemagglutinin (HA) gene of 17 H9N2 subtype AIVs isolated from 2012 to 2015 were amplified by RT-PCR.Sequencing and phylogenetic analysis were performed, and the molecular characteristics of the motif of HA protein cleavage site, the receptor binding sites and potential glycosylation sites were analyzed.The results showed that the HA gene of 17 strains shared nucleotide and deduced amino acid homologies ranging from 87% to 100% and 75% to 100%, respectively.All strains belonged to Eurasian lineage, Y280-like sublineage.The cleavage sites motif of 17 isolates lacked multiple basic amino acids, which made them low pathogenic strains.There were amino acid variations at sites 149, 198, 234 and 235 of the receptor binding sites, while 16 isolates had a leucine residue at site 234, indicative of the characteristics of human influenza virus-like receptor.The analysis results of potential glycosylation site indicated that a glycosylation site deletion took place at amino acid site 218 of 11 isolates and site 492 of 4 isolates, while an additional potential glycosylation site were found at amino acid site 313 of 17 isolates.In summary, the epidemiological investigation of H9N2 subtype AIV should be enhanced and the difference between isolated virus and vaccine virus should be paid more attention.

The study was carried out to investigate the effects of various lysine (Lys) dipeptides on gene expression related to milk protein and amino acid transporters in bovine mammary tissue.Mammary tissues were obtained from midlactating Holstein dairy cows and cultured under lactogenic hormones (prolactin, hydrocortisone and insulin) with different ratios of various Lys-peptide (Lys-Lys (KK), Lys-His (KH), Lys-Phe (KF), Lys-Leu (KL)and Lys-Thr (KT)).They isometrically substituted 0, 5%, 10%, 15% and 20% of free Lys (total concentration of Lys was 210 μg/mL), respectively.After 48 h treatment, mammary tissues were harvested for gene expression analysis by Real-time PCR.The results showed that:Compared with the free Lys group with no Lys substitution, when the KH replaced Lys at ratio of 15%, the KF replacement ratio was 10%, KK, KL and KT replacement ratio was 5%, the CSN1S1 mRNA abundance were the highest (P<0.05);When the KK replaced Lys at ratios of 5%, 10%, 15% and 20%, KH replacement ratios were 10% and 15%, KF replacement ratio was 15%, KL replacement ratios were 5% and 20% and KT replacement ratios were 5%, 10% and 15%, the SLC15A2 gene expression was significantly enhanced (P<0.05); when the KH replaced lysine at ratio of 15%, KK and KL replacement ratio was 10%, KF and KT replacement ratio was 5%, the abundance of SLC6A14⁺ mRNA was the highest (P<0.05);When the KH replaced Lys at ratio of 15% and 20%, KK replacement ratios were 5%, 10% and 15%, KL replacement ratio was 5% and 10%, the mRNA abundance of mTOR was significantly increased (P<0.05).The results indicated that the Lys dipeptides might be able to promote amino acid uptake and milk protein related gene expression in bovine mammary gland tissues.

In order to study the rumen microbial diversity in Yunnan gayals (Bos frontalis), we analyzed the diversity of microbes using largely assembled sequence data obtained by metagenomic sequencing DNA isolated from rumen samples of eight adult galyals. The genome primary sequence (total metagenome size was >3 Gb) was assembled with short reads.Based on the gayal rumen microbial metagenomic sequencing, we received a total of 13 546 196 EGTs. Because of the relatively large amount, 10 387 566 EGTs (76.68% of all EGTs ) were randomly selected to analyze microbial communities by BLAST search of the NCBI-NR database. Microbial species annotations analysis revealed that microbial communities from gayals rumen were mainly composed of the bacterial domain, eukarya domain, archaea domain and unknown microbial composition. The majority microbes mainly belonged to ten phylums, and 94% EGTs belonged to Bacteroidetes, Firmicutes, Proteobacteria and Actinobacteria, of which the highest abundance phylums were Bacteroidete and Firmicutes, accounting for 48% and 42%, respectively. In addition, the main cellulolytic bacteria in rumen of gayals were Ruminococcus flavefacie, Butyrivibrio fibrisolvens, Ruminococcus albus and Fibrobacter succinogenes. They accounted for 1.60%, 1.20%, 0.86% and 0.52% of the total bacteria, respectively. These results further indicated that EGTs data could be used to analyze and explain environmental microbial community structure and function. There was great significance to study the composition of microorganisms in special environments.

In order to detect the procine pseudorabies virus (PRV) infection and differentiate virulent and avirulent PRV, a duplex PCR method was established with two pairs of specific primers designed based on the conserved sequences of gE and gB genes of PRV.Optimize the reaction conditions by adjusting concentration of primers from 0.2 to 1.4 μL, each time adding 0.2 μL and temperature of annealing from 56 to 60 ℃, added 1 ℃ each time, and so on.The results showed that the suitable annealing temperature was 56 ℃ and the suitable primer dose was 1 μL.Two special fragments of 316 bp (PRV gE) and 432 bp (PRV gB) were amplified by the optimized duplex PCR.The specific tests showed that no PCR products were detected for PCV2, PTV, CSFV, PPV, PRRSV and E.coli.The sensitivity test showed that DNA used for the duplex PCR might be as low as 4.4×10³ copy/μL for PRV gE and 3.3×10³ copy/μL for PRV gB.The sensitivity of the duplex PCR was close to the single PCR.Fifty-six clinical samples of sick pigs from different areas in Guangdong and Guangxi province were detected by the duplex PCR and the virulent detection rate was 53.6% (30/56) for PRV and the negative rate was 46.4%(26/56), and there was no attenuated PRV infection.

Porcine circovirus type 2 (PCV2) is a disease caused by porcine circovirus, which can damage the host's immune system and exacerbate the clinical symptoms of many bacterial and viral infections.It has caused increasingly larger losses in the pig industry wordwide.The genomic DNA of PCV2 contains 11 open reading frames (ORFs) and at least seven potential ORFs-encoding proteins larger than 5 ku.However, only five virally encoded proteins, including Rep, Rep', Cap, ORF3 and ORF4, had been identified in PCV2 replication.In this review, we discussed the progress on structure and function of genomic DNA of PCV2 in order to provide insight into the scientific basis of the pathogenesis of PCV2 and prevent its infection.

Feline herpesvirus type 1 (FHV-1) is the pathogeny of feline infectious rhinotracheitis.Based on the sequence of gD gene of FHV-1, one pair of primers(FHV-1F and FHV-1R) and FHV-1Probe were designed for the quantitative Real-time PCR detection method.The results showed that the method could detect FHV-1 DNA specifically and the sensitivity was 75 fg/μL.The reaction efficiency reached 107%, and the R² value was 0.9965.The coefficient of variance(CV) was below 2%.And the method was more sensitive than common PCR.It was specific, stable, and suitable for clinical diagnosis, detection and epidemiological investigation.Using this method, the Shanghai area pet clinic censorship suspected FHV-1 infected samples were detected, and the positive rate reached 27.78%, which indicated that the incidence of FHV-1 in Shanghai area was at a high level.

PRDM protein family which has a PR domain and varying amounts of zinc finger domains (except PRDM11), widely present in primates, rodents, birds and amphibians, involve in regulation of epigenetic proteins.Some PR domains possess transferase activity of histone lysine (HMT), affect epigenetics features and the Zinc finger domains may recognize and bind DNA, RNA, protein;The other PR domains of PRDM without HMT activity, play a key role in carcinogenesis, specification of primordial germ cell, neurogenesis, hematopoiesis, vascular development, and embryonic development.PRDM protein family participate in cell specialization, and disease regulation, regulating gene expression and modifying chromatin structure by methyl transfer, or recruited chromosome structure reshape compounds.This review summarized the latest research progress on structure and function of PRDM protein family.

This study was aimed to investigate the effects of lactic acid bacteria (LAB) on the growth performance, nutrient apparent digestibility and slaughter performance of broilers fed diets contaminated with AFB₁.A total of 240 male AA broilers at one day old were randomly allotted into 4 groups with 6 replicates per group and 10 chicks per replicate.Positive control group, no AFB₁ in the diet;Negative control group, 1-21 d, positive control group diet＋40 μg/kg AFB₁, 22-42 d, positive control group diet＋80 μg/kg AFB₁;LAB group, negative control diet＋1.5×10¹¹ CFU/kg LAB;HSCAS group, negative control diet＋3 g/kg HSCAS.The trial lasted for 42 days.The results showed that the average daily feed intake and body weight gain in negative control group were significantly decreased (P<0.05), and F/G was significantly increased (P<0.05), and the diets supplemented with LAB or HSCAS could significantly improved the growth performance (P<0.05), and the LAB had better effects than HSCAS.Comparing with negative group at 1 to 21 days, the nutrient apparent digestibility of DM, energy, EE, Ca and P were increased by 9.6% (P<0.05), 9.5% (P<0.05), 10.1% (P<0.05), 15.2% (P<0.05) and 18.9% (P<0.05) in LAB group, respectively.Whereas the nutrient apparent digestibility of DM, energy, EE and P were increased by 9.7% (P<0.05)、6.6% (P<0.05)、6.3% (P<0.05) and 17.4% (P<0.05) in HSCAS group, respectively.And the nutrient apparent digestibility of energy, Ca, eviscerated yield percentage and breast muscle percentage in LAB group were significantly higher than that in HSCAS group (P<0.05).LAB used in the present trial could significantly improved the growth performance, nutrient apparent digestibility and slaughter performance of broilers fed diets contaminated with AFB₁, and had better effects than HSCAS.

The experiment was conducted to study the effect of magnetized water on the growth performance, digestion metabolism and slaughter performance of lamb.12 weaned male lambs with average body weight of 5.57 kg±0.36 kg were selected and randomly divided into 2 groups, 6 lambs in each group.2 groups were provided with the same basal diet while lambs in experiment group were given with magnetized water (MW), and that in control group were given with normal water.The digestion metabolism experiments were carried out at 95-110 days and 215-230 days, respectively, and the slaughter performance of lamb was determined at 240 day to study the effects of drinking MW on the growth performance, digestion metabolism and slaughter performance of lamb.The results showed that in the earlier phase, comparing with control group, dry matter voluntary intake and the apparent digestibility of dry matter, organic matter, crude protein, cellulose, hemi-cellulose, calcium and phosphorous of lambs in MW group at 95-110 days were increased by 25.5% (P>0.05), 15.2% (P>0.05), 14.4% (P<0.05), 13.5% (P<0.05), 66.3% (P<0.05), 52.3% (P>0.05), 16.4% (P<0.05)and 1.3% (P>0.05), respectively.In the later phase, that of MW group were increased by 15.3% (P>0.05), 9.0% (P<0.05), 8.8% (P<0.05), 7.7% (P<0.05), 20.6% (P<0.05), 33.5% (P<0.05), 9.8% (P<0.05) and 14.5% (P<0.05), respectively.During 20-220 days, the average daily body weight gain of lambs in WM group was increased by 40.8% (P<0.01)and the live body weight, carcass weight, carcass net meat percentage, carcass lean weight were increased by 40.8% (P<0.01), 52.3% (P<0.01), 8.8% (P<0.01) and 66.2% (P<0.01), respectively.In conclusion, the digestibility of nutrient and daily body weight gain of lamb could be increased and the slaughter performance of lamb be improved to a certain degree by drinking MW.

This experiment was conducted to study the effect of different rearing conditions of indoor and outdoor in winter and summer on growth performance and immune function of Heijiao MA chicken.In the winter and summer, 180 Heijiao MA chicken with the age of 35-day-old were chosen and randomly divided into two groups with three replicates per group and 30 chickens per replicate.The Heijiao MA chicken were reared at outdoor and indoor, respectively, and at 49, 63, 77, 91 days the immune organ indexes, serum antibody titers and growth performance were measured.The results showed that:①Whether it was winter or summer, the body weight of Heijiao MA chicken rearing at indoor were higher than that of outdoor, and the difference was significant at experimental stages in winter (P<0.05).There was significant difference only at 77 days old of Heijiao MA chicken in summer (P<0.05), the rest were not significant (P>0.05);②The ADG of outdoor group in summer was higher than that in winter, while the F/G of outdoor group in summer were significantly lower than that in winter (P<0.05).The ADFI and ADG of indoor group were not significantly different between winter and summer (P>0.05).③On immune function, immune organ indexes and ND antibody titer of Heijiao MA chicken of indoor group in winter were higher than outdoor group, while part of immune organ indexes, AI (H5) and ND antibody titers of outdoor group in summer were higher than that of indoor group;And ND and AI (H5) antibody titers in winter were higher than summer.In conclusion, The climate and season were major factors to influence the growth performance and immune function of Heijiao MA chicken.

This experiment was conducted to study the effects of Lactobacillus fermentum on growth performance, nutrient apparent digestibility, faecal microflora number and serum immune indices of weaned piglets.A total of 160 weaned piglets with an average age of (30±2) days and averge body weight of (7.85±0.68) kg were randomly distributed into 4 groups with 4 replicates per group and 10 piglets per replicate feeding basal diet, 0.1%, 0.2%, and 0.3% Lactobacillus fermentum supplementation diets, respectively.The experiment lasted for 35 d.The results showed that, compared with the control group, the average daily gain, the feed conversion ratio, the apparent digestibility of crude protein, calcium and total phosphorusin of Lactobacillus fermentum groups were all increased in varying degrees.The optimum supplemental level of Lactobacillus fermentum should be 0.2%, and at this level, the average daily gain was significantly increased by 8.70% (P<0.05), the F/G was decreased by 4.62% (P<0.05), the apparent digestibility of crude protein, calcium and total phosphorus were significantly increased (P<0.05), the number of faecal E.coli was significantly decreased (P<0.05), and the serum IgG was significantly increased by 8.39% (P<0.05).The results indicated that Lactobacillus fermentum supplementation could improve the immune function, growth performance, the apparent digestibility of crude protein, calcium and total phosphorus and reduce the number of faecal E.coli of weaned piglets.

This experiment was conducted to study the effects of ampelopsis grossedentata flavonoids on serum biochemical indexes and immune function of piglets.24 healthy piglets were selected and divided into 4 groups for 40 days feeding experiment.Control group was fed with basal diet and treatment groups were fed with 0.25%, 0.5% and 1% AGF adding to basal diet, respectively.At the end of feeding experiment, two piglets from each group were selected and blood samples were collected to analyze the physiological and biochemical indexes of serum.The results showed that AGF could significantly improve the content of ALB in serum (P<0.01), and decrease the serum BUN content in piglets, 0.5% and 1% groups had extremely significant difference with control group (P<0.01);TG content in treatment groups were decreased and 0.25% and 0.5% groups had significant difference with control group (P<0.05).AST content in serum of treatment groups significantly reduced comparing with the control group (P<0.05);ALP content of treatment groups were increased and 0.5% group was extremely significantly different from control group (P<0.01);AGF could extremely significantly improve serum IgM content (P<0.01), extremely significantly increase the IgG content of 0.5% and 1% groups (P<0.01), and improve serum complement C3 and C4 contents of 0.25% and 0.5% groups.In conclusion, AGF could enhance the piglet metabolism ability and immune function.

The aim of the study was to isolate and identify S.cerevisiae in the dairy feed and analyze antibacterial activity of S.cerevisiae fermentation brothe in vitro.5 forage samples and 5 feed samples were collected to isolate yeast by YPD solid medium.Biochemical identification and 26S rDNA sequence were used to identify the isolated strains.The antibacterial activity of S.cerevisiae strains was tested by cylinder plate method.A total of 12 yeast belonged to 4 species were isolated and identified, including 3 S.cerevisiae (Y1, Y7 and Y11), 4 Debaryomyces hansenii, 3 Cryptococcus flavescens and 2 Torulaspora debrueckii.The antibacterial activities of the 3 S.cerevisiae fermentation brothe to Staphylococcus aureus, E.coli and Streptococcus agalactiae were different.Y7 and Y11 fermentation brothe showed antibacterial activities to all the tested pathogens, while Y1 inhibit E.coli alone.This study laid the foundations for further research of probiotics containing S.cerevisiae with excellent performance.

The study was carried out to study the effects of Lonicera fulvotomentosa extract on growth performance, diarrhea rate and anti-oxidation performance in mice.90 mice were randomly divided into five groups:Blank group, experimental groups Ⅰ, Ⅱ, Ⅲ and control group.The blank group was fed with basal diet, the experimental groups were added the 0.02, 0.04 and 0.06 mg/g BW of Lonicera fulvotomentosa extract to basal diet, and the diet of control group was added the colistin sulfate observing the diarrhea rate in mice with folium sennae perfusion.The results showed that comparing with blank group, the finial weight of mice in experimental groups Ⅱ, Ⅲ and control group were significantly improved (P<0.05);The Lonicera fulvotomentosa extract had effects on growth performance in the experimental groups, there was no significant effects on the liver weight (P<0.05);The diarrhea rate in the experimental groups were decreased with the increase of the concentration of Lonicera fulvotomentosa extract, and the diarrhea rate in the control group was significantly lower than blank group (P<0.05).Compared with the blank group, the activity of GSH-Px and CAT in experimental groups (except group Ⅰ) were increased, and the serum SOD activity in experimental group Ⅲ was significantly increased (P<0.05).In conclusion, the growth performance and anti-oxidation performance of mice were increased while the diarrhea rate was decreased by adding the Lonicera fulvotomentosa extract, and adding 0.06 mg/g BW concentration had the best effects.

The flavor of mutton affects the acceptance of consumers.Adipose tissue is the most obvious source of mutton flavor, and the compounds presenting mutton flavor mainly include branched chain fatty acids, aldehyde compounds, phenolic compounds, ketone substance and so on.Mutton flavor is affected by genetic factors such as breed, sex, age, nutrition factors such as the nutrient level of diet and additive, feeding model and disease, etc.The effect of breed on the flavor may be achieved by the genetic control on fat composition and metabolism, and the amount of fat oxidation products is the main reason for different flavor among species.There are differences in fatty content and composition because of different sex, and it also has an effect on the flavor of mutton.Along with the growth of sheep, the deposits of subcutaneous fat, the proportion of saturated fatty acid, and the concentration of the lipid oxide increase, all these led to strong flavor of the lamb.Each parts of the lamb has different flavor because of the differences kind and content of the volatile compounds.The types of diet have a certain influence on mutton flavor, such as high protein feed, high energy feed, flavor plant, seeds with high content of unsaturated fatty acid and other additives.Feeding mode and disease factor also can make a change of mutton flavor.Other factors, such as the feeding density, illumination and so on, also have effects on the flavor of mutton in varying degrees.The flavor of mutton can be improved through breed selection, nutrition regulation and scientific feeding and management during sheep production.

In order to study and evaluate the feeding environment of the beef cattle farms in Xinjiang, the contents of heavy mentals in the soil from 8 beef cattle farms in Xinjiang Aksu, Yili and Tacheng regions were tested according to the Field of Livestock and Poultry Environment Quality Standard (NY/T 388—1999).The atomic absorption spectroscopic method was used to determine the contents of As, Hg, Pb, Cd, and Cr.The mean contents of As, Hg, Pb, Cd, and Cr in 8 beef cattle farms soil were 0.389, 0.568, 22.708, 0.573 and 149.449 mg/kg, respectively, and they were all below the upper limit of NY/T 388—1999.The comprehensive heavy metal pollution index of 8 beef cattle farms were 0.577 to 0.994, and the index were 0.7 to 1 of two observation points in Yili and one observation point in Tacheng.The soil environment quality of these 3 farms were at the level of slight cleaning.The comprehensive heavy metal pollution index of other five observation points were ≤0.7, the soil environment was in a clean level.The soil environment of beef cattle farms in these three regions conformed to the standards of livestock for environment quality and safe products.

Meat quality means the characteristics such as appearance is related to palatability in fresh meat or manufactured meat, it includes meat color, meat structure, hardness, marbling, water holding capacity and so on.The physical characteristics of mutton quality determines the consumer acceptability of meat and there is a close relationship between chemical composition of mutton quality and nutrient substance.With the improvement of living standards, consumers are increasingly high demand for mutton.From quantitative to qualitative change, we need to continuously improve mutton quality.At present, the production of high quality animal products has become a hot topic.This paper is a summary of the effects of breed, gender, age, environment and feed nutrition on mutton quality, and aims to provide a scientific basis for improving of mutton quality with nutritional regulation measures.

This experiment was conducted to discuss the nuclear morphology and cortical granule distribution of sheep oocyte in vivo.In this study, the sheep antral follicles were divide into early period antral follicles group (1 mm≤diameter≤2.5 mm) and middle period antral follicles group(2.5 mm

To investigate the expression of IGF-2 and IGF-1R protein in endometria of canine pyometra and analyze the possible correlation with the incidence of canine pyometra.Immunohistochemistry PV method and Western blotting were used to measure the IGF-2 and IGF-1R expression characteristic of 5 normal endometria and 19 endometria of canine pyometra.The results showed that the IGF-2 level of endometria from pyometra group was extremely significant higher than that of control group by immunohistochemistry and Western blotting (P<0.01).The positive expression rate of IGF-1R in endometria of canine pyometra compared with that in the normal endometria was extremely significant higher by immunohistochemistry and Western blotting (P<0.01).The correlation analysis of IGF-2 and IGF-1R in canine pyometra showed that the result of immunohistochemistry was low correlation (ｒ=0.423), the result of Western blotting was high correlation (ｒ=0.927).The data suggested that increased expression of IGF-2 and IGF-1R played an important role in occurrence of canine pyometra.

To investigate the effects of Toll-like receptors (TLRs) mediated signaling pathway in mouse macrophage RAW264.7 cells on Streptococcus equi subspecies equi (S.equi) infection, RAW264.7 cells were infected with S.equi for different time intervals.The mRNA expression levels of TLR1, TLR2, TLR6, myeloid differentiation factor 88 (MyD88), IL-1, IL-6, IL-10, IL-12 and TNF-α were detected by fluorescence quantitative Real-time PCR.The results showed that at 6 h post-infection, the expression levels of TLR1, TLR2, TLR6 and MyD88 in S.equi infected RAW264.7 cells had no significant difference compared with control group (P>0.05).At 12 h post-infection, the mRNA expression levels of TLR1, TLR2 and TLR6 had no obviously change(P>0.05), while MyD88 mRNA expression level was extremely significantly increased (P<0.01).At 24 h post-infection, the mRNA levels of TLR1, TLR2, TLR6 were extremely significantly increased (P<0.01), the mRNA level of MyD88 had no significant difference (P>0.05), meanwhile, compared with uninfected cells, IL-10 and IL-12 mRNA expression were extremely significant increased in S.equi infected RAW264.7 cells (P<0.01), but IL-1, IL-6 and TNF-α mRNA levels were extremely significantly decreased (P<0.01).These results indicated that the signaling pathway mediated by TLRs participated in the immune response against S.equi.

Tetraspanins with special four-times acrossing membrane structure can contact outer, intrinsic membrane and transmembrane proteins, which forms a channel to connect the internal and external membrane signal.CD81, as one member of Tetraspanins, has a variety of biological activities and is widely dispersed and participates in the organism physiological responses.CD81 plays an important role in many cell process, and is closely related to the pathogenesis of many human diseases, and so widely watched.But the molecular structure, space conformation and physiological mechanisms of CD81 are still not entirely clear.We still need to do further research and discussion, especially, the comprehensively understanding of precise role of CD81 in the process of HCV into a host cell, it is vital for clinical application.This article would focus on the research progresses of molecular structure and biochemical characteristics, cell fusion effect, the virus-identified mechanism, the role in the immune system and clinical application on CD81.In summary, clarifing the function and role of CD81 is important to study the clinical pathogenesis and treatment of various diseases.

The study was conducted to explore the possibility that CLPG (Callipyge) and MSTN (Myostatin) genes which could be the candidate genes of sheep growth traits, and investigate the molecular genetic markers related to sheep growth traits.133 (Austrilian White sheep×Dorper sheep×Hu sheep) hybid-sheep were chosen as subjects, the technology of direct sequencing of PCR products and PCR-RFLP were used to detect the single nucleotide polymorphism of CLPG and MSTN genes, then the association of the SNPs different genotypes and combined genotypes with sheep growth traits were analyzed by the GLM statistical model of SPSS 22.0.Sequencing results showed that the SNP of C/T which called C1 was detected at position 232 bp of the STS sequence in CLPG gene.The SNP of G/A which called M1 was detected in the 3'UTR of MSTN gene.PCR-RFLP analysis showed that two genotypes CC and CT were in C1 site, two genotypes GG and GA were in M1 site.Association analysis revealed that C1 site was significantly or extremely significantly associated with backfat thickness and loin muscle area (P<0.05;P<0.01), M1 site was significantly or extremely significantly associated with body weight, tube girth, backfat thickness and loin muscle area (P<0.05;P<0.01).Meanwhile, the combined genotype was extremely significantly associated with body weight, backfat thickness and loin muscle area (P<0.01).The conclusions indicated that SNPs and combined genotype of CLPG and MSTN genes had effects on growth traits in sheep.C1 and M1 sites could be considered as effective genetic markers for sheep growth traits.

To explore the functional genes affecting the growth and development of Xiang pig, a single nucleotide polymorphism (SNP) site rs80894395, located at intron 10 of polypeptide N-acetylgalactosaminyltransferase 2 (GALNT2) gene was screened out by Illumina Porcine SNP60K chip, which showed a weak association with the abdomen circumference of pig.Further, the genotypes of site rs80894395 in 520 female Xiang pigs were detected using allele-specific PCR(AS-PCR) method, taking 43 Yorkshire pigs and 36 Kele pigs as control groups.All of CC, CT and TT genotypes were detected from the three pig breeds.The dominant genotype of Xiang pig was CT genotype and it was positively weak-associated with its body measurement indexes including body weight, body length, and abdomen circumference.The CC genotype was dominant genotype in Yorkshire pig, and the frequency of T allele was extremely significant decreased compared with that of Xiang pig (P<0.01).There was no difference in the frequencies of CC and CT genotypes in Kele pig, and its allele frequencies was not significantly different compared with that of Xiang or Yorkshire pig breeds (P>0.05).A splice-site nearby site rs80894395 was found out using bioinformatics prediction software, which might affect the splicing process of GALNT2 gene.It suggested that site rs80894395 might have a connection with the growth and development of Xiang pig.

This experiment was designed to investigate the application of estrus synchronization and Ovsynch-timed artificial insemination(TAI) in dairy heifers.1 046 Holstein dairy heifers imported from Australia were randomly assigned into two groups.The heifers in the control group were checked for spontaneous heat, and the heifers in experimental group were treated by prostaglandin F_2α(PGF_2α) to induce heat synchronization or treated by different TAI programs (Ovsynch or Ovsynch+CIDR program).The estrus synchronization rate, 24 d-non-return rate after AI, first AI conception rate and 21-day pregnancy rate were analyzed in each group.The results showed that there was no significant difference in 24 d-non-return rate and the first AI conception rate between the experimental group heifers and control group (P>0.05), but the 21 d pregnancy rate of the experimental group was significantly higher than that of control group (53.1% and 35.3%;P<0.05).There was no significant difference of the estrus synchronization rate between different PG treatments (PG, PG-7-PG and PG-11-PG) (76.1%, 81.7% and 84.6%;P>0.05).There was no significant difference in 24 d-non-return rate, the first AI conception rate and 21 d pregnancy rate in Ovsynch (GPG) and Ovsynch+CIDR (GPG+CIDR) programs (P>0.05).The first AI conception rate of dairy heifers was significantly affected by different AI technicians (P<0.05), but there was no effect of frozen semen from different bulls on the first AI conception rate (P>0.05).Estrus synchronization and TAI programs could increase the submission rate as a result increasing the 21 d pregnancy rate by inducing dairy heifers to heat in a short time, and accelerate the efficiency of AI to reduce the feeding cost effectively.

In order to reveal the polymorphism of MyoD1 gene promoter in pigs, three pig breeds(wild boar Xiang pigs, commercial crossbred pigs and Guizhou Zongdi Hua pigs) were selected as the experimental subjects.By constructing DNA pool and using the technology of direct sequencing to screen the single nucleotide polymorphism on MyoD1 gene promoter region in three groups.Variety bioinformatics softwares were used to predict the core region of the promoter, transcription factors binding sites and CpG island.The results showed that the experiment had totally found three SNPs in MyoD1 gene, among these SNPs, A-39G was located in 5'UTR, T+150C and C+227G were located in exon 1.It was identified that various new transcription factors binding sites emerged and some significant transcription factors binding sites disappeared at the region nearby A-39G site.The software of CpG island searcher indicated that SNP mutation could change the range of CpG island and the content of GC.Therefore, A-39G site locating MyoD1 gene 5'UTR had an important effect on regulating the functional element of promoter.

To investigate the genetic diversity and genetic data of Gazella subgutturosa from Xinjiang Ebinur lake National Wetland Nature Reserves, the Ebinur lake goose antelope 35 feces samples were analyzed.Using noninvasive DNA technology, the mitochondrial DNA D-Loop 878 bp fragment was successfully amplified.Sequences results for GenBank database BLAST comparison and Clustal W and DNASP software analysis.The results showed that the sequences of the above fragments were successfully amplified, total 55 polymorphic loci were detected, which were not found insert and deficiency.This study found 15 variable sites, including 14 transversion sites and 1 top transposition.The average A, T, C and G contents in D-Loop were 28.03%, 31.57%, 24.84% and 15.56%, respectively.The contents of A+T (59.6%) were higher than G+C (40.4%).In addition, a total of 16 mtDNA haploid type were detected, haploid type polymorphism (h) was 0.886 ±0.037, nucleotide diversity degree (π) was 0.037±0.01528.The results showed that Ebinur lake Gazelle subgutturosa maintained high variation in mitochondrial D-Loop sequences.

The purpose of this study was to accelerate the application of molecular markers in genetic breeding and other aspects of Black quail to develop EST-SSR markers for Black quail.100 Black quail were randomly chosen and blood samples were collected to develop EST-SSR new markers and determined the genetic polymorphism using bioinformatics.The results showed that there were 6 EST-SSR markers in the Black quail, the number of observed alleles in EST-SSR was 2 to 6.The average PIC of 6 EST-SSR markers in the Back quail was 0.4962 and the average He was 0.5759.Markers P1 and P2 were highly polymorphic markers among 6 EST-SSR and the rest EST-SSR markers were moderately polymorphic markers.These developed markers could be used for genetic diversity analysis of Black quail and the EST-SSR markers had some relationship with the carbohydrate metabolism, synthesis of melanin in Black quail, but the mechanism needed the further analysis.

It is believed that active metabolism in male reproductive tract is contributed to production of ROS (reactive oxygen species).Low physiological ROS plays a fundamental role in mammalian reproduction, but the excessive accumulation of potentially damaging ROS can affect the function of sperm.Thus, it is important to maintain normal reproductive ability by a protective mechanism against oxidative stress.Here, we review the progress of antioxidant enzymes and redox systems in male reproductive systems, in order to provide references for physiology and pathology researches in male animals.

Here an improved HPLC-PAD method was developed and demonstrated suitable for studying the clinical residue elimination of oxytetracycline in milk from postpartum cows with uterine diseases after intrauterine treatment with oxytetracycline.In this study, 0.1 mol/L Na₂EDTA-Mcllvaine buffer solution was used to extract oxytetracycline from milk, n-hexane for degreasing, and a 3 CC Oasis HLB solid-phase extraction column for purification and concentration.A reversed phase chromatographic column Symmetry Shield RP C18 (250 mm×4.6 mm, 5 μm) with acetonitrile and 0.048% phosphoric acid solution (pH 2.5) as mobile phase by gradient eluting was applied to as the chromatographic separation condition.The results showed that the retention time of oxytetracycline was 6.5 min with a symmetrical and sharp peak, which was well separated from the impurity in the milk sample.Oxytetracycline standard solutions within the concentrations ranging from 5 to 2 000 μg/kg, had a good linearity between peak areas and the concentrations, R²=0.9999.The recoveries of oxytetracycline at three levels of 10, 20, 100 μg/kg were 87.9%, 90.5% and 87.8%, respectively, with the coefficient of variation ranged from 2.2% to 5.8% in intra-day and 4.0% to 5.1% in inter-day.The LOD was 5 μg/kg and the LOQ was 10 μg/kg.All of those parameters suggested that the optimized method was suitable for the study on the clinical residue elimination of oxytetracycline.29 postpartum cows with uterus disease were randomly divided into four different groups in terms of dosage, 2, 3, 4 and 5 g, and treated with oxytetracycline by intrauterine infusion administration, and the residues of oxytetracycline in milk were detected to study its clinical residue elimination.The results showed the oxytetracycline in milk fell to the MRL during 72 h after treatment at 4 g and below dose group.However, in 5 g dose group, residue levels of oxytetracycline in milk were above the MRL at 72 h after treatment.Therefore, 5 g and the higher were not recommended for intrauterine infusion administration.

In a certain area of Shandong province, Marek's disease (MD) occurred in diseased chickens that had been vaccinated by turkey herpesvirus.In order to isolate the virus strain and detect the virus pathogenicity, agar diffusion test, cell culture and indirect immunofluorescence assay (IFA) were used to isolate the Marek's virus from chicken's blood and feather marrow.The isolated strain was adapted to grow in chick embryo fibroblasts (CEF).Genes involved in pathogenesis of MDV, such as meq, pp38 and 132 bp repeat sequence were amplified by PCR.The obtained sequences were compared with that of standard strains published in GenBank by DNAStar software.The results showed that pp38 gene of the SDAU-1 shared homology from 100% with standard virulent sequence.Analysis of 132 bp repeat sequence and meq gene sequences of the viral genome showed that the isolated virus belongs to the highly virulent MDV strains.

To determine the effect of non viral nucleotide on the rescue of infectious PRRSV and optimize the reverse genetics system of PRRSV, two full-length PRRSV plasmids were constructed containing the CMV promoter, and inserted ribozyme sequences in the downstream of CMV promoter in this study.Two non viral nucleotide sequences GG and GCTAGC were inserted into a position between the ribozyme sequence and the 5' UTR of the PRRSV genome, respectively, resulting two full-length PRRSV infectious cDNA clones, Pgg-PRRSV and PNhe-PRRSV.The plasmids were transfected into BHK-21 cells.The M protein of PRRSV was detected by indirect immunofluorescence 48 h after transfection.Two infectious viruses, Pgg-PRRSV and PNhe-PRRSV, had been successfully generated.The titers of the two viruses were determined to be 3.0 and 1.25 TCID₅₀/mL, respectively.And the titer of the two rescued viruses reached a peak as high as 6.5 TCID₅₀/mL after passaged for 3 generations in Marc-145 cells.The results demonstrated that the presence of the two non viral nucleotides (GG) could enhance the rescue in a higher level.Nevertheless, the effect of the two nucleotide sequences was indistinguishable when the viruses passaged for 3 generations.Together, our result indicated that the additional nucleotide sequence was effective only in the initial stage of rescue.

The probiotics tested in the experiment were isolated from the intestinal of weaning piglets.The isolated probiotics and E.coli K88 were inoculated into the culture of intestinal porcine epithelial cell-1 (IPEC-1).The activity of lactate dehydrogenase (LDH) in the supernatant was measured after incubating for 2.5 h.At the same time, the probiotics and E.coli K88 were co-cultured in vitro, the number of E.coli K88 was counted and the probiotics which could be resistant to the E.coli K88 were selected 2.5 h later.The results showed that the Lactobacillus casei and Bacillus coagulans could significantly reduce LDH activity (P<0.05), decrease the damage of E.coli K88; Bacillus coagulans could inhibit the growth of E.coli K88.At the same time, Bacillus coagulans could resist high temperature, acid and bile salt.The results showed that Bacillus coagulans strains had great potential as the application of probiotics strains.The methods could be used as a model of screen probiotics which could inhibit the growth of E.coli K88 in vitro.

For developing immunoglobulin yolk powder products to prevent and treat the diarrheal piglets, and appling the advanced technology of egg yolk antibody to the husbandry and aquaculture, two types of compound immunoglobulin yolk powder were used to prevent and treat the diarrhea piglets which were challenged with ETEC, PEDV and TGEV.The creep feed added with 0.4% typeⅠimmunoglobulin yolk powder was provided to experimental piglets.There was no death after challenging the piglets with ETEC or virus, only 5 minor diarrhea in piglets in the early infection.As feeding continuing, the 5 piglets diarrhea quickly brought under control, conversely, the piglets in negative control group were totally dead.The immunoglobulin yolk powder type Ⅱ mixed with GNS at the rate of 1∶3 was used to cure the diarrhea piglets, 20 mL per piglet and 2 times a day.After curing for 3 days, the diarrhea was greatly improved, for 5 days the piglets diarrhea was basic recovery, and the survival rate could reach 84% to 88%.Dectected the infected of surviving piglets, compared to the drug treatment control group, prevention group had the lowest pathogen amounts, and the drug treatment control group had the most serious infected.

To identify the infection agents from Ningxia Hui Autonomous region, where feedlot cattle indicated bovine respiratory disease complex (BRDC), the M gene of the bovine parainfluenza virus type 3 was amplified by RT-PCR.The PCR product was ligated to pMD18-T vector and cloned to E.coli DH5α.The positive clones were sequenced and compared with the reference strains in GenBank by the molecular biology software.Sequence alignment results showed that a BPIV3 strain was isolated from the samples and named NX49, the M gene of NX49 included 1 056 nucleotides.Evolutionary analysis showed that the NX49 belonged to BPIV3 C genotype and shared 99.4% nucleotide identity with that of the SD0835 isolated in Shandong province.The characterization of the NX49 demonstrated that it was sensitive to temperature, acid and organic matter.The presence of Mg²⁺ showed no protection against the treatment at high temperature.The HA test suggested that the NX49 enables to agglutinate the guinea pig RBC at 4 ℃ and the titer was 1∶4.The study isolated a BPIV3 genotype C strain successfully, which facilitate the study of molecular evolution and epidemiology of BPIV3 in China.

1-day-old SPF chickens and commercial Jingfen chickens were vaccinated with IBD immune complex(IC) vaccine, NDV La Sota vaccine were inoculated simultaneously every one week and every two weeks.NDV La Sota immunization alone was as the control group.At the 2nd, 3rd, 4th and 5th week post inoculation, blood samples were taken and the ND HI antibody were tested.Experimental chickens were challenged with high virulent NDV at the 5th week post inoculation, the protective rate of each group was calculated.The results showed the ND HI antibody were not significant different in the combined immunization of IBD IC vaccine priming and NDV La Sota vaccine boost and NDV La Sota vaccine alone immunization (P>0.05).The results indicated that IBD IC vaccine has no immunosuppression on NDV La Sota vaccine in SPF chickens and commercial Jingfen chickens.

In this experiment, the primer pairs were designed, and polymerase chain reaction (PCR) technique to amplify the segments of the partial sex linked gene chromo-helicase-DNA-binding (CHD) to identificate pigeon's sex using feather pulps and blood.We found that two bands were demonstrated in all female egg-laying pigeons, and the sizes of them were 400 and 700 bp, respectively;In all male egg-laying pigeons, only one band appeared with length of 700 bp.100 unknown sex samples were successfully identified (52 males and 48 females).Based on the checking results between molecular electrophoresis of pigeons and the truly sex of maturity pigeons, we detected CHD gene in 2 928 early age pigeons using PCR amplification and electrophoresis in agarose gel.The PCR amplification results showed 1 517 males and 1 411 females.

This study was conducted to evaluate the nutritive value of unconventional feed in Anhui province and the surrounding areas using Cornell net carbohydrate and protein system (CNCPS).Total 21 unconventional feeds were collected from different sites in Anhui (Hefei, Lu'an, Anqing, Tongling, Bozhou and Suzhou citys), Henan (Xinyang city), Heilongjiang province as well as Gansu province, and the carbohydrate and the protein ingredients of feeds were calculated respectively using the provided formula in CNCPS.The results showed that, SCP/CP of peanut stalk from Suzhou was the highest (55.92%) and that of wheat stalk from Hefei was the lowest (6.85%); Rape stalk from Hefei had the highest value of ADL (23.16%) while straw from Hefei had the lowest value (4.14%).As to the true protein, shoot shell from Hefei was in the highest level (99.83%), while wheat stalk from Suzhou had the lowest value (44.96%); In terms of unavailable fiber content, oray from Suzhou was the highest (47.94%), and takenoko pocket from Hefei was the lowest (10.74%).The experimental results showed that the CNCPS could comprehensively evaluate the nutritive value of unconventional feed and reflecte the pros and cons of unconventional feed in Anhui province and the surrounding areas.It was suggested that the method of CNCPS could be used in the evaluation of feedstuff in Anhui province.