The aim of the study was to analyze the intramuscular fat (IMF) content, fatty acid composition and the related traits of Lulai Black pigs, and to provide data for understanding germplasm characteristics of Lulai Black pigs. In the study, we measured the backfat thickness, free water, IMF content and fatty acid composition of 487 Lulai Black pigs, analyzed the effects of sex and body weight on these traits, and also performed correlation analyses among different traits. The result indicated that Lulai Black pigs had high capability in fat deposition, and the average IMF content was 5.25%. But the population variation of IMF content was rather large, with variation coefficient of 66.04%, suggesting efforts should be made to strengthen the breeding and improve group uniformity. Except cis-11-eicosenoic acid, the sex effects on the other traits were all insignificant (P>0.05). The slaughter weight had significant effect on backfat thickness at the level of 0.01 (P<0.01), on IMF content at the level of 0.05 (P<0.05), and no significant effect on free water and fatty acids composition(P>0.05). Among these traits, the correlation between IMF content and free water was the highest, with the correlation coefficient of -0.86. Additionally, the regression equation between them had high adjusted R² (0.929 4), and could be used as a model to predict IMF content based on free water. Moreover, we also observed high correlation relationships among backfat thicknesses at different parts, between IMF content and some fatty acids, and among some fatty acids. This study contributes to further understand germplasm characteristics of Lulai Black pigs, which would be beneficial to their meat quality breeding in the future.

How to use less and optimized genetic markers to achieve convenient and efficient parentage determination for cows was our research purpose, so as to effectively apply them into paternity test and pedigree construction. Eighteen microsatellite markers with high polymorphisms in cattle recommended on the web of ISAG were selected in this study, 8 markers that could be efficiently amplified were genotyped in 300 Chinese Holstein cows from the Jiabao Farm, based on fluorescent primer and automatic sequencing techniques. The results of paternity testing efficiency showed that the average of allele number, polymorphism information content (PIC), observed heterozygosity (Ho), and expected heterozygosity (He) were 14.63, 0.747, 0.718 and 0.773, respectively. In 3 different conditions of known and/or unknown parent-offspring, their combined probabilities of exclusion (CPEs) were 0.990, 0.999 and 0.999, respectively. The results of paternity testing from 8 father-son showed that the 8 markers could successively be used to identify correct parentage and eliminate error records in the pedigree. Combined probability of exclusion analysis showed that when the number of microsatellite markers increased to 4 (TGLA227, TGLA122, BMC1207, BM103), all CPEs under 3 different situations were 0.949 or higher, which met the requirements of paternity tests in individual case. While, the results of molecular pedigree tree building at population level revealed that when the number of microsatellite sites increased to 6 (TGLA227, TGLA122, BMC1207, BM103, INRA037 and INRA134), inbreeding coefficient and total inbreeding coefficient within population were significantly increased, suggested that the 6 markers could efficiently be applied for pedigree tree construction and/or paternity testing for Chinese Holstein cows population(<200 individuals).

The aims of this study were to determine the genetic diversity of the GH, GHR and GHSR genes in Maiwa yak, to reveal the association between different genotypes and growth traits, to provide the theoretical foundation for the expression regulation of the candidate gene in yak and to look for the molecular marker that can be used for the assistant selection for genetic breeding. The DNA pool technology, PCR-RFLP and the direct sequencing were adopted to study the genetic polymorphism of the GH, GHR and GHSR genes in Maiwa yak and to analyze the association between the polymorphism of the candidate genes and the growth traits, such as withers height, body length, chest circumference, cannon circumference and body weight. The results showed that: 1)The GH, GHR and GHSR genes of the Maiwa yak were all with the polymorphism, among which the GH gene had 2 SNP sites(A757G and T949C). Three mutation sites are found on the GHR gene(T2416C, T3490C and A7500G). T1387C and T3006C mutations existed on the GHSR gene. 2)The fitness test indicated that the A7500G of the GHR gene in the pink lip group and the T3006C of the GHSR gene in the pure black group all deviated from the Hardy-Weinberg equilibrium while the other sites all conformed to the Hardy-Weinberg equilibrium. 3)The different significance test indicated that the T2416C, T3490C and A7500G of the GHR gene were significantly related to the cannon circumference of the Maiwa yak (P<0.01), while the T1387C of the GHSR gene was significantly related to body weight (P<0.05). The results indicate that the GH, GHR and GHSR genes of the Maiwa yak are all with the genetic polymorphism, and it is deduced that the T2416C, T3490C and A7500G of the GHR gene as well as the T1387C of the GHSR gene may be the major effect genes influencing the cannon circumference and body weight of the Maiwa yak or the loci may be closely linked to the major effect genes and they can be regarded as the genetic markers for the assistant selection.

To screen the differentially expressed proteins in different parts of sika deer velvet antler，the parts of sika deer velvet antlers of Lapian, Xuepian and Gupian were selected as experimental material.The differentially expressed proteins were identified by two-dimensional gel electrophoresis (2-DE) and MALDI-TOF-TOF-MS, and analyzed using bioinformatics methods. The result showed that 37 differentially expressed proteins were detected, which were involved in the biological process of multicellular organismal process, detoxification, cellular component organization or biogenesis, cell aggregation, localization, response to stimulus, biological adhesion, developmental process, single-organism process and immune system. There were 18, 5 and 14 up-regulated proteins in the Lapian, Xuepian, Gupian，respectively. We found that EFB1 and CRABP1 might play vital role in the process of velvet antler accelerated growth, and APOA1, FABP4, TTR might play vital role in the process of velvet antler ossification. Taken together, the result provide a basis for further research on the molecular mechanisms involved in the accelerated growth and ossification of deer velvet antler.

To further explore the theoretical basis of the microRNAs in regulating the non-breeding season estrous in sheep, characterization of ovarian microRNAs were constructed and analyzed in estrous and anestrous of non-breeding season induced by nutrition by Solexa sequencing. In this study, the Kazakh sheep with different nutrition levels were used as the research object. In non-breeding season,3 estrous (EN) and 3 anestrous (AN) sheep were slaughtered with their ovary tissue(O) collected, respectively. Two miRNA libraries were constructed and analyzed by the bioinformatics methods. The real-time quantitative PCR (qRT-PCR) was used for validating the differentially expressed miRNAs betweent the 2 periods in ovary. The results showed that the clean reads of 8 665 978 (OEN) and 9 071 102 (OAN) were obtained in the two libraries, and most of the small RNA sequences were between 21 and 23 nt. Compared the two ovary (OEN vs OAN) libaries, the 9 known and 104 novel miRNAs had significant expression difference (P<0.01). The expression level of 6 randomly selected miRNAs by qRT-PCR was the same as the result by Solexa sequencing. KEGG pathway analysis showed that these target genes of difference miRNAs were primarily involved in nutrition regulatory related pathways, such as glycine, serine and threonine metabolism, amino sugar and nucleotide sugar metabolism, D-arginine and D-ornithine metabolism, glycerol phospholipid metabolism, and so on. The differentially expressed miRNAs may play an important role in non-breeding season estrous of sheep through nutrition metabolism pathway by the analysis of predicted target and enriched pathway analysis.

In order to study function of ovine NYD-SP27,BHK-21 cell line stably expressing ovine NYD-SP27 was constructed and relative phenotypic characteristics were analyzed, which lay a foundation for function analysis of ovine NYD-SP27 gene. The ovine NYD-SP27 gene was cloned and inserted into eukaryotic expressing vector pDsRed1-C1. To co-express Red fluorescent protein gene and NYD-SP27 gene, both of them were linked with porcine teschovirus-1 2A peptide (P2A). The recombinant plasmid pDsRed-P2A-Flag-NYDSP was transfected into BHK-21 cells and selected with G418. Positive cells were identified using RT-PCR, indirect immunofluorescence assay (IFA) and Western blot. The RT-PCR results indicated that NYD-SP27 gene was stably integrated into genome of BHK-21 cells. The NYD-SP27 protein could be expressed in BHK-21 cell line and the molecular weight was approximately 63 ku, which suggested that the self-cleavage of P2A realized during the translation process of NYD-SP27 protein and Red fluorescent protein. The eukaryotic expression vectors were confirmed successfully and the BHK-21 cell lines stably expressing ovine NYD-SP27 was successfully established in this study.

This study explored the influence of dilution and low-temperature preservation on chicken semen quality and fertility, aiming at optimizing the short-term preservation conditions for the chicken semen. The semen samples collected from 30 Beijing-You chickens were mixed gently and divided into 4 groups: Group A: original semen as a control group; Group B: diluted with BPSE (1:1); Group C:diluted with BPSE (1:1) after removing seminal plasma; Group D: further diluted with BPSE (1:1)based on group C.Semen of each group was further divided into two proportions. One was used for the semen quality estimation and artificial insemination (AI) and the other was stored at 4℃ for semen quality evaluation at 8, 16, and 24 hours post storage and also AI at 24 hours post storage. For each group, around 120 eggs were collected for hatching to evaluate the fertility and hatching performance.As a result of different dilution groups, sperm motility of group C and D were lower than that of group A and B. Group A, B, and C did not show any difference in fertility or hatchability. The group D, however, showed significantly lower fertility and hatchability (P<0.01). The results of 4℃ storage revealed that, sperm motility of 4 groups decreased significantly (P<0.01). Group C and D decreased more quickly than group A and B. The sperm deformity were also increased during the storage (P<0.05), which were mainly characterized by swelling heads, bending necks, and cracking tails. Fertility and hatchability of group A, B, and C decreased significantly after 24 hours of storage at 4℃ (P<0.05). The fertility and hatchability of group D after storage were 71% and 82%, respectively, which were as high as those of before storage. The results in the present study indicated that, firstly, dilution of semen is feasible to increase the AI efficiency without decreasing the fertility; secondly, removing semen plasma and diluting before 4℃ storage may maintain the sperm motility and guarantee higher fertility.

This study was aimed to elucidate the possible functions of sperm adhesion molecule 1 (SPAM1) on the sperm/cumulus interaction during fertilization and explore its potential mechanism. Genomic DNA was extracted from the tails of mice and genotype for Spam1 null mutation was confirmed by PCR analysis. The protein of the screened wild type (WT) and Spam1-deficient (KO) sperm were extracted and confirmed by Western blot, and the hyaluronidase activity was measured by the colorimetric method. The epididymal sperm were capacitated by incubation for 2 h and subjected to the assays of sperm motility, sperm/cumulus interaction or in vitro fertilization (IVF). These results showed that the SPAM1 protein was readily absent in the KO sperm, and the hyaluronic acid-hydrolyzing enzymes activity was significantly reduced in the KO sperm, compared to that of the WT sperm (P<0.01). There were no significant differences in sperm motility between the KO and WT groups after capacitation (P>0.05). However, the loss of SPAM1 significantly influenced the percentage of acrosome-reacted sperm in the extracellular matrix of the cumulus (P<0.01), resulted in a markedly decrease in sperm entry into and/or penetration of the cumulus mass, only a few sperm succeeded in access to the surface of the oocyte zona pellucida (ZP) and contrastly a remarkably great deal of sperm accumulated on the surface or outer edge of the cumulus (P<0.01). In addition, sperm lacking SPAM1 caused the delayed cumulus disperse and fertilization at IVF 2 h (P<0.05). Therefore, mouse sperm hyaluronidase SPAM1 may be associated with the acrosome reaction and affect the sperm/cumulus interaction, which reveal that SPAM1 may play other possible roles in sperm access to the oocyte ZP except the hyaluronan-degrading activity.

The aim of the present study was to evaluate the effects of low protein level diets with essential amino acids and cysteamine (CS) supplementation on the meat quality and related genes expression of growing pigs. A total of 120 barrows ((42.18±0.70) kg) were arranged in a 2×2 factorial arrangement in 4 treatments with 5 replicates of 6 each. The main effects were crude protein levels (16% and 12%) and CS supplemental levels (0 and 100 mg·kg^-1). The duration of the experiment was 31 days. The results showed that the low protein level diets supplemented with essential amino acids decreased the shear force value (P<0.05) and increased the intramuscular fat content of Longissimus dorsi (P<0.05). The low protein level diets supplemented with essential amino acids increased mRNA abundance of sterol regulatory element binding protein 1c (SREBP-1c), fatty acid synthase (FAS), stearoyl-CoA desaturase (SCD) and μ-calpain in muscle(P<0.05), decreased the mRNA abundance of hormone-sensitive lipase (HSL), carnitine palmitoyl transferase-1 (CPT-1) and calpastatin(P<0.05). CS supplementation decreased the mRNA abundance of calpastatin(P<0.05). No significant interaction effect between dietary protein levels and CS supplementation on the meat quality of growing pigs was observed (P>0.05). The results indicate that low protein level diets supplemented with essential amino acids increase the intramuscular fat content and then improve meat tenderness of Longissimus dorsi in growing pigs, possibly by up-regulating lipogenic genes expression and down-regulating lipolytic genes expression; however, CS supplementation did not show any significant influence on meat quality.

The purpose of the experiment was to investigate the effects of different humidity on thermoregulation and blood parameters of broilers, and to provide data support for the reasonable regulation of the humidity in the poultry house. Two hundred and seventy 1-day-old AA healthy broilers were randomly divided into 3 groups with 6 replicates of 15 broilers per replicate.The treatments were normal control group (60% RH), low humidity group (30% RH) and high humidity group (90% RH). The experiment was conducted in an artificial climate chamber of state key laboratory of animal nutrition and lasted for 42 d. The rectal temperature, respiratory frequency, blood indices and immune organ indices were measured at the 21 and 42 days of age, respectively. The results showed that: 1) Compared with the control group, at the 21 days of age, rectal temperature of low humidity group and high humidity group was significantly increased (P<0.05); the respiratory frequency of the low humidity group and high humidity group had a trend to rise(P<0.1); Rectal temperature of low humidity group significantly decreased at 42 days of age (P<0.05). 2) At 21 days of age, compared with the control group, low humidity and high humidity significantly reduced the number of white blood cells and lymphocyte count (P<0.05); The number of platelets were significantly higher in low humidity group than that in high humidity group(P<0.05). At 42 days of age, the number of white blood cells were significantly lower in low humidity and high humidity groups than that in the control group (P<0.05). 3) At 21 days of age, the serum urea nitrogen content in the low humidity group and the high humidity group was significantly higher than that in the control group (P<0.05), while the total cholesterol content in the high humidity group was significantly lower than that in the control group and low humidity group (P<0.05). At 42 days of age, the serum total protein, uric acid and urea nitrogen contents in the low humidity group and high humidity group were higher than those in the control group, while the content of serum albumin, lysozyme, total cholesterol and triglyceride contents were lower than the control group. 4) The spleen index of broilers in low humidity group was significantly lower than that of high humidity group at 21 days of age (P<0.05). Compared with the control group, at the age of 42 days, the content of IgA in low humidity group and the content of IgM in high humidity group were significantly lower (P<0.05). In conclusion, long-term low humidity or high humidity affected the rectal temperature and respiratory frequency of broilers, changed the number of white blood cells, total cholesterol and blood urea nitrogen content and other blood physiological and biochemical parameters, reduced immune function, and it was not beneficial to healthy growth of broilers.

This experiment aimed to investigate the effects of Lys, Met, Thr and Trp in starters on growth performance, nitrogen utilization and serum indices of weaned lambs, and to determine limiting sequence and proportion of amino acids by partial deduction method. One hundred healthy male Hu lambs at 50 days old with an average weight of 11.00 kg were randomly divided into 5 groups and each group had 4 replicates with 5 lambs per replicate according to single factor design. The control group was fed starter with balanced amino acid (PC) with Lys (1.16%), Met (0.38%), Thr (0.58%) and Trp (0.16%), respectively. The 4 experimental groups were fed the starter deducting Lys, Met, Thr and Trp at 30%, respectively compared with control group (PD-Lys, PD-Met, PD-Thr and PD-Trp group). The experiment lasted for 70 days; During 80-90 and 110-120 days old, nitrogen balance of lambs was determined. The results showed as follows: 1) The BW, ADG and F/G in PD-Met group were significantly lower than that in PC, PD-Thr and PD-Trp groups (P<0.05) on 120 days old. 2) Urinary nitrogen emission in PD-Lys and PD-Met groups was significantly higher than that in PC (P<0.05) during 80-90 days old. Nitrogen deposition, nitrogen retention ratio and nitrogen apparent biological value in PD-Met group were significantly lower than that in PC group (P<0.05) during 80-90 days old, and nitrogen deposition, nitrogen retention ratio, nitrogen apparent digestibility and nitrogen apparent biological value in PD-Met group were significantly lower than that in PC group(P<0.05) during 110-120 days old. 3) The limiting sequence of 4 amino acids were Met > Lys > Thr > Trp and Met > Lys > Trp > Thr from 80 to 90 days old and from 110 to 120 days old, respectively, the proper ratio of Lys, Met, Thr and Trp was 100:37:45:12 and 100:41:39:12 when obtaining the maximum NR. 4) Glucose concentration in PD-Met group was significantly higher than that in PC (P<0.05) on 90 days old. Serum total protein, globulin and cholesterol in PD-Met group was significantly lower than that in PC (P<0.05) on 120 days old. In conclusion, the proper ratios of Lys, Met, Thr and Trp in starter of weaned lambs during 60-120 days old was 100:(37-41):(39-45):12. The limiting sequence were Met > Lys > Thr > Trp(80-90 days old) and Met > Lys > Trp > Thr(110-120 days old), respectively.

This study was conducted to evaluate the accuracy of 6 classic models to predict the enteric methane emissions in beef cattle and analyze the factors affecting predictive accuracy of models. Seventeen Xiangzhong Black cattle were selected in the Hunan Wangcheng beef farm, to measure body weight, dry matter and nutrients intake, enteric methane emissions in two stages. The selected 6 classic models included: equations developed by dry matter intake (DMI), such as Model 1 [CH₄(MJ·d^-1)=1.246×DMI(kg·d^-1)+0.996] and Model 2 [CH₄(MJ·d^-1)=-2.07+2.636×DMI(kg·d^-1)-0.105×DMI²(kg·d^-1)]; equations developed by fiber intake, such as Model 3 [CH₄(MJ·d^-1)=5.58+0.848×NDF(kg·d^-1)] and Model 4 [CH₄(MJ·d^-1)=3.41+0.520×DMI(kg·d^-1)-0.996×ADF(kg·d^-1)+1.15×NDF(kg·d^-1)]; equations developed by gross energy intake (GEI), such as Model 5 [CH₄(MJ·d^-1)=0.065×GEI(MJ·d^-1)] and Model 6 [CH₄(MJ·d^-1)=0.081×GEI(MJ·d^-1)-0.024]. Mean Squared Prediction Error (MSPE) and Consistent Correlation Coefficient (CCC) methods were employed to evaluate the prediction accuracy, and factors influencing the accuracy were also discussed. The result showed that: Model 5 (CCC=0.86) had the highest prediction accuracy among 6 models, next for Model 1 (CCC=0.74) and 6 (CCC=0.79), the lowest for Model 2 (CCC=0.66), 3 (CCC=0.22) and 4 (CCC=0.54). Model 1 and 2 had overall bias of 48.8% and 70.3%, respectively. Model 3 had greatest deviation of regression slope from unity (47.6%), while Model 4 had 28.6% deviation of regression slope from unity and 29.2% overall bias. The current results indicate that Model 5 developed by GEI based on world-wide data by IPCC (2006) Tier 2 is the most accurate model to predict the enteric methane emissions in beef cattle.

Bordetella avium (B. avium), one of the members of Bordetella genus, Alcaligenes Branch, mainly causes birds cough and other respiratory symptoms. To explore its invasive procedure by respiratory tract and regular pattern of lesions development, SPF chicks were challenged by nasal infection with a screened virulent B. avium strain. After infection, bacteria quantity in trachea and lungs were detected at different times. Tracheas colonization and lesions were observed by SEM. Lungs colonization pattern were detected by indirect immunoenzymatic staining. Histopathological changes in lung and bronchus were detected by pathological examination. It was found that bacterial infection of the trachea was in larger quantities, and only a small amount in the lungs at the beginning. And then the amount of bacteria in trachea and lungs showed a clear upward trend. B. avium had colonized on the cilia of trachea, and cilia began to fall off on 1 d after infection. To 5 d after infection, tracheal mucosal surface mottled, cilia fell off in a large area, and even holes appeared. Lungs colonization were detected on 2 d after infection, and B. avium were mainly distributed in the cytoplasm of lung housing and respiratory capillary walls. Positive signals were increasing with the extension of time. Pathological changes in the lungs and bronchi appeared on 5 d after infection, mainly showed congestion. To 10 d after infection, a large number of cell necrosis appeared. In conclusion, this B. avium isolate has a strong tropism on chick respiratory tract. Especially it has priority colonization on cilia of tracheal epithelial cells, and subsequently causes a series of histopathological changes in the trachea, bronchi and lungs.

The aim of the present study was to isolate and characterize the phage that could lyse Enterococcus faecium from fecal samples of dairy cattle in Shihezi, Xinjiang, and provide the basic data for the phage therapy as well as prevention. The phage was isolated and purified from dairy cattle fecal samples by double-layer agar plate method. And its plaque characteristics was observed. The purified phage was concentrated, negatively stained with phosphotungstic acid and observed by transmission electron microscopy. The genome of the isolated phage was sequenced. Its genetic and evolutional history were analyzed. The host range, optimal multiplicity of infection (MOI), one-step growth curve, temperature, and pH stability of the phage were investigated by double-layer agar plate methods. A lytic phage of Enterococcus faecium was isolated and purified, and named vB_EfaM_XJ3. The phage plaques were circular, clear and transparent with smooth edge, and its diameter was 1.2-1.5 mm. The electron microscope observation showed that the phage particles, whose head was twenty-sided, head length was about 53.15 nm, tail length was about 14.42 nm×199.62 nm. The genome of the phage was comprised of double strand DNA, the size was 157.189 kb with GC content of 50.27%. The homology of amino acid sequence of phage vB_EfaM_XJ3 with Salmonella phage SKML-39 was 98%. The study of biological characteristics showed that the vB_EfaM_XJ3 only crack its host bacteria at present. The optimal MOI was 0.001, the incubation period was 15 min, the burst period was 175 min and the burst size was 84. The phage could withstand the temperature up to 50℃ and keep stable titer under pH 5.0-11.0. A lytic phage of Enterococcus faecium was isolated and identified. Its biological characteristics and genetic were analyzed. The results laid the foundation for the further research and application of the antibacterial agent for Salmonella and Enterococcus faecium.

In order to investigate whether the genetic variation in Psoroptes cuniculi be affected by species of rabbit, temperature zone and geographical origin, the complete sequence of mitochondrial cytochrome b (cytb) gene of 68 P. cuniculi samples from 6 different provinces, including isolates from rex-rabbit (Shaanxi, Hebei, Henan and Hubei) and meat-type rabbit (Shandong and Sichuan), were amplified and sequenced for the first time. Then, we analyzed the genetic variability of those P. cuniculi isolates, inferred by cytb sequences. The full length of cytb gene from 68 P. cuniculi isolates were all 1 100 bp. These sequences contained 96 variable sites and 46 haplotypes. The values of haplotypes diversity (Hd=0.959 2) and nucleotide diversity (π=0.012 0) in overall China population indicated a high genetic diversity. The negative value of neutrality tests (Tajima's D=-0.444 01，Fu's Fs=-1.454 25)and the phenomenon of mismatch distribution curve with multiple peak suggested that P. cuniculi population in China occurred expansion event in the past. There was a certain genetic differentiation (Fst=0.262 2) and low gene flow (Nm=0.7) between different geographic populations. An analysis of molecular variance (AMOVA) showed that the genetic differentiation mainly occurred within the population. Haplotype network and NJ phylogenetic tree were both showed that P. cuniculi isolates from China did not cluster according to the species of rabbit, temperature zone and geographical origin. The results indicated that P. cuniculi population from 6 different provinces of China had a high level of genetic diversity and a certain genetic differentiation, however, the genetic structure based on the species of rabbit, temperature zone and geographical origin has not found.

The gene of NADH:ubiquinone oxidoreductase domain containing protein (NDUDC) of Haemonchus contortus was selected to clone, express, and the functions of this protein were studied in this paper. The gene of NDUDC was cloned by RT-PCR, then expression vector was constructed to obtain the recombinant protein. The antigenicity of the protein was analyzed by Western blot. Peripheral blood mononuclear cells (PBMCs) were separated from goat and cultured in vitro with the recombinant NDUDC to check the binding of the protein to PBMCs by indirect immunofluorescence assay. Different concentrations (0, 10, 20, 40 μg·mL^-1) of the recombinant protein were used to stimulate PBMCs, and the effects of cell proliferation, cell migration and NO secretion were tested. The results showed that the recombinant protein NDUDC could stimulate cell proliferation effectively, also significantly enhance cell migration and increase the secretion of NO. These results indicate that NDUDC protein has strong antigenicity, and can stimulate cells to exert immune function.

This study aimed to investigate regulation of calcium dependent protein kinase 9 of Toxoplasma gondii to the macrophages of mouse. The laser confocal microscopy was utilized to check whether CDPK9 was able to bind to Ana-1 macrophages. After co-incubation with CDPK9, the proliferation of macrophages was checked by cell counting kit-8 (CCK-8). Then apoptosis and phagocytosis of macrophages were calculated by flow cytometry. NO production of cells was evaluated by Total Nitric Oxide Assay kit and expressions of TNF-α, TGF-β1, IL-10 and IL-1β were detected by ELISA research reagent. The pictures of laser confocal microscopy showed that CDPK9 could bind to macrophages. After co-incubation with CDPK9, the proliferation of macrophages was suppressed. Moreover, both apoptosis and phagocytosis rates of cells were increased. However, NO production was up-regulated by CDPK9. Concerning cytokines, production of IL-1β and TNF-α was increased, while the production of TGF-β1 and IL-10 was decreased. Our study demonstrated that CDPK9 was able to bind to macrophages and exert its effects on the activity and functions of Ana-1 macrophages via different pathways.

The aim of the present study was to investigate the toxic effects and mechanism of salinomycin on chicken cardiomyocytes. The cardiomyocytes were purified and cultured through differential adhesion and inhibition of 5-bromodeoxyuridine (Brdu). The purity of cardiomyocytes was identified by α-actinin immunofluorescence staining. The chicken cardiomyocytes were exposed to 1, 5, 10, 20 and 50 μg·mL^-1 of salinomycin for 24-72 h. Cell vialibity and cell morphological changes were measured by MTT and microscopy. Membrane integrity (LDH assay) and creatine kinase (CK) activity were determined by colorimetric method. Apoptosis and mitochondrial membrane potential (MMP) were analyzed using flow cytometry. The ultrastructural structures of cells were observed and imaged through transmission electron microscopy and the levels of intracellular ROS were observed by fluorescence microscope. The activities of Caspase-3, Caspase-8 and Caspase-9 were determined by colorimetry. The transcription of several apoptosis-related genes, Bcl-2, Bax, Cytochrome-C (Cyt-C), Caspase-3, Caspase-8 and Caspase-9 mRNA were determined by qRT-PCR. Our result showed that cardiomyocytes purity was more than 90%. Compared with control group, salinomycin significantly inhibited cell growth in a concentration- and time-dependent manner (P<0.01). The release of LDH and CK activity in cardiomyocytes exposed to salinomycin were significantly increased, respectively (P<0.01). Salinomycin induced apoptosis and disrupted mitochondrial function by decreasing mitochondrial membrane potential (MMP) in a concentration-dependent manner. We also found that mitochondrias of cardiomyocytes were severely swollen, vacuoles and the cristae dissolved or even disappeared through transmission electron microscopy. Salinomycin induced the oxidative stress of cardiomyocytes, the levels of intracellular ROS were increased. Activities of Caspase-3, Caspase-8 and Caspase-9 were increased significantly (P<0.01). The relative transcription of Bax, Caspase-3, Caspase-8, Caspase-9 and Cyt-C mRNA were all upregulated (P<0.01), however, the transcription of Bcl-2 mRNA was downregulated (P<0.01). In summary, salinomycin caused chicken cardiomyocytes death through apoptosis, which might involve mitochondial pathway and death-receptor pathway.

This study was conducted to evaluate the effects of dietary glutamine (Gln) supplementation on the immunity in the lipopolysaccharide (LPS)-challenged weaned piglets. A total of twenty-four healthy piglets were weaned at 28 days, and randomly assigned into three treatment groups (8 pigs per treatment): (1)control group (basal diet); (2) LPS group (basal diet); (3)Gln group (basal diet+1.0% glutamine).The experiment lasted for 30 days. On day 22, 25, 28 and 30 of the trial, piglets in the LPS and Gln groups were administered intraperitoneally with LPS (100 μg·kg^-1 body weight), whereas pigs in the control group were injected intraperitoneally with the same volume of sterile saline. After the whole feeding period, blood samples were taken from jugular vein, then pigs were bled by exsanguination to collect samples, which were used to determine the immune parameters. The results showed that, compared with the control group, the concentration of serum IgM in the Gln and LPS groups remarkably increased by 40.00% and 62.86% (P<0.05), respectively. After LPS challenge, the concentration of serum interleukin-8 (IL-8) in the LPS group was significantly decreased by 7.11% (P<0.05), but there was no difference between the Gln group and control group (P>0.05). No matter with LPS challenge or not, Gln supplementation was remarkably increased serum interleukin-17 (IL-17) content(P<0.05).Compared with the control group, in the duodenum, the mRNA expression of IL-8 in LPS group and Gln group was remarkably decreased by 35.06% (P<0.05) and 44.16% (P<0.05). The mRNA expression of IL-12β in the jejunum and ileum of Gln group were higher than that in the LPS group (P>0.05). In the duodenum and jejunum, there were differences in the mRNA expression of IL-17α among the three groups (P<0.05), and the expression of IL-17α in the Gln group was the greatest in the duodenum and ileum; in the jejunum, the mRNA expression of IL-17α in LPS group and Gln group was remarkably decreased by 58.16% (P<0.05) and 56.07% (P<0.05). Collectively, dietary supplemented with 1% Gln could improve the immunity of weaned piglets challenged with LPS to some degree, thereby alleviating immunological stress.

Zearalenone(ZEN) is one of the main mycotoxin endangering animal health. The aim of this research was to screen bacteria with the ability to degrade ZEN efficiently, and detect their degradation characteristics and toxicity. ZEN was used as the sole carbon source for the preliminary screening. And then the strains fermentation broth degradation rate was as the index for the second selection. The 16S rRNA sequence analysis was used to identify the strain with the highest ZEN degradation rate. The ZEN degradation characteristics of the obtained strain was studied, and the activity of the ZEN degradation product was tested in MCF-7 cells. Forty strains were screened out in the preliminary experiment, and strain H6 with the highest ZEN degradation rate was selected after the secondary screening. Strain H6 was identified as the Bacillus amyloliquefaciens by 16S rRNA sequence analysis. At 37℃, 180 r·min^-1, 72 h, the degradation rates of strain H6 fermentation liquor to 1 and 20 μg·mL^-1 ZEN were 95.6% and 85.8%, respectively. Among that, the degradation rates of the culture supernatant and cells to 1 μg·mL^-1 ZEN were 68.93% and 28.70%, respectively. The ZEN degradation activity of strain H6 culture supernatant was significantly decreased after treated with proteinase K, proteinase K combined with SDS and heating, respectively. Compared with the control group, the proliferation rate of MCF-7 cell was significantly reduced when treated with ZEN degradation product. In conclusion, strain H6 with the highest ZEN degradation rate was identified as Bacillus amyloliquefaciens by 16S rRNA sequence analysis. The extracellular enzymes secreted by strain H6 could degrade ZEN, and significantly reduce the estrogen activity.

The aim of the present study was to explore the impact of high temperature on Huainan partridge chickens’ blood-brain barrier. One hundred and twenty 107-day old Huainan partridge chickens were randomly divided into four groups: chickens in group Ⅰ and Ⅱ were raised at normal room temperature (21-23℃, RH 60%-70%), which in Group Ⅲ and Ⅳ subjected to hyperthermia (34-37℃, RH 45%-50%) for 3 hours daily. The treatment lasted for two weeks. Five chickens were randomly selected from each group on day 7 and 14, respectively, chickens in group Ⅱ and Ⅳ were received a single intravenous dose of Gamma-aminobutyric acid (GABA) (8.87 mg·kg^-1 body weight), chickens in group Ⅰ and Ⅲ received same dose of saline, brain tissue samples were collected at time 0.5 h and analyzed GABA using ELISA assay. The brain tissue samples were collected on day 14, and the ultrastructure of the blood brain barrier were detected using transmission electron microscopy (TEM), the mRNA and protein expression of ZO-1 and Occludin in the brain tissues were detected by qRT-PCR and Western blot. The results showed that, On day 7 and 14, no significant differences of brain GABA concentration were found between group Ⅰ and Ⅱ (P>0.05). Brain GABA concentration of chicks in Group Ⅳ were higher than that in group Ⅲ on day 7 and 14, and the differences were very significant on day 7 (P<0.01). Compared with normal temperature, high temperature destroyed the blood-brain barrier structure of chickens. Mitochondrial cristae of Cerebral cortical capillary endothelial cells were damaged, some broken, some degeneration. Capillary endothelial cell tight junctions gap widened. Microvascular basement membrane became loose and thick. The mRNA transcription of ZO-1 and Occludin were very significantly (P<0.01) and significantly lower (P<0.05) than those in normal temperature group, respectively. The protein relative expression of ZO-1 and Occludin were very significantly (P<0.01) lower on days 14 than those in normal temperature group. High temperature destroyed the chicken blood-brain barrier, ZO-1 and Occludin down-regulation may be one of the mediators of it.

The RAW264.7 cells activation of Codonopsis pilosula polysaccharide (CPPS) was investigated through the detection of cell proliferation, TNF-α and IL-6 secretion and mRNA transcription, NF-кB signaling pathway activation. CPPS were obtained by water extraction and ethanol precipitation, then proteins were removed by Sevage method and the content was determined by Phenol-Sulfuric acid method. The proliferation of RAW264.7 cells incubated with different concentrations CPPS (2-fold serially diluted from 31.25 to 4 000 μg·mL^-1) was detected by MTT method. RAW264.7 cells incubated with various concentrations of CPPS (25, 50, 100, 150 and 200 μg·mL^-1), TNF-α and IL-6 secretion were measured using ELISA kits; The TNF-α and IL-6 mRNA transcription were evaluated by RT-PCR. As well, expression of NF-кB p65 and IкB-α was determined by Western blot. The content of CPPS was 83.97%; CPPS (31.25-250 μg·mL^-1) exerted proliferation effect on RAW264.7 cells, and the CPPS concentration of 125 μg·mL^-1 proliferated significantly (P<0.01). While the concentration of CPPS higher than 500 μg·mL^-1 inhibited the proliferation of RAW 264.7 cells. The CPPS (100 and 150 μg·mL^-1) could increase the secretion of TNF-α (P<0.01) and IL-6 (P<0.05), and significantly increased the transcription of TNF-α (P<0.001) and IL-6 (P<0.001). Moreover, CPPS concentration of 100 μg·mL^-1 increased NF-кB p65 expression in nuclear (P<0.01) and decreased IкB-α expression in cytoplasm (P<0.001). CPPS can not only stimulate the RAW264.7 cells proliferation, possibly through activation of NF-кB signaling pathway to the secretion of TNF-α and IL-6 increased, which are involved in immune regulation.