| Ganoderma lucidum is a precious traditional medicinal fungus in China,one of its main active components is the polysaccharide of G lucidum which has the effects of anti-tumor,anti-aging,essential hypertension and improving immune function.Using JUNCAO technology to cultivate G.lucidum is a new technology with independent intellectual property rights in China.After a harvest,a large number of mycelia of G.lucidum cultivated with JUNCAO still remain in the spent mushroom substrate(SMS),there are also many nutritional ingredients left in the SMS,such as polysaccharide.Then hot water extract(HWE)was extracted from SMS of G.lucidum(SMSG)through the method of boiling water,and it contained 15.79%polysaccharide in SMSG.This study was prepared HWE as the test material,and preliminarily determined the dose and the effect that HWE can improve the immune function of mice through intragastric administration with different of doses of HWE.Then the further research was studied to provide the immune theory for HWE as feed additive in dairy cows using the immune-deficient mouse model.The following research were conducted to determine the effects of HWE on rumen fermentation and rumen microorganism in dairy cows based on the immune function of HWE in mice,prepared the dairy cows with permanent fistula as the test animals.And the final part was studied to determine the effects of HWE on production performance,hematology parameters,the immunoglobulins of milk and serum for forty dairy cows in milking period.The main results were listed as follows:1.This study was designed to offer HWE with 0 g/kg BW(Control),0.14 g/kg BW,0.28 g/kg BW,0.84 g/kg BW,1.68 g/kg BW by intragastric administration for 30 days.The results showed that,there were no significant difference in body weight?spleen index and thymus index of mice in different groups.HWE(0.84 g/kgBW)can enhance markedly the spleen lymphocytes proliferation caused by ConA,delayed typed hypersensitivity induced by DNFB,on chicken RBC phagocytizing ability of peritoneal macrophages,the ability of charcoal particles clearance in mice,improve the formation of antibody-producing cells and natural killer cell activity in mice.The results indicated that HWE can enhance the immune function in normal mice.2,This experiment was to study the immunity in immune-deficient mice caused by cyclophosphamide(CY)based on that HWE can enhance the immune function in normal mice.The dose of HWE for CK1,CK2,CY,CL,CM,CH was 0 g/kg BW,0 g/kg BW,1.00 g/kg BW,0.50 g/kg BW,1.00 g/kg BW,2.00 g/kg BW,respectively for 28 days.The results showed that:compared to CY,serum IL-1? of CK1 and CK2 were significantly decreased,and serum TNF-? of CL,CM,CH were decreased,serum IFN-? of CL,CM,CH were markedly decreased.According to the liver pathology,the situation of inflammatory cell infiltration in liver was very serious and the liver lobules of CY occurred necrosis,meanwhile the liver of other groups is no obvious pathological phenomenon.The SOD activity,the CAT activity,the T-AOC ability of CL,CM,CH groups were clearly higher than those in CY group,the MDA concentration in CL,CM,CH groups was significantly lower than those in CY group.And the results indicated that HWE can enhance the spleen index and thymus index and inhibit the concentration of IL-1?,IFN-?,TNF-? in serum,HWE can improve the T-AOC ability when the mice were injected into cyclophosphamide.3.Choosing the spleens of mice and extracting total RNA,the difference of gene expression were determined with different doses of HWE based on the RNA-seq analysis and the method of qRT-PCR.The results of RNA-seq analysis showed that there were clearly different among CK1,CK2,CY,CH on gene expression.Compared to CK1,the gene CHP2 coding tumor associated antigen gene of CK2,CH was up-regulated,while the gene coding co-inhibitor,MHC,co-stimulator,inflammatory chemokine were down-regulated.Besides,the down-regulated gene of CK1,CK2,CH were more compared with CY,such as gene CDKN1 A,CDKN1B,MAPK10,VASH1 and TN-C coding tumor associated gene.The results of qRT-PCR showed that the expression of gene IL-1?,TNF-? and IFN-? in CK1,CK2,CM,CH were lower than CY,that were agreement with the results of the measurement of IL-1?,TNF-? and IFN-? in serum of mice and the results of RNA-seq analysis,and then indicated that HWE can inhance the immune function and offer the theoretical basic theory for HWE as feed additive in dairy cows,provide the theory support for the further study.4.The study was designed to investigate the effects of HWE on rumen fermentation,and subsequently provide the data and references to exploring the effects of HWE on ruminal fermentation.3 adult dairy cows with permanent rumen fistula were used in this experiment and were feed with HWE 0 g/d(CK),100.00 g/d(EG1),200.00 g/d(EG2).The test period was 30days.The results showed that there were not significantly different in TVFA,acetic acid and butyric acid,however,the concentration of propionic acid in EG1 was markedly higher than those in CK.The number of rumen protozoa in CK was higher than those in EG1,EG2.Those results indicated that HWE can increase the concentration of propionic acid,bring down the number of protozoa.5.To obtain a deep understanding of ruminal microorganism involved in HWE,Illumina MiSeq sequencing platform and qRT-PCR were used to generate gene sequencing data from genomic DNA of ruminal microorganism associated with 3 adult dairy cows with permanent rumen fistula were used in this experiment and were fed with HWE.Multi-comparison and species abundances analysis on kingdom,phylum,class,order,family,genus and specie demonstrated that the distribution patterns of rumen microorganism among CK,G1,G2 were significantly different.The results showed that dominant microorganism is Bacteria,and the species abundances of G1,G2 was higher than CK.The bacterias belonging to Bacteroidetes,Proteobacteria,Firmicutes were the dominant rumen microorganism,the species abundances of Bacteroidetes,Proteobacteria,Firmicutes were higher than CK,that was agreement with the results of qRT-PCR.Five bacteria species of top10 belonged to Prevotella in the figure of species abundances among CK,G1,G2,and the abundances of five bacteria species in G1 and G2 were higher than CK.Besides,the abundance of Fibrobacter succinogenes was higher than CK that was agreement with the results of qRT-PCR.There was no significant difference in species in different groups,but there were significant difference in abundances of those species,and the results also showed that there were special species in every group,which belong to Bacteria and Archaea,most of special species belong to Bacteroidetes,Firmicutes and Actinobacteria.6.Each dairy cow was offered a basal diet supplemented with HWE for 0 g/d(CK),33.30g/d(Test Groupl),66.70g/d(Test Group2),100.00 g/d(Test Group3),the physiological index,immunoglobulins,total anti-oxidant capacity in serum,the immunoglobulins in serum and somatic cell counts in milk were to investigate after HWE supplementation in dairy cows.The study showed that,there were significant differences in ALT and TG among the groups,and there were no significant differences in other indexes of serum.There were significant differences in IgA of serum among four groups.The results indicated that HWE can decrease the activity of ALT,improve the total anti-oxidant capacity in dairy cow serum,decrease the somatic cell count in milk,then indicated HWE has the potential to enhance the immune function of dairy cows and can improve the quality of milk.7.The study was designed to investigate the effects of HWE on production performance of dairy cows and to offer the scientific basis for the use of HWE as feed additive with HWE for 0 g/d(Control),33.30g/d(Test Group 1),66.70g/d(Test Group 2),100.00 g/d(Test Group3).The results showed that the feed intake of Test Group 3 was obviously higher than this of Control.The milk yield of Control,Test Group 1,Test Group 2,Test Group 3 was 30.87 kg/d,30.93 kg/d,32.11 kg/d and 31.71 kg/d,respectively,there was significant difference in milk yield among four groups.The concentration of milk protein in Test Group 3 was markedly 4.49%higher than this of Control.The result indicated that HWE improved feed intake,milk yield and milk protein,and HWE may has the potential use as additive. |
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