| Sheep is a kind of economic animal for skin,wool and meat.With the continuous expansion of feeding scale,some pathogenic diseases such as E.coli and coccidiosis are frequently occurred.Although antibiotics or chemical drugs can prevent and treat these diseases,long-term use of sheep will destroy the balance of normal flora in the gastrointestinal tract of animals,and cause gastrointestinal flora disorder.On the other hand,it will make animal body produce Drug resistance.In recent years,probiotics have been widely concerned by the animal husbandry industry,and also attracted great attention of the maj ority of scientific researchers.Probiotics have incomparable advantages in animal disease prevention and treatment,which can not only enhance the immunity of animals and have remarkable effect,but also greatly improve the economic benefits of feeding animals.In this study,the selected Lactobacillus plantarum,Lactococcus lactis,Bacillus subtilis and yeast were mixed to make Probiotics,which were added to the diet of weaned Hu sheep to analyze the effects of Probiotics on growth performance,gastrointestinal microflora and body immunity of weaned Hu sheep;and this study analyzed the effects of probiotics on body weight,fecal status,oocyst excretion and the changes of comprehensive immune index in the experiment of artificial infection with coccidia in lambs.This study is divided into three experimental parts.Experiment 1:Isolation,identification and functional determination of the tested strains.In this experiment,the strains were isolated from the intestines of healthy sheep and laboratory.The isolated strains were identified by morphological and biochemical tests,16S or 26S rRNA gene amplification and other molecular biological methods.The growth curve,acid resistance,bile salt resistance,enzyme production ability,inhibition ability,drug sensitivity and safety of the isolated strains were determined.The results showed that the isolated strains ECL1.2,R,13 and J were Bacillus subtilis,Lactobacillus plantarum,Lactococcus lactis and Pichia manshurica,respectively,and the logarithmic growth periods of ECL1.2,R,13 and J were 1624h,8-20h,12-24h and 8-24h,respectively.The four strains had good acid and bile salt tolerance.Strains ECL1.2 had good cellulase,protease and α-amylase production ability.Strains ECL1.2,R and 13 could inhibit pathogenic bacteria E.coli,Salmonella and Staphylococcus aureus.Strains ECL1.2,R and 13 are sensitive to eight kinds of common antibiotics（norfloxacin,vancomycin,lincomycin,rifampicin,clarithromycin,gentamicin,penicillin,cefazolin）.The probiotics made from the mixture of strains ECL1.2,R,13 and J are safe and non-toxic to mice.Experiment 2:The effect of probiotics on growth performance,gastrointestinal flora and immunity in weaned lambs.Twenty healthy Hu sheep with similar body weight and age were randomly divided into 4 groups with 5 sheep in each group.Weaned lambs were fed with low（4×108 CFU/individual/day）,medium（4×109 CFU/individual/day）and high(4×1010CFU/individual/day)doses of probiotics respectively,and were slaughtered 28 days later.The growth performance,blood routine indexes and immune related indexes of weaned lambs were detected.The alpha diversity and community composition of bacterial community in rumen,duodenum,cecum and rectum contents of weaned lambs were analyzed by Illumina Miseq high-throughput sequencing technology.The results showed that:1.Adding probiotics to the diet can increase the average daily gain of weaned lambs,reduce the feed to weight ratio and improve the feed conversion rate.2.Adding different doses of probiotics can increase the number of neutrophils,basophils,leukocytes,lymphocytes and monocytes in plasma of weaned lambs.3.Adding probiotics could increase the diversity of rumen microbial community of weaned lambs;reduce the diversity of duodenum and cecum microbial community of weaned lambs,make the composition structure of duodenum and cecum more concentrated;have little effect on the diversity of rectal microbial community of weaned lambs;improve the relative abundance of beneficial microbial community in rumen,duodenum,cecum and rectum of weaned lambs and reduce the relative abundance of harmful bacteria.4.Adding probiotics could improve thymus index,increase serum IFN-γ,IL-2 and IgG contents,and reduce serum TNF-α,TNF-β and IL-4 contents of weaned lambs.5.The average crypt depth of duodenum,ileum and jejunum of weaned lambs with probiotics was significantly increased（p<0.01）;and had no significant effect on the density of the duodenum,ileum and jejunum（p>0.05）.Experiment 3:Effect of probiotics on coccidia infection by artificially in weaned lambs.Thirty healthy Hu sheep with similar body weight and age were randomly divided into 6 groups:low（4×108 CFU/individual/day）doses probiotics group,medium（4×109 CFU/individual/day）doses probiotics group,high(4×1010 CFU/individual/day)doses probiotics group,coccidia group,treatment group and blank control group,with 5 sheep in each group.After 14 days,105 Eimeria oocysts were artificially infected in the other 5 groups except the blank control group.The treatment group was given diclazuril solution 1mg/kg for two consecutive days at the peak of ovulation.The feces of each group were collected every two days within three weeks after challenge.The condition of coccidial oocysts excreted by each group was observed under microscope.The feces status,weight,blood routine indexes and immune indexes of sheep were detected during the period.The results showed that:1.Coccidia infection can reduce the weight of sheep,increase the feed to weight ratio and cause diarrhea.Adding probiotics to sheep diet can reduce the decrease of growth performance and diarrhea caused by coccidiosis.The effect of medium dose probiotics group is the best.2.Probiotics can reduce the risk of infection coccidiosis and the number of oocysts in feces of weaned lambs.3.After artificial infection with coccidia,the number of white blood cells and neutrophils increased,the number of red blood cells and the concentration of hemoglobin decreased in weaned lambs.Adding probiotics can reduce the increase of white blood cells and lymphocytes,and reduce the decrease of red blood cells and hemoglobin caused by artificial infection with coccidia,and the medium dose of ecological preparation had the best effect.4.Compared with the blank control group,the content of serum IFN-α in sheep infected with coccidia was significantly decreased（p<0.05）.Compared with the coccidium group,the serum IFN-y and IL-2 levels of weaned lambs with probiotics were significantly increased（p<0.05）,and the serum TNF-α,TNF-β and IL-4 contents were significantly reduced（p<0.05）.In conclusion,the probiotics prepared by the mixture of Bacillus subtilis,Lactobacillus plantarum,Lactococcus lactis and Pichia manshurica added to the diet of weaned lambs can improve the growth performance and blood quality of lambs;have certain effects on the diversity of microbial flora in rumen,duodenum,cecum and rectum of lambs,increase the number of beneficial bacteria and reduce the number of harmful bacteria in rumen and intestines;enhance the immune function of lambs and the absorption function of intestinal tract;improve the ability of anti coccidiosis of weaned lambs.The results of this study provide data support for the application of microecological agents in actual production,and provide theoretical basis for the healthy development of animal husbandry. |
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