Effects Of Bacillus Coagulans On Laying Performance,immunity And Intestinal Health In Late Laying Hens

The purpose of this experiment was to study the effect of Bacillus coagulans strain with high spore production on laying performance,egg quality and diarrhea rate of laying hens at the later stage of egg production and to determine the appropriate dosage.By measuring the he organ index,immunity,serum biochemistry,serum hormones,intestinal structure,digestive enzymes,intestinal flora and intestinal barrier function were determined and so on,the mechanism was analyzed to provide a basis for further research and application.Experiment 1:In this experiment,810 Jingfen No.1 laying hens（health,weight and laying rate were basically the same）at the later stage of egg laying（66 weeks）were selected and randomly divided into 5groups,with 6 replicates in each group and 27 hens in each replicate.The negative control group was fed with non-resistant basal diet.The experimental group I,II and III were fed with 3.33x10⁶cfu/g,1x10⁷cfu/g,3.33x10⁷cfu/g coagulation Bacillus respectively in the basal diet.The positive control group was fed with Bacillus subtilis 1x10⁷ cfu/g for 1 week’s pre-feeding,and formal feeding for 8 weeks.On the 28th day and56th day,30 eggs（5 eggs per repeat）were randomly selected from each group,and the egg quality index detection was completed within 6 hours.The results showed that adding Bacillus coagulans to the diet could improve the production performance and egg quality of laying hens at the later laying stage.Production performance:Compared with the negative control group,the experimental group could significantly increase the laying rate（P<0.05）and the weight of qualified eggs（P<0.05）.It could also significantly reduce the rate of inferior eggs（P<0.05）,diarrhea rate（P<0.05）and feed-egg ratio（P<0.05）.Egg quality:Compared with the negative control group,the yolk color of the experimental group I and III increased significantly at 28th day and 56th days（P<0.05）,and both groups were significantly higher than that of Bacillus subtilis group（P<0.05）.The thickness of eggshell in experimental group I at 28th day was significantly increased（P<0.05）,and Bacillus subtilis group also had similar effect.The weight,height,Harrington unit,egg grade and strength of eggshell had the tendency of increasing at 28th day and 56th day of test,but the difference was not significant（P>0.05）.Experiment 2:On the basis of experiment 1,the next day after the end of the experiment,12 laying hens of the same size were randomly selected from each group（2 in each repetition）to weigh on an empty stomach（12 hours after feeding breakage）.Bloodletting of carotid artery andnaturally precipitated serum were sequentially sampled and stored in accordance with the standard,and analyzed and determined according to the measurement method of each index.The results showed that adding Bacillus coagulans to the diet could improve the organ index and serum immune index of laying hens,and affect the serum biochemical index.Visceral index:Compared with negative control group,spleen index in test group II was significantly increased（P<0.05）,but the difference of heart index,liver index and pancreas index of test group II were not significant（P>0.05）.Immune index:Compared with negative control group,IL-4（P<0.05）,IL-10（P<0.05）,IgM（P<0.05）,IgA（P<0.05）,IgG（P<0.05）,IL-2（P<0.05）were significantly increased.Compared with negative and positive control group,TNF-alpha in group I and group II was significantly increased（P<0.05）,IFN-gamma level in group I and III was significantly decreased（P<0.05）.Serum biochemical indicators:compared with negative control group,alkaline phosphatase activity（P<0.05）and serum calcium（P<0.05）were significantly increased,and triglyceride（P<0.05）and urea in group I and group II（P<0.05）were significantly decreased.Compared with the positive control group,blood phosphorus in group II significantly increased（P<0.05）.The total protein TP,albumin ALB,globulin glob,albumin ratio A/G,glucose Glu had the tendency of increasing,and the total cholesterol TC had the tendency of decreasing,but the difference were not significant（P>0.05）.Experiment 3:On the basis of Experiment 1,serum and ovary were taken to determine the expression of serum hormones and ovarian receptor mRNA,respectively.The results showed that supplementation of Bacillus coagulans in diet could increase the expression of serum hormones and ovarian receptor mRNA in laying hens at late laying stage.Compared with the negative control group,the serum hormone FSH in the test group was significantly increased（P<0.05）,which was significantly higher than that in the positive control group（P<0.05）,and the serum hormone E₂ in the test group was significantly increased（P<0.05）,and the serum hormone LH in the test group III was significantly higher than that in the positive control group（P<0.05）,but the difference was not significant（P>0.05）.The expression of FSHR in ovaries of laying hens in test control group was significantly increased（P<0.05）,while the expression of LHR was similar to that of FSHR,but the difference was not significant（P>0.05）.Experiment 4:On the basis of experiment 1,the serum,intestinal mucosa,intestinal slices and cecum contents were analyzed and determined according to each index determination method,and the DNA V3-V4 region of cecum contents of laying hens was analyzed by high throughput sequencing technology based on Illumina PE300 sequencing platform,and the effect of Bacillus coagulans on cecum flora of laying hens in late laying period was analyzed.The results showed that dietary supplementation of Bacillus coagulans could significantly improve the intestinal health of laying hens in the late laying period.Compared with negative control group,sIgA in the small intestine,height of villi in the small intestine,and ratio of villi length to crypt depth were significantly increased（P<0.05）and crypt depth was decreased（P<0.05）.Amylase,lipase and trypsin activities were significantly increased（P<0.05）and intestinal barrier function of laying hens was significantly decreased compared with negative control group.Serum D-lactic microflora and improved the structure of intestinal microflora in laying hens.The relative abundance of Bacteroidetes in cecum contents was the highest,followed by Firmicutes and Proteobacteria.Compared with the positive control group,the relative abundance of beneficial bacteria such as Ruminococcus torques group（P<0.05）,Ruminococcaceae_uncultured（P<0.05）and Bacteroidetes（P<0.05）,Bacillales（P<0.05）,Lactobacillus（P<0.05）were significantly increased in the negative control group.It reduced the number of harmful bacteria such as Spirochaetes（P<0.05）,Enterococcaceae（P<0.05）,thus promoting intestinal health,improving intestinal digestion and absorption and mucosal immune function,guaranteeing the normal development of organs and systems,and enabling the body to maintain normal levels.Bacillus subtilis group also had similar effects.In conclusion,the dietary supplementation of Bacillus coagulans at the late laying stage can improve intestinal health and production performance.Including improving laying performance,egg quality,immune function,intestinal structure,intestinal barrier function,digestive enzyme activity,promoting the expression and release of gonadal hormones,improving serum biochemical indicators,improving intestinal flora structure,and the effect is better than that of Bacillus subtilis.The short-term feeding effect of the high-dose group was good,but the long-term feeding effect was not stable as that of the low-dose group.