Effects Of Dietary Comnination Of Bacillus Subtilis And Zinc Bacitracin By Stages On Growing Performance And Intestinal Health Of Layer Chickens During 0?16 Weeks

Bacillus subtilis(B.subtilis)as one of the alternatives to antibiotic growth promoters(AGP)has been proven to be useful in improving growth proformance and intestinal health in poultry.Although B.subtilis is widely used in food processing and disease control and prevention,Zinc Bacitracin(ZB)hasn't been totally replaced in livestock on account of the dependence on it in the short term.B.subtilis plays an important role in maintaining microbiological balance and promoting intestinal health,supplement of which cannot exert an influence on hosts steadily,while the addition of AGP will lead to altering of epithelial homeostasis in the gut,thus,ZB mixed with B.subtilis may exert synergistic effect on intestinal health.Therefore,the objective of the present study was to test the hypothesis that if dietary inclusion of ZB alone or in combination with B.subtilis by stages(0~3,4~6,7~12,13~16 weeks)will affect cecal microbiota composition and intestinal health from 0 to 16 weeks age,and if the effect of short-term simultaneous supplementation is superior to supplementation with overall ZB in order to reduce the addition of ZB meanwhile maintaining the growth so as to provide theoretical basis for application modes and phases of B.subtilis in poultry production.The test consists of three parts: Exp.1 Effects of the addition of B.subtilis and ZB by stages on growing performance of layers before laying eggsWe conducted the trial with 630 one-day-old Hy-Line Brown layers in five equal groups with seven repeats per treatment and each repeat included 18 layers.The feeding experiment lasted for 16 weeks and treatments were designed as follows:(1)AGP group: basal diet + Zinc Bacitracin(20 mg/kg,the same below)for 0~16 weeks;(2)BA3 group: basal diet + Zinc Bacitracin + B.subtilis(500 g/t,the same below)for 0~3 weeks in basal diet,basal diet for 4~16 weeks;(3)BA6 group: Zinc Bacitracin + B.subtilis for 0~6 weeks in basal diet,basal diet for 7~16 weeks;(4)BA12 group: Zinc Bacitracin + B.subtilis for 0~12 weeks in basal diet,basal diet for 13~16 weeks;(5)BA16 group: Zinc Bacitracin + B.subtilis for 0~16 weeks in basal diet.Results showed: 1)The addition of Zinc Bacitracin + B.subtilis by stages in 0~16 weeks had no effects on average body weight,average feed intake,average daily gain and feed conversion ratio of layers(P>0.05);2)There were no significant differences among groups in terms of immune and digestive organ indexes and the activity of amylase and lipase in 0~6 weeks(P>0.05),there were no significant differences was observed in skeleton growth in 7~12 weeks and abdominal fat deposition in 13~16 weeks among groups(P>0.05),tibia lenghth were all around 10 cm in all groups.The result suggested that a mixture of Zinc Bacitracin and B.subtilis that supplemented in a short period(0~3 weeks)could make the body weight and size up to the standard before laying period and seemed to be an alternative to long-term use of Zinc Bacitracin for maintaining growth performance in 0~16 weeks.Exp.2 Effects of the addition of B.subtilis and ZB by stages on intestinal health of layers before laying eggsAt 3,6,12 and 16 weeks in the trial,the mucosa and segments of duodenum,jejunum and ileum were collected.The segments were made in paraffin section,while the mRNA levels of disaccharidase,ZO-1,occludin and mucin2 were quantified by Real-time quantitative PCR and sIgA contents were detected by ELISA on mucosa.Results showed: 1)In BA3 group at 0~3 periods,crypt depth was significantly decreased in duodenum and jejunum(P<0.05),and villus height/crypt depth ratio tended to be higher comparing with AGP group(P=0.058),while significantly decrease in villus height in jejunum and ileum was observed(P<0.05),but villus height in ileum was significantly decreased(P<0.05).At 7~12 periods,villus height was significantly increased(P<0.05)in birds of BA3 groups compared to BA6 and AGP groups,in the ileum of birds exposed to mixture of AGP and B.subtilis at any age period,villus height and VH/CD ratio was significantly higher and crypt depth was significantly shallower than those of the AGP group(P<0.05).At 13~16 periods,villus height was significantly increased in birds of BA16 groups compared to BA6 and AGP groups(P<0.05);2)In 4~6 weeks,the levels of sucrase were found to be elevated significantly on duodenum and ileum mucosa in birds of BA3 treatments compared to BA6(P<0.05),in 7~12 weeks,sucrase expression was significantly increased in the BA3 group compared to AGP and BA6 in jejunum(P<0.05),no other changes were observed(P>0.05);3)No significant differences were observed in the expression of ZO-1,occludin and mucin2 on intestinal mucosa in any of the treatment groups from 0 to 16 weeks(P>0.05).4)In jejunum,the sIgA contents on mucosa of birds were significantly increased in BA3 treatment compared to AGP and BA6 treatments in 13~16 weeks(P<0.05).The result suggested that a mixture of AGP and B.subtilis that supplemented in 0~3 weeks could have effect on digestion and absorption in small intestine at overall periods,digestion of sucrase and mucosal immunity at week 16 to maintain overall growth performance.Exp.3 Effects of the addition of B.subtilis and ZB by stages on cecum microbiota of layers before laying eggsAt 3,6,12 and 16 weeks in the trial,the cotents of cecum were collected for determination of microbial community structure and evolutionary changes by 16 S rRNA sequencing analysis and determination of the bacterial number by absolute quantitative real-time PCR(qPCR).Results showed: 1)In 0~3 weeks,the major microbes of BA3 group were the phylum Firmicutes(91.6%)and Tenericutes(5.7%),while the phylum Firmicutes(81.9%),Bacteroidetes(10.0%)and Tenericutes(5.3%)were dominants in AGP group.In 4~6 weeks,Firmicutes(85.8%)and Tenericutes(9.9%)comprised the major bacterial phylum present in the cecum of BA3 group,Bacteroidetes took only 2.6%,while Firmicutes(67.1~68.1%),Bacteroidetes(26.7~28.8%)and Tenericutes(2.2~2.3%)were dominants in AGP and BA6 groups.In 7~16 weeks,the microbial community structure in cecum tended to be stable and dominated by Firmicutes and Bacteroidetes in all groups.2)Relative abundance of Anaerotruncus was increased significantly in the BA3 group in 0~3 weeks(P<0.05).In 4~6 weeks,the abundance of Alistipes,Bacteroides,Odoribacter,and Dehalobacterium was the lowest in the BA3 group(P<0.05),and Lactobacillus,Dorea,Oscillospira and Ruminococcus and Anaeroplasma were the highest(P<0.05),and the abundance of Sutterella in BA3 group was lower than BA6(P<0.05).In 7~12 weeks,the abundance of [Ruminococcus] in BA12 group was higher than AGP and BA6 groups(P<0.05),in addition,BA6 group was found higher Bilophila and lower Oscillospira abundance than other groups(P<0.05).While in 13~16 weeks,the abundance of Prevotella and Sutterella in BA3 was higher than other groups(P<0.05).3)PCoA analysis showed that the cecum microbiota developed and changed by periods and the mixture of Zinc Bacitracin and B.subtilis had better effect on the microbial community than AGP.4)At week 6,the number of species E.coli in BA3 group was higher than BA6(P<0.05).At week 12,the number of species B.fragilis in AGP group was significantly decreased compared to other groups(P<0.05),and group C.leptum cluster was significantly increased in BA3 and BA6 groups compared with AGP(P<0.05).No changes were observed at week 16(P>0.05).The result suggested that a mixture of Zinc Bacitracin and B.subtilis that supplemented in 0~3 weeks could increase beneficial bacterium and decrease the bacterium associated with intestinal inflammation to regulate and stabilizing gastrointestine microbiota in earlier stage(0~6 weeks)while maintaining the overall growth and development,therefore the use of AGP was reduced on the basis of having negative effect on development.In conclusion,the addition of AGP reduced the total bacteria in intestine to promote growth of the host and may give rise to microbial dysbiosis and inflammation.Zinc Bacitracin and B.subtilis added simultaneously to diet for early 3 weeks regulated the cecum microbiota,especially at week 6,promoted beneficial bacterium such as Lactobacillus,Oscillospira and Ruminococcus,decrease the bacterium associated with intestinal inflammation to maintain the intestinal health and development of host.On account of the establishment of layers' cecum microbiota was characteristic with life stages,the early nutrition of layers could had influence on overall performance until later periods,to promote intestinal morphology,up-regulating sucrase expression and elevating sIgA content on intestinal mucosa at later stages(7~16 weeks)to be up to the standard before laying eggs.