| This experiment was conducted to study the effects of long-term addition of two probiotic additives Bacillus subtilis(BS)and Enterococcus faecium(EF)on the production performance and intestinal health of laying hens,and to provide theoretical guidance and scientific support for improving the production capacity of laying hens and maintaining intestinal health.In this experiment,a total of 528Loghman laying hens at one day old were chosen for the study,and a two-factor completely randomized experimental design with a 2×2 matrix was employed.The control group was fed a basal diet;in the Bacillus subtilis group,Bacillus subtilis(6×10~6 cfu/g)was added to the basal diet.In the Enterococcus faecium group,Enterococcus faecium(4×10~5 cfu/g)was added to the basal diet;in the combined group of Bacillus subtilis and Enterococcus faecium,Bacillus subtilis(6×10~6 cfu/g)and Enterococcus faecium(4×10~5 cfu/g)were added to the basal diet;there were 4treatments with 6 replicates per treatment and 22 chickens per replicate.The duration of the experiment spanned 30 weeks.The feed-to-weight ratio of the Bacillus subtilis group was significantly lower than that of the control group during weeks 1～8(P<0.05).The simultaneous addition of Bacillus subtilis and Enterococcus faecium had a tendency to reduce the feed conversion ratio of laying hens from 1 to 8 weeks(P=0.065).Enterococcus faecium group significantly increased the laying rate of laying hens at 23～26 weeks(P<0.05).The laying rate of laying hens at 23～30 weeks was increased(P<0.05).E.faecium supplementation in the diet increased the average egg weight of laying hens at 27～30 weeks and 23～30 weeks(P<0.05).B.subtilis and E.faecium had a significant interaction effect on the laying rate of laying hens at23～26 weeks(P<0.05)and 23～30 weeks(P<0.01).When B.subtilis and E.faecium were added to the diet at the same time,the laying rate of laying hens at23～26 weeks and 23～30 weeks was increased compared with the control group.Total serum protein levels were significantly higher in the Enterococcus faecalis group than in the control group at 30 weeks(P<0.05).Bacillus subtilis group significantly increased the content of Ig A,Ig G and Ig M in serum of laying hens at 30 weeks,and decreased the content of TNF-α(P<0.05).Enterococcus faecium group significantly increased the content of Ig A,Ig G and Ig M in serum of laying hens at 30 weeks,and decreased the content of IL-8(P<0.05).The mixed addition of Bacillus subtilis and Enterococcus faecium significantly increased the content of Ig A,Ig G and Ig M in serum of laying hens at 30 weeks,and decreased the content of IL-8 in serum.Bacillus subtilis significantly increased the concentration of anti-mullerian hormone(AMH)in serum of laying hens at 30 weeks(P<0.01).The levels of estradiol(E2),follicle stimulating hormone(FSH)and progesterone(PROG)in the Enterococcus faecium group were significantly higher than those in the control group at 30 weeks(P<0.01).Bacillus subtilis and Enterococcus faecium had a significant interaction on the content of AMH(P<0.01)in the serum of laying hens at 30 weeks,which was reflected in the simultaneous addition of the two increased the content of AMH in the serum.Bacillus subtilis group significantly reduced the crypt depth of duodenum and increased the villus height/crypt depth ratio of duodenum and jejunum(P<0.05).Enterococcus faecium group significantly increased the villus height of duodenum and jejunum of laying hens,and increased the villus height ratio of duodenum,jejunum and ileum of laying hens(P<0.05).There was a significant interaction between Bacillus subtilis and Enterococcus faecium.When Bacillus subtilis and Enterococcus faecium were added to the diet,compared with the control group,the villus height of duodenum(P<0.01)and jejunum(P<0.01)was significantly increased,the crypt depth of jejunum(P<0.01)was decreased,and the villus height of duodenum(P<0.05),jejunum(P<0.01)and ileum(P<0.05)was increased.Bacillus subtilis significantly reduced the relative expression of IL-1βm RNA and TNF-αm RNA in the jejunum of laying hens(P<0.05),and Enterococcus faecium group significantly reduced the relative expression of TNF-αm RNA in the jejunum of laying hens(P<0.05);there was an interaction between Bacillus subtilis and Enterococcus faecium.When Bacillus subtilis and Enterococcus faecium were added to the diet at the same time,the relative expression of TNF-αm RNA(P<0.05)in the jejunum of laying hens was significantly reduced compared with the control group.Enterococcus faecium group significantly increased the relative expression of Claudin-2 m RNA in jejunum of laying hens(P<0.05).At 8 weeks,the Shannon,Chao1 and ACE indexes of the B.subtilis group were significantly decreased(P<0.01),and there was a significant interaction between B.subtilis and E.faecium.When both B.subtilis and E.faecium were added to the diet,the Chao index(P<0.01)and ACE index(P=0.03)were significantly decreased compared with the control group.At 20 weeks of age,compared with the control group,dietary supplementation of B.subtilis significantly increased Chao1 index(P=0.03)and ACE index(P<0.05),and E.faecium significantly increased Chao1index(P=0.01)and ACE index(P<0.01).There was a significant interaction between B.subtilis and E.faecium.When B.subtilis and E.faecium were added to the diet at the same time,Chao index and ACE index were significantly increased compared with the control group(P=0.03).At 30 weeks,dietary Bacillus subtilis significantly increased ACE index(P<0.01),and dietary Enterococcus faecium significantly increased Chao1 index(P=0.05).ACE index decreased(P=0.01).At 8 weeks,the dominant bacteria at the phylum level were Firmicutes,Bacteroidota,Proteobacteria and Campylobacterota.Bacillus subtilis significantly increased the relative abundance of Bacteroidota(P<0.01).There was an interaction between Bacillus subtilis and Enterococcus faecium(P<0.05).The relative abundance of Bacteroidota was significantly higher than that of the control group when the two were mixed.At 20 weeks of age,the dominant phylum of cecal chyme microorganisms in laying hens were Bacteroidota,Proteobacteria,Firmicutes and Verrucomicrobiota.The addition of Enterococcus faecium in the diet significantly increased the relative abundance of Firmicutes(P<0.05).At 30 weeks,The dominant bacteria at the phylum level were Firmicutes,Bacteroidota,Proteobacteria and Actinobacteriota.Bacteroides,Lactobacillus.At 8 weeks,the dominant bacteria in cecal digesta of laying hens at the genus level were Alistipes,Faecalibacterium,Parabacteroides and Barnesiella.Bacillus subtilis significantly increased the relative abundance of Alistipes(P<0.01).Enterococcus faecium significantly increased the relative abundance of Helicobacter(P<0.01).At 20 weeks of age,The dominant microflora of cecal chyme microorganisms at the genus level were Bacteroides,Akkermansia,Alistipes and Barnesiella.Bacillus subtilis significantly reduced the relative abundance of Alistipes(P<0.05).The relative abundance of Alistipes in Enterococcus faecium group was significantly lower than that in the control group(P<0.01).The relative abundance of[Ruminococcus]＿torques＿group was significantly increased when Bacillus subtilis and Enterococcus faecium were mixed,and there was an interaction between them(P<0.05).At 30 weeks,Limosilactobacillus and Megamonas were the dominant bacteria at the genus level.In summary,Bacillus subtilis improved feed utilization,enhanced immune function,improved intestinal morphology,enhanced barrier function and relative abundance of dominant flora.Enterococcus faecium increased the laying rate of laying hens,inhibited the expression of inflammatory factors,enhanced immunity,improved intestinal morphology and maintained the stability of intestinal flora by increasing the content of reproductive hormones in serum.The combination of the two can promote the laying performance,reproductive hormones and immune function of laying hens,improve the intestinal morphology and reduce the expression of inflammatory factors,but the improvement effect on intestinal flora is not obvious,and its potential still needs to be further explored. |
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