| With the improvement of living standards,people have higher and higher requirements for food safety.Most people prefer choosing healthy foods that are green,organic and free of antibiotic residues.With the promulgation of the national "anti-resistance order" in 2020,it is particularly important to find non-toxic,harmless,and residue-free feed additives in livestock and poultry production.Fulvic acid is a feedstuff additive which is healthy without residual.It can improve the growth performance of growing-finishing pigs,reduce the thickness of back fat and improve the redness of flesh color and the marble score,prevent pigs from nutritional deficiencies,joint cysts and other diseases.It can also avoid the many hazards caused by antibiotic residues to human health.The application of fulvic acid in animal husbandry can provide more realistic basis for non-resistant breeding.In the current study,450 42-week-old Jinghong No.1 laying hens’ parent stock were selected as the test animal and divided into five groups,each with 90 chickens.Different concentrations of fulvic acid were added in the diet.Group A was the control group,0.025%fulvic acid in group B,0.05% in group C,0.1% in group D,and 0.2% in group E for for 10 weeks.The egg production and health status of different groups were recorded every day.On the last day of the first,fourth,seventh,and tenth weeks of the test period,30 blood samples were taken from each group and separated the serum;each group collects 50 eggs for the egg quality investigation;at the end of the experiment,15 animals were slaughtered in each group.Liver,spleen tissue and cecal contents were collected.HE staining method was used to make paraffin sections of liver and spleen samples.The effects of different doses of fulvic acid on histomorphology of liver and spleen of laying hens were analyzed.The influence of chicken liver and spleen tissue morphology,the serum IL-2,TLR-2,IgA,IgG,IgM,IgY and other immune indicators were measured by ELISA method;16S r RNA sequencing was used for analyzing the microbial diversity in the cecal contents of each group.The results were as follows:1.The results of production performance analysis: The average egg weight of treatment groups was significantly higher than that in group A(control group)(P<0.05),group A was62.69g;the egg production rate of group B(0.025%)and group E(0.200%)are significantly lower than the group A,group C(0.050%)and group D(0.10%)(P <0.05);The laying rate of group C was significantly lower than that in group A,B,E(P <0.05),The soft broken egg rate and fecal-egg rate of each group were not significantly different;the feed intake of the group C and E was significantly lower than that in group A,and the feed-egg ratio of group C was the lowest,and significantly lower than that in the group A,which was 1.37(P <0.05).2.The results of egg quality analysis: At 43 weeks of age,the color of egg yolk in group E was significantly higher than the other four groups(P<0.05);The weight of egg yolk in the group A and B was significantly lower than that of the group D(P<0.05).At 46 weeks of age,the egg shape index of the group A and B was significantly lower than that of the group C,D,and E(P<0.05);the egg shell thickness of the group B and C was significantly lower than that of the group A(P<0.05);group C had the highest egg yolk color,and group D had the lowest eggshell weight;group E had significantly higher eggshell weight than that of the groups C、D(P<0.05).At 49 weeks of age,the egg shape index of group B was significantly higher than that of group A and E(P<0.05);the protein height of group A and B was significantly higher than that of group D and E(P<0.05);Yolk color in Control group was significantly lower than that of treatment groups(P<0.05);Group A and B had significantly higher Haugh unit than groups D and E(P<0.05);The egg yolk weight of treatment groups was significantly higher than that of control group(P<0.05);the weight of eggshell of group C was significantly lower than that of group B,D,and E(P<0.05),and the weight of eggshell of group D was significantly higher than that of A,B,C three groups(P<0.05).At 52 weeks of age,the eggshell thickness of group B was significantly lower than that of group A,D,and E(P<0.05);The Albumen height of group D was significantly lower than that of group A(P<0.05);the color of egg yolk in group C and E was significantly higher than that of the other three groups(P<0.05);the Haugh unit of group D was significantly lower than that of group A(P<0.05);The egg yolk weight of treatment groups was significantly lower than control group(P<0.05),which was 16.71 g.2.Histology results of liver and spleen.The liver tissues of the five groups were all dark brown,with smooth surface and no bleeding or other lesions.The results of liver tissue sections showed that the hepatocyte plates of group D were neatly arranged,focal lymphocytic infiltration occurred locally;the hepatocytes of group E were irregular,mild congestion of liver sinusoids,focal lymphocytic infiltration occurred locally.The spleen of each group were reddish-brown,with smooth surface,without bleeding or other lesions.The germinal center of spleen nodules in group D was larger;the red pulp of spleen in group E had a slight degree of congestion.3.Immune Index(1)IgA: At 43 weeks of age,the IgA level of treatment groups was significantly higher than that in control group A(P<0.05);at 46 weeks of age,group B and C were significantly higher than that in group A,D and E(P<0.05);at 52 weeks of age,group C was significantly higher than that in the group A and B(P<0.05).(2)IgG: At 43 weeks of age,the IgG level of treatment group was significantly higher than that of group A(P<0.05);at 46 weeks of age,the level of group B was significantly higher than that in group A and D(P<0.05);at 49 weeks of age,the IgG level of treatment groups was higher than that in the control group;at 52 weeks of age,group C was significantly higher than that in group A(P<0.05).(3)IgM: At 46 weeks,the group E was significantly higher than that in the group D(P<0.05);at 52 weeks,the fulvic acid supplemented groups were lower than the control group.(4)IgY: At 43 weeks of age,IgY of group B was significantly higher than that of group A(P<0.05);at 46 weeks of age,group C was significantly higher than that of group A and B(P<0.05);at 49 weeks of age,the three groups of B,D,and E were significantly higher than group A(P<0.05);at 52 weeks of age,treatment groups were significantly higher than group A(P<0.05).(5)IL-2: At 43 weeks of age,the IL-2 of group A was significantly lower than that of group C,D,and E(P<0.05);at 46 weeks of age,group D was significantly higher than that of group A and D(P<0.05),treatment groups were higher than control group;at 49 weeks of age,treatment groups were significantly higher than the group A(P<0.05);at 52 weeks of age,The three groups of C,D,E were significantly higher than the control group.(6)TLR-2: At 46 weeks of age,group D was significantly higher than the three groups of A,B,and C(P<0.05);at 49 weeks of age,group B was significantly higher than group A(P<0.05),treatment groups were higher than group A;at 52 weeks of age,group B was significantly higher than the two groups of A and C(P<0.05).4.Intestinal microbiota: The dominant species in the cecum content of each group at the phylum level were Bacteroides and Firmicutes,but the abundances of all phyla were different.At the genus level,and the top three dominant species in the cecum were the same.Fulvic acid(0.05%)increased the abundance of Bacteroides.The shannon index of group C was the largest,and the Chao index was the smallest.0.05% fulvic acid can increase the diversity and richness of intestinal microbes.β diversity results showed that the microbial composition difference among five groups was small.Function cluster results showed that the gene expression level of amino acid transportation and metabolism was the highest.The results of KEGG analysis showed that proper concentration fulvic acid can increase the effect of signaling molecules and interactive metabolic pathways.In summary,0.05% fulvic acid can improve laying performance,egg quality,and immune indexes of laying hens,increase the diversity of intestinal microbiota,the abundance of Bacteroides,and increase the role of signal molecules and interaction metabolic pathways.The addition of fulvic acid over 0.2% will cause slight pathology in the liver and spleen. |
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