Study On The Regulation Mechanism Of Vitamin B₁₂ On Egg Quality Based On Omics Technology

Vitamin B₁₂,a micronutrient that must be obtained through food,has been shown to be highly effective in transferring vitamin B₁₂ from the diet to eggs.Vitamin B₁₂ deficiency caused by inadequate diet has become a global problem.The use of vitamin B₁₂ has been transformed from treatment to prevention,and the production of fortified food has become a trend.However,studies on the changes of egg nutrient composition and the mechanism of vitamin B₁₂ absorption and transfer in laying hens and the molecular mechanism affecting the changes of egg nutrient composition has not been reported.Therefore,this experiment took Jinghong laying hens as the research object and systematically studied the regulation mechanism of vitamin B₁₂ on egg nutrient composition by means of microbiome,transcripome and metabolome technology through short-term and whole laying period feeding experiments,providing theoretical basis and reference for the application of vitamin B₁₂ in the production of laying hens.1 Effects of vitamin B₁₂ on performance and egg utrient composition of laying hensA total of 756 30-week-old Jinghong laying hens were randomly divided into 7 groups with 6replicates per group and 18 hens per replicate.Hens in the control group were fed a basal diet,and hens in the experimental groups were fed the basal diet supplemented with 25,50,100,400,800,1200 μg/kg vitamin B₁₂,respectively.The pre-experiment lasted for 2 weeks and the experiment lasted 6 weeks.Results are as follows:1.1 Dietary vitamin B₁₂ supplementation had no significant effect on laying performance of laying hens during peak laying period（P > 0.05）;The eggshell ratio was significantly increased when the supplemental level was equal to or higher than 400 μg/kg（P < 0.05）;The eggshell thickness of 50,100,400 μg/kg group was significantly higher than that of low dose group（P < 0.05）.1.2 Compared with the control group,the serum Holo TC content was significantly increased,and the content of homocysteine decreased significantly in the vitamin B₁₂ supplementation group at or above 100μg/kg（P < 0.05）;The serum TCN1 content in 100 μg/kg group was the highest;The liver TCN1 content in100、400 μg/kg group was the highest（P < 0.05）,and the triglyceride content in 100 μg/kg group was the lowest（P < 0.05）,hepatic cholesterol content decreased significantly with the increase of vitamin B₁₂ supplemental level（P < 0.05）;vitamin B₁₂ in liver serum and egg yolk was not significantly increased in groups with or above 100 μg/kg（P < 0.05）,The conversion rate of vitamin B₁₂ in yolk of 100 μg/kg group was the highest.1.3 Dietary vitamin B₁₂ had no significant effect on routine egg composition.Total fatty acid content,monounsaturated fatty acid content and total polyunsaturated fatty acid content in yolk were significantly increased in vitamin B₁₂ supplemental group（P < 0.05）,the nutritional score of egg white was improved when vitamin B₁₂ was added 100 or 400 μg/kg,and improve the overall nutritional value of eggs.2 Study on the regulation mechanism of different dietary Vitamin B₁₂ levels on egg nutrient compositionDifferent part of intestine,liver,kidney,follicle and cecal digesta in the contronl group,25,100,400μg/kg group were studied by fluorescence quantification and omics techniques.The results show as follows:2.1 The m RNA expression of ileum and cecum receptor proteins AMN and CUBN,ATP binding cassette subfamily D member 4（ABCD4）,lysosomal membrane protein（LYZ）in ileum,ATP binding cassette subfamily C member（ABCC1）and ABCC6 in duodenum,ABCC2,ABCC3 and ABCC6 in the ileum,ABCC2,ABCC6 in the cecum,cobalamin transporter receptor CD320（cluster of differentiation 320）in the duodenum and ileum,low density lipoprotein receptor-related protein 2（LRP2）in kidney were significantly upregulated in experimental groups.2.2 The results of cecal microbiome of laying hens showed that dietary vitamin B₁₂ did not regulate the diversity of cecal microbiome of laying hens,but changed the structure of the microbiome.The relative abundance of Faecalibacterium,Paraprevotella,Blvii28,Perlucidibaca,Lactobacillus,Butyricicoccus and Succinatimonas was significantly increased,while The relative abundance Acinetobacter,Bilophila decreased significantly（P < 0.05）.2.3 Liver transcriptome results showed that 118,517,1089 differentially expressed genes were screened out in 25,100,400 μg/kg group compared with the control group.In the 100 μg/kg group,there was a significant enrichment pathway in KEGG,namely the cell cycle pathway,and the pathways of tyrosine metabolism,phenylalanine metabolism,glycerol phospholipid metabolism,sugar synthesis and decomposition,folic acid-carbon metabolism,retinol metabolism,glutathione metabolism were significantly changed in 400 μg/kg group.The results of liver metabolomics showed that 11,24,46 metabolites with significant difference were screened from 25,100,400 μg/kg group,which were classified as lipid amino acid vitamin and coenzyme nucleic acid metabolites.In 25 μg/kg group,the KEGG metabolism pathway was significantly affected by vitamin B6 metabolism,primary cholic acid biosynthesis,fatty acid biosynthesis and unsaturated fatty acid biosynthesis.The pathways in 100 or 400 μg/kg groups were significantly affected including pyrimidine metabolism,purine metabolism,vitamin B6 metabolism,glycerophospholipid metabolism,sphingolipid metabolism,arginine biosynthesis,cysteine and methionine metabolism lysosome and other metabolic pathways.3 Effects of vitamin B₁₂ on health of laying hens during whole laying periodTwo laying hens with a total population of 16 000 each were selected to carry out the whole laying period feeding experiment.From 21 weeks of age,the control group and experimental group were fed the basal diet supplemented with 25 and 100 μg/kg vitamin B₁₂,respectively,until 70 weeks of age.The results showed that adding 100 μg/kg vitamin B₁₂ during laying period increased laying rate of laying hens.RBC,HGB,HCT,ALB,GSP and SOD values increased（P < 0.05）,indicating that the experimental group improved hematopoietic function and enhanced antioxidant effect of laying hens.In conclusion,add the appropriate vitamin B₁₂ can improve the nutritional value of eggs,increase the absorption of vitamin B₁₂ in the body of transhipment,promote the growth of cecum beneficial bacteria,and inhibit the growth of harmful bacteria,change the metabolism of liver enzymes and vitamins,fat metabolism,nucleotide metabolism,amino acid metabolism and sugar metabolism related metabolic pathways,not adverse effects to health of laying hens,to add 100 μg/kg vitamin B₁₂ is appropriate.