| Milk containing antibiotics is fed to calves on some dairy farms to avoid wasting.Therefore,the basic composition and characteristics of rumen bacteria community,the function and metabolism,as well as the relationship between rumen bacteria community and calf growth should be further explored.Currently,lots of researches related with rumen bacteria have been done,but calves rumen bacteria and bacterial gene function,especially the genes involved in material synthesis metabolic pathway are still unclear.How does the growth of calves,particularly the resistance genes of bacteria community be affected? This has become a common issue for China’s dairy production,animal welfare and food safety to solve.This study was conducted to investigate the basic feeding status and the utilization of milk containing antibiotics on dairy farms in Heilongjiang province.Total of 54 newborn calves with no significant body weight difference were selected and randomly divided into three groups according the rule of similar birth day.Each group contained 6 calves and was set 3 repeats.Three groups were as followed: calves fed with fresh milk without antibiotics(CK,B1),milk containing antibiotics(AM,B2)and pasteurized milk with antibiotics(APM,B3).The experiment period lasted 180 d.The body weight at 1d,30 d,60 d,90 d and 180 d and the average daily gain in each stage of 1-180 days were determined.Body size index,visceral weight and p H value were measured to calculate diarrhea rate.Rumen fluid of calves at 60 d(T1),90 d(T2)and 180 d(T3)were collected to construct of rumen bacteria metagenomic library by using Illumina Mi Seq platform.Bioinformatics analysis and functional annotation of the sequenced results were conducted in KEGG,NOG,CAZy and ARDB databases.The results:(1)Currently,the phenomenon calves fed with milk containing antibiotics,primarily β-lactam antibiotics,is common on Heilongjiang farms,accounting for 94.6%.Weight at 30 d of calves fed with milk containing antibiotics were significantly reduced(p<0.05).Throughout the breeding period,the body height,body slanting length,chest circumference,pipe circumference,body straight length and chest deep among CK,AM and APM groups were not significantly different(p>0.05).The weight of rumen and reticulum was less affected by the milk at the whole feeding stage.Different milks produced obvious effects on the p H at different positions of the gastrointestinal tract,of which had a significant effect on jejunal p H value XII(p<0.05),and the influence of different milks on rumen p H value disappeared in a month after weaning.Milk with antibiotics or pasteurized didn’t produce effect on the development of stomach and abomasum.Calves diarrhea incidence was the lowest in APM group.(2)A total of 3,498,534 contigs and 262,753 scaffolds were obtained in the present trial.The study found that calves rumen bacterium group structure was affected by the different months and milk sources,calves rumen bacteria distribution was selective after antibiotic treatment.In the 9 samples,the highest abundance of bacteria was Bacteroidetes(69.8%),the second was Firmicutes(9.8%).In addition,the abundance of Proteobacteria(9.2%),Fibrobacteres(4.9%)and Actinobacteria(2.8%)were also relatively high.In species level,the abundance of Prevotella was highest,accounting for 63.28%,then were Bacteroides(12.36%);Fibrobacter(1.49%);Barnesiella(2.68%)and Alistipes(1.47%).And the trial found Spirochaetes has antibiotic resistance.The digestion effect of Bacteroidetes and Fibrobacteres was stronger in the rumen;Bacteroidetes was more sensitive to pasteurized milk with antibiotics.The result of PCA analysis showed that rumen bacterium structure of calves at 6 months had significant difference with that of 2,3 months,and milk containing antibiotics would have a great influence on rumen microbial community.(3)A total of 86,145 genes and the functional annotations in NOG database,33,980 genes mainly covering 9 functional classes in CAZyme database were obtained.A total of 318,1398,30 and 24 DEGs were identified for the T1 versus T2,T1 versus T3,B1 versus B2 and B1 versus B3 comparison groups,respectively.Cell cycle including glycogen synthase kinase-3β(GSK3β),cyclin-dependent kinase 2(CDK2),and cyclin-dependent kinase 7(CDK7)and biosynthesis of amino acids were significantly enriched for the DEGs in the T1 versus T2 and T1 versus T3 comparison groups.This study detected a total of 6 types of antibiotic resistance genes,19 genotypes,including 5 kinds of tetracyclines antibiotic resistance genes(tet37、tet32、teto、tetw、tetx);5 aminoglycosides antibiotic resistance genes(aphbid、a Phiiia、aph33ib、antbia、accbie).5 macrolides antibiotics antibiotic resistance genes(ermb、ermf、ermg、ermq、mema);2 β-lactam antibiotic resistance genes(bl2b_rob、bl2e_cfxa),1 coli peptide antibiotic resistance gene(baca);1 sulfonamides antibiotic resistance gene(sul2).Further analysis of antibiotic resistance mechanisms showed the first factor for antibiotic resistance was efflux pump,accounting for 56.57%;the second was antibiotic deactivation,accounting for 26.26%;the third was cellular protection,accounting for 12.64%.Using microbial molecular ecology and metagenomics,this trial studied the effect of milk containing β-lactam class antibiotics on calves’ growth,the basic component of rumen bacteria,the corresponding dynamic change of metabolic pathways,the resistance genes and production mechanism.The results showed milk containing antibiotics had influence on rumen microbial community and might lead to antibiotic residues-associated food safety problems through affecting pathogenic Spirochaetes.Moreover,Cell cycle(GSK3β,CDK2 and CDK7)and biosynthesis of amino acids pathways significantly were enriched for the DEGs in the T1 versus T2 and T1 versus T3 comparison groups.This study describes the effect of milk containing β-lactams antibiotic on calves rumen bacteria group,bacteria resistance genes and causes,and analyzes differentially expressed genes involved in the metabolic pathways,in order to provide certain theoretical basis for taking advantage of milk containing antibiotics and improving calves health. |
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