| Due to the incomplete development of their intestinal tract and the fact that the intestinal flora has not yet been established,newborn calves are highly susceptible to infestation by pathogenic bacteria such as Escherichia coli,which results in an increase in intestinal permeability,destruction of the intestinal mucosa,and an increase in the inflammatory response,which in turn triggers diarrhea.In this study,we investigated the effects of an early intervention probiotic complex on growth performance,blood immuno-antioxidant indices,intestinal flora structure and function,and plasma differential metabolites in newborn Holstein and newborn Simmental calves by a combination of 16 s r RNA genes high-throughput sequencing,metabolomics,and metagenomics.To further reveal the mechanism of action of probiotic complexes on the maintenance of immunity and growth promotion of newborn calves,and to provide a scientific basis for the wide application of probiotic complexes in the healthy and safe breeding of calves.Experiment 1: Effect of probiotic complex on growth performance,immunity and antioxidant function of newborn Holstein calves.Forty newborn Holstein female calves(36±2.53 kg)born within 3 days of age,with similar body weight and same litter size were selected as test animals for this experiment.They were randomly divided into control group(D),lactobacillus complex group(L),yeast complex group(S)and lactobacillus + yeast complex group(LS),with 10 heads in each group for 15 days of the test period.Complex probiotic powders were mixed between them into milk replacer powder in a 1:1 ratio,i.e.,5 g/d(1 g each of lactobacillus and yeast complex)in LS group,3 g/d(1 g each of lactobacillus complex)in L group,2 g/d(1 g each of yeast complex)in S group,Group D 0g/d.The results showed that the effect of probiotic complex was not significant(P > 0.05)in increasing the ADG of calves in the experimental group,but the rate of diarrhea in the LS group was lower than that of the other groups during the experimental period.Serum levels of T-AOC,SOD,and GSHPx were significantly higher in the LS group and were significantly higher in the LS group than in the L and S groups at both day 15 and day 45,and MDA levels were significantly lower(P < 0.05).The serum levels of Ig M and Ig G in the LS group were significantly higher on day 45,and there was also a trend of increase in Ig A.The serum levels of IL-10 in the LS group were significantly higher on day 45,and there was a trend of increase in IL-4 but the difference was not significant,and the levels of IL-6,IL-8,and TNF-α were significantly lower;the levels of IL-2 in the LS group were significantly higher on day 15(P < 0.05).Experiment 2: Effect of probiotic complex on metabolism,diversity and structure of intestinal flora in newborn Holstein calves.Data analysis of gut flora and plasma metabolites of newborn Holstein calves at 15 days by 16 s RNA high-throughput sequencing and LC-MS metabolomics showed that the richness of the calf gut microbiota was higher in the LS group than in the other groups,and that the rectal contents microbiota of the newborn calves was relatively tightly clustered in the LS,L,and S groups,whereas it was segregated in the D group,as indicated by the UPGMA clustering analysis and the PCo A principal component analysis.The abundance of Actinobacteria phylum was significantly higher in group L and LS than in group D and S(P < 0.05).At the genus level,the top four genera in relative abundance in the intestinal flora were Faecalibacterium,Collinsella,Blautia,and Bifidobacterium,and the abundance of Bifidobacterium was significantly higher in the LS group than in the L,S,and D groups(P < 0.05).Plasma metabolomics analyses showed significant changes in plasma metabolites and metabolic pathways in calves from the LS group,involving arginine and proline metabolism,nicotinic acid and nicotinamide metabolism and glycerophospholipid metabolism,and these three metabolic pathways were significantly enriched between calves from the LS group and those from the D group.The LS vs D metabolic set had the most differential metabolites.Creatinine,Pyrroline hydroxycarboxylic acid,Nicotinamide,Indole-3-carboxaldehyde,L-carnitine,C17 sphingomyelin,Acetylcholine,and Arachidonic acid were screened as potential markers.Experiment 3: Changes in body metabolism,intestinal flora diversity and structure of newborn Holstein calves after cessation of probiotic feeding.There were no significant differences in gut flora richness and diversity between calves in group LS and calves in group D after 1 month of cessation of feeding probiotic complex.However,there was a significant difference in the structure of microbiota between calves of LS,L,S and D groups.According to UPGMA cluster analysis,the microbiota of rectal contents of calves from groups LS,L,and S remained tightly clustered and were significantly separated from those of group D.At the phylum level,the abundance of Actinobacteria phylum in LS,L and S groups was significantly higher than that in D group(P < 0.05).At the genus level,the relative abundance of Bifidobacterium,Blautia,and Collinsella in LS,L,and S groups was significantly higher than that in the control group(P < 0.05).The plasma metabolome showed that pathways of ornithine and arginine metabolism,phenylalanine,tyrosine and tryptophan biosynthesis,Dglutamine and D-glutamate metabolism,tryptophan metabolism,and fatty acid biosynthesis were significantly enriched between the two groups of calves.3-Indoleacetic Acid,13-HPODE,Glutamine,Hexanoylcarnitine,Lyso PC,Phenylalanine,Ornithine,and Tyrosine were screened as potential metabolic markers for calves at this stage.Experiment 4: Effect of probiotic complex on growth performance,immune and antioxidant functions of newborn Simmental calves.Twelve newborn Simmental female calves(40.73±4.44 kg)born within 3 days of age,with similar body weights and the same number of births were selected as test animals for this experiment.The 12 newborn Simmental female calves were randomly divided into control group(Con),lactobacillus + yeast complex group(Pro),6 cows in each group,and the test period was 30 days.The probiotic complex powder was mixed with saline in the ratio of 1:1to be instilled through a syringe and the calves in the Con group were instilled with equal amount of saline.The results showed that the probiotic complex significantly increased the ADG of calves from 0 to 30 days,and the weight and body size indexes of calves in the Pro group were significantly higher than those of calves in the Con group with the increase of days of age(P<0.05).Complex probiotics significantly reduced the serum COR concentration of calves,and the serum COR level of calves in the Pro group reached the lowest at 30 days,and significantly increased the serum levels of GSH-Px,SOD,T-AOC,and reduced the MDA level of calves(P < 0.05).The serum levels of Ig G and Ig M of calves in the Pro group were significantly higher than those of the Con group,and the serum levels of IL-2 and IL-4 in the calves in the Pro group were significantly higher than those of calves in the Con group at 30 days,while the levels of pro-inflammatory cytokines,such as IL-6,TNF-α and IFN-γ,were significantly lower than those of the Con group(P < 0.05).The concentrations of IGF-1,GH,and INS in calf serum were also significantly increased(P < 0.05).Experiment 5: Effect of a probiotic complex on plasma metabolites and differential metabolic pathways in newborn Simmental calves.Calf plasma was analysed by untargeted metabolomics after 30 days of early intervention with the probiotic complex,and 45 plasma differential metabolites were screened from all identified metabolites between the two groups.36 of these metabolites were significantly up-regulated and 9metabolites were significantly down-regulated in the probiotic complex group.Metabolic pathways such as tyrosine metabolism,lipoic acid metabolism,lipid metabolism and fatty acid biosynthesis differed between the two groups of calves.Maltol,Sebacic Acid,Octanoate,Homovanillic acid,N-Acetyl-D-mannosamine,and Indol sulfuric acid were also screened as potential marker metabolites.According to the results,the differential metabolites involved were shown to protect calf intestinal health and reduce the risk of oxidative stress caused by environmental and other factors on the calf organism.Experiment 6: Effects of a probiotic complex on the structure and function of the intestinal flora of newborn Simmental calves.Metagenomic sequencing was used to analyse the rectal contents of calves at 0 days,30 days and 3 months.The results showed that shannon and chao1 indices increased significantly over time in both groups of calves,and calves in the Pro group were higher than those in the Con group.At 30 days,the simpson,shannon and chao1 indices of calves in the Pro group were significantly higher than those in the Con group(P < 0.05).The abundance of harmful genera such as Actinobacteria,g＿＿unclassified＿＿f＿＿Enterobacteriaceae,Clostridium and Escherichia in calves of the Pro group was significantly reduced over time,and the abundance of Prevotella,Parabacteroides,Phocaeicola,Lactobacillus and Limosilactobacillus was significantly increased(P < 0.05).Pathways such as thiamine metabolism,glycine,serine and threonine metabolism were significantly enriched in the Pro group;significantly increased the abundance of glycoside hydrolases and decreased the abundance of glycosyl transferases in calves in the Pro group;and significantly decreased the resistance of the intestinal flora to multiple antibiotics(P <0.05).Experiment 7: Effect of early intervention probiotic complex on blood parameters,apparent digestibility and short chain fatty acids in weaned Simmental calves.When all the test calves reached the weaning age,we found that the effect of early intervention probiotic complex on the growth performance of weaned Simmental calves was not obvious,but the weight,ADG,body height and slant length of calves in the Pro group were still higher than those in the Con group.The Pro group had a more significant apparent digestibility of NDF(P<0.05).It also enhanced the antioxidant capacity of calves during weaning(P < 0.05).It reduces the release of pro-inflammatory factors,enhances immunity,promotes the digestive utilisation of roughage and protein deposition in calves,and significantly increases the levels of SCFAs in the calf intestine to defend against pathogen invasion of the intestine. |
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