Effect of a complex feed additive on the composition and function of the Oryctolagus dominis caecum microbiome

There is a growing interest in the study of natural multicomponent feed additives to regulate gut microbiome composition and improve immune and physiological status of rabbits. In the present work, for the first time, it was bioinformatically found that a complex probiotic biological product affects the change in the predicted metabolic pathways of the rabbit intestinal microbiome. The aim of the work was to study the joint action of a complex containing minerals and a probiotic on physiological status, composition and functional potential of gut microbiome in rabbits. For the study (the vivarium of the FGBU VO SPKhFU of the Ministry of Health of Russia, St. Petersburg, 2021), ten Soviet chinchilla rabbits of 2.5 months of age (5.37-5.53 kg bw) were allocated to two groups of five rabbits each. Control group I received the recommended basal diet (BD, RAAS norms 2003), test group II was fed with the BD supplemented with a complex feed additive (30 mg per animal day-1) consisting of the microelement preparation Silaccess at 5 mg/kg of bodyweigh (LLC TECHNOLOG 2D, Russia) and the probiotic strain Bacillus subtilis 1-85. On days 30 and 60, the animals were weighed before morning feeding, and blood was sampled to evaluate natural resistance parameters (bactericidal function, including lysozyme activity, and phagocytic activity of neutrophils). Chyme samples of the caecum for microbiome studies aseptically collected at the end of the experiment were immediately placed in sterile plastic tubes. Total DNA was isolated using the Genomic DNA Purification Kit (Thermo Fisher Scientific, Inc., USA). The bacterial community was assessed by NGS sequencing on a MiSeq automated sequencer (Illumina, Inc., USA) using primers to the V3-V4 region of the 16S rRNA gene which allows us to identify microorganisms to the species level: the forward primer 5´-TCGTCGGCAGCGTCAGATGTGTATAAGAGACAGCCTACGGGNGGCWGCAG-3´ and the reverse primer, 5´-GTCTCCGTGGGCTCGGAGATGTGTATAAGAGACAGGACTACHVGGGTAT-CTAATCC-3´. The reconstruction and prediction of the functional content of the metagenome was performed using the PICRUSt2 (v.2.3.0) software package (https://github.com/picrust/picrust2). Mathematical and statistical processing was carried out by the multivariate analysis of variance procedure using Microsoft Excel XP/2003, R-Studio (Version 1.1.453) (https://rstudio.com). In group II compared to control, the phagocytic index was higher (p ≤ 0.05) by 1.8 %, the phagocytic number by 32.3 % (p ≤ 0.05). NGS sequencing revealed the values of the Chao1, Shannon and Simpson biodiversity indices to be higher (p ≤ 0.05) in group II compared to group I. Taxonomic analysis of caecum microbial community disclosed 12 phyla of the kingdom Bacteria among which representatives of the phylum Firmicutes dominated (80.2±6.2 % in group I, 78.2±7.4 % in group II). In group II, there was a 1.3-2.6-fold increase in the abundance of phyla Verrucomicrobiota , Actinobacteriota , Patescibacteria , Proteobacteria , Desulfobacterota and a 4.8-fold decrease in the abundance of the phylum Campylobacterota (p ≤ 0.05). In the caecum of test rabbits, the genus Bacillus spp. increased 2.82 times compared to control (p ≤ 0.05). Staphylococcus sciuri was found in group I (0.075±0.006 %) but not in group II. Data processing using the PICRUSt2 software tool (v.2.3.0) revealed 370 predictable metabolic pathways in the rabbit gut microbial community, 36 of which differed (p ≤ 0.05) between the groups. In group II, the intestinal microbiome pathways related to the degradation of aromatic compounds and xenobiotics, protein, carbohydrate, and energy metabolism, alcohol biosynthesis, photorespiration, assimilation of formaldehyde, degradation of myo-, chiro- and scillo-inositol, cell wall synthesis and spore formation activated compared to group I (p £ 0.05). The dominant proportion (15 pathways) of enhanced potential metabolic pathways was associated with the degradation of aromatic compounds and xenobiotics. Thus, a complex dietary additive based on the probiotic strain of Bacillus subtilis 1-85 and microelements has a multiple positive effect both on gut microorganisms (fewer pathogens, metabolic regulation) and on the macroorganism (higher values of immunity parameters, a better growth performance of Soviet chinchilla rabbits).