The microbiota structure in the cecum of laying hens contributes to dissimilar H2S production.

BACKGROUND: Host genotype plays a crucial role in microbial composition of laying hens, which may lead to dissimilar odor gas production. The objective of this study was to investigate the relationship among layer breed, microbial structure and odor production. RESULTS: Thirty Hy-Line Gray and thirty Lohmann Pink laying hens were used in this study to determine the impact of cecal microbial structure on odor production of laying hens. The hens were managed under the same husbandry and dietary regimes. Results of in vivo experiments showed a lower hydrogen sulfide (H2S) production from Hy-Line hens and a lower concentration of soluble sulfide (S2-) but a higher concentration of butyrate in the cecal content of the Hy-Line hens compared to Lohmann Pink hens (P < 0.05), which was consistent with the in vitro experiments (P < 0.05). However, ammonia (NH3) production was not different between genotypes (P > 0.05). Significant microbial structural differences existed between the two breed groups. The relative abundance of some butyrate producers (including Butyricicoccus, Butyricimonas and Roseburia) and sulfate-reducing bacteria (including Mailhella and Lawsonia) were found to be significantly correlated with odor production and were shown to be different in the 16S rRNA and PCR data between two breed groups. Furthermore, some bacterial metabolism pathways associated with energy extraction and carbohydrate utilization (oxidative phosphorylation, pyruvate metabolism, energy metabolism, two component system and secretion system) were overrepresented in the Hy-Line hens, while several amino acid metabolism-associated pathways (amino acid related enzymes, arginine and proline metabolism, and alanine-aspartate and glutamate metabolism) were more prevalent in the Lohmann hens. CONCLUSION: The results of this study suggest that genotype of laying hens influence cecal microbiota, which in turn modulates their odor production. Our study provides references for breeding and enteric manipulation for defined microbiota to reduce odor gas emission.

The tigecycline resistance gene tetX has an expensive fitness cost based on increased outer membrane permeability and metabolic burden in Escherichia coli.

Livestock-derived tetX-positive Escherichia coli with tigecycline resistance poses a serious risk to public health. Fitness costs, antibiotic residues, and other tetracycline resistance genes (TRGs) are fundamental in determining the spread of tetX in the environment, but there is a lack of relevant studies. The results of this study showed that both tetO and tetX resulted in reduction in growth and an increased in the metabolic burden of E. coli, but the presence of doxycycline reversed this phenomenon. Moreover, the protection of E. coli growth and metabolism by tetO was superior to that of tetX in the presence of doxycycline, resulting in a much lower competitiveness of tetX-carrying E. coli than tetO-carrying E. coli. The results of RNA-seq showed that the increase in outer membrane proteins (ompC, ompF and ompT) of tetX-carrying E. coli resulted in increased membrane permeability and biofilm formation, which is an important reason for fitness costs. Overall, the increased membrane permeability and metabolic burden of E. coli is the mechanistic basis for the high fitness cost of tetX, and the spread of tetO may limit the spread of tetX. This study provides new insights into the rational use of tetracycline antibiotics to control the spread of tetX.

Bacillus coagulans R11 consumption influenced the abundances of cecum antibiotic resistance genes in lead-exposed laying hens.

Bacillus coagulans is regarded as a clean, safe and helpful probiotic additive in the production of livestock and poultry breeds. Some studies have also shown that Bacillus coagulans can adsorb heavy metals in water, even in the gut of animals. However, whether Bacillus coagulans feeding influences antibiotic resistance gene (ARG) abundance in the gut of lead-exposed laying hens is unknown. To better apply such probiotics in the breeding industry, the present study employed Bacillus coagulans R11 and laying hens in model experiments to test ARG changes in the cecum of laying hens under lead exposure and B. coagulans R11 feeding. The results showed that there was the trend for ARG abundance decreasing in feeding B. coagulans R11 without lead exposure to laying hens in the cecum; however, feeding B. coagulans R11 to laying hens exposed to lead obviously increased the abundances of aminoglycoside and chloramphenicol ARGs. Further experiment found that hydroquinone, dodecanedioic acid, gibberellin A14, alpha-solanine, jasmonic acid and chitin were involved in the abundances of ARGs in the cecum, in addition the abundances of these compounds were also significantly enhanced by lead exposure or combination effects of lead and B. coagulans R11. As a result, the ARG hazards increased with feeding B. coagulans R11 to laying hens exposed to lead, and the key compounds which influenced by the combination effects of lead and B. coagulans R11 might influence the ARGs abundance.

Microbial Diversity and Community Variation in the Intestines of Layer Chickens.

The intestinal microbiota is increasingly recognized as an important component of host health, metabolism and immunity. Early gut colonizers are pivotal in the establishment of microbial community structures affecting the health and growth performance of chickens. White Lohmann layer is a common commercial breed. Therefore, this breed was selected to study the pattern of changes of microbiota with age. In this study, the duodenum, caecum and colorectum contents of white Lohmann layer chickens from same environment control farm were collected and analyzed using 16S rRNA sequencing to explore the spatial and temporal variations in intestinal microbiota. The results showed that the diversity of the microbial community structure in the duodenum, caecum and colorectum increased with age and tended to be stable when the layer chickens reached 50 days of age and the distinct succession patterns of the intestinal microbiota between the duodenum and large intestine (caecum and colorectum). On day 0, the diversity of microbes in the duodenum was higher than that in the caecum and colorectum, but the compositions of intestinal microbes were relatively similar, with facultative anaerobic Proteobacteria as the main microbes. However, the relative abundance of facultative anaerobic bacteria (Escherichia) gradually decreased and was replaced by anaerobic bacteria (Bacteroides and Ruminococcaceae). By day 50, the structure of intestinal microbes had gradually become stable, and Lactobacillus was the dominant bacteria in the duodenum (41.1%). The compositions of dominant microbes in the caecum and colorectum were more complex, but there were certain similarities. Bacteroides, Odoribacter and Clostridiales vadin BB60 group were dominant. The results of this study provide evidence that time and spatial factors are important factors affecting the intestinal microbiota composition. This study provides new knowledge of the intestinal microbiota colonization pattern of layer chickens in early life to improve the intestinal health of layer chickens.

A novel method for extraction of polypropylene microplastics in swine manure.

With the development of modern industry and agriculture, plentiful microplastics (MPs) were produced as a result of the abuse of plastic. The widespread presence of MPs in soils has caused coastal ecological environment pollution. Previous research has shown that fertilizer is one pathway for the entry of MPs into agricultural soils. Meanwhile, livestock manure is a major fertilizer for crops, and the application of livestock manure compost creates a potential pathway for MPs to enter soils. Thus, MPs may exist in livestock manure from the process of livestock breeding and ultimately contaminate agricultural soils. Based on the increasing attention to MP pollution, manure-born MPs will attract more interest in the future. Thus, the present study compares the extraction effects of centrifugation with fractional distillation, and an improved method is introduced to extract polypropylene (PP) from different types of swine manure. The numbers of particles and fibers were determined using a camera (MS60) connected to a stereomicroscope (Mshot MZ62), and the results showed that the recovery rate of plastic particles in swine manure based on different added numbers ranged from 71.43% ± 8.36 to 96.67% ± 3.33 with the centrifugation method, and only 31.11% ± 10.56 to 43.33% ± 12.56 using fractional distilling. The recovery rate for fibers was generally higher than for particles, especially using centrifugation, and ranged from 95.67% ± 1.58 to 100% ± 0, while the rate of fiber recovery using fractional distillation ranged from 39.44% ± 10.66 to 39.44 ± 10.66. The results of recovery rates using the two methods show that the effect of extraction by centrifugation is superior to the method of fractional distillation, with a recovery rate of approximately 100% for fibers and 90% for particles. The recovery number of microplastics evaluated with a line regression model was acceptable. Graphical abstract.

Breed differences in the expression levels of gga-miR-222a in laying hens influenced H2S production by regulating methionine synthase genes in gut bacteria.

BACKGROUND: The microbiota in the cecum of laying hens is crucial for host digestion, metabolism, and odor gas production. The results of recent studies have suggested that host microRNAs (miRNAs) can regulate gene expression of the gut microbiota. In the present study, the expression profiles of host-derived miRNAs in the cecal content of two laying hen breeds; Hy-line Gray and Lohmann Pink, which have dissimilar H2S production, were characterized; and their effects on H2S production by regulating the expression of gut microbiota-associated genes were demonstrated. RESULTS: The differential expression of microbial serine O-acetyltransferase, methionine synthase, aspartate aminotransferase, methionine-gamma-lyase, and adenylylsulfate kinase between the two hen breeds resulted in lower H2S production in the Hy-line hens. The results also revealed the presence of miRNA exosomes in the cecal content of laying hens, and an analysis of potential miRNA-target relationships between 9 differentially expressed miRNAs and 9 differentially expressed microbial genes related to H2S production identified two methionine synthase genes, Odosp_3416 and BF9343_2953, that are targeted by gga-miR-222a. Interestingly, in vitro fermentation results showed that gga-miR-222a upregulates the expression of these genes, which increased methionine concentrations but decreased H2S production and soluble sulfide concentrations, indicating the potential of host-derived gga-miR-222a to reduce H2S emission in laying hens. CONCLUSION: The findings of the present study reveal both a physiological role by which miRNAs shape the cecal microbiota of laying hens and a strategy to use host miRNAs to manipulate the microbiome and actively express key microbial genes to reduce H2S emissions and breed environmentally friendly laying hens. Video Abstract.

[Measurement of methane and carbon dioxide emissions from ruminants based on the NDIR technique].

Methane (CH4) production in the rumen represents a loss of energy for the host animal; in addition, methane eructated by ruminants may contribute to a greenhouse effect or global warming. The dinumal CH4 and carbon dioxide (CO2) emissions from sheep were continuously recorded using the flow-through chamber method. A type new type of non-disperse infrared (NDIR) gas sensors based on pulse IR source was introduced, and by using the high performance pyroelectric IR sensor with built in interference filter and the "single light and two wavelengths" technology, CH4 and CO2 measurement from ruminants was achieved. Animals were given dry oat hay as the basic diet and supplemented concentrate with the ratio of 7 : 3. The results showed that the recovery was 96.7% and 96.2% for CH4 and CO2, respectively. Methane and carbon dioxide output from sheep respectively averaged 15.6 g per day and 184.7 g per day, equivalent to 6.8 and 71.1 kg per animal. Diurnal fluctuations in hourly rates of CH4 and CO2 production in hourly of methane increased during day light to reach a peak at or near sunset and then declined towards sunrise, and consideration was given to the dry matter intake of the animals used in these studies and its possible effects on CH4 production.

Metabolic activity of Bacillus coagulans R11 and the health benefits of and potential pathogen inhibition by this species in the intestines of laying hens under lead exposure.

Probiotics are widely used in agricultural breeding for care and maintenance of animal health, especially Bacillus coagulans, a new and popular species that could replace Lactobacillus. However, lead contamination in feed might influence the beneficial function. In the present study, Bacillus coagulans R11 was used as a model bacterium to investigate the effect of lead on changes in metabolites and genes, which could influence the beneficial function on laying hen. At the laboratory scale, transcriptomics and metabolomics were used to screen the main metabolites and related genes under lead exposure. The results showed that 4-acetamidobutanoic acid, dodecanoic acid, L-3-phenyllactic acid, apigenin and daidzein, which are antioxidants and antibacterial agents, were the main metabolites, even in the 100 ppm lead exposure group (the levels of these metabolites were 1.17-, 1.10-, 4.80-, 1.43- and 1.67-fold higher in the 100 ppm group than in pure culture medium). Twenty-three genes associated with the syntheses of the above 5 main metabolites were identified. Further animal experiments showed that B. coagulans R11 feeding of laying hens under lead exposure could prevent oxidative damage by increasing T-AOC and T-SOD activity and reducing the MDA concentration in serum and reducing the abundances of potential pathogens (Escherichia coli, Pseudomonas aeruginosa and Salmonella). Further analysis also showed that the inhibition of pathogen growth was due to the regulation of gene expression, as observed by transcriptomics, and these genes were associated with the abovementioned 5 main metabolites. However, the laying rate decreased by 10.53% compared with that of the control group when the lead exposure concentration was 100 mg/kg. The present study suggested that Bacillus coagulans R11 could help prevent oxidative damage and inhibit pathogen growth in laying hens to maintain a healthy intestinal environment for daily breeding, but under high-lead conditions, Bacillus coagulans R11 feeding could decrease the laying rate.

The Combination of Lead and Bacillus coagulans R11 Increased the Concentration of Alpha-Solanine in the Cecum of Laying Hens and the Pathogens Abundance Decreased.

Alpha-solanine is an alkaloid that can inhibit the growth of pathogens and cancer cells, the present study proved that feeding with Bacillus coagulans R11 increases the concentration of alpha-solanine in the cecum of laying hens, which also decreases the abundance of potential pathogens. In addition, the bacteria genera, metabolism pathways and its proteins involved in the biosynthesis of alpha-solanine in the cecum were also characterized. The results showed that B. coagulans R11 feeding could increase the concentration of alpha-solanine, even with lead exposure. Mevalonic acid and MEP/DOXP pathways were both participated in the biosynthesis of alpha-solanine; at the same time, the gut metabolites (S)-2-amino-6-oxohexanoate, N2-succinyl-L-ornithine and the bacteria proteins atoB, ispH were shown to be crucial role in the biosynthesis of alpha-solanine in the gut. The genera Faecalibacterium sp. An77 and Faecalibacterium sp. An58 2 were important in the biosynthesis of alpha-solanine, which provided the key proteins atoB and ispH. In addition, alpha-solanine could decrease the abundance of Prevotella sp. 109 and Prevotella marshii. In conclusion, alpha-solanine could be biosynthesized by cecal microorganisms with the stimulation of B. coagulans R11 in the intestine of laying hens, in addition, alpha-solanine was the main compound which also decreased the abundance of gut potential.

Bacillus coagulans R11 maintained intestinal villus health and decreased intestinal injury in lead-exposed mice by regulating the intestinal microbiota and influenced the function of faecal microRNAs.

Lead contamination is an environmental problem, especially in developing countries; due to the nondegradable characteristics of lead, it is easily deposited in human and animal bodies by the food chain. Probiotics are regarded as a good tool to remove lead ions in the intestine and maintain gut health conditions, but previous studies failed to elucidate the relationship among probiotics, the host and the gut microbiota. In the present study, B. coagulans R11 was employed as the "lead removal tool" in lead-exposed mouse, and the effects of B. coagulans R11 on intestinal cells, the microbiota and faecal microRNAs were tested. The results indicated that B. coagulans R11 had no negative effects on mouse intestine model cells and helped keep cells in a normal proliferation ratio and reduce the reactive oxygen species and apoptosis ratios under lead exposure conditions. An in vivo mouse experiment also showed that B. coagulans R11 feeding could reduce the intestinal villi damage caused by lead through adjusting the microbiota structure and function, such as increasing the genus abundance of Akkermansia and Alistipes, decreasing the genus abundance of Alloprevotella, Lachnospiraceae, Parabacteroides and Ruminiclostridium, and keeping the protein dltD existing. Host faecal microRNAs may be influenced by lead and B. coagulans R11, which may change the microbiota structure. Thus, B. coagulans R11 has the potential to be developed and considered as the probiotic that protects the host gut against villi damage and gut microbiota structure and function disorders during lead exposure.

The metabolism and morphology mutation response of probiotic Bacillus coagulans for lead stress.

Lead is among the most common toxic heavy metals and its contamination is of great public concern. Bacillus coagulans is the probiotic which can be considered as the lead absorption sorbent to apply in the lead contaminant water directly or indirectly. A better understanding of the lead resistance and tolerance mechanisms of B. coagulans would help further its development and utilization. Wild-type Bacillus coagulans strain R11 isolated from a lead mine, was acclimated to lead-containing culture media over 85 passages, producing two lead-adapted strains, and the two strains shown higher lead intracellular accumulation ability (38.56-fold and 19.36-fold) and reducing ability (6.94-fold and 7.44-fold) than that of wild type. Whole genome sequencing, genome resequencing, and comparative transcriptomics identified lead resistance and tolerance process significantly involved in these genes which regulated glutathione and sulfur metabolism, flagellar formation and metal ion transport pathways in the lead-adapted strains, elucidating the relationships among the mechanisms regulating lead deposition, deoxidation, and motility and the evolved tolerance to lead. In addition, the B. coagulans mutants tended to form flagellar and chemotaxis systems to avoid lead ions rather than export it, suggesting a new resistance strategy. Based on the present results, the optimum lead concentration in environment should be considered when employed B. coagulans as the lead sorbent, due to the bacteria growth ability decreased in high lead concentration and physiology morphology changed could reduce the lead removal effectiveness. The identified deoxidization and compound secretion genes and pathways in B. coagulans R11 also are potential genetic engineering candidates for synthesizing glutathione, cysteine, methionine, and selenocompounds.

Biosorption of lead (Pb2+) by the vegetative and decay cells and spores of Bacillus coagulans R11 isolated from lead mine soil.

The lead (Pb2+) bioaccumulation capacities and mechanisms of three different physiological structures (vegetative cells, decay cells and spores) of B. coagulans R11 isolated from a lead mine were examined in this study. The results showed that the total Pb2+ removal capacity of vegetative cells (17.53 mg/g) was at its optimal and higher than those of the spores and decay cells at the initial lead concentration of 50 mg/L. However, when the initial lead concentration surpassed 50 mg/L, Pb2+ removal capacity of decay cells was more efficient. Zeta potential, Fourier transform infrared (FTIR) and functional group modification analyses demonstrated that the electrostatic attraction and chelating activity of the functional groups were the primary pathways involved in the extracellular accumulation of Pb2+ by the vegetative cells and spores. However, the primary Pb2+ binding pathway in the decay cells was hypothesized to be due to physical adsorption, which easily led to Pb2+ desorption. Based on these results, we conclude that the vegetative cell is the ideal lead sorbent. Therefore, it is important to inhibit the transformation of the vegetative cells into decay cells and spores, which can be achieved by culturing the bacteria under anaerobic conditions to prevent spore formation. Heat stimulation can effectively enhance spore germination to generate vegetative cells.