Shengyong Mao

Characterising the bacterial microbiota across the gastrointestinal tracts of dairy cattle: membership and potential function

The bacterial community composition and function in the gastrointestinal tracts (GITs) of dairy cattle is very important, since it can influence milk production and host health. However, our understanding of bacterial communities in the GITs of dairy cattle is still very limited. This study analysed bacterial communities in ten distinct GIT sites (the digesta and mucosa of the rumen, reticulum, omasum, abomasum, duodenum, jejunum, ileum, cecum, colon and rectum) in six dairy cattle. The study observed 542 genera belonging to 23 phyla distributed throughout the cattle GITs, with the Firmicutes, Bacteroidetes and Proteobacteria predominating. In addition, data revealed significant spatial heterogeneity in composition, diversity and species abundance distributions of GIT microbiota. Furthermore, the study inferred significant differences in the predicted metagenomic profiles among GIT regions. In particular, the relative abundances of the genes involved in carbohydrate metabolism were overrepresented in the digesta samples of forestomaches, and the genes related to amino acid metabolism were mainly enriched in the mucosal samples. In general, this study provides the first deep insights into the composition of GIT microbiota in dairy cattle, and it may serve as a foundation for future studies in this area.

Microbiome-metabolome analysis reveals unhealthy alterations in the composition and metabolism of ruminal microbiota with increasing dietary grain in a goat model.

Currently, knowledge about the impact of high-grain (HG) feeding on rumen microbiota and metabolome is limited. In this study, a combination of the 454 pyrosequencing strategy and the mass spectrometry-based metabolomics technique was applied to investigate the effects of increased dietary grain (0%, 25% and 50% maize grain) on changes in whole ruminal microbiota and their metabolites using goat as a ruminant model. We observed a significant influence of HG feeding in shaping the ruminal bacterial community structure, diversity and composition, with an overall dominance of bacteria of the phylum Firmicutes along with a low abundance of Bacteriodetes in the HG group. High-grain feeding increased the number of ciliate and methanogens, and decreased the density of anaerobic fungi and the richness of the archaeal community. The metabolomics analysis revealed that HG feeding increased the levels of several toxic, inflammatory and unnatural compounds, including endotoxin, tryptamine, tyramine, histamine and phenylacetate. Correlation analysis on the combined datasets revealed some potential relationships between ruminal metabolites and certain microbial species. Information about these relationships may prove useful in either direct (therapeutic) or indirect (dietary) interventions for ruminal disorders due to microbial compositional shifts, such as ruminal acidosis.

Effects of alfalfa and cereal straw as a forage source on nutrient digestibility and lactation performance in lactating dairy cows.

This study was conducted to investigate the nutrient digestibility and lactation performance when alfalfa was replaced with rice straw or corn stover in the diet of lactating cows. Forty-five multiparous Holstein dairy cows were blocked based on days in milk (164 ± 24.8 d; mean ± standard deviation) and milk yield (29.7 ± 4.7 kg; mean ± standard deviation) and were randomly assigned to 1 of 3 treatments. Diets were isonitrogenous, with a forage-to-concentrate ratio of 45:55 [dry matter (DM) basis] and contained identical concentrate mixtures and 15% corn silage, with different forage sources (on a DM basis): 23% alfalfa hay and 7% Chinese wild rye hay (AH), 30% corn stover (CS), and 30% rice straw (RS). The experiment was conducted over a 14-wk period, with the first 2 wk for adaptation. The DM intake of the cows was not affected by forage source. Yield of milk, milk fat, protein, lactose, and total solids was higher in cows fed diets of AH than diets of RS or CS, with no difference between RS and CS. Contents of milk protein and total solids were higher in AH than in RS, with no difference between CS and AH or RS. Feed efficiency (milk yield/DM intake) was highest for cows fed AH, followed by RS and CS. Cows fed AH excreted more urinary purine derivatives, indicating that the microbial crude protein yield may be higher for the AH diet than for RS and CS, which may be attributed to the higher content of fermentable carbohydrates in AH than in RS and CS. Total-tract apparent digestibilities of all the nutrients were higher in cows fed the AH diet than those fed CS and RS. The concentration of rumen volatile fatty acids was higher in the AH diet than in CS or RS diets, with no difference between CS and RS diets. When the cereal straw was used to replace alfalfa as a main forage source for lactating cows, the shortage of fermented energy may have reduced the rumen microbial protein synthesis, resulting in lower milk protein yield, and lower nutrient digestibility may have restricted milk production.

Comparative studies of the composition of bacterial microbiota associated with the ruminal content, ruminal epithelium and in the faeces of lactating dairy cows.

The objective of this research was to compare the composition of bacterial microbiota associated with the ruminal content (RC), ruminal epithelium (RE) and faeces of Holstein dairy cows. The RC, RE and faecal samples were collected from six Holstein dairy cows when the animals were slaughtered. Community compositions of bacterial 16S rRNA genes from RC, RE and faeces were determined using a MiSeq sequencing platform with bacterial‐targeting universal primers 338F and 806R. UniFrac analysis revealed that the bacterial communities of RC, RE and faeces were clearly separated from each other. Statistically significant dissimilarities were observed between RC and faeces (P = 0.002), between RC and RE (P = 0.003), and between RE and faeces (P = 0.001). A assignment of sequences to taxa showed that the abundance of the predominant phyla Bacteroidetes was lower in RE than in RC, while a significant higher (P < 0.01) abundance of Proteobacteria was present in RE than in RC. When compared with the RC, the abundance of Firmicutes and Verrucomicrobia was higher in faeces, and RC contained a greater abundance of Bacteroidetes and Tenericutes. A higher proportions of Butyrivibrio and Campylobacter dominated RE as compared to RC. The faecal microbiota was less diverse than RC and dominated by genera Turicibacter and Clostridium. In general, these findings clearly demonstrated the striking compositional differences among RC, RE and faeces, indicating that bacterial communities are specific and adapted to the harbouring environment.

Effect of dietary forage sources on rumen microbiota, rumen fermentation and biogenic amines in dairy cows.

BACKGROUND Fifteen lactating Holstein dairy cows were assigned to three diets in a 3 × 3 Latin square design to evaluate the effects of dietary forage sources on rumen microbiota, rumen fermentation and biogenic amines. Diets were isonitrogenous and isocaloric, with a forage/concentrate ratio of 45:55 (dry matter basis) but different main forage sources, namely cornstalk (CS), Leymus chinensis (LC) or alfalfa hay (AH). RESULTS Pyrosequencing of the V3-V6 hypervariable coding region of 16S rRNA revealed that the rumen microbiota was significantly affected by forage sources. AH feeding increased the proportion of genera Prevotella and Selenomonas compared with the CS diet, while CS feeding increased the proportion of genera Anaerotruncus, Papillibacter, Thermoactimoyces, Bacillus and Streptomyces compared with the LC or AH diet. AH and LC feeding both increased the propionate concentration compared with the CS diet. AH feeding decreased the concentrations of tyramine, putrescine and histamine compared with the LC diet. CONCLUSION These results indicate that a high proportion of alfalfa hay in the ration is beneficial for milk yield and a healthy and balanced rumen microbiota in lactating cattle. This can be attributed to the higher degradation of rumen organic matter and the more balanced carbohydrates and proteins for optimal rumen microbial growth.

Characterization of bacterial community of raw milk from dairy cows during subacute ruminal acidosis challenge by high‐throughput sequencing

BACKGROUND Four cannulated primiparous Holstein dairy cows (84 ± 25 DIM) were used in a 2 × 2 crossover experimental design. The two diets contained 40% (low-concentrate diet, or control diet, LC) and 70% (high-concentrate diet, or SARA induction diet, HC) concentrate feeds respectively. Milk samples were collected on days 17, 18 and 19 of each experimental period. DNA was extracted from each milk sample, and pyrosequencing was applied to analyse the milk microbial community. RESULTS Regardless of diet, the bacterial community of milk was dominated by Actinobacteria, Firmicutes, Proteobacteria and Bacteroidetes. HC feeding showed a higher proportion of some mastitis-causing pathogen bacteria, such as Stenotrophomonas maltophilia, Streptococcus parauberis and Brevundimonas diminuta, as well as of psychrotrophic bacteria, such as Pseudomonas, Brevundimonas, Sphingobacterium, Alcaligenes, Enterobacter and Lactobacillus. However, the diversity of the milk bacterial microbiota (ACE, Chao, and Shannon index) was not affected by HC feeding. CONCLUSION To the best of our knowledge, this is the first report on the use of pyrosequencing for evaluating the impacts of nutrition on changes in the composition of milk microbiota. These findings indicate that HC feeding may increase the risk of dairy cows suffering from mastitis, decrease the organoleptic quality of raw milk and dairy products, and limit the shelf life of processed fluid milk.

Grain-rich diets altered the colonic fermentation and mucosa-associated bacterial communities and induced mucosal injuries in goats.

Remarkably little information is available about the impact of high-grain (HG) feeding on colonic mucosa-associated bacteria and mucosal morphology. In the present study, 12 male goats were randomly assigned to either a hay diet (n = 6) or an HG diet (65% grain; n = 6) to characterise the changes in the composition of the bacterial community in colonic mucosa and the mucosal morphology of the colon. The results showed that HG feeding decreased the colonic pH and increased the concentrations of total short chain fatty acids and lipopolysaccharides in colonic digesta. The principal coordinate analysis results showed that the HG diet altered the colonic mucosal bacterial communities, with an increase in the abundance of genus Blautia and a decrease in the abundance of genera Bacillus, Enterococcus, and Lactococcus. The HG-fed goats showed sloughing of the surface layer epithelium, intercellular tight junction erosion, cell mitochondrial damage, and upregulation of the relative mRNA expression of IL-2 and IFN-γ in colonic mucosa. Collectively, our data indicate that HG feeding induced changes in colonic mucosal morphology and cytokines expression that might be caused by excessive fermentation and dramatic shifts in the bacterial populations in the colon.

Effects of Ruminal Infusion of Garlic Oil on Fermentation Dynamics, Fatty Acid Profile and Abundance of Bacteria Involved in Biohydrogenation in Rumen of Goats

This study aimed to investigate the effects of ruminal infusion of garlic oil (GO) on fermentation dynamics, fatty acid (FA) profile, and abundance of bacteria involved in biohydrogenation in the rumen. Six wethers fitted with ruminal fistula were assigned to two groups for cross-over design with a 14-d interval. Each 30-d experimental period consisted of a 27-d adaptation and a 3-d sample collection. Goats were fed a basal diet without (control) or with GO ruminal infusion (0.8 g/d). Ruminal contents collected before (0 h) and at 2, 4, 6, 8, and 10 h after morning feeding were used for fermentation analysis, and 0 h samples were further used for FA determination and DNA extraction. Garlic oil had no influence on dry matter intakes of concentrate and hay. During ruminal fermentation, GO had no effects on total VFA concentration and individual VFA molar proportions, whereas GO increased the concentrations of ammonia nitrogen and microbial crude protein (p<0.05). Compared with control, GO group took a longer time for total VFA concentration and propionate molar proportion to reach their respective maxima after morning feeding. The ratio of acetate to propionate in control reduced sharply after morning feeding, whereas it remained relatively stable in GO group. Fatty acid analysis showed that GO reduced saturated FA proportion (p<0.05), while increasing the proportions of C18, t11-18:1 (TVA), c9,t11-conjugated linoleic acid (c9,t11-CLA), t10,c12-CLA, and polyunsaturated FA (p<0.05). The values of TVA/(c9,t11-CLA+TVA) and C18:0/(TVA+ C18:0) were reduced by GO (p<0.05). Real-time PCR showed that GO tended to reduce Butyrivibrio proteoclasticus abundance (p = 0.058), whereas GO had no effect on total abundance of the Butyrivibrio group bacteria. A low correlation was found between B. proteoclasticus abundance and C18:0/(TVA+C18:0) (p = 0.910). The changes of fermentation over time suggested a role of GO in delaying the fermentation process and maintaining a relatively modest change of ruminal environment. The inhibitory effects of GO on the final step of biohydrogenation may be related to its antibacterial activity against B. proteoclasticus and other unknown bacteria involved.

Characterization and comparison of the temporal dynamics of ruminal bacterial microbiota colonizing rice straw and alfalfa hay within ruminants

Three ruminally cannulated Holstein cows were used to characterize the dynamics of bacterial colonization of rice straw and alfalfa hay and to assess the differences in the composition and inferred gene function of the colonized microbiota between these 2 forages. Nonincubated (0h) rice straw and alfalfa hay samples and residues in nylon bags incubated for 0.5, 2, 6, 16, and 48h were analyzed for dry matter and were used for DNA extraction and MiSeq (Illumina Inc., San Diego, CA) sequencing of the 16S rRNA gene. The microbial communities that colonized the air-dried and nonincubated (0h) rice straw and alfalfa hay were both dominated by members of the Proteobacteria (contributing toward 70.47% of the 16S RNA reads generated). In situ incubation of the 2 forages revealed major shifts in the community composition: Proteobacteria were replaced within 30min by members belonging to the Bacteroidetes and Firmicutes, contributing toward 51.9 and 36.6% of the 16S rRNA reads generated, respectively. A second significant shift was observed after 6h of rumen incubation, when members of the Spirochaetes and Fibrobacteria phyla became abundant in the forage-adherent community. During the first 30min of rumen incubation, ~20.7 and 36.1% of the rice straw and alfalfa hay, respectively, were degraded, whereas little biomass degradation occurred between 30min and 2h after the rice straw or alfalfa hay was placed in the rumen. Significant differences were noted in attached bacterial community structure between the 2 forage groups, and the abundances of dominant genera Anaeroplasma, Butyrivibrio, Fibrobacter, and Prevotella were affected by the forage types. Real-time PCR results showed that the 16S rRNA copies of total bacteria attached to these 2 forages were affected by the forage types and incubation time, and higher numbers of attached bacterial 16S rRNA were observed in the alfalfa hay samples than in the rice straw from 0.5 to 16h of incubation. The metagenomes predicted by phylogenetic investigation of communities by reconstruction of unobserved states (PICRUSt) revealed that the forage types significantly affected 21 metabolic pathways identified in the Kyoto Encyclopedia of Genes and Genomes, and 33 were significantly changed over time. Collectively, our results reveal a difference in the dynamics of bacterial colonization and the inferred gene function of microbiota associated with rice straw and alfalfa hay within the rumen. These findings are of great importance for the targeted improvement of forage nutrient use efficiency in ruminants.

Intravenous lipopolysaccharide challenge alters ruminal bacterial microbiota and disrupts ruminal metabolism in dairy cattle.

In the present study, three primiparous lactating Holstein cows (260-285 d in lactation) were used in a 3 × 3 Latin square design to assess the effects of three doses (0.0, 0.4 and 0.8 μg/kg body weight) of lipopolysaccharide (LPS, Escherichia coli 0111:B4) on changes in ruminal microbiota and ruminal fermentation. Ruminal pH was linearly decreased (P< 0.001) by LPS challenge, and the concentrations of acetate, propionate, butyrate, total volatile fatty acids and amino N increased linearly (P< 0.001) according to the LPS dose. LPS infusion linearly decreased (P< 0.001) the organic matter degradability of alfalfa hay and soyabean meal in the rumen, but did not affect (P>0.10) the gene expression of Na⁺/K⁺-ATPase and monocarboxylic acid transporter-1, -2 and -4. A plot of principal coordinate analysis based on unweighted UniFrac values and analysis of molecular variance revealed that the structure of ruminal bacterial communities in the control was distinct from that of the ruminal microbiota in the cattle exposed to LPS. At the phylum level, when compared with the control group, LPS infusion in the tested cows linearly increased (P< 0.05) the abundance of Firmicutes, and linearly decreased (P< 0.05) the percentage of Bacteroidetes, Tenericutes, Spirochaetes, Chlorobi and Lentisphaerae. To our knowledge, this is the first study to report that intravenously LPS challenge altered the ruminal bacterial microbiota and fermentation profiles. The present data suggest that systemic LPS could alter ruminal environment and ruminal microbiota composition, leading to a general decrease in fermentative activity.