Supplemental tables and figures for the paper "Changes in Rumen Epithelial Morphology and Transcriptome, Rumen Metabolome, and Blood Biochemical Parameters in Lactating Dairy Cows with Subacute Rumen Acidosis Following Rumen Content Transplantation".

Supplemental Table S1. Ingredients and nutritional composition of the conventional diet (CON) and the high-grain diet (HG)Figure S1 Principal component (PC) analysis of total genes in the rumen epithelial papilla between the donor-recipient (DR) and the self-recipient (SR) groups. PERMANOVA results with 9999 permutations are shown.Figure S2 Heatmap-like functional classification of the differentially expressed genes (DEGs) in rumen epithelial papilla between the donor-recipient (DR) and the self-recipient (SR) groups. Expression patterns of the DEGs corresponding to the (A) significantly enriched Gene Ontology biological process terms and (B) KEGG pathways at a P value of Figure S3 The orthogonal partial least squares discriminant analysis (OPLS-DA) of rumen metabolites between the donor-recipient (DR) and the self-recipient (SR) groups. The score scatter plot of the OPLS-DA model in the (A) positive and (B) negative ion modes. Permutation test plots of 200 iterations in the (C) positive and (D) negative ion modes. R2 and Q2 are fitness power and predictive power of the model, respectively.Figure S4 Metabolic pathway analysis of KEGG for differential metabolites between the donor-recipient (DR) and the self-recipient (SR) groups.</p

Coordinated response of milk bacterial and metabolic profiles to subacute ruminal acidosis in lactating dairy cows

Abstract Background Bovine milk is an important source of nutrition for human consumption, and its quality is closely associated with the microbiota and metabolites in it. But there is limited knowledge about the milk microbiome and metabolome in cows with subacute ruminal acidosis. Methods Eight ruminally cannulated Holstein cows in mid lactation were selected for a 3-week experiment. The cows were randomly allocated into 2 groups, fed either a conventional diet (CON; 40% concentrate; dry matter basis) or a high-concentrate diet (HC; 60% concentrate; dry matter basis). Results The results showed that there was a decreased milk fat percentage in the HC group compared to the CON group. The amplicon sequencing results indicated that the alpha diversity indices were not affected by the HC feeding. At the phylum level, the milk bacteria were dominated by Proteobacteria, Actinobacteria, Bacteroidetes, and Firmicutes both in the CON and HC groups. At the genus level, the HC cows displayed an improved proportion of Labrys (P = 0.015) compared with the CON cows. Results of both the principal components analysis and partial least squares of discriminant analysis of milk metabolome revealed that samples of the CON and HC groups clustered separately. A total of 31 differential metabolites were identified between the two groups. Of these, the levels of 11 metabolites decreased (α-linolenic acid, prostaglandin E2, L-lactic acid, L-malic acid, 3-hydroxysebacic acid, succinyladenosine, guanosine, pyridoxal, L-glutamic acid, hippuric acid, and trigonelline), whereas the levels of the other 20 metabolites increased in the HC group with respect to the CON group (P < 0.05). Conclusion These results suggested that subacute ruminal acidosis less impacted the diversity and composition of milk microbiota, but altered the milk metabolic profiles, which led to the decline of the milk quality

High‐production dairy cattle exhibit different rumen and fecal bacterial community and rumen metabolite profile than low‐production cattle

Abstract Our aim was to simultaneously investigate the gut bacteria typical characteristic and conduct rumen metabolites profiling of high production dairy cows when compared to low‐production dairy cows. The bacterial differences in rumen fluid and feces were identified by 16S rDNA gene sequencing. The metabolite differences were identified by metabolomics profiling with liquid chromatography mass spectrometry (LC‐MS). The results indicated that the high‐production dairy cows presented a lower rumen bacterial richness and species evenness when compared to low‐production dairy cows. At the phylum level, the high‐production cows increased the abundance of Proteobacteria and decreased the abundance of Bacteroidetes, SR1, Verrucomicrobia, Euryarchaeota, Planctomycetes, Synergistetes, and Chloroflexi significantly (p < 0.05). At the genus level, the rumen fluid of the high‐production group was significantly enriched for Butyrivibrio, Lachnospira, and Dialister (p < 0.05). Meanwhile, rumen fluid of high‐production group was depleted for Prevotella, Succiniclasticum, Ruminococcu, Coprococcus,YRC22, CF231, 02d06, Anaeroplasma, Selenomonas, and Ruminobacter significantly (p < 0.05). A total of 92 discriminant metabolites were identified between high‐production cows and low‐production cows. Compared to rumen fluid of low‐production dairy cows, 10 differential metabolites were found up‐regulated in rumen fluid of high‐production dairy cows, including 6alpha‐Fluoropregn‐4‐ene‐3,20‐dione, 3‐Octaprenyl‐4‐hydroxybenzoate, disopyramide, compound III(S), 1,2‐Dimyristyl‐sn‐glycerol, 7,10,13,16‐Docosatetraenoic acid, ferrous lactate, 6‐Deoxyerythronolide B, vitamin D2, L‐Olivosyl‐oleandolide. The remaining differential metabolites were found down‐regulated obviously in high‐production cows. Metabolic pathway analyses indicated that most increased abundances of rumen fluid metabolites of high‐yield cows were related to metabolic pathways involving biosynthesis of unsaturated fatty acids, steroid biosynthesis, ubiquinone and other terpenoid‐quinone biosynthesis. Most down‐regulated metabolic pathways were relevant to nucleotide metabolism, energy metabolism, lipid metabolism and biosynthesis of some antibiotics

Fecal microbial gene transfer contributes to the high-grain diet-induced augmentation of aminoglycoside resistance in dairy cattle

ABSTRACTA high-grain (HG) diet can rapidly lower the rumen pH and thus modify the gastrointestinal microbiome in dairy cattle. Although the prevalence of antibiotic resistance is strongly linked with the gut microbiome, the influences of HG diet on animals’ gut resistome remain largely unexplored. Here, we examined the impact and mechanism of an HG diet on the fecal resistome in dairy cattle by metagenomically characterizing the gut microbiome. Eight lactating Holstein cattle were randomly allocated into two groups and fed either a conventional (CON) or HG diet for 3 weeks. The fecal microbiome and resistome were significantly altered in dairy cattle from HG, demonstrating an adaptive response that peaks at day 14 after the dietary transition. Importantly, we determined that feeding an HG diet specifically elevated the prevalence of resistance to aminoglycosides (0.11 vs 0.24 RPKG, P < 0.05). This diet-induced resistance increase is interrelated with the disproportional propagation of microbes in Lachnospiraceae, indicating a potential reservoir of aminoglycosides resistance. We further showed that the prevalence of acquired resistance genes was also modified by introducing a different diet, likely due to the augmented frequency of lateral gene transfer (LGT) in microbes (CON vs HG: 254 vs 287 taxa) such as Lachnospiraceae. Consequently, we present that diet transition is associated with fecal resistome modification in dairy cattle and an HG diet specifically enriched aminoglycosides resistance that is likely by stimulating microbial LGT.IMPORTANCEThe increasing prevalence of antimicrobial resistance is one of the most severe threats to public health, and developing novel mitigation strategies deserves our top priority. High-grain (HG) diet is commonly applied in dairy cattle to enhance animals’ performance to produce more high-quality milk. We present that despite such benefits, the application of an HG diet is correlated with an elevated prevalence of resistance to aminoglycosides, and this is a combined effect of the expansion of antibiotic-resistant bacteria and increased frequency of lateral gene transfer in the fecal microbiome of dairy cattle. Our results provided new knowledge in a typically ignored area by showing an unexpected enrichment of antibiotic resistance under an HG diet. Importantly, our findings laid the foundation for designing potential dietary intervention strategies to lower the prevalence of antibiotic resistance in dairy production

Guidelines for the use and interpretation of assays for monitoring autophagy

In 2008 we published the first set of guidelines for standardizing research in autophagy. Since then, research on this topic has continued to accelerate, and many new scientists have entered the field. Our knowledge base and relevant new technologies have also been expanding. Accordingly, it is important to update these guidelines for monitoring autophagy in different organisms. Various reviews have described the range of assays that have been used for this purpose. Nevertheless, there continues to be confusion regarding acceptable methods to measure autophagy, especially in multicellular eukaryotes. A key point that needs to be emphasized is that there is a difference between measurements that monitor the numbers or volume of autophagic elements (e.g., autophagosomes or autolysosomes) at any stage of the autophagic process vs. those that measure flux through the autophagy pathway (i.e., the complete process); thus, a block in macroautophagy that results in autophagosome accumulation needs to be differentiated from stimuli that result in increased autophagic activity, defined as increased autophagy induction coupled with increased delivery to, and degradation within, lysosomes (in most higher eukaryotes and some protists such as Dictyostelium) or the vacuole (in plants and fungi). In other words, it is especially important that investigators new to the field understand that the appearance of more autophagosomes does not necessarily equate with more autophagy. In fact, in many cases, autophagosomes accumulate because of a block in trafficking to lysosomes without a concomitant change in autophagosome biogenesis, whereas an increase in autolysosomes may reflect a reduction in degradative activity. Here, we present a set of guidelines for the selection and interpretation of methods for use by investigators who aim to examine macroautophagy and related processes, as well as for reviewers who need to provide realistic and reasonable critiques of papers that are focused on these processes. These guidelines are not meant to be a formulaic set of rules, because the appropriate assays depend in part on the question being asked and the system being used. In addition, we emphasize that no individual assay is guaranteed to be the most appropriate one in every situation, and we strongly recommend the use of multiple assays to monitor autophagy. In these guidelines, we consider these various methods of assessing autophagy and what information can, or cannot, be obtained from them. Finally, by discussing the merits and limits of particular autophagy assays, we hope to encourage technical innovation in the field