Supplementary File 1 Li et al

RNA-seq comparisons of all data sets<br

Image_1_Analysis of serum antioxidant capacity and gut microbiota in calves at different growth stages in Tibet.pdf

IntroductionThe hypoxic environment at high altitudes poses a major physiological challenge to animals, especially young animals, as it disturbs the redox state and induces intestinal dysbiosis. Information about its effects on Holstein calves is limited.MethodsHere, serum biochemical indices and next-generation sequencing were used to explore serum antioxidant capacity, fecal fermentation performance, and fecal microbiota in Holstein calves aged 1, 2, 3, 4, 5, and 6 months in Tibet.Results and DiscussionSerum antioxidant capacity changed with age, with the catalase and malondialdehyde levels significantly decreasing (p  0.05) in total volatile fatty acid levels were noted between the groups. In all groups, Firmicutes, Bacteroidetes, and Actinobacteria were the three most dominant phyla in the gut. Gut microbial alpha diversity significantly increased (p < 0.05) with age. Principal coordinate analysis plot based on Bray–Curtis dissimilarity revealed significant differences (p = 0.001) among the groups. Furthermore, the relative abundance of various genera changed dynamically with age, and the serum antioxidant capacity was associated with certain gut bacteria. The study provides novel insights for feeding Holstein calves in high-altitude regions.</p

Image_2_Analysis of serum antioxidant capacity and gut microbiota in calves at different growth stages in Tibet.png

IntroductionThe hypoxic environment at high altitudes poses a major physiological challenge to animals, especially young animals, as it disturbs the redox state and induces intestinal dysbiosis. Information about its effects on Holstein calves is limited.MethodsHere, serum biochemical indices and next-generation sequencing were used to explore serum antioxidant capacity, fecal fermentation performance, and fecal microbiota in Holstein calves aged 1, 2, 3, 4, 5, and 6 months in Tibet.Results and DiscussionSerum antioxidant capacity changed with age, with the catalase and malondialdehyde levels significantly decreasing (p  0.05) in total volatile fatty acid levels were noted between the groups. In all groups, Firmicutes, Bacteroidetes, and Actinobacteria were the three most dominant phyla in the gut. Gut microbial alpha diversity significantly increased (p < 0.05) with age. Principal coordinate analysis plot based on Bray–Curtis dissimilarity revealed significant differences (p = 0.001) among the groups. Furthermore, the relative abundance of various genera changed dynamically with age, and the serum antioxidant capacity was associated with certain gut bacteria. The study provides novel insights for feeding Holstein calves in high-altitude regions.</p

Data_Sheet_1_Effect of Limit-Fed Diets With Different Forage to Concentrate Ratios on Fecal Bacterial and Archaeal Community Composition in Holstein Heifers.docx

<p>Limit-feeding of a high concentrate diet has been proposed as an effective method for improving feed efficiency and reducing total manure output of dairy heifers; meanwhile the effects of this method on hindgut microbiota are still unclear. This study investigated the effects of a wide range of dietary forage:concentrate ratios (F:C) on the fecal composition of bacteria and archaea in heifers using next-generation sequencing. Four diets with different F:C (80:20, 60:40, 40:60, and 20:80) were limit-fed to 24 Holstein heifers, and the fecal fermentation parameters and bacterial and archaeal communities were investigated. With increasing dietary concentrate levels, the fecal dry matter output, neutral detergent fiber (NDF) content, and proportion of acetate decreased linearly (P < 0.01), while the fecal starch content and proportions of propionate, butyrate, and total branched-chain volatile fatty acids (TBCVFAs) were increased (P ≤ 0.05). An increased concentrate level linearly increased (P = 0.02) the relative abundance of Proteobacteria, and linearly decreased (P = 0.02) the relative abundance of Bacteroidetes in feces. At the genus level, the relative abundance of unclassified Ruminococcaceae and Paludibacter which may have the potential to degrade forage decreased linearly (q ≤ 0.02) with increasing dietary concentrate levels, while the relative abundance of Roseburia and Succinivibrio which may be non-fibrous carbohydrate degrading bacteria increased linearly (q ≤ 0.05). Some core microbiota operational taxonomic units (OTUs) also showed significant association with fecal VFAs, NDF, and/or acid detergent fiber (ADF) content. Meanwhile, the relative abundance of most detected taxa in archaea were similar across different F:C, and only Methanosphaera showed a linear decrease (P = 0.01) in high concentrate diets. Our study provides a better understanding of fecal fermentation parameters and microbiota under a wide range of dietary F:C. These findings support the potential for microbial manipulation by diet, which could enhance feed digestibility and relieve environmental problems associated with heifer rearing.</p

Additional file 1 of Repeated inoculation with rumen fluid accelerates the rumen bacterial transition with no benefit on production performance in postpartum Holstein dairy cows

Additional file 1: Table S1. Diet ingredients and chemical composition of TMR diet

Image_2_Effect of Limit-Fed Diets With Different Forage to Concentrate Ratios on Fecal Bacterial and Archaeal Community Composition in Holstein Heifers.TIF

<p>Limit-feeding of a high concentrate diet has been proposed as an effective method for improving feed efficiency and reducing total manure output of dairy heifers; meanwhile the effects of this method on hindgut microbiota are still unclear. This study investigated the effects of a wide range of dietary forage:concentrate ratios (F:C) on the fecal composition of bacteria and archaea in heifers using next-generation sequencing. Four diets with different F:C (80:20, 60:40, 40:60, and 20:80) were limit-fed to 24 Holstein heifers, and the fecal fermentation parameters and bacterial and archaeal communities were investigated. With increasing dietary concentrate levels, the fecal dry matter output, neutral detergent fiber (NDF) content, and proportion of acetate decreased linearly (P < 0.01), while the fecal starch content and proportions of propionate, butyrate, and total branched-chain volatile fatty acids (TBCVFAs) were increased (P ≤ 0.05). An increased concentrate level linearly increased (P = 0.02) the relative abundance of Proteobacteria, and linearly decreased (P = 0.02) the relative abundance of Bacteroidetes in feces. At the genus level, the relative abundance of unclassified Ruminococcaceae and Paludibacter which may have the potential to degrade forage decreased linearly (q ≤ 0.02) with increasing dietary concentrate levels, while the relative abundance of Roseburia and Succinivibrio which may be non-fibrous carbohydrate degrading bacteria increased linearly (q ≤ 0.05). Some core microbiota operational taxonomic units (OTUs) also showed significant association with fecal VFAs, NDF, and/or acid detergent fiber (ADF) content. Meanwhile, the relative abundance of most detected taxa in archaea were similar across different F:C, and only Methanosphaera showed a linear decrease (P = 0.01) in high concentrate diets. Our study provides a better understanding of fecal fermentation parameters and microbiota under a wide range of dietary F:C. These findings support the potential for microbial manipulation by diet, which could enhance feed digestibility and relieve environmental problems associated with heifer rearing.</p

KO^<i>t</i>Bu-Mediated Coupling of Indoles and [60]Fullerene: Transition‑Metal-Free and General Synthesis of 1,2-(3-Indole)(hydro)[60]fullerenes

Direct coupling of indoles with C₆₀ has been achieved for the first time. Transition-metal-free KO^<i>t</i>Bu-mediated reaction of indoles to [60]­fullerene has been developed as a practical and efficient method for the synthesis of various 1,2-(3-indole)­(hydro)[60]­fullerenes that are otherwise difficult to direct synthesize in an efficient and selective manner. This methodology tolerates sensitive functionalities such as chloro, ester, and nitro on indole and builds molecular complexity rapidly, with most reactions reaching completion in <1 h. A plausible reaction mechanism is proposed to explain the high regioselectivity at the 3-position of the indoles and the formation of 1,2-(3-indole)­(hydro)[60]­fullerenes

Data_Sheet_2_Effect of Limit-Fed Diets With Different Forage to Concentrate Ratios on Fecal Bacterial and Archaeal Community Composition in Holstein Heifers.xlsx

<p>Limit-feeding of a high concentrate diet has been proposed as an effective method for improving feed efficiency and reducing total manure output of dairy heifers; meanwhile the effects of this method on hindgut microbiota are still unclear. This study investigated the effects of a wide range of dietary forage:concentrate ratios (F:C) on the fecal composition of bacteria and archaea in heifers using next-generation sequencing. Four diets with different F:C (80:20, 60:40, 40:60, and 20:80) were limit-fed to 24 Holstein heifers, and the fecal fermentation parameters and bacterial and archaeal communities were investigated. With increasing dietary concentrate levels, the fecal dry matter output, neutral detergent fiber (NDF) content, and proportion of acetate decreased linearly (P < 0.01), while the fecal starch content and proportions of propionate, butyrate, and total branched-chain volatile fatty acids (TBCVFAs) were increased (P ≤ 0.05). An increased concentrate level linearly increased (P = 0.02) the relative abundance of Proteobacteria, and linearly decreased (P = 0.02) the relative abundance of Bacteroidetes in feces. At the genus level, the relative abundance of unclassified Ruminococcaceae and Paludibacter which may have the potential to degrade forage decreased linearly (q ≤ 0.02) with increasing dietary concentrate levels, while the relative abundance of Roseburia and Succinivibrio which may be non-fibrous carbohydrate degrading bacteria increased linearly (q ≤ 0.05). Some core microbiota operational taxonomic units (OTUs) also showed significant association with fecal VFAs, NDF, and/or acid detergent fiber (ADF) content. Meanwhile, the relative abundance of most detected taxa in archaea were similar across different F:C, and only Methanosphaera showed a linear decrease (P = 0.01) in high concentrate diets. Our study provides a better understanding of fecal fermentation parameters and microbiota under a wide range of dietary F:C. These findings support the potential for microbial manipulation by diet, which could enhance feed digestibility and relieve environmental problems associated with heifer rearing.</p

Effects of Diets Supplemented with Ensiled Mulberry Leaves and Sun-Dried Mulberry Fruit Pomace on the Ruminal Bacterial and Archaeal Community Composition of Finishing Steers

<div><p>This study investigated the effects of ensiled mulberry leaves (EML) and sun-dried mulberry fruit pomace (SMFP) on the ruminal bacterial and archaeal community composition of finishing steers. Corn grain- and cotton meal-based concentrate was partially replaced with EML or SMFP. The diets had similar crude protein (CP), neutral detergent fiber (NDF), and metabolizable energy. Following the feeding trial, the steers were slaughtered and ruminal liquid samples were collected to study the ruminal microbiome. Extraction of DNA, amplification of the V4 region of the 16S rRNA gene, and Illumina MiSeq pyrosequencing were performed for each sample. Following sequence de-noising, chimera checking, and quality trimming, an average of 209,610 sequences were generated per sample. Quantitative real-time PCR was performed to examine the selected bacterial species in the rumen. Our results showed that the predominant phyla were <i>Bacteroidetes</i> (43.90%), <i>Firmicutes</i> (39.06%), <i>Proteobacteria</i> (4.31%), and <i>Tenericutes</i> (2.04%), and the predominant genera included <i>Prevotella</i> (13.82%), <i>Ruminococcus</i> (2.51%), <i>Butyrivibrio</i> (2.38%), and <i>Succiniclasticum</i> (2.26%). Compared to the control group, EML and SMFP groups had a higher abundance of total bacteria (<i>p</i> < 0.001); however, the bacterial community composition was similar among the three groups. At the phylum level, there were no significant differences in <i>Firmicutes</i> (<i>p</i> = 0.7932), <i>Bacteroidetes</i> (<i>p</i> = 0.2330), <i>Tenericutes</i> (<i>p</i> = 0.2811), or <i>Proteobacteria</i> (<i>p</i> = 0.0680) levels among the three groups; however, <i>Fibrobacteres</i> decreased in EML (<i>p</i> = 0.0431). At the genus level, there were no differences in <i>Prevotella</i> (<i>p</i> = 0.4280), <i>Ruminococcus</i> (<i>p</i> = 0.2639), <i>Butyrivibrio</i> (<i>p</i> = 0.4433), or <i>Succiniclasticum</i> (<i>p</i> = 0.0431) levels among the groups. Additionally, the dietary treatments had no significant effects on the archaeal community composition in the rumen. Therefore, EML and SMFP supplementation had no significant effects on the ruminal bacterial or archaeal community composition of finishing steers.</p></div

Additional file 1: of Effects of a wide range of dietary forage-to-concentrate ratios on nutrient utilization and hepatic transcriptional profiles in limit-fed Holstein heifers

Table S1. Ingredient and chemical composition of experimental diets. 1 OM, organic matter; CP, crude protein; EE, ether extract; NDF, neutral detergent fiber; NFC, nonfiberous carbohydrate; ME, metabolizable energy. 2 Pre-experimental diet. 3 Contained 18.50% Ca; 6.00% P; 4.2% Mg; 1.4% K; 2.6% S; 7.5% Na; 12.0% Cl; 30 mg/kg of Se; 0.25% Zn; 0.25% Fe; 0.25% Mn; 1100 mg/kg Cu; 15 mg/kg I; 265,000 IU/kg vitamin A; 110,200 IU/kg vitamin D; and 2300 IU/kg vitamin E. 4 NFC = 100 − (NDF + CP + ether extract + ash). 5 Estimated as ME = total digestible nutrients × 0.04409 × 0.82, according to NRC (2001). Table S2. PCR primers for qRT-PCR validation of five randomly selected genes. Table S3. Nutrient intake of Holstein dairy heifers fed diets containing differing forage levels. Table S5. Gene Ontology analysis of total differentially expressed genes. Table S6. Differentially expressed genes clustered in Short Time-series Expression Miner (STEM) profiles 17, 19, and 4. (DOCX 71 kb