Early-life ruminal microbiome-derived indole-3-carboxaldehyde and prostaglandin D2 are effective promoters of rumen development.

BACKGROUND: The function of diverse ruminal microbes is tightly linked to rumen development and host physiology. The system of ruminal microbes is an excellent model to clarify the fundamental ecological relationships among complex nutrient-microbiome-host interactions. Here, neonatal lambs are introduced to different dietary regimes to investigate the influences of early-life crosstalk between nutrients and microbiome on rumen development. RESULTS: We find starchy corn-soybean starter-fed lambs exhibit the thickest ruminal epithelia and fiber-rich alfalfa hay-fed lambs have the thickest rumen muscle. Metabolome and metagenome data reveal that indole-3-carboxaldehyde (3-IAld) and prostaglandin D2 (PGD2) are the top characteristic ruminal metabolites associated with ruminal epithelial and muscular development, which depend on the enhanced ruminal microbial synthesis potential of 3-IAld and PGD2. Moreover, microbial culture experiment first demonstrates that Bifidobacterium pseudolongum is able to convert tryptophan into 3-IAld and Candida albicans is a key producer for PGD2. Transcriptome sequencing of the ruminal epithelia and smooth muscle shows that ruminal epithelial and muscular development is accompanied by Wnt and Ca2+ signaling pathway activation. Primary cell cultures further confirm that 3-IAld promotes ruminal epithelial cell proliferation depending on AhR-wnt/ß-catenin signaling pathway and PGD2 accelerates ruminal smooth muscle cell proliferation via Ca2+ signaling pathway. Furthermore, we find that 3-IAld and PGD2 infusion promote ruminal epithelial and musculature development in lambs. CONCLUSIONS: This study demonstrates that early-life ruminal microbiome-derived 3-IAld and PGD2 are effective promoters of rumen development, which enhances our understanding of nutrient-microbiome-host interactions in early life.

Early concentrate starter introduction induces rumen epithelial parakeratosis by blocking keratinocyte differentiation with excessive ruminal butyrate accumulation.

INTRODUCTION: Rumen epithelial parakeratosis, a common disease in ruminants caused by abnormalities in the ruminal stratified squamous epithelial keratinization process, negatively impacts ruminant health and performance. However, we still lack a comprehensive perception of the underlying mechanisms and the predisposing factors for this disorder. OBJECTIVES: Here, we investigated rumen epithelial cell heterogeneity, differentiation trajectories, and cornification to clarify the rumen epithelial keratinization process and discern the key ruminal metabolites contributing to rumen epithelial parakeratosis. METHODS: Twenty-four 14-day-old lambs were divided into three groups, including only milk feeding, milk plus alfalfa hay feeding, and milk plus corn-soybean concentrate starter feeding. At 42 days of age, the lambs were slaughtered, and rumen tissues were collected for single-cell RNA-sequencing (scRNA-seq), immunofluorescence, and quantitative real-time PCR (qRT-PCR) analyses. Ruminal fluid samples were collected for metabolomic analyses. Rumen epithelial organoid was used to verify the key ruminal metabolites contributing to parakeratosis. RESULTS: As expected, we observed that concentrate starter introduction resulted in rumen epithelial parakeratosis. Moreover, scRNA-seq analysis revealed a developmental impediment in the transition from differentiated keratinocytes to terminally differentiated keratinocytes (TDK) in lambs with concentrate starter introduction. Immunofluorescence and qRT-PCR analyses further verified the location and expression of marker genes of TDK. Metabolomic analysis showed a robust positive correlation between ruminal butyrate levels and rumen epithelial keratinization. More importantly, we successfully established a rumen organoid model capable of facilitating the study of the keratinization process in the rumen epithelia and further confirmed that high dose butyrate indeed contributed to rumen epithelial parakeratosis. CONCLUSION: Collectively, concentrate starter introduction induces ruminal epithelial parakeratosis by blocking keratinocyte differentiation with excessive ruminal butyrate accumulation in a neonatal lamb model. These findings enhance our understanding of rumen epithelial keratinization and provide valuable insights for addressing rumen epithelial parakeratosis using early nutritional intervention strategies.

Alterations in nutrient digestion and utilization associated with different residual feed intake in Hu sheep.

Improving feed efficiency is crucial to the animal industry. Residual feed intake (RFI) is now regarded as an index of feed efficiency evaluation and is independent of growth characteristics. Our study aims to explore the alterations in growth performance and nutrient digestion in Hu sheep with different RFI phenotypes. Sixty-four male Hu sheep (body weight = 24.39 ± 1.12 kg; postnatal days = 90 ± 7.9) were selected for the study. After an evaluation period of 56 days and power analysis, samples were collected from 14 low RFI (L-RFI group, power = 0.95) and 14 high RFI sheep (H-RFI group, power = 0.95). The L-RFI sheep yielded a lower (P < 0.05) feed conversion ratio and dry matter intake; however, both groups exhibited similar average daily gain (P > 0.05). The acid detergent fiber, neutral detergent fiber, organic matter, and crude protein apparent digestibility were higher (P < 0.05) in L-RFI sheep. N intake and fecal N output (% of N intake) were lower (P < 0.05) and N retention (% of N intake) was higher (P < 0.05) in L-RFI sheep, whereas no difference (P > 0.05) was found in urine N output (% of N intake) between the 2 groups. Furthermore, L-RFI sheep gave lower (P < 0.05) serum glucose concentrations and higher (P < 0.05) non-esterified fatty acid concentrations. Meanwhile, a lower ruminal acetate molar proportion (P < 0.05) and higher propionate molar proportion (P < 0.05) were observed in L-RFI sheep. In summary, these results revealed that despite having lower dry matter intake, L-RFI sheep possess higher nutrient digestibility, N retention, ruminal propionate production and serum glucose utilization, in order to meet energy demands. Selection for low RFI sheep could reduce feed costs, which in turn provides economic benefits to the sheep industry.

Effects of whole maize high-grain diet feeding on colonic fermentation and bacterial community in weaned lambs.

High-grain diet is commonly used in intensive production to boost yield in short term, which may cause adverse effects such as rumen and colonic acidosis in ruminants. Maize is one of the key components of high-grain diet, and different processing methods of maize affect the digestive absorption and gastrointestinal development of ruminants. To investigate the effects of maize form in high-grain diets on colonic fermentation and bacterial community of weaned lambs, twenty-two 2.5-month-old healthy Hu lambs were fed separately a maize meal low-grain diet (19.2% grain; CON), a maize meal high-grain diet (50.4% grain; CM), and a whole maize high-grain diet (50.4% grain; CG). After 7 weeks of feeding, the total volatile fatty acid concentration (P = 0.035) were significantly higher in lambs from CM than that from CON. The sequencing results of colonic content microbial composition revealed that the relative abundance of genera Parasutterella (P = 0.028), Comamonas (P = 0.031), Butyricicoccus (P = 0.049), and Olsenella (P = 0.010) were higher in CM than those in CON; compared with CM, the CG diet had the higher relative abundance of genera Bacteroides (P = 0.024) and Angelakisella (P = 0.020), while the lower relative abundance of genera Olsenella (P = 0.031) and Paraprevotella (P = 0.006). For colonic mucosal microbiota, the relative abundance of genera Duncaniella (P = 0.024), Succiniclasticum (P = 0.044), and Comamonas (P = 0.012) were significantly higher in CM than those in CON. In comparison, the relative abundance of genera Alistipes (P = 0.020) and Campylobacter (P = 0.017) were significantly lower. And the relative abundance of genera Colidextribacter (P = 0.005), Duncaniella (P = 0.032), Christensenella (P = 0.042), and Lawsonibacter (P = 0.018) were increased in the CG than those in the CM. Furthermore, the CG downregulated the relative abundance of genes encoding infectious-disease-parasitic (P = 0.049), cancer-specific-types (P = 0.049), and neurodegenerative-disease (P = 0.037) in colonic microbiota than those in the CM. Overall, these results indicated that maize with different grain sizes might influence the colonic health of weaned lambs by altering the composition of the colonic bacterial community.

Single-cell transcriptomic landscape of the sheep rumen provides insights into physiological programming development and adaptation of digestive strategies.

As an important evolutionary innovation and unique organ, the rumen has played a crucial role in ruminant adaptation to complex ecological environments. However, the cellular basis of its complex morphology and function remains largely unknown. In this study, we identified eight major cell types from seven representative prenatal and postnatal rumen samples using ~56 600 single-cell transcriptomes. We captured the dynamic changes and high heterogeneity in cellular and molecular profiles before, during, and after the appearance of keratinized stratified squamous epithelium with neatly arranged papillae and functional maturity. Basal cells, keratinocytes, differentiating keratinocytes, terminally differentiated keratinocytes, and special spinous cells provided the cellular basis for rumen epithelium formation. Notably, we obtained clear evidence of two keratinization processes involved in early papillogenesis and papillae keratinization and identified TBX3 as a potential marker gene. Importantly, enriched stratum spinosum cells played crucial roles in volatile fatty acid (VFA) metabolism and immune response. Our results provide a comprehensive transcriptional landscape of rumen development at single-cell resolution, as well as valuable insight into the interactions between dietary metabolism and the rumen.

Proteomic and parallel reaction monitoring approaches to evaluate biomarkers of mutton tenderness.

Intensive fattening usually results in the changes of meat quality. Tenderness is a central attribute for mutton sensory qualities and consumers' choice. Here, we reported that intensive fattening mutton was more tender than that of traditionally raised sheep. By proteomic approach, we found 49 differentially expressed proteins in longissimus dorsi muscle. After bioinformatics analysis, 5 cytoskeletal proteins, 3 protein binding proteins and 7 metabolic enzymes were identified as potential biomarkers for mutton tenderness. Finally, we verified the expression of these abundant proteins by parallel reaction monitoring (PRM). Collectively, our results reveal that the mutton of sheep raised by intensive fattening is more tender than that of traditionally raised sheep. Myosin-2, myosin-13, vimentin, carbonic anhydrase, carbonic anhydrase-2, Glutathione S-transferase and Microtubule-associated protein 4 isoform X1 can be candidate biomarkers for mutton tenderness. Our data also indicate a central role of cytoskeletal proteins and metabolic enzymes in determining mutton tenderness.

Toward Selective Oxidation of Contaminants in Aqueous Systems.

The presence of diverse pollutants in water has been threating human health and aquatic ecosystems on a global scale. For more than a century, chemical oxidation using strongly oxidizing species was one of the most effective technologies to destruct pollutants and to ensure a safe and clean water supply. However, the removal of increasing amount of pollutants with higher structural complexity, especially the emerging micropollutants with trace concentrations in the complicated water matrix, requires excessive dosage of oxidant and/or energy input, resulting in a low cost-effectiveness and possible secondary pollution. Consequently, it is of practical significance but scientifically challenging to achieve selective oxidation of pollutants of interest for water decontamination. Currently, there are a variety of examples concerning selective oxidation of pollutants in aqueous systems. However, a systematic understanding of the relationship between the origin of selectivity and its applicable water treatment scenarios, as well as the rational design of catalyst for selective catalytic oxidation, is still lacking. In this critical review, we summarize the state-of-the-art selective oxidation strategies in water decontamination and probe the origins of selectivity, that is, the selectivity resulting from the reactivity of either oxidants or target pollutants, the selectivity arising from the accessibility of pollutants to oxidants via adsorption and size exclusion, as well as the selectivity due to the interfacial electron transfer process and enzymatic oxidation. Finally, the challenges and perspectives are briefly outlined to stimulate future discussion and interest on selective oxidation for water decontamination, particularly toward application in real scenarios.

Transcriptome profiling of hepatic and renal mRNAs and lncRNAs under a nutritional restriction during pregnancy in a sheep model.

This study aimed to investigate the transcriptome profiles of liver and kidney in pregnant sheep under a nutritional restriction. Twenty Hu sheep were segregated into control group (CON) and severe feed restriction (FR) group. Results showed that the concentration of insulin decreased, whereas glucagon, epinephrine, and norepinephrine increased in the FR group. Histological morphology showed no apparent difference in terms of fat deposition in the kidney. In addition, FR significantly decreased the hepatic gene expression of gluconeogenic genes. However, in the kidney, the relative mRNA expression levels of gluconeogenic genes and glucose transporter 1 were observed to increase while the mRNA expression of sodium-glucose co-transporter 1 were decreased by FR. The differentially expressed genes in the liver were associated with fatty acid metabolism and inflammation. In the kidney, FR mainly activated the gluconeogenesis improving negative energy balance. These results provide a better understanding of the consequences of starvation during pregnancy.

Mimicking associative learning using an ion-trapping non-volatile synaptic organic electrochemical transistor.

Associative learning, a critical learning principle to improve an individual's adaptability, has been emulated by few organic electrochemical devices. However, complicated bias schemes, high write voltages, as well as process irreversibility hinder the further development of associative learning circuits. Here, by adopting a poly(3,4-ethylenedioxythiophene):tosylate/Polytetrahydrofuran composite as the active channel, we present a non-volatile organic electrochemical transistor that shows a write bias less than 0.8 V and retention time longer than 200 min without decoupling the write and read operations. By incorporating a pressure sensor and a photoresistor, a neuromorphic circuit is demonstrated with the ability to associate two physical inputs (light and pressure) instead of normally demonstrated electrical inputs in other associative learning circuits. To unravel the non-volatility of this material, ultraviolet-visible-near-infrared spectroscopy, X-ray photoelectron spectroscopy and grazing-incidence wide-angle X-ray scattering are used to characterize the oxidation level variation, compositional change, and the structural modulation of the poly(3,4-ethylenedioxythiophene):tosylate/Polytetrahydrofuran films in various conductance states. The implementation of the associative learning circuit as well as the understanding of the non-volatile material represent critical advances for organic electrochemical devices in neuromorphic applications.

Transcriptomic analysis reveals the molecular mechanisms of rumen wall morphological and functional development induced by different solid diet introduction in a lamb model.

BACKGROUND: This study aimed to elucidate the molecular mechanisms of solid diet introduction initiating the cellular growth and maturation of rumen tissues and characterize the shared and unique biological processes upon different solid diet regimes. METHODS: Twenty-four Hu lambs were randomly allocated to three groups fed following diets: goat milk powder only (M, n = 8), goat milk powder + alfalfa hay (MH, n = 8), and goat milk powder + concentrate starter (MC, n = 8). At 42 days of age, the lambs were slaughtered. Ruminal fluid sample was collected for analysis of concentration of volatile fatty acid (VFA) and microbial crude protein (MCP). The sample of the rumen wall from the ventral sac was collected for analysis of rumen papilla morphology and transcriptomics. RESULTS: Compared with the M group, MH and MC group had a higher concentration of VFA, MCP, rumen weight, and rumen papilla area. The transcriptomic results of rumen wall showed that there were 312 shared differentially expressed genes (DEGs) between in "MH vs. M" and "MC vs. M", and 232 or 796 unique DEGs observed in "MH vs. M" or "MC vs. M", respectively. The shared DEGs were most enriched in VFA absorption and metabolism, such as peroxisome proliferator-activated receptor (PPAR) signaling pathway, butanoate metabolism, and synthesis and degradation of ketone bodies. Additionally, a weighted gene co-expression network analysis identified M16 (2,052 genes) and M18 (579 genes) modules were positively correlated with VFA and rumen wall morphology. The M16 module was mainly related to metabolism pathway, while the M18 module was mainly associated with signaling transport. Moreover, hay specifically depressed expression of genes involved in cytokine production, immune response, and immunocyte activation, and concentrate starter mainly altered nutrient transport and metabolism, especially ion transport, amino acid, and fatty acid metabolism. CONCLUSIONS: The energy production during VFA metabolism may drive the rumen wall development directly. The hay introduction facilitated establishment of immune function, while the concentrate starter enhanced nutrient transport and metabolism, which are important biological processes required for rumen development.

Urine metabolome alterations in malnutrition and the impact of glycerol or rumen-protected choline chloride supplementation in advanced pregnant ewes.

The objective of this study was to explore the metabolic profiles of pregnancy malnutrition induced by feed restriction (FR) and the counteracting effects of glycerol and rumen-protected choline chloride supplementation. Two feeding trials were conducted. In the first experiment, twenty pregnant Hu sheep carrying multiple fetuses with a gestation period of 108 d were randomly divided into two groups. The ewes in the control (CON) group were offered 100 % of their nutritional requirements as recommended by the National Research Council (NRC), while the FR group was offered 30 % of feed intake of CON for 15 d. In the second experiment, eighteen pregnant Hu sheep were offered a feed intake comprising 30 % of the NRC-recommended nutritional requirements twice daily. The sheep were randomly divided into three groups: the FR group in the second experiment (FR2), with no supplementation, the glycerol (GLY) group, which received 40 ml of glycerol per d, and the rumen-protected choline chloride (RPC) group, which received 10 g of rumen-protected choline chloride per d for 9 d. In the first experiment, the urine metabolome of sixteen ewes showed significant difference between the CON group and FR group. Compared with the CON group, FR decreased the level of d-glucose, lactic acid, levoglucosan, α-ketoglutarate, phosphohydroxypyruvic acid, glucose 6-phosphate and the methyl donors, while increasing the level of pyruvate, fumaric acid and carnitines in urine. Both the GLY and RPC treatments counteracted some of these changes and modulated the urine metabolome in advanced pregnant ewes suffering from malnutrition.

The Effect of Oligo(Ethylene Oxide) Side Chains: A Strategy to Improve Contrast and Switching Speed in Electrochromic Polymers.

Solution-processable electrochromic polymers (ECPs) with high performance are urgently needed for extensive applications. Nevertheless, they suffer from slow switching speed because of low ionic conductivities. Herein, we present an effective strategy to improve the contrast and switching speed in ECPs via facile side-chain engineering. A novel electrochromic thieno[3,2-b]thiophene-based polymer (PmOTTBTD) is designed and successfully synthesized by introducing oligo(ethylene oxide) side chains with high ionic conductivity. Compared to the counterpart POTTBTD without modification by oligo(ethylene oxide) chains, PmOTTBTD demonstrates nearly double contrast (42 % vs. 24 %) with a fast oxidation switching process that just takes half of the time when detected under 400 nm, as well as much higher coloration efficiencies (e. g. 239.04 cm2 C-1 vs. 226.26 cm2 C-1 @ 400 nm and 314.04 cm2 C-1 vs. 174.00 cm2 C-1 @ 650∼700 nm). Besides, PmOTTBTD exhibits excellent stability with negligible decay after 3000 cycles. Our work suggests a facile strategy that could be adopted to realize high-performance ECPs via molecular design tuning.

Dynamic Changes of Plasma Metabolome in Response to Severe Feed Restriction in Pregnant Ewes.

Maternal metabolic disorders in ewes induced by energy deficiency have a detrimental effect on the maternal health and lambs. However, the dynamic processes of metabolic disorders are unknown. Therefore, this study attempted to explore the dynamic changes of maternal metabolism based on metabolomics approach during energy deficiency in pregnant ewes. Twenty pregnant Hu sheep were fed a basic diet or a 70% restricted basic diet. The HPLC-MS platform was applied to identify blood metabolites. Principal component analysis of blood samples based on their metabolic profile showed that blood samples of feed restriction group differed after the treatment. In particular, when comparing both groups, there were 120, 129, and 114 differential metabolites at day 5, day 10, and day 114 between the two groups, respectively. Enrichment analysis results showed that four metabolic pathways (glycerophospholipid metabolism, linoleic acid metabolism, arginine and proline metabolism, and aminoacyl-tRNA biosynthesis) at day 5, four metabolic pathways (aminoacyl-tRNA biosynthesis, aminoacyl-tRNA biosynthesis, glycerophospholipid metabolism, and citrate cycle) at day 10, and nine metabolic pathways (aminoacyl-tRNA biosynthesis, synthesis and degradation of ketone bodies, glycerophospholipid metabolism, butanoate metabolism, linoleic acid metabolism, citrate cycle, alanine, aspartate and glutamate metabolism, valine, leucine and isoleucine biosynthesis, and arginine and proline metabolism) at day 15 were significantly enriched between the two groups. These findings revealed temporal changes of metabolic disorders in pregnant ewes caused by severe feed restriction, which may provide insights into mitigation measures.

Effects of Disodium Fumarate on In Vitro Rumen Fermentation, The Production of Lipopolysaccharide and Biogenic Amines, and The Rumen Bacterial Community.

The effect of disodium fumarate (DF) on the ruminal fermentation profiles, the accumulation of lipopolysaccharide (LPS) and bioamines, and the composition of the ruminal bacterial community was investigated by in vitro rumen fermentation. The addition of DF increased the total gas production; the concentrations of propionate, valerate, total volatile fatty acids, and ammonia-nitrogen; and the rumen pH after a 24 h fermentation. By contrast, DF addition decreased the ratio of acetate to propionate and the concentrations of lactate, lipopolysaccharide, methylamine, tryptamine, putrescine, histamine, and tyramine (P < 0.05). Principal coordinates analysis and molecular variance analysis showed that DF altered the ruminal bacterial community (P < 0.05). At the phylum level, DF decreased the proportion of Proteobacteria, and increased the proportions of Spirochaetae and Elusimicrobia (P < 0.05). At the genus level, DF decreased the percentage of Ruminobacter, while increasing the percentage of Succinivibrio and Treponema (P < 0.05). Overall, the results indicate that DF modified rumen fermentation and mitigated the production of several toxic compounds. Thus, DF has great potential for preventing subacute rumen acidosis in dairy cows and for improving the health of ruminants.

Rumen microbial and fermentation characteristics are affected differently by acarbose addition during two nutritional types of simulated severe subacute ruminal acidosis in vitro.

Little information is available on whether or not the effect of an alpha-glucosidase inhibitor on the prevention of ruminal acidosis is influenced by the type of diet during ruminant feeding. This study was conducted to explore the effect of acarbose addition on the prevention of severe subacute ruminal acidosis induced by either cracked wheat or beet pulp in vitro. Cracked wheat and beet pulp were fermented in vitro by rumen microorganisms obtained from three dairy cows. When cracked wheat was used as the substrate and fermented for 24 h, compared with the control, acarbose addition decreased the concentrations of acetate, propionate, butyrate, total volatile fatty acids, and lactate (P < 0.05), while linearly increased the ratio of acetate to propionate, pH value, and the ammonia-nitrogen level (P < 0.05). Applying Illumina MiSeq sequencing of a fragment of the 16S rRNA gene revealed that the relative abundance of Firmicutes and Bacteroidetes as well as the ACE (abundance-based coverage estimator) value, Chao 1 value, and Shannon index increased significantly (P < 0.05), while there was a significant reduction (P < 0.05) in the relative abundance of Tenericutes as well as Proteobacteria after adding acarbose compared to the control. On the other hand, when beet pulp was used as the substrate, acarbose addition had no significant effects (P > 0.05) on the fermentation parameters and the Chao 1 value, the Shannon index, and the proportion of Firmicutes and Bacteroidetes. In general, these findings indicate that acarbose had more effects on ruminal fermentation when wheat was used as the substrate, whereas it exhibited little effect on ruminal fermentation when beet pulp was used as the substrate.

Effects of Acarbose Addition on Ruminal Bacterial Microbiota, Lipopolysaccharide Levels and Fermentation Characteristics In vitro.

This study investigated the effects of acarbose addition on changes in ruminal fermentation characteristics and the composition of the ruminal bacterial community in vitro using batch cultures. Rumen fluid was collected from the rumens of three cannulated Holstein cattle fed forage ad libitum that was supplemented with 6 kg of concentrate. The batch cultures consisted of 8 mL of strained rumen fluid in 40 mL of an anaerobic buffer containing 0.49 g of corn grain, 0.21 g of soybean meal, 0.15 g of alfalfa and 0.15g of Leymus chinensis. Acarbose was added to incubation bottles to achieve final concentrations of 0.1, 0.2, and 0.4 mg/mL. After incubation for 24 h, the addition of acarbose linearly decreased (p<0.05) the total gas production and the concentrations of acetate, propionate, butyrate, total volatile fatty acids, lactate and lipopolysaccharide (LPS). It also linearly increased (p<0.05) the ratio of acetate to propionate, the concentrations of isovalerate, valerate and ammonia-nitrogen and the pH value compared with the control. Pyrosequencing of the 16S rRNA gene showed that the addition of acarbose decreased (p<0.05) the proportion of Firmicutes and Proteobacteria and increased (p<0.05) the percentage of Bacteroidetes, Fibrobacteres, and Synergistetes compared with the control. A principal coordinates analysis plot based on unweighted UniFrac values and molecular variance analysis revealed that the structure of the ruminal bacterial communities in the control was different to that of the ruminal microbiota in the acarbose group. In conclusion, acarbose addition can affect the composition of the ruminal microbial community and may be potentially useful for preventing the occurrence of ruminal acidosis and the accumulation of LPS in the rumen.