Flavonoids from citrus peel display potential synergistic effects on inhibiting rumen methanogenesis and ammoniagenesis: a microbiome perspective.

Flavonoids have been recognized as potential phytochemicals to reduce enteric methane (CH4) production and improve rumen nitrogen efficiency in ruminants. We evaluated whether naringin, hesperidin, their combination, or a mixed citrus flavonoid extract (CFE) as additives can inhibit methanogenesis and ammoniagenesis in dairy cows using an in vitro rumen batch refermentation system. The rumen inocula from dairy cows were incubated in batch cultures with five groups: no addition (CON), hesperidin (20 g/kg DM), naringin (20 g/kg DM), hesperidin + naringin (10 g/kg DM of hesperidin + 10 g/kg DM of naringin), and CFE (20 g/kg DM). The combination of naringin plus hesperidin and CFE achieved greater reductions in CH4 and ammonia production compared to either naringin or hesperidin alone. Microbiome analysis revealed that the decrease in CH4 emissions may have been caused by both the direct inhibitory impact of citrus flavonoids on Methanobrevibacter and a simultaneous decrease in protozoa Isotricha abundance. The relatively lower proportion of Entodinium in naringin plus hesperidin or CFE was responsible for the lower ammonia concentration. These results suggest that citrus flavonoids possess potential synergistic effects on mitigating ruminal CH4 emissions by cows and improving nitrogen utilization.

From organic fertilizer to the soils: What happens to the microplastics? A critical review.

In recent, soil microplastic pollution arising from organic fertilizers has been of a great increasing concern. In response to this concern, this review presents a comprehensive analysis of the occurrence and evolution of microplastics in organic fertilizers, their ingress into the soil, and the subsequent impacts. Organic fertilizers are primarily derived from solid organic waste generated by anthropocentric activities including urban (daily-life, municipal wastes and sludge), agricultural (manure, straw), and industrial (like food industrial waste etc.) processes. In order to produce organic fertilizer, the organic solid wastes are generally treated by aerobic composting or anaerobic digestion. Currently, microplastics have been widely detected in the raw materials and products of organic fertilizer. During the process of converting organic solid waste materials into fertilizer, intense oxidation, hydrolysis, and microbial actions significantly alter the physical, chemical, and surface biofilm properties of the plastics. After the organic fertilizer application, the abundances of microplastics significantly increased in the soil. Additionally, the degradation of these microplastics often promotes the adsorption of organic pollutants and affects their retention time in the soil. These microplastics, covered by biofilms, also significantly alter soil ecology due to the unique properties of the biofilm. Furthermore, the biofilms also play a role in the degradation of microplastics in the soil environment. This review offers a new perspective on the soil environmental processes involving microplastics from organic fertilizer sources and highlights the challenges associated with further research on organic fertilizers and microplastics.

Bioconversion of citrus waste by long-term DMSO-cryopreserved rumen fluid to volatile fatty acids and biogas is feasible: A microbiome perspective.

Preserving rumen fluid as the inoculum for anaerobic digestion of food waste is necessary when access to animal donors or slaughterhouses is limited. This study aims to compare two preservation methods relative to fresh ruminal inoculum: (1) cryoprotected with 5% dimethyl sulfoxide (DMSO) and stored at -20 °C and (2) frozen at -20 °C, both for 6 months. The fermentation activity of different inoculum was evaluated by rumen-based in vitro anaerobic fermentation tests (volatile fatty acids, biomass digestibility, and gas production). Citrus pomace was used as the substrate during a 96-h fermentation. The maximum volatile fatty acids, methane production, and citrus pomace digestibility from fresh rumen fluid were not significantly different from rumen fluid preserved with DMSO. Metagenome analysis revealed a significant difference in the rumen microbial composition and functions between fresh rumen fluid and frozen inoculum without DMSO. Storage of rumen fluid using -20 °C with DMSO demonstrated the less difference compared with fresh rumen fluid in microbial alpha diversity and taxa composition. The hierarchical clustering tree of CAZymes showed that DMSO cryoprotected fluid was clustered much closer to the fresh rumen fluid, showing more similarity in CAZyme profiles than frozen rumen fluid. The abundance of functional genes associated with carbohydrate metabolism and methane metabolism did not differ between fresh rumen fluid and the DMSO-20 °C, whereas the abundance of key functional genes significantly decreased in frozen rumen fluid. These findings suggest that using rumen liquid preserved using DMSO at -20 °C for 180 days is a feasible alternative to fresh rumen fluid. This would reduce the need for laboratories to maintain animal donors and/or reduce the frequency of collecting rumen fluid from slaughterhouses.

Potential modulating effects of Allium mongolicum regel ethanol extract on rumen fermentation and biohydrogenation bacteria of dairy cows in vitro.

The objective of this study was to evaluate the potential modulating effects of Allium mongolicum regel ethanol extract (AME) on rumen fermentation and biohydrogenation (BH) bacteria in vitro. Four Holstein cows were used as donors for the rumen fluid used in this study. In experiment 1, five treatments (supplemented with 0 mg/g, 1 mg/g, 2 mg/g, 3 mg/g, and 4 mg/g of AME based on fermentation substrate, respectively) were conducted to evaluate the effects of different levels of AME on fermentation status in vitro. The results showed that after 24 h of fermentation, MCP was reduced with AME supplementation (p < 0.05), and the multiple combinations of different combinations index (MFAEI) value was the highest with 3 mg/g of AME. In experiment 2, six treatments were constructed which contained: control group (A1); the unsaturated fatty acid (UFA) mixture at 3% concentration (A2); the mixture of A2 and 3 mg/g of AME (A3); 3 mg/g of AME (A4); the UFA mixture at 1.5% concentration (A5); the mixture of A5 and 3 mg/g of AME (A6). The abundance of bacterial species involved in BH was measured to evaluate the potential modulating effect of AME on rumen BH in vitro. Compared with the A1 group, the A3, A4, and A6 groups both showed significant decreases in the abundance of rumen BH microbial flora including Butyrivibrio proteoclasticus, Butyrivibrio fibrisolvens, Ruminococcus albus and Clostridium aminophilum (p < 0.01). The A3 group was less inhibitory than A4 in the abundance of B. proteoclasticus, B. fibrisolvens, and R. albus, and the inhibitory effect of the A6 group was higher than that of A4. In conclusion, the supplementation with 3 mg/g of AME could modulate the rumen fermentation and affect BH key bacteria, which suggests that AME may have the potential to inhibit the rumen BH of dairy cows.

Could natural phytochemicals be used to reduce nitrogen excretion and excreta-derived N2O emissions from ruminants?

Ruminants play a critical role in our food system by converting plant biomass that humans cannot or choose not to consume into edible high-quality food. However, ruminant excreta is a significant source of nitrous oxide (N2O), a potent greenhouse gas with a long-term global warming potential 298 times that of carbon dioxide. Natural phytochemicals or forages containing phytochemicals have shown the potential to improve the efficiency of nitrogen (N) utilization and decrease N2O emissions from the excreta of ruminants. Dietary inclusion of tannins can shift more of the excreted N to the feces, alter the urinary N composition and consequently reduce N2O emissions from excreta. Essential oils or saponins could inhibit rumen ammonia production and decrease urinary N excretion. In grazed pastures, large amounts of glucosinolates or aucubin can be introduced into pasture soils when animals consume plants rich in these compounds and then excrete them or their metabolites in the urine or feces. If inhibitory compounds are excreted in the urine, they would be directly applied to the urine patch to reduce nitrification and subsequent N2O emissions. The phytochemicals' role in sustainable ruminant production is undeniable, but much uncertainty remains. Inconsistency, transient effects, and adverse effects limit the effectiveness of these phytochemicals for reducing N losses. In this review, we will identify some current phytochemicals found in feed that have the potential to manipulate ruminant N excretion or mitigate N2O production and deliberate the challenges and opportunities associated with using phytochemicals or forages rich in phytochemicals as dietary strategies for reducing N excretion and excreta-derived N2O emissions.

Integrated multi-omics analysis reveals the positive leverage of citrus flavonoids on hindgut microbiota and host homeostasis by modulating sphingolipid metabolism in mid-lactation dairy cows consuming a high-starch diet.

BACKGROUND: Modern dairy diets have shifted from being forage-based to grain and energy dense. However, feeding high-starch diets can lead to a metabolic disturbance that is linked to dysregulation of the gastrointestinal microbiome and systemic inflammatory response. Plant flavonoids have recently attracted extensive interest due to their anti-inflammatory effects in humans and ruminants. Here, multi-omics analysis was conducted to characterize the biological function and mechanisms of citrus flavonoids in modulating the hindgut microbiome of dairy cows fed a high-starch diet. RESULTS: Citrus flavonoid extract (CFE) significantly lowered serum concentrations of lipopolysaccharide (LPS) proinflammatory cytokines (TNF-α and IL-6), acute phase proteins (LPS-binding protein and haptoglobin) in dairy cows fed a high-starch diet. Dietary CFE supplementation increased fecal butyrate production and decreased fecal LPS. In addition, dietary CFE influenced the overall hindgut microbiota's structure and composition. Notably, potentially beneficial bacteria, including Bacteroides, Bifidobacterium, Alistipes, and Akkermansia, were enriched in CFE and were found to be positively correlated with fecal metabolites and host metabolites. Fecal and serum untargeted metabolomics indicated that CFE supplementation mainly emphasized the metabolic feature "sphingolipid metabolism." Metabolites associated with the sphingolipid metabolism pathway were positively associated with increased microorganisms in dairy cows fed CFE, particularly Bacteroides. Serum lipidomics analysis showed that the total contents of ceramide and sphingomyelin were decreased by CFE addition. Some differentially abundant sphingolipid species were markedly associated with serum IL-6, TNF-α, LPS, and fecal Bacteroides. Metaproteomics revealed that dietary supplementation with CFE strongly impacted the overall fecal bacterial protein profile and function. In CFE cows, enzymes involved in carbon metabolism, sphingolipid metabolism, and valine, leucine, and isoleucine biosynthesis were upregulated. CONCLUSIONS: Our research indicates the importance of bacterial sphingolipids in maintaining hindgut symbiosis and homeostasis. Dietary supplementation with CFE can decrease systemic inflammation by maintaining hindgut microbiota homeostasis and regulating sphingolipid metabolism in dairy cows fed a high-starch diet. Video Abstract.

Organic fertilizer facilitates the soil microplastic surface degradation and enriches the diversity of bacterial biofilm.

The land-use of organic fertilizers is considered as an important sustainable method for resource utilization, which may have an impact on the microplastic behaviors in the soil. Here, a 240-d dark culture experiment was conducted to reveal the degradation and biofilm characteristics of degradable and refractory granule microplastics in soil and soil-fertilizer systems. The results indicated that microplastics generally exhibited a weak weight loss as well as a specific etiolation on the surface after the culture, especially polyvinyl-chloride and polyhydroxyalkanoates (PHA). Increase in carbon-oxygen functional groups and the changes of oxygen/carbon ratios were noticed, which implied that oxidation and degradation occurred on the surface of microplastics during the cultural process. The changes were more intense on the degradable PHA, and the fertilized-soil treatment than those of the refractory microplastics and the pure soil. Moreover, the addition of organic fertilizers enriched the community diversity of bacterial biofilm on multiple microplastic surfaces. In this regard, the animal fertilizers provided a stronger effect than the plant fertilizers. Overall, the soil, fertilizer and microplastic types affected the community structure and diversity of bacterial biofilm. The outcomes of this study would provide a theoretical basis for the utilization of organic matters for agricultural soil applications.

Assimilation behaviors and metabolite formations of estrone sulfate sodium (E1-3S) and 17ß-estradiol-3-O-sulfate sodium (E2-3S) in the wheat.

The conjugated steroid estrogens (CSEs), including estrone sulfate sodium (E1-3 S) and 17ß-estradiol-3-O-sulfate sodium (E2-3 S), exhibit distinct metabolic behaviors in the aqueous and soil environments. However, their assimilation behaviors and metabolite formations in plant bodies (shoots and roots) remain poorly understood. Therefore, this study used a modified plant hydroponic system to explore the efficiency with which wheat (Triticum acstivnm L.) assimilated the two estrogen conjugates, E1-3 S and E2-3 S. Results indicated the potential of wheat to absorb E1-3 S and E2-3 S, with their assimilation in the root being significantly higher (104-105 ng/g dw) than in the shoot (103-104 ng/g dw). E1-3 S de-sulfated and transformed to estrone (E1) at a rate of 4%-45% in the root's oxidative environment, whereas E2-3 S converted to E1-3 S at 210%-570%. However, the root-to-shoot transfer was impeded by a less potent metabolic activity within the shoot system. The co-exposure treatment revealed that E1 or 17ß-estradiol (E2) affects the assimilation of E1-3 S and E2-3 S by wheat, with E1 inhibiting E1-3 S assimilation and E2 promoting E2-3 S assimilation in wheat bodies. Nonetheless, free-form steroid estrogens (FSEs), which typically have a significant hormone action, can oxidative-damage the wheat tissues, producing a progressive wilting of wheat leaf and so limiting the transpiration process. Co-exposure initially increased the assimilation amounts of E1-3 S (particularly in shoots) and E2-3 S (in both roots and shoots), but these values rapidly declined as exposure duration increased. The combined effects of E1-3 S and E2-3 S exposure also increased their assimilation. These findings suggest the need for further investigation into the cumulative impact of environmental estrogen contaminants. The findings of present study can potentially guide the development of strategies to prevent and manage steroid estrogen contamination in agricultural contexts.

Multi-omics analysis reveals that the metabolite profile of raw milk is associated with dairy cows' health status.

The metabolic status of dairy cows directly influences the nutritional quality and flavor of raw milk. A comprehensive comparison of non-volatile metabolites and volatile compounds in raw milk from healthy and subclinical ketosis (SCK) cows was performed using LC-MS, GC-FID, and HS-SPME/GC-MS. SCK can significantly alter the profiles of water-soluble non-volatile metabolites, lipids, and volatile compounds of raw milk. Compared with healthy cows, milk from SCK cows had higher contents of tyrosine, leucine, isoleucine, galactose-1-phosphate, carnitine, citrate, phosphatidylethanolamine species, acetone, 2-butanone, hexanal, dimethyl disulfide and lower content of creatinine, taurine, choline, α-ketoglutaric acid, fumarate, triglyceride species, ethyl butanoate, ethyl acetate, and heptanal. The percentage of polyunsaturated fatty acids in milk was lowered in SCK cows. Our results suggest that SCK can change milk metabolite profiles, disrupt the lipid composition of milk fat globule membrane, decrease the nutritional value, and increase the volatile compounds associated with off-flavors in milk.

New Insights of Biological Functions of Natural Polyphenols in Inflammatory Intestinal Diseases.

The intestine is critically crucial for nutrient absorption and host defense against exogenous stimuli. Inflammation-related intestinal diseases, including enteritis, inflammatory bowel disease (IBD), and colorectal cancer (CRC), are heavy burdens for human beings due to their high incidence and devastating clinical symptoms. Current studies have confirmed that inflammatory responses, along with oxidative stress and dysbiosis as critical pathogenesis, are involved in most intestinal diseases. Polyphenols are secondary metabolites derived from plants, which possess convincible anti-oxidative and anti-inflammatory properties, as well as regulation of intestinal microbiome, indicating the potential applications in enterocolitis and CRC. Actually, accumulating studies based on the biological functions of polyphenols have been performed to investigate the functional roles and underlying mechanisms over the last few decades. Based on the mounting evidence of literature, the objective of this review is to outline the current research progress regarding the category, biological functions, and metabolism of polyphenols within the intestine, as well as applications for the prevention and treatment of intestinal diseases, which might provide ever-expanding new insights for the utilization of natural polyphenols.

Citrus flavonoid extracts alter the profiling of rumen antibiotic resistance genes and virulence factors of dairy cows.

Citrus flavonoid extracts (CFE) have the potential to reduce rumen inflammation, improve ruminal function, and enhance production performance in ruminants. Our previous studies have investigated the effects of CFE on the structure and function of rumen microbiota in dairy cows. However, it remains unclear whether CFE affects the prevalence of antibiotic resistance genes (ARG) and virulence factors genes (VFG) in the rumen. Therefore, metagenomics was used to identify the rumen ARG and VFG in lactating dairy cows fed with CFE diets. The results showed that CFE significantly reduced the levels of Multidrug and Antiphagocytosis in the rumen (p < 0.05) and increased the levels of Tetracycline, Iron uptake system, and Magnesium uptake system (p < 0.05). Furthermore, the changes were found to have associations with the phylum Lentisphaerae. It was concluded that CFE could be utilized as a natural plant product to regulate virulence factors and antibiotic resistance of rumen microbiota, thereby improving rumen homeostasis and the health of dairy cows.

Feeding citrus flavonoid extracts decreases bacterial endotoxin and systemic inflammation and improves immunometabolic status by modulating hindgut microbiome and metabolome in lactating dairy cows.

The objectives of this study were to determine the effects of dietary supplementation with citrus flavonoid extracts (CFE) on milk performance, serum biochemistry parameters, fecal volatile fatty acids, fecal microbial community, and fecal metabolites in dairy cows. Eight multiparous lactating Holstein cows were used in a replicated 4 × 4 Latin square design (21-day period). Cows were fed a basal diet without addition (CON) or basal diet with added CFE at 50 (CFE50), 100 (CFE10), and 150 g/d (CFE150). Feeding CFE up to 150 g/d increased milk yield and milk lactose percentage. Supplementary CFE linearly decreased milk somatic cell count. Serum cytokines interleukin-1ß (IL-1ß), IL-2, IL-6, and tumor necrosis factor-α (TNF-α) concentrations decreased linearly as the levels of CFE increased. Cows in CFE150 had lower serum lipopolysaccharide and lipopolysaccharide binding protein compared with CON. These results indicate feeding CFE decreased systemic inflammation and endotoxin levels in dairy cows. Furthermore, feeding CFE linearly increased the concentrations of total volatile fatty acids, acetate, and butyrate in feces. The relative abundances of beneficial bacteria Bifidobacterium spp., Clostridium coccoides-Eubacterium rectale group, and Faecalibacterium prausnitzii in feces increased linearly with increasing CFE supplementation. The diversity and community structure of fecal microbiota were unaffected by CFE supplementation. However, supplementing CFE reduced the relative abundances of genera Ruminococcus_torques_group, Roseburia, and Lachnospira, but increased genera Bacteroides and Phascolarctobacterium. Metabolomics analysis showed that supplementary CFE resulted in a significant modification in the fecal metabolites profile. Compared with CON, fecal naringenin, hesperetin, hippuric acid, and sphingosine concentrations were greater in CFE150 cows, while fecal GlcCer(d18:1/20:0), Cer(d18:0/24:0), Cer(d18:0/22:0), sphinganine, and deoxycholic acid concentrations were less in CFE150 cows. Predicted pathway analysis suggested that "sphingolipid metabolism" was significantly enriched. Overall, these results indicate that citrus flavonoids could exert health-promoting effects by modulating hindgut microbiome and metabolism in lactating cows.

Effect of Grape Seed Proanthocyanidins on Fat Metabolism and Adipocytokines in Obese Rats.

This study aimed to investigate the effect of Grape Seed Proanthocyanidin (GSP) on fat metabolism and adipocytokines in obese rats. Fifty 5-week-old rats were randomly assigned to five groups (n = 10 per group) and given either a basal diet, a high-fat diet, or a high-fat diet supplemented with GSP (25, 50, and 100 mg/d) per group. The experiment lasted for five weeks, including a one-week adaptation period and a four-week treatment period. At the end of the experimental period, serum and adipose tissue samples were collected and analyzed. Additionally, we co-cultured 3T3-L1 preadipocytes with varying concentrations of GSP to explore its effect on adipocyte metabolism. The results demonstrated that GSP supplementation reduced weight, daily gain, and abdominal fat weight coefficient (p < 0.05). It also decreased levels of glucose, cholesterol (TC) (p < 0.05), triglycerides (TG) (p < 0.05), low-density lipoprotein (LDL), cyclooxygenase-2 (COX-2), and interleukin-6 (IL-6) in adipose tissue. Furthermore, GSP addition caused adipocyte crumpling in vitro and reduced the mRNA expression of COX-2, LEP, and TNF-α in adipocytes in vitro. These findings provide compelling evidence for exploring the role of GSP in the prevention and treatment of obesity and related diseases.

Characterization of the Dynamic Changes of Ruminal Microbiota Colonizing Citrus Pomace Waste during Rumen Incubation for Volatile Fatty Acid Production.

Rumen microorganisms are promising for efficient bioconversion of lignocellulosic wastes to biofuels and industrially relevant products. Investigating the dynamic changes of the rumen microbial community colonizing citrus pomace (CtP) will advance our understanding of the utilization of citrus processing waste by rumen fluid. Citrus pomace in nylon bags was incubated in the rumen of three ruminally cannulated Holstein cows for 1, 2, 4, 8, 12, 24, and 48 h. Results showed that total volatile fatty acids concentrations and proportions of valerate and isovalerate were increased over time during the first 12 h. Three major cellulose enzymes attached to CtP rose initially and then decreased during the 48-h incubation. Primary colonization happened during the initial hours of CtP incubation, and microbes compete to attach CtP for degrading easily digestible components and/or utilizing the waste. The 16S rRNA gene sequencing data revealed the diversity and structure of microbiota adhered to CtP were distinctly different at each time point. The increased abundance of Fibrobacterota, Rikenellaceae_RC9_gut_group, and Butyrivibrio may explain the elevated volatile fatty acids concentrations. This study highlighted key metabolically active microbial taxa colonizing citrus pomace in a 48-h in situ rumen incubation, which could have implications for promoting the biotechnological process of CtP. IMPORTANCE As a natural fermentation system, the rumen ecosystem of ruminants can efficiently degrade plant cellulose, indicating that the rumen microbiome offers an opportunity for anaerobic digestion to utilize biomass wastes containing cellulose. Knowledge of the response of the in situ microbial community to citrus pomace during anaerobic fermentation will help improve the current understanding of citrus biomass waste utilization. Our results demonstrated that a highly diverse rumen bacterial community colonized citrus pomace rapidly and continuously changed during a 48-h incubation period. These findings may provide a deep understanding of constructing, manipulating, and enriching rumen microorganisms to improve the anaerobic fermentation efficiency of citrus pomace.

Dietary citrus flavonoid extract improves lactational performance through modulating rumen microbiome and metabolites in dairy cows.

The effects of dietary supplementation with citrus flavonoid extract (CFE) on milk performance, rumen fermentation, rumen microbiome, rumen metabolome, and serum antioxidant indexes were evaluated. Eight multiparous lactating cows were allocated to a replicated 4 × 4 Latin square with 25-d periods consisting of 20 d of adaptation and 5 d of sampling. Experimental treatments included a control diet (CON) and CON supplemented with 50 g d-1 (CFE50), 100 g d-1 (CFE100), and 150 g d-1 (CFE150). Feeding CFE to dairy cows increased milk production and milk lactose. Milk somatic cell count linearly reduced with increasing CFE amount. Supplementing CFE linearly increased the ruminal concentrations of total volatile fatty acids, acetate, propionate, butyrate, and microbial crude protein. Ruminal lipopolysaccharide linearly decreased with increasing CFE amount. Compared with CON, CFE150 cows exhibited a greater abundance of Firmicutes and a low abundance of Bacteroidetes. Cellulolytic bacteria (genera Ruminococcus, Clostridium, and Butyrivibrio) and carbohydrate metabolism were enriched in the CFE150 cows. For archaea and viruses, major methanogens (genera Methanobacterium and Methanosarcina) and phylum Uroviricota were inhibited in the CFE150 cows. Compared with CON, the ruminal concentrations of tyrosine, proline, pyruvate, glucose, and glucose-6-phosphate were higher in the CFE150 cows. The metabolites of citrus flavonoids, such as hippuric acid, hesperetin, and naringenin, were increased in the CFE150 cows. Supplementing CFE significantly improved the antioxidant capacity of the dairy cows. This study highlighted that dietary supplementation with CFE led to significant changes in the rumen microbial composition and metabolites, and consequently resulted in an improved lactational performance of dairy cows.

Ceramide on the road to insulin resistance and immunometabolic disorders in transition dairy cows: driver or passenger?

Dairy cows must undergo profound metabolic and endocrine adaptations during their transition period to meet the nutrient requirements of the developing fetus, parturition, and the onset of lactation. Insulin resistance in extrahepatic tissues is a critical component of homeorhetic adaptations in periparturient dairy cows. However, due to increased energy demands at calving that are not followed by a concomitant increase in dry matter intake, body stores are mobilized, and the risk of metabolic disorders dramatically increases. Sphingolipid ceramides involved in multiple vital biological processes, such as proliferation, differentiation, apoptosis, and inflammation. Three typical pathways generate ceramide, and many factors contribute to its production as part of the cell's stress response. Based on lipidomic profiling, there has generally been an association between increased ceramide content and various disease outcomes in rodents. Emerging evidence shows that ceramides might play crucial roles in the adaptive metabolic alterations accompanying the initiation of lactation in dairy cows. A series of studies also revealed a negative association between circulating ceramides and systemic insulin sensitivity in dairy cows experiencing severe negative energy balance. Whether ceramide acts as a driver or passenger in the metabolic stress of periparturient dairy cows is an unknown but exciting topic. In the present review, we discuss the potential roles of ceramides in various metabolic dysfunctions and the impacts of their perturbations. We also discuss how this novel class of bioactive sphingolipids has drawn interest in extrahepatic tissue insulin resistance and immunometabolic disorders in transition dairy cows. We also discuss the possible use of ceramide as a new biomarker for predicting metabolic diseases in cows and highlight the remaining problems.

Microbiome-Metabolomics Insights into the Milk of Lactating Dairy Cows to Reveal the Health-Promoting Effects of Dietary Citrus Peel Extracts on the Mammary Metabolism.

The effects of dietary supplementation with citrus peel extract (CPE) on milk biochemical parameters, milk bacterial community, and milk metabolites were evaluated. Eight lactating cows were allocated to a replicated 4 × 4 Latin square. Experimental treatments included the control diet (CON), and CON supplemented with CPE at 50 g/d (CPE50), 100 g/d (CPE100), and 150 g/d (CPE150). Supplementing with CPE linearly decreased milk interleukin-6 and malondialdehyde concentrations and linearly increased lysozyme activity and 1,1-diphenyl-2-picrylhydrazyl radical scavenging activity. Compared with CON, the milk of CPE150 cows had fewer abundances of several opportunistic pathogens and psychrotrophic bacteria, such as Escherichia-Shigella, Sphingobacterium, Alcaligenes, Stenotrophomonas, and Ochrobactrum. Supplementing with CPE significantly altered the metabolic profiling in the milk. The metabolites of flavonoids were enriched in the milk of cows fed CPE150, while some proinflammation compounds were decreased compared with CON. Correlation analysis showed that the change in the bacterial community might partly contribute to the alteration in the expression of milk cytokines. In conclusion, CPE exerts health-promoting effects (e.g., antioxidant, anti-microbial, and anti-inflammatory) in the mammary metabolism of cows due to its flavonoid compounds, which also provide additional value in terms of milk quality improvement.

Rcs phosphorelay affects the sensitivity of Escherichia coli to plantaricin BM-1 by regulating biofilm formation.

Introduction: Plantaricin BM-1 is a class IIa bacteriocin produced by Lactobacillus plantarum BM-1 that exerts significant antibacterial activity against many foodborne bacteria. Studies have shown that class IIa bacteriocins inhibit Gram-positive bacteria via the mannose phosphotransferase system; however, their mechanism of action against Gram-negative bacteria remains unknown. In this study, we explored the mechanism through which the Rcs phosphorelay affects the sensitivity of Escherichia coli K12 cells to plantaricin BM-1. Methods and Results: The minimum inhibitory concentrations of plantaricin BM-1 against E. coli K12, E. coli JW5917 (rcsC mutant), E. coli JW2204 (rcsD mutant), and E. coli JW2205 (rcsB mutant) were 1.25, 0.59, 1.31, and 1.22 mg/ml, respectively. Growth curves showed that E. coli JW5917 sensitivity to plantaricin BM-1 increased to the same level as that of E. coli K12 after complementation. Meanwhile, scanning electron microscopy and transmission electron microscopy revealed that, under the action of plantaricin BM-1, the appearance of E. coli JW5917 cells did not significantly differ from that of E. coli K12 cells; however, cell contents were significantly reduced and plasmolysis and shrinkage were observed at both ends. Crystal violet staining and laser scanning confocal microscopy showed that biofilm formation was significantly reduced after rcsC mutation, while proteomic analysis identified 382 upregulated and 260 downregulated proteins in E. coli JW5917. In particular, rcsC mutation was found to affect the expression of proteins related to biofilm formation, with growth curve assays showing that the deletion of these proteins increased E. coli sensitivity to plantaricin BM-1. Discussion: Consequently, we speculated that the Rcs phosphorelay may regulate the sensitivity of E. coli to plantaricin BM-1 by affecting biofilm formation. This finding of class IIa bacteriocin against Gram-negative bacteria mechanism provides new insights.

Rumen-protected glucose supplementation alters fecal microbiota and its metabolic profiles in early lactation dairy cows.

Introduction: Negative energy balance (NEB) is the pathological basis of metabolic disorders in early lactation dairy cows. Rumen-protected glucose (RPG) is a feed additive to relieve NEB of cows in early lactation. The aims of the current study were to evaluate the impact of different doses of RPG supply on fecal microbiota and metabolome in early lactation dairy cows, and their correlation with each other. Methods: A total of 24 multiparous Holstein dairy cows in early lactation were randomly assigned to one of four treatments for the first 35 days of the early lactation period, as follows: control group, a basal diet without RPG (CON); low RPG, a basal diet plus 200 g/d RPG (LRPG); medium RPG, a basal diet plus 350 g/d RPG (MRPG); or HRPG, high RPG, a basal diet plus 500 g/d RPG (HRPG). After 35 days, fecal samples were obtained from cows in all groups individually and using 16S rRNA gene sequencing to evaluate their microbiotas, while their metabolites were evaluated through metabolomics. Results: As expected, Firmicutes and Bacteroidetes were the core bacteria phyla. After RPG supplementation, there were an increase in Firmicutes and a decrease in Bacteroidetes. MRPG increased the relative abundance of cellulolytic bacteria, including Ruminococcaceae_UCG-005, Lachnospiraceae_UCG-008, Lachnospiraceae_FCS020_group, and Ruminiclostridium_9, while it decreased the relative abundance of Alistipes, Prevotellaceae_UCG-003, and Dorea. RPG supplementation could regulate the carbohydrate metabolism and amino acid metabolism pathway significantly and relieve lipolysis in dairy cows. Correlation analysis of fecal microbiome and metabolome showed that some major differential bacteria were the crucial contributors to differential metabolites. Conclusion: In conclusion, RPG supplementation can affect the fecal microbial components and microbial metabolism, and 350 g RPG might be the ideal dose as a daily supplement.

Development of a recombinase polymerase amplification assay with lateral flow dipstick (RPA-LFD) for rapid detection of Shigella spp. and enteroinvasive Escherichia coli.

Shigella spp. and enteroinvasive Escherichia coli (EIEC) are widely distributed and can cause serious food-borne diseases for humans such as dysentery. Therefore, an efficient detection platform is needed to detect Shigella and EIEC quickly and sensitively. In this study, a method called recombinase polymerase amplification combined with lateral flow dipstick (RPA-LFD) was developed for rapid detection of Shigella and EIEC. RPA primers and LFD detection probes were designed for their shared virulence gene ipaH. Primers and probes were screened, and the primer concentration, and reaction time and temperature were optimized. According to the optimization results, the RPA reaction should be performed at 39°C, and when combined with LFD, it takes less than 25 min for detection with the naked eye. The developed RPA-LFD method specifically targets gene ipaH and has no cross-reactivity with other common food-borne pathogens. In addition, the minimum detection limit of RPA-LFD is 1.29×102 copies/µL. The detection of food sample showed that the RPA-LFD method was also verified for the detection of actual samples.