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Abstract This study evaluated the effects of supplementing the diet of lactating cows with Acremonium terrestris culture (ATC) on milk production, serum antioxidant capacity, inflammatory indices, and serum lipid metabolomics. Over 90 days, 24 multiparous Chinese Holstein cows in mid-lactation (108 ± 10.4 days in milk, 637 ± 25 kg body weight, 30.23 ± 3.7 kg/d milk yield) were divided into either a control diet (CON) or a diet supplemented with 30 g of ATC daily. All the data were analyzed using Student’s t test with SPSS 20.0 software. The results showed that compared with CON feeding, ATC feeding significantly increased milk yield, antioxidant capacity, and immune function. Lipidome screening identified 143 lipid metabolites that differed between the two groups. Further analysis using “random forest” machine learning revealed three glycerophospholipid serum metabolites that could serve as lipid markers with a predictive accuracy of 91.67%. This study suggests that ATC can be a useful dietary supplement for improving lactational performance in dairy cows and provides valuable insights into developing nutritional strategies to maintain metabolic homeostasis in ruminants.

Mastitis affects almost all mammals including humans and dairy cows. In the dairy industry, bovine mastitis is a disease with a persistently high incidence, causing serious losses to the health of cows, the quality of dairy products, and the economy of dairy farms. Although local udder infection caused by the invasion of exogenous pathogens into the mammary gland was considered the main cause of mastitis, evidence has been established and continues to grow, showing that nutrition factors and gastrointestinal microbiome (GM) as well as their metabolites are also involved in the development of mammary inflammatory response. Suboptimal nutrition is recognized as a risk factor for increased susceptibility to mastitis in cattle, in particular the negative energy balance. The majority of data regarding nutrition and bovine mastitis involves micronutrients. In addition, the dysbiotic GM can directly trigger or aggravate mastitis through entero-mammary gland pathway. The decreased beneficial commensal bacteria, lowered bacterial diversity, and increased pathogens as well as proinflammatory metabolites are found in both the milk and gastrointestinal tract of mastitic dairy cows. This review discussed the relationship between the nutrition (energy and micronutrient levels) and mastitis, summarized the role of GM and metabolites in regulating mastitis. Meanwhile, several non-antibiotics strategies were provided for the prevention and alleviation of mastitis, including micronutrients, probiotics, short-chain fatty acids, high-fiber diet, inulin, and aryl hydrocarbon receptor.

Bovine mastitis is a global problem and is mainly treated with antimicrobial agents that can significantly affect milk quality and threaten human health. The mammary gland can be damaged by the accumulation of reactive oxygen species (ROS), particularly during the perinatal period. Dihydromyricetin (DHM) has a wide range of pharmacological activities and is considered safe. However, its poor solubility, absorption, and bioavailability have limited its application. To address this issue, we designed and synthesized new chitosan-derivative nanoparticles (HACC-NPs) loaded with DHM, which have the potential to overcome its bioabsorption problems and enhance its antibacterial and antioxidant effects. We used the emulsification/crosslinking method to load DHM into chitosan-derivative nanoparticles (DHM/HACC-NPs); then, SEM, TEM and FTIR analyses were used to show a denser, more tightly packed matrix for the DHM/HACC-NPs, which were successfully prepared. The size (176.4 nm), polydispersity index (PDI, 0.21), and absolute value of the zeta potential (+26.3 mV) confirmed the stability of the DHM/HACC-NPs. More importantly, DHM/HACC-NPs had a stronger antibacterial effect on S. aureus (MIC = 0.52 mg/mL and MBC = 1.02 mg/mL) than on E. coli (MIC = 1.02 mg/mL and MBC = 2.05 mg/mL). Notably, DHM/HACC-NPs were concentration-dependent scavengers of DPPH and hydroxyl radicals. In summary, DHM/HACC-NPs exhibited superior biocompatibility and antimicrobial and antioxidant activities, offering new insights into the development of a robust, udder-stable drug platform as a promising strategy for treating dairy cow mastitis.

Abstract 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.

This study investigates the impact of propylene glycol (PRG) on ketotic cows, focusing on alleviating oxidative stress and enhancing immunity through modulating amino acid and lipid metabolism. Ketosis, a prevalent metabolic disease in dairy cows, negatively affects productivity and health. PRG, known for its gluconeogenic properties, was administered to cows with ketosis daily for three days and compared to an untreated group. Serum samples were taken to measure the biochemical parameters, and metabolomic and lipidomic analyses were performed with ultra-high-performance liquid chromatography–mass spectrometry. The results showed significant reductions in serum non-esterified fatty acids, beta-hydroxybutyrate, and C-reactive protein levels, alongside increased glucose, anti-inflammatory factor interleukin-10, superoxide dismutase, and glutathione peroxidase activities. Metabolomic and lipidomic analyses revealed significant alterations, including increased levels of glucogenic amino acids like glutamate and proline, and decreased levels of ceramide species. A pathway analysis indicated that PRG affects multiple metabolic pathways, including alanine, aspartate, glutamate metabolism, and sphingolipid metabolism. These findings suggest that PRG not only mitigates oxidative stress, but also enhances immune function by restoring metabolic homeostasis. This study provides valuable insights into the biochemical mechanisms underlying PRG’s therapeutic effects, offering potential strategies for the effective management and treatment of ketosis in dairy cows.

Abstract 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

Dairy cows face metabolic challenges around the time of calving, leading to a negative energy balance and various postpartum health issues. Adipose tissue is crucial for cows during this period, as it regulates energy metabolism and supports immune function. Naringin, one of the main flavonoids in citrus fruit and their byproducts, is a potent antioxidant and anti-inflammatory phytoconstituent. The study aimed to evaluate the effects of supplemental naringin on performance, systemic inflammation, oxidative status, and adipose tissue metabolic status. A total of 36 multiparous Holstein cows (from ~21 d prepartum through 35 d postpartum) were provided a basal control (CON) diet or a CON diet containing naringin (NAR) at 30 g/d per cow. Supplemental NAR increased the yield of raw milk and milk protein, without affecting dry matter intake. Cows fed NAR showed significantly lower levels (p < 0.05) of serum non-esterified fatty acid (NEFA), C-reactive protein, IL-1β, IL-6, malonaldehyde, lipopolysaccharide (LPS), aspartate aminotransferase, and alanine aminotransferase, but increased (p < 0.05) glutathione peroxidase activity relative to those fed CON. Supplemental NAR increased (p < 0.05) adipose tissue adiponectin abundance, decreased inflammatory responses, and reduced oxidative stress. Lipidomic analysis showed that cows fed NAR had lower concentrations of ceramide species (p < 0.05) in the serum and adipose tissue than did the CON-fed cows. Adipose tissue proteomics showed that proteins related to lipolysis, ceramide biosynthesis, inflammation, and heat stress were downregulated (p < 0.05), while those related to glycerophospholipid biosynthesis and the extracellular matrix were upregulated (p < 0.05). Feeding NAR to cows may reduce the accumulation of ceramide by lowering serum levels of NEFA and LPS and increasing adiponectin expression, thereby decreasing inflammation and oxidative stress in adipose tissue, ultimately improving their systemic metabolic status. Including NAR in periparturient cows’ diets improves lactational performance, reduces excessive lipolysis in adipose tissue, and decreases systemic and adipose tissue inflammation and oxidative stress. Integrating lipidomic and proteomic data revealed that reduced ceramide and increased glycerophospholipids may alleviate metabolic dysregulations in adipose tissue, which in turn benefits systemic metabolic status.

Ketosis, especially its subclinical form, is frequently observed in high-yielding dairy cows and is linked to various diseases during the transition period. Although adipose tissue plays a significant role in the development of metabolic disorders, its exact impact on the emergence of subclinical ketosis (SCK) is still poorly understood. The objectives of this study were to characterize and compare the profiling of transcriptome and lipidome of blood and adipose tissue between SCK and healthy cows and investigate the potential correlation between metabolic disorders and lipid metabolism. We obtained blood and adipose tissue samples from healthy cows (CON, n = 8, β-hydroxybutyric acid concentration < 1.2 mmol/L) and subclinical ketotic cows (SCK, n = 8, β-hydroxybutyric acid concentration = 1.2–3.0 mmol/L) for analyzing biochemical parameters, transcriptome, and lipidome. We found that serum levels of nonesterified fatty acids, malonaldehyde, serum amyloid A protein, IL-1β, and IL-6 were higher in SCK cows than in CON cows. Levels of adiponectin and total antioxidant capacity were higher in serum and adipose tissue from SCK cows than in CON cows. The top enriched pathways in whole blood and adipose tissue were associated with immune and inflammatory responses and sphingolipid metabolism, respectively. The accumulation of ceramide and sphingomyelin in adipose tissue was paralleled by an increase in genes related to ceramide biosynthesis, lipolysis, and inflammation and a decrease in genes related to ceramide catabolism, lipogenesis, adiponectin production, and antioxidant enzyme systems. Increased ceramide concentrations in blood and adipose tissue correlated with reduced insulin sensitivity. The current results indicate that the lipid profile of blood and adipose tissue is altered with SCK and that certain ceramide species correlate with metabolic health. Our research suggests that disruptions in ceramide metabolism could be crucial in the progression of SCK, exacerbating conditions such as insulin resistance, increased lipolysis, inflammation, and oxidative stress, providing a potential biomarker of SCK and a novel target for nutritional manipulation and pharmacological therapy.

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-1β (IL-1β), 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.

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.

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

ABSTRACT: Mastitis is generally considered a local inflammatory disease caused by the invasion of exogenous pathogens and resulting in the dysbiosis of microbiota and metabolites in milk. However, the entero-mammary pathway theory may establish a possible link between some endogenous gut bacteria and the occurrence and development of mastitis. In the current study, we attempted to investigate differences in the gut microbiota profile and metabolite composition in gut and serum from healthy cows and those with subclinical mastitis and clinical mastitis. Compared with those of healthy cows, the microbial community diversities in the feces of cows with subclinical mastitis (SM) and clinical mastitis (CM) were lower. Lower abundance of Bifidobacterium, Romboutsia, Lachnospiraceae_NK3A20_group, Coprococcus, Prevotellaceae_UCG-003, Ruminococcus, and Alistipes, and higher abundance of the phylum Proteobacteria and the genera Escherichia-Shigella and Streptococcus were observed in CM cows. Klebsiella and Paeniclostridium were significantly enriched in the feces of SM cows. Several similarities were observed in feces and serum metabolites in mastitic cows. Higher levels of proinflammatory lipid products (20-trihydroxy-leukotriene-B4, 13,14-dihydro-15-keto-PGE2, and 9,10-dihydroxylinoleic acids) and lower levels of metabolites involved in secondary bile acids (deoxycholic acid, 12-ketolithocholic acid), energy (citric acid and 3-hydroxyisovalerylcarnitine), and purine metabolism (uric acid and inosine) were identified in both SM and CM cows. In addition, elevated concentrations of IL-1β, IL-6, tumor necrosis factor-α and decreased concentrations of glutathione peroxidase and superoxide dismutase were detected in the serum of SM and CM cows. Higher serum concentrations of triglyceride and total cholesterol and lower concentrations of high-density lipoproteins in mastitic cows might be related to changes in the gut microbiota and metabolites. These findings suggested a significant difference in the profile of feces microbiota and metabolites in cows with different udder health status, which might increase our understanding of bovine mastitis.

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

This study investigated the effects of inulin on rumen fermentation parameters, ruminal microbiome and metabolites, as well as lactation performance and serum indexes in dairy cows. Sixteen Holstein dairy cows with similar body conditions were randomly divided into 2 groups (n = 8 per group), with inulin addition at 0 and 200 g/d per cow. The experiment lasted for 6 weeks, including a 1-week adaptation period and a 5-week treatment period. At the end of the experimental period, the milk, serum and rumen fluid were sampled and analyzed. The microbiome and metabolome in the rumen fluid were analyzed via 16S rRNA sequencing and untargeted metabolomics, respectively. The results showed that supplementation with inulin (200 g/d per cow) increased the milk yield (P = 0.001), milk protein (P = 0.032), lactose rate (P = 0.004) and proportion of saturated fatty acids (SFA) in milk (P

Objective An experiment was conducted to evaluate the effects of bamboo leaf extract (BLE) on the production performance, rumen fermentation parameters, and rumen bacterial communities of heat-stressed dairy cows. Methods The experiment comprised a 14-day adaptation period and a 21-day experimental period and was conducted in a high-temperature and humidity environment (daily mean ambient temperature = 33.5°C±1.3°C; daily mean relative humidity = 64.9%±0.8%, daily mean temperature-humidity index = 86.2±0.4). Twelve Holstein dairy cows were randomly allocated into two groups. A total mixed ration supplemented with BLE at 0 (CON) and 1.3 g/kg dry matter (DM) were fed, respectively. Feed intake and milk yield were recorded daily. Milk samples were collected on 1, 11, and 21 d of the experimental period to analyze milk performance. Rumen fluid samples were collected on 21 d of the experimental period to analyze rumen fermentation parameters and rumen bacterial communities. Results Compared with the control group, supplementation of BLE increased milk yield (p

This study was conducted to evaluate the effects of two glucogenic diets (C: ground corn and corn silage; S: steam-flaked corn and corn silage) and a lipogenic diet (L: sugar beet pulp and alfalfa silage) on the ruminal bacterial and archaeal structures, the metabolomic products, and gas production after 48 h in vitro fermentation with rumen fluid of dairy cows. Compared to the C and S diets, the L dietary treatment leaded to a lower dry matter digestibility (DMD), lower propionate production and ammonia-nitrogen concentration. The two glucogenic diets performed worse in controlling methane and lactic acid production compared to the L diet. The S diet produced the greatest cumulative gas volume at any time points during incubation compared to the C and L diet. The metabolomics analysis revealed that the lipid digestion especially the fatty acid metabolism was improved, but the amino acid digestion was weakened in the L treatment than in other treatments. Differences in rumen fermentation characteristics were associated with (or resulting from) changes in the relative abundance of bacterial and archaeal genera. The rumen fluid fermented with L diet had a significantly higher number of cellulolytic bacteria, including the genera of Ruminococcus, Butyrivibrio, Eubacterium, Lachnospira, unclassified Lachnospiraceae, and unclassified Ruminococcaceae. The relative abundances of amylolytic bacteria genera including Selenomonas_1, Ruminobacter, and Succinivibrionaceae_UCG-002 were higher in samples for diets C and S. The results indicated that the two glucogenic diets leaded to a higher relative abundance of bacteria which functions in succinate pathway resulting in a higher propionate production. The steam-flaked corn diet had a higher gas production and lower level of metabolites in fatty acids and amino acids. Most highly abundant bacteria were observed to be not sensitive to dietary alterations of starch and fiber, except for several amylolytic bacteria and cellulolytic bacteria. These finding offered new insights on the digesting preference of ruminal bacteria, which can assist to improve the rumen functioning.

IntroductionNegative 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.MethodsA 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.ResultsAs 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.ConclusionIn 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.

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.

BackgroundBovine metabolism undergoes significant changes during subclinical mastitis, but the relevant molecular mechanisms have not been elucidated. In this study we investigated the changes in milk microbiota and metabolites after intramammary infusion of matrine-chitosan hydrogels (MCHs) in cows with subclinical mastitis.MethodsInfusions were continued for 7 days, and milk samples were collected on days 1 and 7 for microbiome analysis by 16S rRNA gene sequencing and metabolite profiling by liquid chromatography-mass spectrometry.ResultsMCHs significantly decreased the somatic cell count on day 7 compared to day 1, and the Simpson index indicated that microbial diversity was significantly lower on day 7. The relative abundance of Aerococcus, Corynebacterium_1, Staphylococcus and Firmicutes was significantly decreased on day 7, while Proteobacteria increased. In the milk samples, we identified 74 differentially expressed metabolites. The MCHs infusion group had the most significantly upregulated metabolites including sphingolipids, glycerophospholipids, flavonoids and fatty acyls. The mammary gland metabolic pathways identified after MCHs treatment were consistent with the known antimicrobial and anti-inflammatory properties of matrine that are associated with glycerophospholipid metabolism and the sphingolipid metabolic signaling pathways.ConclusionThese insights into the immunoregulatory mechanisms and the corresponding biological responses to matrine demonstrate its potential activity in mitigating the harmful effects of bovine mastitis.

The sesquiterpene lactone, artemisinin, is a primary component of the medicinal plant Artemisia annua L., which has anti-inflammatory, antibacterial and antioxidant activities. However, the potential effects of artemisinin on the mammary gland of dairy cows and the underlying molecular mechanisms remain unclear. Here, we utilized systematic network pharmacology and proteomics to elucidate the mechanism by which artemisinin affects milk production and the proliferation of bovine mammary epithelial cells (BMECs). Nineteen bioactive compounds and 56 key targets were identified through database mining. To delineate the mechanism of artemisia’s activity, a protein-protein interaction network and integrated visual display were generated from bioinformatics assays to explore the relationships and interactions among the bioactive molecules and their targets. The gene ontology (GO) terms and kyoto encyclopedia of genes and genomes annotation suggested that the apoptotic process, cell division, p53 pathway, prolactin and PI3K-Akt pathways played vital roles in mammary gland development. Using proteomics analysis, we identified 122 up-regulated and 96 down-regulated differentially significant expressed proteins (DSEPs). The differentially significant expressed proteins had multiple biological functions associated with cell division, apoptosis, differentiation, and migration. Gene ontology enrichment analysis suggested that differentially significant expressed proteins may promote cell proliferation and regulate apoptosis in bovine mammary epithelial cells. Kyoto encyclopedia of genes and genomes pathway analysis indicated that several biological pathways, such as those involved in antigen processing and presentation, cell adhesion molecules and ribosomes, played significant roles in the effects of artemisinin on bovine mammary epithelial cells. These findings contribute to a comprehensive understanding of the mechanism by which artemisinin affects bovine mammary epithelial cells to improve mammary gland turnover by inducing cell proliferation and mammary gland development.

Nutricines, the nutritionally active substances in feed, play a vital role in enhancing immune function, antioxidant activity, and feed efficiency in dairy cows. Identifying nutricines in total mixed ration (TMR) provides insights into feed quality and their impact on dairy cow health. However, due to the structural diversity of nutricines, data mining using multivariate variable models faces challenges in exploring their relationships. To address this, this study established a hierarchical clustering and optimization factor strategy for 13 common flavonoid peaks detected using apparent data and HPLC-DAD. The establishment of the flavonoid fingerprint of TMR diet in dairy cows detected 13 common peaks, five of which were found using standard products: p-coumaric acid, sinapic acid, tricin, and diosmetin. In vitro fermentation results using different TMR samples in substrate fermentation indicated that the dry matter disappearance rate, NH3-N, acetate, propionate, butyrate, isovalerate, and valerate changes varied significantly (p < 0.05). In spectrum–activity relationship studies, P2, P6, P8, P9, P10, and P11 were all considered possible factors causing this effect. In the analysis of optimization factor strategy, the peak spectrum model of four fermentation parameters, i.e., pH, dry matter digestibility, NH3-N, and acetate, was constructed after optimization (p < 0.05), and the data model is listed in the main text. In structure–activity relationship studies, ferulic acid, isoferulic acid, methyl sinapic acid, methyl 4-hydroxycinnamate, and p-hydroxybenzalacetone may serve as candidate references for compound 10 and may play an important role in affecting the digestibility of dry matter in in vitro fermentation. These findings highlight the role of flavonoids in TMR feed as key factors in maintaining dairy cow health and differentiating nutritional value. This study proposes a novel method for future TMR diet formulation and quality evaluation, with potential implications for improving dairy cow health and performance. Further research is needed to validate these findings and elucidate the mechanisms underlying nutricine effects on dairy cow nutrition and health.

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.

Abstract Background Due to the high prevalence and complex etiology, bovine mastitis (BM) is one of the most important diseases to compromise dairy cow health and milk quality. The shift in milk compositions has been widely investigated during mastitis, but recent studies suggested that gastrointestinal microorganism also has a crucial effect on the inflammation of other peripheral tissues and organs, including the mammary gland. However, research focused on the variation of rumen inner-environment during mastitis is still limited. Therefore, the ruminal microbial profiles, metabolites, and milk compositions in cows with different udder health conditions were compared in the present study. Furthermore, the correlations between udder health status and ruminal conditions were investigated. Based on the somatic cell counts (SCC), California mastitis test (CMT) parameters and clinical symptoms of mastitis, 60 lactating Holstein dairy cows with similar body conditions (excepted for the udder health condition) were randomly divided into 3 groups (n = 20 per group) including the healthy (H) group, the subclinical mastitis (SM) group and the clinical mastitis (CM) group. Lactation performance and rumen fermentation parameters were recorded. And rumen microbiota and metabolites were also analyzed via 16S rRNA amplicon sequencing and untargeted metabolomics, respectively. Results As the degree of mastitis increased, rumen lactic acid (LA) (P

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.

The behaviors of dairy cows, such as feeding, ruminating, running, resting (standing, lying), head-shaking, drinking, and walking, can indicate their health status. In this study, a multi-sensor was used to collect data of cow’s multi-behaviors for research on behavior recognition. Firstly, a collar style data acquisition system equipped with geomagnetic and acceleration sensors to collect the behavioral data of dairy cows during their daily activities was designed. Secondly, the dairy cow behavioral recognition fusion model based on K-Nearest-Neighbors (KNN) and Random Forest (RF) models were used for behavior classification. To verify the accuracy of the fusion model, the algorithms of KNN, RF, Gradient Boosting Decision Tree (GBDT), Support Vector Machine (SVM), and Learning Vector Quantization (LVQ) were introduced for comparative recognition experiments with different algorithms. The KNN-RF fusion model had the highest average recognition accuracy of 98.51%, followed by the KNN model with an average recognition accuracy of 95.37%, and the LVQ model had the lowest average recognition accuracy of 80.81%. For the recognition and verification of each behavior, the KNN-RF fusion model had the most obvious improvement in the recognition of dairy cow feeding behavior, with a recognition accuracy of 99.34%, followed by the KNN model with a recognition accuracy of 95.07%. All six models had the lowest recognition accuracy for cow head-shaking behavior: a recognition accuracy of 89.11% with the KNN-RF model followed by the RF model with a recognition accuracy of 85.14%. The system can quickly and continuously collect cow behavior information, accurately recognize individual behaviors, and provide a scientific basis for the optimal design and efficient management of digital facilities and equipment for dairy cows.

The large-scale development of herbivorous animal husbandry in China has increased the demand for forage products. However, due to scarce land resources and poor soil quality, forage is in short supply. In particular, high-quality forage in China heavily relies on imports. The contradiction between supply and demand for forage grass products is increasingly notable. Therefore, the development of indigenous new forage resources with a strong ecological adaptability and a high nutritional value is a key to solving this problem. Jerusalem artichoke (JA, Helianthus tuberosus L.), a perennial herb of the genus Helianthus, has advantageous growth traits such as resistance to salinity, barrenness, drought, cold, and disease. The contents of crude protein, crude fiber, and calcium in the optimal harvest period of forage-type JA straw are comparable to those of alfalfa hay at the full bloom stage and the straw of ryegrass and corn at the mature stage. Inulin in JA tubers is a functional ingredient that has prebiotic effects in the gastrointestinal tract of monogastric animals and young ruminants. In addition, some bioactive substances (e.g. flavonoids, phenolic acids, sesquiterpenes, polysaccharides, and amino acids) in JA leaves and flowers have antibacterial, anti-inflammatory, and antioxidant functions as well as toxicities to cancer cells. These functional ingredients may provide effective alternatives to antibiotics used in livestock production. In this review, we summarized the potentials of JA as a feed ingredient from the aspects of nutritional value and fermenting characteristics of the straw, the functions of physiological regulation and disease prevention of inulin in the tubers, and bioactive substances in the leaves and flowers.

The study was conducted to evaluate the effects of Anemoside B4 on diarrhea incidence, serum indices, and fecal microbial of suckling calves. Sixty newborn Chinese Holstein calves with similar body weight (43.7 ± 3.9 kg) were randomly divided into four groups with 15 calves each, fed the diet which was supplied 0 (CON), 15 (A1), 30 (A2), and 45 (A3) mg/day of Anemoside B4, respectively. The trial period is 56 days. The blood and fecal samples were collected at 28 and 56 days of age. Results show that during the whole trial period, the diarrhea incidence in Group A1, A2, and A3 was significantly lower than that in Group CON (p < 0.05). Compared with the Group CON, Anemoside B4 supplementation significantly decreased the contents of serum D-lactic acid and diamine oxidase at 28-day-old (p < 0.05). At 56-day-old, the content of serum D-lactic acid in Group A3 tended to be higher (0.05 < p < 0.01), and the content of serum diamine oxidase in Group A3 increased significantly, in comparison with Group CON (p < 0.05). Group A3 increased the level of Chao1 and Simpson indices at 28-day-old (0.05 < p < 0.01), and Chao1, Observed_species, Shannon, and Simpson indices at 56-day-old (p < 0.05), in comparison to Group CON. Compared with Group CON, 45 mg / day Anemoside B4 supplementation significantly increased the contents of Bacteroidota (at the phylum level), Prevotella (at the genus level) at 28-day-old (p < 0.05), and the content of Sutterella (at the genus level) at 56-day-old (p < 0.05), promoted the processes of energy metabolism, glycan biosynthesis and metabolism, metabolism of cofactors and vitamins (p < 0.05). A positive correlation was observed between Prevotella and metabolism of cofactors and vitamins, energy metabolism, and glycan biosynthesis and metabolism. A positive correlation was observed between Sutterella and energy metabolism. In conclusion, Anemoside B4 could effectively alleviate calf diarrhea, protect the integrity of intestinal mucosa, and change the structure of intestinal microbiota, indicating the potential value of Anemoside B4 in regulating intestinal microbiota and the prevention of intestinal diseases.

The occurrence and development of mastitis is linked to dysbiostic gastrointestinal microbiota. Inulin is a dietary prebiotic that improves the profile of intestinal flora. Our previous study showed that inulin supplementation could improve the ruminal microbes of subclinical mastitis (SCM) cows. The current study attempted to further investigate the response of hindgut (fecal) microbiome and metabolites, serum metabolism, and protein expression to inulin in the in SCM cows. Different levels of inulin (0, 100, 200, 300, and 400 g/day per cow) were supplemented in SCM cows. Compared with control group, Bacteroides and Bifidobacteria were increased, and Paeniclostridium, Ruminococcaceae, Coprococcus, and Clostridia were decreased in the feces of inulin groups, and accompanied with elevated propionate and butyrate concentrations, while secondary bile acid (SBA) metabolites were increased and proinflammatory lipid oxidation products were dropped in both feces and serum. In serum, inulin intake suppressed the levels of triglyceride (TG) and low-density lipoprotein (LDL). Serum proteome analysis found that CD44 antigen, phosphatidylinositol-glycan-specific phospholipase D, apolipoprotein A-II, and superoxide dismutase [Cu-Zn] were upregulated, while cathelicidin-1, haptoglobin, serpin A3, inter-alpha-trypsin inhibitor heavy chain H4 were downregulated in inulin groups. These findings suggested further evidence for inulin supplementation in amelioration of inflammatory symptoms in SCM cows, which might provide alternative treatment for mastitis.

At present, research on high-concentrate (HC) diets mostly focused on the rumen, and there is a paucity of information on the hindgut microbiota of dairy cows. In the present study, a 2 × 2 crossover design with four healthy Holstein cows was used, and the metagenomics approach was adopted to reveal the remodeling effects of HC diets on hindgut microbiota and their metabolic functions. Results showed that, compared with the low-concentrate (LC) diets, HC diets have markedly decreased (p < 0.05) the abundance of cellulolytic bacteria (such as Fibrobacter, Ruminococcus, and Ruminiclostridium) and methanogens (such as Methanobrevibacter, Methanosarcina, and Methanosphaera); and correspondingly, HC diets have significantly reduced (p < 0.05) the abundance of carbohydrate-active enzymes (CAZy) related to hemicellulases (GH10, GH11, and GH54) and cellulases (GH1, GH44, and GH45) and increased the abundance of one oligosaccharide-degrading enzyme (GH32). Furthermore, 62 Kyoto Encyclopedia of Genes and Genomes (KEGG) pathways of hindgut microbiota were affected (p < 0.05) by different dietary treatments, and the major pathways altered by HC diets were “Methane metabolism” (enriched in the LC group), “Lipid metabolism” (enriched in the HC group), and several sub-pathways in “Amino acid metabolism” (such as Phenylalanine metabolism, and Phenylalanine, tyrosine, and tryptophan biosynthesis). Also, the microbial genes involved in the pathways “Methane metabolism” (except 1 gene), “Tryptophan metabolism”, and “Phenylalanine metabolism” were all decreased (p < 0.05) in the present study. These findings suggested that HC diets caused the remodeling of hindgut microbiota and its potential functions, and these results may benefit in gaining a deeper understanding of the impact of HC diets on the hindgut microbiota of dairy cows.

Abstract Streptococcus agalactiae is one of the causative agents of subclinical mastitis, a common disease of dairy cows that causes great economic losses in the industry worldwide. It is thought that pathology is mainly due to inflammatory damage of bovine mammary epithelial cells (bMECs); however, the mechanism by which S. agalactiae damages the bMECs is not clear. The aim of this study was to evaluate the inflammatory effects of S. agalactiae on bMECs and the resulting changes in protein profiles. The bMECs were incubated with S. agalactiae for different times and assayed for cell viability by MTT assay, apoptosis by annexin V and propidium iodide dual staining, and morphological and ultrastructural changes by scanning and transmission electron microscopy. Quantitative real-time PCR was used to determine the effect of S. agalactiae on expression of mRNA of inflammatory factors in bMECs and protein levels were quantitated by liquid chromatography/mass spectrometry. Exposure to S. agalactiae significantly decreased the cell viability and triggered apoptosis, as well as up-regulating TNF-α, IL-1β and IL-6 mRNA, and inhibiting IL-8 expression. S. agalactiae also induced morphological and ultrastructural changes. Furthermore, we identified 325 up-regulated and 704 down-regulated proteins in the treated vs control group. All significant differentially expressed proteins (DSEPs) were classified into three major areas by function: biological processes, cellular components and molecular functions. These differentially expressed proteins included enzymes and proteins associated with various metabolic processes and cellular immunity. Pathway enrichment analysis showed that eight down-regulated signaling pathways were significantly enriched. Exposure to even subclinical levels of S. agalactiae can lead to inflammation and bMEC damage. Our data suggest some possible molecular mechanisms for the harmful effects of subclinical mastitis in dairy cows.

Abstract This study aimed to explore the effects of artemisinin (ART) on the milk microbiome and metabolites of dairy cow. A total of 12 mid-lactation Holstein dairy cows with similar parity, days in milk were randomly divided into 2 groups receiving either a total mixed ration (TMR) as the control group or this TMR and 120 g/d/head ART as the ART group. The milk samples were collected weekly to determine the contents, and end-of-trial (week 8) milk samples were used to identify microbial species and metabolite profiles by 16S rRNA sequencing and LC–MS analyses, respectively. We observed that the milk fat content significantly increased by ART treatment (P 0.05). Compared with its abundance in the control (CON) group, Firmicutes was significantly decreased, whereas Proteobacteria was significantly increased. Furthermore, in the ART group, the relative abundances of the genera Aerococcus, Staphylococcus, Corynebacterium_1 and Facklamia were significantly lower (P

Objective An experiment was conducted to evaluate the effects of Lonicera japonica extract (LJE) on milk production, rumen fermentation and blood biomarkers of energy metabolism, inflammation and oxidative stress during the perinatal period of Holstein dairy cows. Methods Eighteen Holstein dairy cows were used in a complete randomized design experiment with 3 dietary treatments and 6 cows per treatment. All cows received the same basal total mixed ration (TMR) including a prepartal diet (1.35 Mcal of net energy for lactation [NEL]/kg of dry matter [DM], 13.23% crude protein [CP]) from −60 d to calving and a postpartal diet (1.61 Mcal of NEL/kg of DM, 17.39% CP) from calving to 30 days in milk (DIM). The 3 dietary treatments were TMR supplemented with LJE at 0 (control), 1 and 2 g/kg DM, respectively. LJE was offered from 21 d before calving to 30 DIM. Dry matter intake (DMI) and milk production were measured daily after calving. Milk and rumen fluid samples were collected on 29 and 30 d after calving. On −10, 4, 14, and 30 d relative to calving, blood samples were collected to analyze the biomarkers of energy metabolism, inflammation and oxidative stress. Results Compared with control diet, LJE supplementation at 1 and 2 g/kg DM increased DMI, milk yield and reduced milk somatic cell count. LJE supplementation also decreased the concentrations of blood biomarkers of pro-inflammation (interleukin-1β [IL-1β], IL-6, and haptoglobin), energy metabolism (nonesterified fatty acid and β-hydroxybutyric acid) and oxidative stress (reactive oxygen metabolites), meanwhile increased the total antioxidant capacity and superoxide dismutase concentrations in blood. No differences were observed in rumen pH, volatile fatty acid, and ammonia-N (NH3-N) concentrations between LJE supplemented diets and the control diet. Conclusion Supplementation with 1 and 2 g LJE/kg DM could increase DMI, improve lactation performance, and enhance anti-inflammatory and antioxidant capacities of dairy cows during perinatal period.

Objective The present study explored the effects of grape seed procyanidin extract (GSPE) on rumen fermentation, methane production and archaeal communities in vitro. Methods A completely randomized experiment was conducted with in vitro incubation in a control group (CON, no GSPE addition; n = 9) and the treatment group (GSPE, 1 mg/bottle GSPE, 2 g/kg dry matter; n = 9). The methane and volatile fatty acid concentrations were determined using gas chromatography. To explore methane inhibition after fermentation and the response of the ruminal microbiota to GSPE, archaeal 16S rRNA genes were sequenced by MiSeq high-throughput sequencing. Results The results showed that supplementation with GSPE could significantly inhibit gas production and methane production. In addition, GSPE treatment significantly increased the proportion of propionate, while the acetate/propionate ratio was significantly decreased. At the genus level, the relative abundance of Methanomassiliicoccus was significantly increased, while the relative abundance of Methanobrevibacter decreased significantly in the GSPE group. Conclusion In conclusion, GSPE is a plant extract that can reduce methane production by affecting the structures of archaeal communities, which was achieved by a substitution of Methanobrevibacter with Methanomassiliicoccus.

Objective In the present study, an liquid chromatography/mass spectrometry (LC/MS) metabolomics approach was performed to investigate potential biomarkers of milk production in high- and low-milk-yield dairy cows and to establish correlations among rumen fluid metabolites. Methods Sixteen lactating dairy cows with similar parity and days in milk were divided into high-yield (HY) and low-yield (LY) groups based on milk yield. On day 21, rumen fluid metabolites were quantified applying LC/MS. Results The principal component analysis and orthogonal correction partial least squares discriminant analysis showed significantly separated clusters of the ruminal metabolite profiles of HY and LY groups. Compared with HY group, a total of 24 ruminal metabolites were significantly greater in LY group, such as 3-hydroxyanthranilic acid, carboxylic acids, carboxylic acid derivatives (L-isoleucine, L-valine, L-tyrosine, etc.), diazines (uracil, thymine, cytosine), and palmitic acid, while the concentrations of 30 metabolites were dramatically decreased in LY group compared to HY group, included gentisic acid, caprylic acid, and myristic acid. The metabolite enrichment analysis indicated that protein digestion and absorption, ABC transporters and unsaturated fatty acid biosynthesis were significantly different between the two groups. Correlation analysis between the ruminal microbiome and metabolites revealed that certain typical metabolites were exceedingly associated with definite ruminal bacteria; Firmicutes, Actinobacteria, and Synergistetes phyla were highly correlated with most metabolites. Conclusion These findings revealed that the ruminal metabolite profiles were significantly different between HY and LY groups, and these results may provide novel insights to evaluate biomarkers for a better feed digestion and may reveal the potential mechanism underlying the difference in milk yield in dairy cows.

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.

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.

ABSTRACT Subclinical mastitis (SCM) is one of the highly infectious diseases in dairy cows with the characteristics of high incidence and nonvisible clinical symptoms. The gastrointestinal microbiota is closely related to mastitis. Inulin is a prebiotic fiber with functions in improving intestinal microbial communities and enhancing the host’s immunity. However, the impact of dietary inulin on the rumen inner environment remains unknown. The current study investigated whether inulin could relieve SCM by affecting the profiles of ruminal bacterial and metabolites in dairy cows. Inulin inclusion rates were 0, 100, 200, 300, and 400 g/day per cow, respectively. Inulin increased milk yield, milk protein, and lactose and reduced the somatic cell counts (SCC) in milk. In serum, the concentration of proinflammatory cytokines, such as interleukin-6 (IL-6), IL-8, tumor necrosis factor α (TNF-α), and malondialdehyde (MDA) were decreased, and IL-4 and superoxide dismutase (SOD) were increased. Meanwhile, inulin increased the concentration of propionate, butyrate, and lactic acid (LA), while it decreased NH3-N in rumen. The propionate- and butyrate-producing bacteria (e.g., Prevotella and Butyrivibrio) and several beneficial commensal bacteria (e.g., Muribaculaceae and Bifidobacterium) as well as metabolites related to energy and amino acid metabolism (e.g., melibiose and l-glutamate) were increased. However, several proinflammatory bacteria (e.g., Clostridia UCG-014, Streptococcus, and Escherichia-Shigella) were decreased, accompanied by the downregulation of lipid proinflammatory metabolites, for example, ceramide(d18:0/15:0) [Cer(d18:0/15:0)] and 17-phenyl-18,19,20-trinor-prostaglandin E2. In the current study, the above indicators showed the best response in the 300 g/day inulin group. Overall, dietary supplementation of inulin could alleviate inflammatory responses in cows with SCM through improving the rumen inner environment. IMPORTANCE The correlation between mastitis and the gastrointestinal microbiome in dairy cows has been demonstrated. Regulating the profile of rumen microorganisms may contribute to remission of subclinical mastitis (SCM). Supplementation of inulin in the diets of cows with SCM could increase the abundance of short-chain fatty acid (SCFA)-producing bacteria and beneficial commensal bacteria in rumen and meanwhile the levels of amino acids and energy metabolism. Conversely, the abundance of ruminal bacteria and metabolites with proinflammatory effects were decreased. Our study suggests that the improvement of the rumen internal environment by inulin supplementation could ameliorate inflammatory responses during SCM in dairy cows and thus improve lactation performance and milk quality. Our results provide a theoretical basis for regulation measures of SCM in dairy cows.

Abstract Background Overfeeding of high-concentrate diet (HC) frequently leads to subacute ruminal acidosis (SARA) in modern dairy cows’ production. Thiamine supplementation has been confirmed to attenuate HC induced SARA by increasing ruminal pH and ratio of acetate to propionate, and decreasing rumen lactate, biogenic amines and lipopolysaccharide (LPS). The effects of thiamine supplementation in HC on rumen bacteria and fungi profile had been detected in our previous studies, however, effects of thiamine supplementation in HC on rumen non-methanogen archaea is still unclear. The objective of the present study was therefore to investigate the effects of thiamine supplementation on ruminal archaea, especially non-methanogens in HC induced SARA cows. Results HC feeding significantly decreased dry matter intake, milk production, milk fat content, ruminal pH and the concentrations of thiamine and acetate in rumen fluid compared with control diet (CON) (P

Effects of dietary supplementation of inulin in dairy cows were investigated in this study. Thirty-six mid-lactation Holstein dairy cows were randomly divided into six groups with six cows per group and offered a total mixed ration supplemented with 0 (control), 50, 150, 200, 250 and 350 g/d inulin per head, respectively. The animals were pre-fed for 2 weeks before rumen fluid, milk and blood samples were collected weekly for 3 weeks. With increasing inulin doses, milk yield (p < 0.01) and milk fat concentration (p = 0.04) were linearly increased, while milk urea nitrogen (p < 0.01) and somatic cell count (p = 0.04) were linearly decreased. Linear increases were also detected in the proportions of milk saturated fatty acids (p < 0.01) and polyunsaturated fatty acids (p = 0.04); however, milk monounsaturated fatty acids were linearly decreased (p = 0.04). The ruminal concentrations of acetate, propionate and butyrate increased, while the ruminal pH and the concentration of NH3-N reduced at a decreasing rate with increasing inulin doses (p < 0.01). Moreover, the concentrations of lactic acid (p = 0.03) and total volatile fatty acids (p < 0.01) were linearly upregulated. There were linear increases in the serum concentrations of superoxide dismutase (p = 0.02), immunoglobin G (p < 0.01), immunoglobin M (p = 0.04), interleukin-2 (p < 0.01) and interleukin-10 (p = 0.04); quadratic increases in serum total protein (p < 0.01) and albumin (p = 0.02) and linear decreases in serum total cholesterol (p = 0.02), triglyceride (p < 0.01) and malondialdehyde (p < 0.01). The results indicated that inulin increased milk production, shifted milk fatty acid profile, upregulated rumen volatile fatty acid concentration, and enhanced antioxidant and immunity function in dairy cows in a dose-dependent manner.

The dual stress of reduced feed intake and increased milk yield in dairy cows early postpartum results in a negative energy balance. Rumen-protected glucose (RPG) has been reported to replenish energy, increase milk yield, and improve gut health. However, early postpartum cows often develop an insulin resistance, implying that RPG may not be well utilized and increased milk production may increase the liver’s fat oxidization burden. This study aimed to investigate the effects of RPG on the hepatic oxidative/antioxidative status and protein profile. Starting 7 d before expected calving, six pairs of cows were supplemented with rumen-protected glucose (RPG, n = 6) or with an equal amount of rumen-protecting coating fat (CON, n = 6). Liver samples were obtained from 10 cows 14 d after calving (d 14). Concentration of malondialdehyde and activity of glutathione peroxidase were increased and the activities of catalase and superoxide dismutase tended to increase in the livers of the RPG cows compared to the CON cows. The revised quantitative insulin sensitivity check index (RQUICKI) was decreased by RPG, but triacylglycerol concentration in liver was increased by RPG supplementation. The overall profiles of hepatic proteins were similar between CON and RPG. A partial least square regression was conducted to identify the proteins associated with liver lipidosis, oxidative stress, and antioxidative capacity. The top twenty proteins, according to their variable importance value, were selected for metabolic pathway enrichment analysis. Eighteen enriched KEGG pathways were identified, including metabolism, the citrate cycle, propanoate metabolism, the peroxisome, and type II diabetes mellitus. Our study showed that RPG supplementation reduced insulin sensitivity but increased the liver triglyceride concentration and the oxidative stress in early postpartum cows. Liver proteins related to lipidosis, oxidative stress, and antioxidative capacity, were positively associated with the glutamine metabolism, citric acid cycle, peroxisome, and type II diabetes pathways, which may indicate an increased risk of liver metabolic disorders caused by RPG supplementation in early postpartum cows.

Steam explosion is an environment-friendly pretreatment method to improve the subsequent hydrolysis process of lignocellulosic biomass. Steam explosion pretreatment improved ruminal fermentation and changed fermentation pattern of corn stover during ruminal fermentation in vitro. The study gave a comprehensive insight into how stream explosion pretreatment shifted archaeal and bacterial community structure to change ruminal fermentation in vitro of corn stover. Results showed that steam explosion pretreatment dramatically improved the apparent disappearance of dry matter (DM), neutral detergent fiber (NDF), and acid detergent fiber (ADF). Steam explosion pretreatment significantly increased the molar proportion of propionate and decreased the ratio of acetate to propionate. At archaeal level, steam explosion pretreatment significantly increased the relative abundance of Methanobrevibacter, which can effectively remove metabolic hydrogen to keep the fermentation continuing. At bacterial level, the shift in fermentation was achieved by increasing the relative abundance of cellulolytic bacteria and propionate-related bacteria, including Spirochaetes, Elusimicrobia, Fibrobacteres, Prevotella, Treponema, Ruminococcus, and Fibrobacter.

Staphylococcus aureus is one of the most important pathogens causing mastitis in dairy cows. The objective of this study was to establish a rat model of mastitis induced by S. aureus infection and to explore changes in the proteomes of mammary tissue in different udder states, providing a better understanding of the host immune response to S. aureus mastitis. On day 3 post-partum, 6 rats were randomly divided into two groups (n = 3), with either 100 μL of PBS (blank group) or a S. aureus suspension containing 2×107 CFU·mL-1 (challenge group) infused into the mammary gland duct. After 24 h of infection, the rats were sacrificed, and mammary gland tissue was collected. Tandem mass tag (TMT)-based technology was applied to compare the proteomes of healthy and mastitic mammary tissues. Compared with the control group, the challenge group had 555 proteins with significant differences in expression, of which 428 were significantly upregulated (FC>1.2 and p0.83 and p

Abstract Ruminal thiamine deficiencies occur when dairy cows are overfed with high-concentrate diet, and thiamine supplementation has been proved to attenuate high-concentrate diet induced SARA. However, there is limited knowledge of the relationship between thiamine supplementation in high-concentrate diets and ruminal fungi. In order to investigate the impacts of thiamine supplementation on ruminal fungi, twelve Chinese Holstein dairy cows were randomly assigned into three treatments: control diet (CON; 20% starch, dry matter basis), high-concentrate diet (HC; 33.2% starch, dry matter basis) and high-concentrate diet supplemented with 180 mg thiamine/kg dry matter intake. Dry matter intake and milk production were recorded during the experimental periods. On day 21, rumen fluid samples were collected at 3 h postfeeding and ruminal pH, thiamine concentration and volatile fatty acids were measured. Metagenome sequencing method was conducted to detect ruminal fungi composition. Feeding HC significantly decreased dry matter intake, milk production, ruminal pH, ruminal acetate and thiamine concentration, however, significantly increased propionate and isovalerate (P

The microbial ecosystem in the udders of dairy cows directly influences the flavor and quality of milk. However, to our knowledge, no published research has analyzed the complex relationship between the udder microbiome and its associated metabolism in animals with subclinical mastitis. We identified the bacterial species and measured relative population numbers in the milk of cows with subclinical Streptococcus agalactiae mastitis (GBS) and compared this information to that from the milk of healthy cows. Metabolite profiles were determined to investigate correlations between the milk microbiota and metabolic factors in healthy vs. GBS dairy cows. Six milk samples from GBS cows and six from healthy cows were subjected to 16S rRNA gene sequencing to identify the microbial species using a MiSeq high-throughput sequencing apparatus. The metabolites present in the milk were identified by gas chromatography time-of-flight mass spectrometry. Both principal component analysis and orthogonal partial least squares discriminant analysis indicated that the metabolites were well-separated from each other in the milk samples from the two groups. GBS dramatically altered microbial diversity, and the GBS group had significantly fewer Proteobacteria, Actinobacteria, and Acidobacteria than the CON group, with greater relative abundance of Firmicutes (p < 0.01). Several bacterial genera, such as Streptococcus, were significantly more abundant in milk from the GBS group than in milk from the CON group, and there was a tendency for greater abundance of Turicibacter (p = 0.07) and Enterococcus spp. (p = 0.07) in the GBS group. The levels of five milk metabolites were significantly higher in the GBS group than in the CON group: phenylpyruvic acid, the homogentisic acid: 4-hydroxyphenylpyruvic acid ratio, the xanthine: guanine ratio, uridine and glycerol. Metabolic pathway analysis of the different metabolites revealed that the following were enriched in both groups: galactose metabolism; pentose and glucuronate interconversion; starch and sucrose metabolism; alanine, aspartate and glutamate metabolism; arginine biosynthesis; citrate cycle (TCA cycle); D-glutamine and D-glutamate metabolism; and the neomycin, kanamycin, and gentamicin biosynthesis pathways. Several typical metabolites were highly correlated with specific ruminal bacteria, such as Streptococcaceae, Lachnospiraceae, Lactobacillaceae and Corynebacteriaceae, demonstrating the functional correlations between the milk microbiome and associated metabolites. These findings revealed that the milk microbiota and metabolite profiles were significantly different between the two groups of cows, raising the question of whether the microbiota associated with the bovine mammary gland could be related to mammary gland health. There was also a relationship between milk quality and the presence of spoilage bacteria. Other bacterial taxa should be investigated, as related information may provide insights into how perturbations in milk metabolomics profiles relate to differences in milk synthesis between healthy cows and those with subclinical mastitis.

The rumen microbial complex adaptive mechanism invalidates various methane (CH4) mitigation strategies. Shifting the hydrogen flow toward alternative electron acceptors, such as propionate, was considered to be a meaningful mitigation strategy. A completely randomized design was applied in in vitro incubation to investigate the effects of replacing forage fiber with non-forage fiber sources (NFFS) in diets on methanogenesis, hydrogen metabolism, propionate production and the methanogenic and bacterial community. There are two treatments in the current study, CON (a basic total mixed ration) and TRT (a modified total mixed ration). The dietary treatments were achieved by partly replacing forage fiber with NFFS (wheat bran and soybean hull) to decrease forage neutral detergent fiber (fNDF) content from 24.0 to 15.8%, with the composition and inclusion rate of other dietary ingredients remaining the same in total mixed rations. The concentrations of CH4, hydrogen (H2) and volatile fatty acids were determined using a gas chromatograph. The archaeal and bacterial 16S rRNA genes were sequenced by Miseq high-throughput sequencing and used to reveal the relative abundance of methanogenic and bacterial communities. The results revealed that the concentration of propionate was significantly increased, while the concentration of acetate and the acetate to propionate ratio were not affected by treatments. Compared with CON, the production of H2 increased by 8.45% and the production of CH4 decreased by 14.06%. The relative abundance of Methanomassiliicoccus was significantly increased, but the relative abundance of Methanobrevibacter tended to decrease in TRT group. At the bacterial phylum level, the TRT group significantly decreased the relative abundance of Firmicutes and tended to increase the relative abundance of Bacteroidetes. The replacement of forage fiber with NFFS in diets can affect methanogenesis by shifting the hydrogen flow toward propionate, and part is directed to H2in vitro. The shift was achieved by a substitution of Firmicutes by Bacteroidetes, another substitution of Methanobrevibacter by Methanomassiliicoccus. Theoretical predictions of displacements of H2 metabolism from methanogenesis to propionate production was supported by the dietary intervention in vitro.

Bacillus subtilis SH21 was observed to produce an antifungal protein that inhibited the growth of F. solani. To purify this protein, ammonium sulfate precipitation, gel filtration chromatography, and ion-exchange chromatography were used. The purity of the purified product was 91.33% according to high-performance liquid chromatography results. Sodium dodecyl sulfate–polyacrylamide gel electrophoresis and liquid chromatography–tandem mass spectrometry (LC–MS/MS) analysis revealed that the molecular weight of the protein is 30.72 kDa. The results of the LC–MS/MS analysis and a subsequent sequence-database search indicated that this protein was a chitosanase, and thus, we named it chitosanase SH21. Scanning and transmission electron microscopy revealed that chitosanase SH21 appeared to inhibit the growth of F. solani by causing hyphal ablation, distortion, or abnormalities, and cell-wall depression. The minimum inhibitory concentration of chitosanase SH21 against F. solani was 68 µg/mL. Subsequently, the corresponding gene was cloned and sequenced, and sequence analysis indicated an open reading frame of 831 bp. The predicted secondary structure indicated that chitosanase SH21 has a typical a-helix from the glycoside hydrolase (GH) 46 family. The tertiary structure shared 40% similarity with that of Streptomyces sp. N174. This study provides a theoretical basis for a topical cream against fungal infections in agriculture and a selection marker on fungi.

To identify relationships between animal, dietary and management factors and the resulting methane (CH4) emissions, and to identify potential mitigation strategies for CH4 production, it is vital to develop reliable and accurate CH4 measurement techniques. This review outlines various methods for measuring enteric CH4 emissions from ruminants such as respiration chambers (RC), sulphur hexafluoride (SF6) tracer, GreenFeed, sniffer method, ventilated hood, facemask, laser CH4 detector and portable accumulation chamber. The advantages and disadvantages of these techniques are discussed. In general, RC, SF6 and ventilated hood are capable of 24 h continuous measurements for each individual animal, providing accurate reference methods used for research and inventory purposes. However, they require high labor input, animal training and are time consuming. In contrast, short-term measurement techniques (i.e., GreenFeed, sniffer method, facemask, laser CH4 detector and portable accumulation chamber) contain additional variations in timing and frequency of measurements obtained relative to the 24 h feeding cycle. However, they are suitable for large-scale measurements under commercial conditions due to their simplicity and high throughput. Successful use of these techniques relies on optimal matching between the objectives of the studies and the mechanism of each method with consideration of animal behavior and welfare. This review can provide useful information in selecting suitable techniques for CH4 emission measurement in ruminants.

Background: Thiamine supplementation in high-concentrate diets (HC) was confirmed to attenuate ruminal subacute acidosis through promoting carbohydrate metabolism, however, whether thiamine supplementation in HC impacts methane metabolism is still unclear. Therefore, in the present study, thiamine was supplemented in the high-concentrate diets to investigate its effects on ruminal methanogens and methanogenesis process. Methods: an in vitro fermentation experiment which included three treatments: control diet (CON, concentrate/forage = 4:6; DM basis), high-concentrate diet (HC, concentrate/forage = 6:4; DM basis) and high-concentrate diet supplemented with thiamine (HCT, concentrate/forage = 6:4, DM basis; thiamine supplementation content = 180 mg/kg DM) was conducted. Each treatment concluded with four repeats, with three bottles in each repeat. The in vitro fermentation was sustained for 48h each time and repeated three times. At the end of fermentation, fermentable parameters, ruminal bacteria and methanogens community were measured. Results: HC significantly decreased ruminal pH, thiamine and acetate content, while significantly increasing propionate content compared with CON (p < 0.05). Conversely, thiamine supplementation significantly increased ruminal pH, acetate while significantly decreasing propionate content compared with HC treatment (p < 0.05). No significant difference of ruminal methanogens abundances among three treatments was observed. Thiamine supplementation significantly decreased methane production compared with CON, while no significant change was found in HCT compared with HC. Conclusion: thiamine supplementation in the high-concentrate diet (HC) could efficiently reduce CH4 emissions compared with high-forage diets while without causing ruminal metabolic disorders compared with HC treatment. This study demonstrated that supplementation of proper thiamine in concentrate diets could be an effective nutritional strategy to decrease CH4 production in dairy cows.

Milk can mediate maternal-neonatal signal transmission by the bioactive component extracellular vesicles (EVs), which select specific types of miRNA to encapsulate. The miRNA profiling of sheep milk EVs was characterized by sequencing and compared with that of cow milk. Nanoparticle tracking analysis revealed that the concentration of sheep milk EVs was 1.3 ± 0.09 × 1012 particles/mL and the diameter was peaked at 131.2 ± 0.84 nm. Sheep milk EVs contained various small RNAs, including tRNA, Cis-regulatory element, rRNA, snRNA, other Rfam RNA, and miRNA, which held about 36% of all the small RNAs. In total, 84 types of miRNA were annotated with Ovis aries by miRBase (version 22.0) in sheep milk EVs, with 75 shared types of miRNAs in all samples. The miR-26a, miR-191, let-7f, let-7b and miR-10b were highly expressed both in cow and sheep milk EVs, and 14 sheep milk EV-miRNAs in the top 20, occupying 98% of the total expression, were immune-related. Although pathway analysis showed different potential functions of cow and sheep milk EV-miRNAs, there were still some shared points: lipid metabolism (phospholipase D, glycerophospholipid and glycosylphosphatidylinositol), calcium metabolism, and nerve conduction (axon guidance and synapse). This study provides reference for the bioactive components in the milk of different species.