Multi-Omics Reveals Disrupted Immunometabolic Homeostasis and Oxidative Stress in Adipose Tissue of Dairy Cows with Subclinical Ketosis: A Sphingolipid-Centric Perspective.

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, ß-hydroxybutyric acid concentration < 1.2 mmol/L) and subclinical ketotic cows (SCK, n = 8, ß-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-1ß, 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.

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.

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.

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.

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.

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.

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.

Lipidomic profiling using GC and LC-MS/MS revealed the improved milk quality and lipid composition in dairy cows supplemented with citrus peel extract.

Eight lactating cows were used to determine the effects of citrus peel extract (CPE) on milk performance, antioxidant properties, and milk lipids composition. CPE supplementation up to 150 g/d (CPE150) increased milk yield and the proportions of unsaturated fatty acids of conjugated linoleic acid. CPE with abundant polyphenol and flavonoids can transfer these bioactive substances to mammary gland and improve the antioxidant properties of milk obtained from cows. Lipidomics revealed that 56 lipid species were altered between CON vs CPE150, and there were five key differential metabolic pathways. In particular, milk phosphatidylethanolamine and phosphatidylcholine were significantly increased with dietary CPE supplementation. In summary, our results provide insights into the modifications in the milk components and milk quality of dairy cows received CPE. The inclusion of CPE in the diet of dairy cows may be an effective and natural way to increase the antioxidant amounts and beneficial lipids in milk.

Matrine attenuates bovine mammary epithelial cells inflammatory responses induced by Streptococcus agalactiae through inhibiting NF-κB and MAPK signaling pathways.

Streptococcus agalactiae is one of the main pathogens associated with bovine mastitis. The invasion of S. agalactiae in bovine mammary epithelial cells (BMECs) has been implicated as a key event in the pathogenesis of mastitis. Matrine is known for its various pharmacological activities, such as immune response regulation and anti-inflammation. The primary aim of the research was to investigate the preventive effect of matrine on S. agalactiae-induced inflammation in BMECs along with underlying molecular mechanisms. Our data showed matrine at the concentrations of 50-100 µg/mL promoted BMECs proliferation without infection, and decreased cytotoxicity induced by S. agalactiae. Subsequently, BMECs were pre-treated with matrine (50, 75, or 100 µg/mL) for 24 h, followed by the infection with S. agalactiae for an additional 6 h. Pretreatment with matrine followed by S. agalactiae treatment decreased cell apoptosis of BMECs. Also, pretreatment of matrine to BMECs prevented the invasion of S. agalactiae. The mRNA abundances of IL-1ß, IL-6, IL-8, and TNF-α were down-regulated in S. agalactiae-infected cells pretreated with matrine. In addition, the greater ratios of protein NF-κB p-p65/p65, p-IκBα/IκBα, p-38/38, and p-ERK/ERK induced by S. agalactiae were attenuated due to matrine treatment. Furthermore, pretreatment of BMECs with matrine impeded the degradation of TAK1 induced by S. agalactiae infection. These results suggest matrine could be a potential modulator in immune response of the mammary gland. In conclusion, matrine prevents cellular damage due to S. agalactiae infection by the modulation of NF-κB and MAPK signaling pathways and pro-inflammatory cytokine production.