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.

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

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

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

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

Consumption of Supplementary Inulin Modulates Milk Microbiota and Metabolites in Dairy Cows with Subclinical Mastitis.

The milk microbiota and mediated metabolites directly affect the health of the udder in dairy cows. Inulin, a dietary prebiotic, can modulate the profile of gastrointestinal microbiota. However, whether the inulin intake affects the milk microbial population and metabolites remains unknown. In this study, 40 subclinical mastitis (SCM) cows were randomly divided into 5 groups. Five inulin addition doses, 0, 100, 200, 300, and 400 g/day per cow, based on the same basal diet, were supplemented. The experiments lasted for 8 weeks. The results showed lower relative abundance of mastitis-causing and proinflammation microbes in milk (i.e., Escherichia-Shigella, Pseudomonas, Rhodococcus, Burkholderia-Caballeronia-Paraburkholderia, etc.) and higher abundances of probiotics and commensal bacteria, such as Lactobacillus, Bifidobacterium, etc., in the cows fed 300 g/day inulin compared to that in the control group. Meanwhile, the levels of arachidonic acid proinflammatory mediators (leukotriene E3, 20-carboxy-leukotriene B4, and 12-Oxo-c-LTB3) and phospholipid metabolites were reduced, and the levels of compounds with antibacterial and anti-inflammatory potential (prostaglandin A1, 8-iso-15-keto-prostaglandin E2 [PGE2], etc.) and participating energy metabolism (citric acid, l-carnitine, etc.) were elevated. These data suggested that inulin intake might modulate the microflora and metabolite level in extraintestinal tissue, such as mammary gland, which provided an alternative for the regulation and mitigation of SCM. IMPORTANCE The profile of the microbial community and metabolic activity in milk are the main determinants of udder health status and milk quality. Recent studies have demonstrated that diet could directly modulate the mammary gland microbiome. Inulin is a probiotic dietary fiber which can improve the microbiota population in the gastrointestinal tract. However, whether inulin intake can further regulate the profile of the microbiota and metabolic activities in milk remains unclear. In subclinical mastitic cows, we found that inulin supplementation could reduce the abundance of Escherichia-Shigella, Pseudomonas, Rhodococcus, and Burkholderia-Caballeronia-Paraburkholderia and the levels of (±)12, 13-DiHOME, leukotriene E3 and 20-carboxy-leukotriene B4 etc., while it elevated the abundance of Lactobacillus, Bifidobacterium, and Muribaculaceae, as well as the levels of prostaglandin A1 (PGA1), 8-iso-15-keto-PGE2, benzoic acid, etc. in milk. These data suggest that inulin intake affects the profile of microorganisms and metabolites in milk, which provides an alternative for the regulation of mastitis.

Discrepancies among healthy, subclinical mastitic, and clinical mastitic cows in fecal microbiome and metabolome and serum metabolome.

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-1ß, 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.

Ruminal Degradation of Rumen-Protected Glucose Influences the Ruminal Microbiota and Metabolites in Early-Lactation Dairy Cows.

Rumen-protected glucose (RPG) plays an important role in alleviating the negative energy balance of dairy cows. This study used a combination of rumen microbes 16S and metabolomics to elucidate the changes of rumen microbial composition and rumen metabolites of different doses of RPG's rumen degradation part in early-lactation dairy cows. Twenty-four multiparous Holstein cows in early lactation were randomly allocated to control (CON), low-RPG (LRPG), medium-RPG (MRPG), or high-RPG (HRPG) groups in a randomized block design. The cows were fed a basal total mixed ration diet with 0, 200, 350, and 500 g of RPG per cow per day, respectively. Rumen fluid samples were analyzed using Illumina MiSeq sequencing and ultrahigh-performance liquid chromatography coupled to quadrupole time-of-flight mass spectrometry. MRPG supplementation increased bacterial richness and diversity, including increasing the relative abundance of cellulolytic bacteria, such as Ruminococcus, Lachnospiraceae_NK3A20_group, Ruminiclostridium, and Lachnospiraceae_UCG-008 MRPG significantly increased the concentrations of acetate, propionate, butyrate, and total volatile fatty acid in the rumen. Ruminal fluid metabolomics analysis showed that RPG supplementation could significantly regulate the synthesis of amino acids digested by protozoa in the rumen. Correlation analysis of the ruminal microbiome and metabolome revealed some potential relationships between major bacterial abundance and metabolite concentrations. Our analysis found that RPG supplementation of different doses can change the diversity of microorganisms in the rumen and affect the rumen fermentation pattern and microbial metabolism and that a daily supplement of 350 g of RPG might be the ideal dose.IMPORTANCE Dairy cows in early lactation are prone to a negative energy balance because their dry matter intake cannot meet the energy requirements of lactation. Rumen-protected glucose is used as an effective feed additive to alleviate the negative energy balance of dairy cows in early lactation. However, one thing that is overlooked is that people often think that rumen-protected glucose is not degraded in the rumen, thus ignoring its impact on the microorganisms in the rumen environment. Our investigation and previous experiments have found that rumen-protected glucose is partially degraded in the rumen. However, there are few reports on this subject. Therefore, we conducted research on this problem and found that rumen-protected glucose supplementation at 350 g/day can promote the development and metabolism of rumen flora. This provides a theoretical basis for the extensive application of rumen bypass glucose at a later stage.

An Antifungal Chitosanase from Bacillus subtilis SH21.

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.

Proteomic analysis of bovine mammary epithelial cells after in vitro incubation with S. agalactiae: potential biomarkers.

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-1ß 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.

Dietary grape seed procyanidins suppressed weaning stress by improving antioxidant enzyme activity and mRNA expression in weanling piglets.

This study was conducted to investigate the effect of grape seed procyanidins (GSP) on growth performance, digestive enzyme activity, antioxidant enzyme activity and mRNA expression in weanling piglets. A total of 96 piglets (Pietrain × Large White) with an average initial body weight (BW) of 8.4 ± 1.7 kg were weaned at 28 days, and randomly divided into 4 groups. Four groups of animals were fed with a basic diet supplemented with various doses of GSP (0, 40, 70 and 100 mg/kg respectively) during the 28-day treatment period. The results showed that the group receiving 40 mg/kg GSP significantly increased the average daily gain (ADG, p < .05) and decrease the feed/gain ratio (F/G, p < .05). Interestingly, the incidence of diarrhoea was significantly reduced in the groups of 40 and 70 mg/kg GSP, but it was increased in the group of 100 mg/kg GSP. Subsequent biochemical studies indicated that dietary GSP significantly increased the activities of digestive enzymes and antioxidant enzymes, including amylase (Amy), lipase(LPS, p < .05), glutathione peroxidase activity (GSH-Px, p < .05), superoxide dismutase activity (SOD, p < .05) and total antioxidant capacity (T-AOC, p < .05) in serum, liver and muscle, increased the expression of GSH-Px, SOD and CAT genes (p < .05) in the liver, and decreased the level of malondialdehyde (MDA, p < .05) in serum, liver and muscle. Taken together, these studies revealed that low GSP supplement in diets can improve growth performance of weaned piglets, which is associated with increased digestive and antioxidant enzyme activities and enhanced resistance to weanling stress.

Effects of Lonicera japonica extract on performance, blood biomarkers of inflammation and oxidative stress during perinatal period in dairy cows.

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-1ß [IL-1ß], IL-6, and haptoglobin), energy metabolism (nonesterified fatty acid and ß-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.

Metabolomics reveals potential biomarkers in the rumen fluid of dairy cows with different levels of milk production.

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.

Illumina MiSeq sequencing reveals the effects of grape seed procyanidin on rumen archaeal communities in vitro.

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.

Metagenomic insights into effects of thiamine supplementation on ruminal non-methanogen archaea in high-concentrate diets feeding dairy cows.

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 < 0.05), while the concentrations of propionate and ammonia-nitrogen (NH3-N) were significantly increased compared with CON (P < 0.05). These changes caused by HC were inversed by thiamine supplementation (P < 0.05). The taxonomy results showed that ruminal archaea ranged from 0.37 to 0.47% of the whole microbiota. Four characterized phyla, a number of Candidatus archaea and almost 660 species were identified in the present study. In which Euryarchaeota occupied the largest proportion of the whole archaea. Furthermore, thiamine supplementation treatment significantly increased the relative abundance of non-methanogens compared with CON and HC treatments. Thaumarchaeota was increased in HC compared with CON. Thiamine supplementation significantly increased Crenarchaeota, Nanoarchaeota and the Candidatus phyla, however decreased Thaumarchaeota compared with HC treatment. CONCLUSIONS: HC feeding significantly decreased ruminal pH and increased the content of NH3-N which led to N loss and the increase of the relative abundance of Thaumarchaeota. Thiamine supplementation increased ruminal pH, improved the activity of ammonia utilizing bacteria, and decreased Thaumarchaeota abundance to reduce the ruminal NH3 content and finally reduced N loss. Overall, these findings contributed to the understanding of thiamine's function in dairy cows and provided new strategies to improve dairy cows' health under high-concentrate feeding regime.

Thiamine status, metabolism and application in dairy cows: a review.

As the co-enzyme of pyruvate dehydrogenase and α-ketoglutarate dehydrogenase, thiamine plays a critical role in carbohydrate metabolism in dairy cows. Apart from feedstuff, microbial thiamine synthesis in the rumen is the main source for dairy cows. However, the amount of ruminal thiamine synthesis, which is influenced by dietary N levels and forage to concentrate ratio, varies greatly. Notably, when dairy cows are overfed high-grain diets, subacute ruminal acidosis (SARA) occurs and results in thiamine deficiency. Thiamine deficiency is characterised by decreased ruminal and blood thiamine concentrations and an increased blood thiamine pyrophosphate effect to >45 %. Thiamine deficiency caused by SARA is mainly related to the increased thiamine requirement during high grain feeding, decreased bacterial thiamine synthesis in the rumen, increased thiamine degradation by thiaminase, and decreased thiamine absorption by transporters. Interestingly, thiamine deficiency can be reversed by exogenous thiamine supplementation in the diet. Besides, thiamine supplementation has beneficial effects in dairy cows, such as increased milk and component production and attenuated SARA by improving rumen fermentation, balancing bacterial community and alleviating inflammatory response in the ruminal epithelium. However, there is no conclusive dietary thiamine recommendation for dairy cows, and the impacts of thiamine supplementation on protozoa, solid-attached bacteria, rumen wall-adherent bacteria and nutrient metabolism in dairy cows are still unclear. This knowledge is critical to understand thiamine status and function in dairy cows. Overall, the present review described the current state of knowledge on thiamine nutrition in dairy cows and the major problems that must be addressed in future research.

Punicalagin induces Nrf2 translocation and HO-1 expression via PI3K/Akt, protecting rat intestinal epithelial cells from oxidative stress.

PURPOSE: Oxidative stress plays a central role in heat stress-induced gastrointestinal injury. Punicalagin (PUN), a major polyphenol abundant in pomegranate fruit, husk and juice, exhibits antioxidative effects. In this study we used a heat stress model to investigate the intestinal protection effect of PUN and the underlying mechanisms. MATERIALS AND METHODS: IEC-6 cells were pretreated with PUN for 6 h and exposed to 42 °C for 6 h. Intracellular reactive oxygen species levels, malondialdehyde, nitrogen oxide, and superoxide dismutase activity were measured. IEC-6 cells were treated with PUN at different times and doses, the protein levels of haeme oxygenase-1 (HO-1) were evaluated. The nuclear translocation of the transcription factor NF-erythroid 2-related factor (Nrf2) and the phosphorylation level of PI3K/Akt were also investigated. RESULTS: PUN significantly decreased the heat stress-induced cell death and apoptosis. Heat stress increased reactive oxygen species, malondialdehyde and nitrogen oxide production, while it decreased superoxide dismutase activity. These effects were markedly inversed when the cells were pretreated with PUN. Furthermore, PUN treatment induced the expression of HO-1 and increased Nrf2 nuclear translocation in a time- and dose-dependent manner. The Nrf2-related cytoprotective effects of PUN were via the PI3K/Akt signalling pathway, as LY294002, a specific PI3K/Akt inhibitor, suppressed the PUN-induced nuclear translocation of Nrf2, the HO-1 up-regulation and the protective effect of PUN against oxidative stress. CONCLUSIONS: PUN protects IEC-6 cells against oxidative stress by up-regulating the expression of HO-1 via a mechanism that involves PI3K/Akt activation and Nrf2 translocation.

Effect of patchouli alcohol on the regulation of heat shock-induced oxidative stress in IEC-6 cells.

Purpose Patchouli alcohol (PA) is used to treat gastrointestinal dysfunction. The purpose of this study was to ascertain the function of PA in the regulated process of oxidative stress in rat intestinal epithelial cells (IEC-6). Materials and methods Oxidative stress was stimulated by exposing IEC-6 cells to heat shock (42 °C for 3 h). IEC-6 cells in treatment groups were pretreated with various concentrations of PA (10, 40, and 80 ng/mL) for 3 h before heat shock. Results Heat shock caused damage to the morphology of IEC-6 cells, and increased reactive oxygen species (ROS) level and malondialdehyde (MDA) content. Moreover, mRNA and protein expression by target genes related to oxidative stress in heat shock were also altered. Specifically, the mRNA expression by HSP70, HSP90, GSH-px, NRF2 nd HO-1were all increased, and Nrf2 and Keap1 protein expression were increased after heat shock. However, pretreatment with PA weakened the level of damage to the cellular morphology, and decreased the MDA content caused by heat shock, indicating PA had cytoprotective activities. Pretreatment with PA at high dose significantly increased generation of intracellular ROS. Compared with the heat shock group alone, PA pretreatment significantly decreased the mRNA expression by HSP70, HSP90, SOD, CAT, GSH-px, KEAP1 and HO-1. Furthermore, the high dose of PA significantly increased Nrf2 protein expression, while both the intermediate and high dose of PA significantly increased HO-1 protein expression. Conclusion Heat-shock-induced oxidative stress in IEC-6 cells, and PA could alleviate the Nrf2-Keap1 cellular oxidative stress responses.

Medicinal herbs as a potential strategy to decrease methane production by rumen microbiota: a systematic evaluation with a focus on Perilla frutescens seed extract.

Mitigation of the methane (CH4) emission from ruminants is needed to decrease the environmental impact of ruminant animal production. Different plant materials and chemicals have been tested, but few are both effective and practical. Medicinal herbs contain biological compounds and antimicrobials that may be effective in lowering the CH4 production. However, few studies have systematically evaluated medicinal herbs for their effect on CH4 production or on the rumen microbiota. In this study, extracts from 100 medicinal herbs were assessed for their ability to decrease CH4 production by rumen microbiota in vitro. The extracts of 12 herbs effectively lowered the CH4 production, with the extract of Perilla frutescens seeds being the most effective. The major components of P. frutescens seed extract were identified, and the effects of the extract on the fermentation characteristics and populations of rumen methanogens, fungi, protozoa, and select bacteria were also assessed. The decreased CH4 production induced by the P. frutescens seed extract was accompanied by an increased abundance of Ruminobacter, Selenomonas, Succinivibrio, Shuttleworthis, Pseudobutyrivbrio, Anaerovibrio, and Roseomonas and a decreased abundance of Methanobrevibacter millerae. The abundance of Pedobacter, Anaeroplasma, Paludibacter, Ruminococcus, and unclassified Lachnospiraceae was positively correlated with the CH4 production, with no effects on volatile fatty acids. This study suggests that medicinal herbs may be used to mitigate the CH4 emission from ruminants.

Microbial release of ferulic and p-coumaric acids from forages and their digestibility in lactating cows fed total mixed rations with different forage combinations.

BACKGROUND: Ferulic acid (FA) and p-coumaric acid (PCA) are widely distributed in graminaceous plant cell walls. This study investigated the in vitro and in vivo digestibility of ester-linked FA (FAest) and PCA (PCAest) in lactating dairy cows. RESULTS: Regarding corn stover, ensiled corn stover, whole corn silage, Chinese wild ryegrass and alfalfa hay with different phenolic acid profiles, the in vitro rumen digestibility of forage FAest and PCAest was negatively correlated with the ether-linked FA content and original PCA/FA ratio in the forages. The concentration of both phenolic acids in culture fluids was low after a 72 h incubation, and the mixed rumen microorganisms metabolized nearly all phenolic acids released into the culture fluids. FAest digestibility in the whole digestive tract was negatively correlated with dietary PCA/FA ratio, but a converse result occurred with dietary PCAest digestibility. The digestibility in either the rumen or the whole digestive tract was greater for FAest than for PCAest. CONCLUSION: Forage PCAest in comparison with FAest is not easily digested in either the rumen or the whole digestive tract, and they were negatively affected by forage FAeth content and lignification extent indicated by the original dietary PCA/FA ratio.

Activation of transcription factor AP-1 in response to thermal injury in rat small intestine and IEC-6 cells.

BACKGROUND: Our previous studies indicated that heat stress can cause significant damage to the intestinal epithelium and induce differential expression of many genes in rat small intestine. The transcription factors AP-1 and NF-κB, which act as important mediators by binding to specific DNA sequences within gene promoters, regulate the transcription of genes associated with immune regulation, stress response and cell fate. METHODS: To determine whether AP-1 and NF-κB are involved in hyperthermia-induced injury in rat small intestine and IEC-6 cells, we investigated their activity, and the expression of related proteins, by electrophoretic mobility shift assays and western blotting, respectively. RESULTS: Heat stress resulted in severe damage to the epithelium of the small intestine. The cell morphology and viability were obviously altered when IEC-6 cell was exposed to hyperthermia. AP-1 was activated in the small intestine of heat-stressed rats, as was phosphorylation of the JNK signaling pathway. In IEC-6 cell line, AP-1 activation in groups exposed to 42 °C for 1 h, 2 h and 4 h was significantly increased. In contrast, NF-κB was not activated in both in vivo and in vitro models. CONCLUSION: These results reveal that AP-1 is likely to play an important role in regulating gene transcription in rat small intestine and IEC-6 cells during exposure to heat stress.

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.