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Ruminating behavior accompanies the development of the rumen and the intake of solid feed in calves. However, few studies have reported on the emergence and development of rumination. In this study, we observed ruminating behavior changes of 56 Holstein calves (body weight at birth = 40.1 ± 3.96 kg; mean ± standard deviation) from birth to 30 d of age under the feeding management of suckling calves that were only fed pelleted concentrate feed and milk. All calves were housed in individual pens equipped with infrared cameras. We explored feed intake within 30 d of age, body weight on 61 d of age, and other apparent indicators, including the age of first eating the bedding, duration of non-nutritive oral behavior at 25 and 30 d of age, total starter feed intake within 30 d of age, average daily starter feed intake within 30 d of age, and duration of ruminating behavior at 25 and 30 d of age for all calves, to further explore the effects of the age of first ruminating behavior (AFR). The AFR fitted the normal distribution and ranged from 15 to 20 d of age for 50% of the experimental population. The AFR was positively correlated with the age of first eating the bedding and duration of non-nutritive oral behavior at 30 d of age. Total starter feed intake within 30 d of age, average daily starter feed intake within 30 d of age, duration of ruminating behavior at 25 and 30 d of age, and duration of eating the bedding at 25 and 30 d of age were negatively correlated with AFR. Overall, to the best of our knowledge, this is the first study that has analyzed the correlation between AFR and other indicators. We found that earlier AFR was associated with shorter duration of non-nutritive oral behavior, longer durations of rumination and eating the bedding, and higher feed intake by 30 d of age.

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 < 0.001), but decreased the proportion of unsaturated fatty acids (USFA) (P = 0.041). Rumen pH (P = 0.040) and the concentration of NH3-N (P = 0.024) were decreased; however, acetate (P < 0.001), propionate (P = 0.003), butyrate (P < 0.001) and lactic acid (LA) (P = 0.043) were increased. The total cholesterol (TC) (P = 0.008) and triglycerides (TG) (P = 0.01) in serum were also reduced. Additionally, inulin addition elevated the relative abundance of several beneficial symbiotic and short-chain fatty acid (SCFA)-producing bacteria, such as Muribaculaceae (false discovery rate [FDR]-adjusted P < 0.01), Acetitomaculum (FDR-adjusted P = 0.043), and Butyrivibrio (FDR-adjusted P = 0.036), while elevating the levels of L-lysine (FDR-adjusted P = 4.24 × 10-3), L-proline (FDR-adjusted P = 0.0158), and L-phenylalanine (FDR-adjusted P = 0.027). In contrast, several pathogens and ruminal bacteria abundant in high-fat diets, such as Escherichia-Shigella (FDR-adjusted P = 0.022), Erysipelotrichaceae __UCG-004 (FDR-adjusted P < 0.01) and RF39 (FDR-adjusted P = 0.042) were decreased along with the reduction of lysophosphatidylcholine (LysoPC) (18:1 (9Z)) (FDR-adjusted P = 1.03 × 10-3), LysoPC (16:0) (FDR-adjusted P = 0.0108), LysoPC (18:2 (9Z, 12Z)) (FDR-adjusted P = 1.65 × 10-3) and 8-methylnonenoate. In conclusion, dietary inulin supplementation could increase the relative abundance of commensal microbiota and SCFA-producing bacteria, upregulate amino acidmetabolism and downregulate lipid metabolism in the rumen of dairy cows, which might further improve lactation performance and the level of serum lipids.

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.

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 P 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

This study aimed to verify the anti-inflammatory effect of soybean isoflavones (SI) on the inflammatory response induced byStreptococcus agalactiae(S. agalactiae) of bovine mammary epithelial cells (bMECs) and to elucidate its possible mechanism. BMECs were pretreated with SI of different concentrations (20, 40, 60, 80, 100 μg/mL) for 0.5, 3, 6, 9, 12, 15, 18, 24 h. And then,S. agalactiaewas used to infect bMECs for 6 h (MOI = 50:1) to establish the inflammation model. Cell viability, growth curves ofS. agalactiae, cytotoxicity, andS. agalactiaeinvasion rate were determined. A proteomics technique was used to further detect differential proteins and enrichment pathways. SI (40 μg/mL) improved the viability of bMECs at 12 h (p p p S. agalactiaegrowth and internalization into bMECs in a time- and dose-dependent manner. In addition, proteomics results showed that 133 proteins were up-regulated and 89 proteins were down-regulated significantly. The differentially significantly expressed proteins (DSEPs) were mainly related to cell proliferation, differentiation, apoptosis, and migration. GO annotation showed that 222 DSEPs were divided into 23 biological processes (BP) terms, 14 cell components (CC) terms, and 12 molecular functions (MF) terms. DSEPs were significantly enriched in 10 pathways, of which the immune pathway was the main enrichment pathway.

The internal environment of the cow's udder directly affects the udder health and milk quality. 16S rDNA sequencing and liquid chromatography-mass spectrometry (LC-MS) methods were used to investigate the significant differences in milk microbial diversity and metabolites among cows that are healthy (H) and those suffering from subclinical mastitis (SM) and clinical mastitis (CM). Results uncovered more than 16 and 192 differently abundant microbiota at the phylum and genus levels, respectively, and 673 different levels of metabolites enriched in 20 pathways in milk among the 3 groups. This study revealed the positive relevance between Staphylococcus and Streptococcus and ceramide in milk from CM cows. Similarly, Acinetobacter and Corynebacterium were positively associated with testosterone glucuronide and 5-methyl-tetrahydrofolate, in milk from SM cows. On the basis of the combined analysis of microbiome and metabolome, this study indicated that, apart from the exogenous pathogens, some beneficial symbiotic bacteria, such as Dietzia, Aeromicrobium, Alistipes, and Sphingobacterium, rarely reported in milk have been found to be significantly reduced during mastitis.

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.

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.

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.

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., 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.

Background Inulin is a kind of fructo-oligosaccharide (FOS) derived mainly from Jerusalem artichoke and chicory tubers, which is also a soluble dietary fiber. Inulin has become a scientifically proven prebiotic product with satisfactory effects in improving the structure of intestinal flora, regulating blood lipid and glycemia, etc., in humans and monogastric. However, unlike monogastric animals, ruminal microbes are the largest microbiota in ruminants. The microflora profile and metabolism activity in the rumen are closely related to the health of dairy cows. 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. A total of 16 Holstein dairy cows with similar body condition were randomly divided into two groups (n = 8 per group), with inulin addition at 0 and 200 g/d per cow, respectively. Experiment was lasted for 6 weeks including 1 week of adaptation period and 5 weeks of treatment period. At the end of the experimental period, the milk, serum and rumen fluid were sampled and analyzed. The rumen microbiota and metabolites were analyzed via 16S rRNA sequencing and untargeted metabolomics, respectively. Results The supplementation of inulin (200 g/d per cow) increased the milk yield (P = 0.001), milk protein (P = 0.032), lactose rate (P = 0.004) and the proportion of saturated fatty acids (SFA) in milk (P P = 0.041). Rumen pH (P = 0.040) and concentration of NH3-N (P = 0.024) were decreased, however, acetate (P P = 0.003), butyrate (P P = 0.043) were increased. The total cholesterol (TC) (P = 0.008) and triglycerides (TG) (P = 0.01) in serum were also reduced. Additionally, the inulin addition elevated the relative abundance of several beneficial symbiotic and short-chain fatty acids (SCFA)-producing bacteria, such as Muribaculaceae (FDR-adjusted P Acetitomaculum (FDR-adjusted P = 0.043), and Butyrivibrio (FDR-adjusted P = 0.036) etc., meanwhile elevated the levels of L-Lysine (FDR-adjusted P = 4.24 × 10− 3), L-Proline (FDR-adjusted P = 0.0158), L-Phenylalanine (FDR-adjusted P = 0.027), etc. By contrast, several pathogens and ruminal bacteria abundant in high-fat diets, such as Escherichia-Shigella (FDR-adjusted P = 0.022), Erysipelotrichaceae__UCG-004 (FDR-adjusted P RF39 (FDR-adjusted P = 0.042) etc., were decreased along with the reduction of LysoPC (18:1(9Z)) (FDR-adjusted P = 1.03 × 10− 3), LysoPC (16:0) (FDR-adjusted P = 0.0108), LysoPC (18:2(9Z, 12Z)) (FDR-adjusted P = 1.65 × 10− 3) and 8-Methylnonenoate etc. Conclusion The results indicated the supplementation of inulin in dietary can increase the relative abundance of commensal microbiota and SCFAs-producing bacteria, meanwhile, upregulate amino acids (AAs) metabolism and downregulate lipid metabolism in rumen of dairy cows, which might further improve lactation performance and the level of serum lipids.

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 P P P = 0.039), Moraxella (FDR-adjusted P = 0.011) and Neisseriaceae (FDR-adjusted P = 0.036), etc., were accompanied by a significant increase in 12-oxo-20-dihydroxy-leukotriene B4 (FDR-adjusted P = 5.97 × 10− 9) and 10beta-hydroxy-6beta-isobutyrylfuranoeremophilane (FDR-adjusted P = 3.88 × 10− 10). Meanwhile, in the rumen of SM cows, the Ruminiclostridium_9 (FDR-adjusted P = 0.042) and Enterorhabdus (FDR-adjusted P = 0.043) were increased along with increasing methenamine (FDR-adjusted P = 6.95 × 10− 6), 5-hydroxymethyl-2-furancarboxaldehyde (5-HMF) (FDR-adjusted P = 2.02 × 10− 6) and 6-methoxymellein (FDR-adjusted P = 2.57 × 10− 5). The short-chain fatty acids (SCFAs)-producing bacteria and probiotics in rumen, including Prevoterotoella_1 (FDR-adjusted P = 0.045) and Bifidobacterium (FDR-adjusted P = 0.035), etc., were significantly reduced, with decreasing 2-phenylbutyric acid (2-PBA) (FDR-adjusted P = 4.37 × 10− 6). Conclusion The results indicated that there was a significant shift in the ruminal microflora and metabolites associated with inflammation and immune responses during CM. Moreover, in the rumen of cows affected by SM, the relative abundance of several opportunistic pathogens and the level of metabolites which could produce antibacterial compounds or had a competitive inhibitory effect were all increased.

Staphylococcus aureus (S. aureus) is the major pathogen responsible for mastitis in dairy cows, an important threat to their health, but prevention of S. aureus infection of the mammary gland remains challenging. Berberine hydrochloride (BH), a naturally occurring phytochemical, exhibits a wide range of activities, including antibacterial effects on S. aureus. In this study, we prepared a novel berberine hydrochloride-carboxymethyl chitosan hydrogel (BH-CMCH) with excellent thermosensitivity, injectability and in vitro antibacterial activity. In a rat model of mastitis induced by S. aureus, mammary duct injection of BH-CMCH reduced the bacterial load in infected mammary gland tissue and protected the tissue from damage from infection. In addition, proteomics analysis showed that mammary duct injection of BH-CMCH enhanced autolysosome degradation and promoted the innate immune response by activating the lysosomal pathway and up-regulating related significantly differentially expressed proteins (SDEPs). Taken together, the findings support the potential of BH-CMCH as an antibacterial agent against S. aureus-induced mastitis.

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.

Applying sewage sludge or animal manure onto agricultural land can result in estrogen pollution, which increases the risk of human exposure to steroid estrogens (SEs) via the food chain. However, the uptake and accumulation mechanism of SEs by plants is still unclear. In this study, the uptake, accumulation, and translocation of 17β-E2, a representative SE, were investigated through a series of wheat hydroponic experiments. Various inhibitors were applied to explore the uptake pathways of 17β-E2 by wheat. In addition, the effects of exposure concentrations, coexisting 17α-ethynylestradiol (EE2) and plant properties on the uptake of 17β-E2 were examined. The results indicated that the accumulation of 17β-E2 in wheat roots mainly resulted from adsorption and active uptake that involved aquaporins and anion channels transport. The chlorophyll and protein contents of plants were positively correlated with the uptake of 17β-E2, whereas competitive inhibition occurred when 17β-E2 and EE2 coexisted in the same solution. Nevertheless, the results of a split-root experiment showed that 17β-E2 absorbed by wheat could further migrate in plant via long-distance transport and ultimately was discharged from plants, suggesting that 17β-E2 was still at risk of being released even though it had been absorbed by plants. These results could provide valuable insights into the risk assessment and risk control of the uptake of SEs by plants.

BackgroundThe metabolic processes of cows undergo significant changes during subclinical mastitis, but their molecular mechanisms have not been clearly elucidated. This study investigated the changes in milk metabolites after intramammary infusion of matrine, a plant alkaloid with anticancer properties, in the form of a chitosan hydrogel into bovine mammary glands with subclinical mastitis. Infusions were continued for seven days, and milk samples were collected on day 1 and day 7 for analysis of the microbiome by 16S rRNA gene sequencing and metabolites by liquid chromatography-mass spectrometry.ResultsMatrine-chitosan hydrogels (MCHs) significantly decreased the somatic cell count on day 7 and the Simpson index indicated that microbial diversity was significantly lower on day 7 than on day 1. On day 7, the numbers of Aerococcus, Corynebacterium_1 and Staphylococcus were significantly lower, while the abundance of Firmicutes was very significantly decreased. The numbers of Probacteria increased, however. In milk samples, we identified 74 differentially expressed metabolites and the MCH infusion group had the most significantly upregulated metabolites including sphingolipids, glycerophospholipids, flavonoids and fatty acyls. Principal component analysis and the orthogonal partial least squares discriminant test confirmed good separation of the milk metabolites. The identification of active milk metabolic pathways after MCH treatment supported the known antimicrobial and anti-inflammatory properties of matrine that are associated with glycerophospholipid metabolism and the sphingolipid metabolic signaling pathways.Conciusions These insights into the mechanisms and the corresponding biological responses to matrine demonstrate its potential immunoregulatory activity and emphasize the need for continued investigation.

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 &lt, 0.05). Conversely, thiamine supplementation significantly increased ruminal pH, acetate while significantly decreasing propionate content compared with HC treatment (p &lt, 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.

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

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 present study aimed to identify metabolic signature changes of the arteriovenous metabolome and the new metabolites that involved in mammary biological process during milk synthesis. GC/MS-based metabolomics profiling of arteriovenous plasma from 30 lactating dairy cows fed three diets identified a total of 144 metabolites. Phenylalanine and tyrosine, involved in aminoacyl-tRNA biosynthesis and phenylalanine metabolism, were shown higher expression in the artery than in the vein based on both GC/MS and targeted analysis for cows fed both alfalfa hay diet and rice straw diet. Mammary uptake or clearance of citric acid, stearic acid, oleic acid, fructose, β-mannosylglycerate, 4-hydroxybutyrate, and D-talose were significantly correlated with milk performance or feed intake, indicating that these metabolites might be newly identified precursors or indicators of milk synthesis. This comprehensive assessment of metabolic changes in the arteriovenous metabolome will provide a fundamental understanding of the key metabolites involved in milk synthesis and shows implications of how metabolites from arteriovenous plasma across MG are involved in biological processes or physiological functions for milk synthesis. The newly identified metabolites from the present study provide potential new targeted insights into the study of physiological process for milk synthesis in the MG.

An experiment was conducted to uncover the effects of increasing dietary grain levels on expression of thiamine transporters in ruminal epithelium, and to assess the protective effects of thiamine against high-grain-induced inflammation in dairy cows. Six rumen-fistulated, lactating Holstein dairy cows (627 ± 16.9 kg of body weight, 180 ± 6 d in milk; mean ± standard deviation) were randomly assigned to a replicated 3 × 3 Latin square design trial. Three treatments were control (20% dietary starch, dry matter basis), high-grain diet (HG, 33.2% dietary starch, DM basis), and HG diet supplemented with 180 mg of thiamine/kg of dry matter intake. On d 19 and 20 of each period, milk performance was measured. On d 21, ruminal pH, endotoxic lipopolysaccharide (LPS), and thiamine contents in rumen and blood, and plasma inflammatory cytokines were detected; a rumen papillae biopsy was taken on d 21 to determine the gene and protein expression of toll-like receptor 4 (TLR4) signaling pathways. The HG diet decreased ruminal pH (5.93 vs. 6.49), increased milk yield from 17.9 to 20.2 kg/d, and lowered milk fat and protein from 4.28 to 3.83%, and from 3.38 to 3.11%, respectively. The HG feeding reduced thiamine content in rumen (2.89 vs. 8.97 μg/L) and blood (11.66 vs. 17.63 μg/L), and the relative expression value of thiamine transporter-2 (0.37-fold) and mitochondrial thiamine pyrophosphate transporter (0.33-fold) was downregulated by HG feeding. The HG-fed cows exhibited higher endotoxin LPS in rumen fluid (134,380 vs. 11,815 endotoxin units/mL), and higher plasma concentrations of lipopolysaccharide binding protein and pro-inflammatory cytokines when compared with the control group. The gene and protein expression of tumor necrosis factor α (TNFα), IL1B, and IL6 in rumen epithelium increased when cows were fed the HG diet, indicating that local inflammation occurred. The depressions in ruminal pH, milk fat, and protein of HG-fed cows were reversed by thiamine supplementation. Thiamine supplementation increased thiamine contents in rumen and blood, and also upregulated the relative expression of thiamine transporters compared with the HG group. Thiamine supplementation decreased ruminal LPS (49,361 vs. 134,380 endotoxin units/mL) and attenuated the HG-induced inflammation response as indicated by a reduction in plasma IL6, and decreasing gene and protein expression of pro-inflammatory cytokines in rumen epithelium. Western bottling analysis showed that thiamine suppressed the protein expression of TLR4 and the phosphorylation of nuclear factor kappa B (NFκB) unit p65. In conclusion, HG feeding inhibits thiamine transporter expression in ruminal epithelium. Thiamine could attenuate the epithelial inflammation during high-grain feeding, and the protective effects may be due to its ability to suppress TLR4-mediated NFκB signaling pathways.

This study was conducted to evaluate the effects of replacing alfalfa hay with rice straw (RS) or corn stover (CS) on amino acid (AA) profiles of gastrointestinal digesta in lactating cows. Eighteen lactating dairy cows were randomly assigned to one of three groups (n = 6) and fed identical concentrate and corn silage with different forages on dry matter basis: (i) 23% alfalfa hay and 7% Chinese wild rye hay (AH); (ii) 30% CS; and (iii) 30% RS. After the 14-week feeding, a total of 18 cows were slaughtered to collect digesta from four representative organs, including rumen, omasum, duodenum and jejunum. The AA profiles of ruminal microbial fraction were similar among the treatments, except for greater Arg in cows fed RS than in cows fed AH or CS. Most of the analysed AA changed under different diets. Significant differences were found among the microbial fraction, rumen fluid and rumen digesta, with greater essential AA in digesta than in microbial fraction or rumen fluid and greater essential AA in microbial fraction than rumen fluid. Significant differences in individual AA profiles of digesta and relevant fluid were found across the four representative digestive tract parts, including rumen, omasum, duodenum and jejunum, showing much lower Leu proportion in CS and RS than in AH in duodenal fluid. In summary, ruminal microbes may prefer using essential AA, rather than non-essential AA. The AA profile of ruminal microbes was constant except for Arg. The AA composition of digesta across the four digestive tracts changed dramatically, which indicated differences in the ability and efficiency of AA absorption. The lower duodenum absorbable Leu proportion in cows fed CS or RS indicated the shortage of Leu in CS or RS diets, which might also restrict the balanced AA absorption.

Two experiments were conducted to reveal the effects of grain-induced subacute rumen acidosis (SARA) on thiamine status in blood and rumen fluid in dairy cows. In both experiments, 6 multiparous, rumen-fistulated Holstein dairy cows were used in a 2-treatment, 2-period crossover design. Each experimental period consisted of 21d (total of 42d). Experiment 1 was to investigate the effects of SARA on thiamine status in blood and rumen fluid. Treatments were either control (20% starch, dry matter basis) or SARA-inducing diet (SAID, 33.2% starch, dry matter basis). In experiment 2, the effects of dietary thiamine supplementation on attenuating SARA and ruminal fermentation characteristics in dairy cows were studied. All cows received the same SAID diet during the whole experimental period; treatments were with or without thiamine (180mg of thiamine/kg of dry matter intake). In both experiments, rumen fluid samples were collected at 0, 3, 6, 9, and 12h after morning feeding on d 21 and 42 of the experiments for measurement of pH, thiamine, volatile fatty acid, and lactate contents. Peripheral blood was also collected at 3h after morning feeding on d 21 and 42 to measure thiamine, carbohydrate metabolites, and enzyme activities. In experiment 1, cows fed the SAID diet had lower ruminal and plasma thiamine concentrations and higher lactate than cows fed the control diet. The ruminal thiamine contents were positively related to pH and the concentrations of acetate in the rumen, and negatively correlated with the lactate contents. Experiment 2 demonstrated that ruminal pH and the concentrations of thiamine, acetate, and total volatile fatty acids in the rumen were increased, whereas ruminal lactate contents were reduced by thiamine supplementation. The concentrations of lactate and the activity of lactate dehydrogenase in blood were reduced in the thiamine supplemented group, and the opposite was true for the nonesterified fatty acids and α-ketoneglutarate dehydrogenase contents. In conclusion, the thiamine status was affected by SARA in dairy cows and ruminal infusion of thiamine could help attenuate SARA by improving theproportions of ruminal volatile fatty acids and reducing lactate contents in rumen fluid and blood.

Simple Summary Kelp powder has been widely used as feed ingredient or additives in monogastric animal production and presented positive effects on production performance and intestinal microbiota because of its abundance in biomass output, novel oligosaccharides, and iodine. However, little information is available for the nutritional effects of kelp on ruminants. Therefore, the objective of the present study was to investigate the effects of kelp partial replacing dietary forage on rumen fermentation and production performance of dairy cows. Results indicated that, a substitution of 5% kelp powder for forage in the diet reduced the fiber content in the feeding diet and then regulated the proliferation of ruminal microbiota, which lead to a significant reduction of NH3-N while increasing acetate, propionate, and total volatile fatty acids (TVFA) concentrations in rumen, and finally partly increasing milk production and milk fat content. The present study may provide more comprehensive cognition of the nutritional value of kelp powder as a dietary raw material. It may further promote the utilization of kelp powder and provide a useful dietary raw material to the husbandry of dairy cows. Abstract Background: Kelp powder, which was rich in novel oligosaccharides and iodine might be utilized by the rumen microbiome, promoted the ruminal fermentation and finally enhanced the lactation performance of dairy cows. Therefore, the purpose of this study was to investigate the effects of kelp powder partially replacing dietary forage on rumen fermentation and lactation performance of dairy cows. (2) Methods: In the present study, 20 Chinese Holstein dairy cows were randomly divided into two treatments, a control diet (CON) and a kelp powder replacing diet (Kelp) for a 35-d long trial. Dry matter intake (DMI), milk production, milk quality, ruminal fermentable parameters, and rumen microbiota were measured to investigate the effects of kelp powder feeding on dairy cows. (3) Results: On the lactation performance, kelp significantly increased milk iodine content and effectively enhanced milk production and milk fat content. On the fermentable aspects, kelp significantly raised TVFA while reducing the ammonia-N content. On the rumen microbial aspect, kelp feeding significantly promoted the proliferation of Firmicutes and Proteobacteria while suppressing Bacteroidetes. (4) Conclusion: kelp powder as an ingredient of feedstuff might promote the rumen fermentation ability and effectively increase milk fat and iodine content, and consequently improve the milk nutritional value.

Bisphenol A (BPA) and its derivatives, including bisphenols S (BPS), F (BPF), E (BPE), B (BPB), Z (BPZ), and AF (BPAF), are widely used in consumer products. Moreover, they are typically detected in the environment, food, and humans. Previous studies have linked BPA to several health risks, but it is still unclear whether BPA replacements are safe. In this study, we developed an in vitro model based on human embryonic stem cells (hESCs) to explore the potential neural toxicity of these compounds. We observed that the bisphenols affected the viability of hESCs and hESC-derived neural stem cells (NSCs) at high concentrations, with BPS being the least cytotoxic and BPAF the strongest cytotoxic compound. At human-relevant concentrations, the bisphenols did not significantly interfere with gene expression and protein levels during hESC differentiation into the neural epithelium, as well as during specification of neuron-like cells from NSCs. Nevertheless, monitoring of cell morphology changes indicated that exposure to BPA and its derivatives impaired neurite length in neuron-like cells. Thus, our findings provide insights into the molecular mechanisms of bisphenol-dependent neurotoxicity at low nanomolar levels and support the view that BPA substitutes may not be sufficiently safe for widespread use as industrial chemicals.

Early lactation dairy cows are vulnerable to negative energy balance (NEB) due to reduced dry matter intake (DMI) and high energy requirements for lactation. This study mainly revealed the mechanism of different doses of rumen-protected glucose (RPG) to alleviate NEB of the early lactating dairy cows through serum untargeted metabolomics, and explored the effects of different doses of RPG on rumen fermentation, lactation performance, and serum biochemical indices associated with NEB. Thirty-two multiparous Holstein cows in early lactation, blocked by parity, liveweight, and milk yield, were allocated to one of four treatments in a randomized block design. Treatments were: control (CON), low RPG (LRPG), medium RPG (MRPG) and high RPG (HRPG) with 0, 200, 350 and 500 g RPG per cow per day, respectively. Treatments were applied from calving to 35 days postpartum. The results showed that compared with other groups, the MRPG group changed the rumen environment, increased milk protein composition on 35 d, and decreased serum concentrations of β-hydroxybutyric acid (BHBA), non-esterified fatty acid (NEFA), aspartate aminotransferase (AST), and could increase glucose (GLU) concentration in serum. Dietary RPG supplementation affected ruminal pH and some volatile fatty acid (VFA) concentrations. The results of serum metabolomics showed that the metabolites with the largest contribution of the first 30 differences after RPG supplementation were mainly involved in lipid metabolism. The results of this study indicated that adding 350 g RPG to a cow’s diet in early lactation could improve energy balance by reducing fat mobilization.

Jujuboside A is a kind of the saponins isolated from the seeds ofZiziphus jujuba, which possesses multiple biological effects, such as antianxiety, antioxidant, and anti-inflammatory effects; however, its mediatory effect on isoproterenol-stimulated cardiomyocytes has not been investigated yet. In this study, we tried to detect the protective effect and potential mechanism of JUA on ISO-induced cardiomyocytes injury. H9C2 cells were treated with ISO to induce cell damage. Cells were pretreated with JUA to investigate the effects on the cell viability, morphological changes, light chain 3 conversion, and the activation of PI3K/Akt/mTOR signaling pathway. Results showed that ISO significantly inhibited the cell viability in a time- and dose-dependent manner. JUA pretreatment could reverse the reduction of cell viability and better the injury of H9C2 cells induced by ISO. Western blot analysis showed that JUA could accelerate the phosphorylation of PI3K, Akt, and mTOR. Results also indicated that JUA could significantly decrease the ratio of microtubule-associated protein LC3-II/I in H9C2 cells. Taken together, our research showed that JUA could notably reduce the damage cause by ISO via promoting the phosphorylation of PI3K, Akt, and mTOR and inhibiting LC3 conversion, which may be a potential choice for the treatment of heart diseases.

Colostrum is a unique source of biological molecules, and the uptake of colostrum immunoglobulin G (IgG) by neonatal calves is related to the success of passive immunity transfer, an important determinant of health and survival. However, studies on colostral IgG uptake in the small intestine by using proteomics approaches have been scarce. In the present study, samples of the duodenum, jejunum and ileum were collected ~2 h after birth from calves not fed colostrum, and 8, 24, and 36 h after birth from calves fed colostrum. Protein samples were extracted and separated by temporal two-dimensional gel electrophoresis, and differential protein spots were identified by mass spectrometry. After colostrum feeding, internalised IgG in the duodenum and jejunum was detected at ~8 and 24 h, and then was barely detected at 36 h after birth. The IgG internalised in the ileum of calves fed colostrum was detected ~24 and 36 h after birth. Beta-lactoglobulin was upregulated in the entire small intestine; these levels were maintained for 24 h and were barely detected thereafter in neonatal calves fed colostrum. Moreover, changes in several proteins in the small intestine were detected after calves received colostrum. The results of the present study showed the distribution and variation of internalised IgG in the duodenum, jejunum and ileum of neonatal calves that received colostrum after birth. These findings indicated that IgG and β-lactoglobulin in the small intestine of calves fed colostrum may be related to their unique bioactive functions, providing a basis for improvements in calf rearing and management.

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.

Heat stress is important in the pathogenesis of intestinal epithelial barrier dysfunction. Ferulic acid (FA), a phenolic acid widely found in fruits and vegetables, can scavenge free radicals and activate cell stress responses. This study is aimed at investigating protective effects of FA on heat stress-induced dysfunction of the intestinal epithelial barrier in vitro and in vivo. Intestinal epithelial (IEC-6) cells were pretreated with FA for 4 h and then exposed to heat stress. Heat stress caused decreased transepithelial electrical resistance (TER) and increased permeability to 4-kDa fluorescein isothiocyanate (FITC)-dextran (FD4). Both effects were inhibited by FA in a dose-dependent manner. FA significantly attenuated the decrease in occludin, ZO-1 and E-cadherin expression observed with heat stress. The distortion and redistribution of occludin, ZO-1 and E-cadherin proteins were also effectively prevented by FA pretreatment. Moreover, heat stress diminished electron-dense material detected in tight junctions (TJs), an effect also alleviated by FA in a dose-dependent manner. In an in vivo heat stress model, FA (50 mg/kg) was administered to male Sprague-Dawley rats for 7 consecutive days prior to exposure to heat stress. FA pretreatment significantly attenuated the effects of heat stress on the small intestine, including the increased FD4 permeability, disrupted tight junctions and microvilli structure, and reduced occludin, ZO-1 and E-cadherin expression. Taken together, our results demonstrate that FA pretreatment is potentially protective against heat stress-induced intestinal epithelial barrier dysfunction.

A novel xylanase gene, xyn-lxy, was cloned from a metagenomic fosmid library, which was previously constructed from the rumen contents of Hu sheep and was functionally characterized in Escherichia coli. The open reading frame was composed of 1923 bp and encoded for 640 amino acids, including a catalytic domain of glycosyl hydrolase family 10 and carbohydrate-binding module 9. The gene showed 97 % identity with uncultured bacterium Contig1552 but low similarity with xylanases from known cellulolytic-degrading microorganisms in the rumen. The recombinant XYN-LXY showed a specific activity of 664.7 U mg(-1). The optimal temperature and pH of the enzyme were 50 °C and 6.0, respectively. Specifically, XYN-LXY was exclusively activated by Mn(2+) among all of the cations and reducing agents tested in this study. An enzymatic hydrolysis assay revealed that XYN-LXY degraded birchwood xylan into xylooligosaccharide with a low degree of polymerization. After incubation for 4 h, the concentration of the dominant product, xylobiose, was 2.297 ± 0.175 mg ml(-1) (74.07 % of total product) followed by xylose with a concentration of 0.656 ± 0.010 mg ml(-1) (21.14 % of total product). The XYN-LXY exhibited deep degradation effects on the xylan substrate, which were rarely observed with endo-xylanase, making it a promising candidate for industrial application, especially in biofuel production.