Proper motility enhances rumen fermentation and microbial protein synthesis with decreased saturation of dissolved gases in rumen simulation technique.

The physiological function of the reticulorumen plays an essential role in ruminant nutrition, and detailed knowledge of rumen motility can further advance understanding of ruminant nutrition and physiology. Rumen motility was simulated by setting different stirrer rotation speeds in a rumen simulation technique (RUSITEC) system. The aim of this study was to investigate the effects of rotation speeds on rumen fermentation, saturation factor of dissolved gases, hydrogen (H2) and methane (CH4) emissions, microbial protein synthesis, and selected microbial population using RUSITEC. The experiment was performed according to a balanced 3 × 3 Latin square design, and each period included 7 d for adaptation and 3 d for sampling. Three motility treatments included 5, 15, and 25 rpm rotation speeds. Daily total gas and H2 and CH4 emissions had quadratic responses to the increasing rotation speed and were highest at 15 rpm. Quadratic and linear responses (highest at 5 rpm) to increasing rotation speed were observed for saturation factors of H2 and CH4, liquid-dissolved H2 and CH4 concentrations, and headspace concentration of H2 in the gas phase, whereas increasing rotation speed linearly decreased saturation factors of CO2 and liquid-dissolved CO2 concentration. Quadratic and linear responses to increasing rotation speed were observed for molar percentages of acetate, ammonia, and microbial protein concentration, whereas increasing rotation speed quadratically increased pH and decreased total volatile fatty acid concentration and acetate-to-propionate ratio. The 15-rpm rotation speed had the highest values of total volatile fatty acids, acetate molar percentage, and microbial protein concentration. Quadratic and linear responses to increasing rotation speed were observed for copy numbers of solid-associated fungi and fluid-associated bacteria, fungi, and protozoa, while increasing rotation speed linearly increased copy numbers of solid-associated protozoa. Rotation at 15 rpm increased populations of fungi and protozoa in the solid rumen contents and the population of bacteria and fungi in the liquid rumen contents. In summary, this study provides insights on the biofunction of proper rumen motility (i.e., at a rotation speed of 15 rpm), such as improving feed fermentation, increasing gas emissions with decreased dissolved gas concentrations and saturation factors, and promoting microbial colonization and microbial protein synthesis, although further increase in rotation speed (i.e., to 25 rpm) decreases feed fermentation and microbial protein synthesis.

Association of fibre degradation with ruminal dissolved hydrogen in growing beef bulls fed with two types of forages.

The present study investigated the association between fibre degradation and the concentration of dissolved molecular hydrogen (H2) in the rumen. Napier grass (NG) silage and corn stover (CS) silage were compared as forages with contrasting structures and degradation patterns. In the first experiment, CS silage had greater 48-h DM, neutral-detergent fibre (NDF) and acid-detergent fibre degradation, and total gas and methane (CH4) volumes, and lower 48-h H2 volume than NG silage in 48-h in vitro incubations. In the second experiment, twenty-four growing beef bulls were fed diets including 55 % (DM basis) NG or CS silages. Bulls fed the CS diet had greater DM intake (DMI), average daily gain, total-tract digestibility of OM and NDF, ruminal dissolved methane (dCH4) concentration and gene copies of protozoa, methanogens, Ruminococcus albus and R. flavefaciens, and had lower ruminal dH2 concentration, and molar proportions of valerate and isovalerate, in comparison with those fed the NG diet. There was a negative correlation between dH2 concentration and NDF digestibility in bulls fed the CS diet, and a lack of relationship between dH2 concentration and NDF digestibility with the NG diet. In summary, the fibre of CS silage was more easily degraded by rumen microorganisms than that of NG silage. Increased dCH4 concentration with the CS diet presumably led to the decreased ruminal dH2 concentration, which may be helpful for fibre degradation and growth of fibrolytic micro-organisms in the rumen.

3-Nitrooxypropanol supplementation had little effect on fiber degradation and microbial colonization of forage particles when evaluated using the in situ ruminal incubation technique.

3-Nitrooxypropanol (3-NOP) is an investigational compound that acts as an enzyme inhibitor to decrease ruminal methanogenesis. We hypothesized that when feeding 3-NOP to cattle fed a high-forage diet, H2 would accumulate in the rumen, which could suppress microbial colonization of feed particles and fiber degradation. Therefore, the study investigated the effects of supplementing a high-forage diet with 3-NOP on ruminal fiber degradability and microbial colonization of feed particles using the in situ technique. Eight ruminally cannulated beef cattle were allocated to 2 groups (4 cattle/group) in a crossover design with 2 periods and 2 dietary treatments. The treatments were control (basal diet) and 3-NOP (basal diet supplemented with 3-NOP, 150 mg/kg of dry matter). The basal diet consisted of 45% barley silage, 45% chopped grass hay, and 10% concentrate (dry matter basis). Samples of dried, ground barley silage and grass hay were incubated in the rumen of each animal for 0, 4, 12, 24, 36, 48, 96, 120, 216, and 288 h to determine neutral detergent fiber (NDF) degradation kinetics. An additional 2 bags were incubated for 4 and 48 h to evaluate the bacterial community attached to the incubated forages. Dietary supplementation of 3-NOP decreased (-53%) the dissolved methane concentration and increased (+780%) the dissolved H2 concentration in ruminal fluid, but did not substantially alter in situ NDF degradation. The addition of 3-NOP resulted in a decrease in the α-diversity of the microbial community with colonizing communities showing reduced numbers of amplicon sequence variants and phylogenetic diversity compared with control diets. Principal coordinate analysis plots indicated that forages incubated in animals fed 3-NOP resulted in highly specific changes to targeted microbes compared with control diets based on unweighted analysis (considering only absence and presence of taxa), but did not alter the overall composition of the colonizing community based on weighted UniFrac distances; unchanged relative abundances of major taxa included phyla Bacteroidetes, Firmicutes, and Fibrobacteres. The effect of 3-NOP on colonizing methanogenic microbes differed depending upon the forage incubated, as abundance of genus Methanobrevibacter was decreased for barley silage but not for grass hay. In conclusion, 3-NOP supplementation of a high-forage diet decreased ruminal methanogenesis and increased dissolved H2 concentration, but had no negative effects on ruminal fiber degradation and only minor effects on relative abundances of the major taxa of bacteria adhered to forage substrates incubated in the rumen.

Liquid hot water treatment of rice straw enhances anaerobic degradation and inhibits methane production during in vitro ruminal fermentation.

Liquid hot water (LHW) treatment can be used to disrupt the fiber structure of rice straw. This in vitro ruminal batch culture study investigated the effect of LHW treatment on feed degradation, methane (CH4) production, and microbial populations. Rice straw was treated by LHW, and in vitro ruminal fermentation was performed using an automatic system with 72 h of incubation. Scanning electron microscopy showed that LHW treatment disrupted the physical structure of rice straw. Liquid hot water treatment decreased neutral detergent fiber and hemicellulose contents of the rice straw and increased neutral detergent solubles, water-soluble carbohydrates, and arabinose contents. Liquid hot water treatment increased dry matter degradation and volatile fatty acid concentration and decreased the acetate:propionate ratio, CH4 production, hydrogen accumulation, neutral detergent fiber degradation, and populations of protozoa, fungi, and cellulolytic bacteria. In summary, LHW treatment disrupted the cellulose-hemicellulose-lignin structure matrix of rice straw, leading to increased substrate degradability and decreased CH4 production. Therefore, the LHW treatment is a potential strategy to improve the nutritive value of forage such as rice straw and decrease the CH4 emissions in ruminants.

Molecular hydrogen produced by elemental magnesium inhibits rumen fermentation and enhances methanogenesis in dairy cows.

Hydrogen is a key metabolite that connects microbial fermentation and methanogenesis in the rumen. This study was to investigate the effects of elevated H2 produced by elemental Mg on rumen fermentation and methanogenesis in dairy cows. Four nonlactating Chinese Holstein dairy cows were employed for this experiment in a replicated crossover design. The 2 dietary treatments included a basal diet supplemented with Mg(OH)2 (14.5 g/kg of feed dry matter) or elemental Mg (6.00 g/kg of feed dry matter). When compared with Mg(OH)2 treatment, cows fed diet with elemental Mg had similar rumen Mg2+ concentration, but higher rumen dissolved H2 and methane concentrations at 2.5 h after morning feeding. Also, elemental Mg supplementation decreased feed digestibility, rumen volatile fatty acid concentration, and relative abundance of group Ruminococcaceae_UCG-014, genus Bifidobacterium, and group Mollicutes_RF9, increased acetate to propionate ratio, succinate concentration, and abundance of family Christensenellaceae. Elemental Mg supplementation increased enteric CH4 emission, altered methanogen community with increased abundance of order Methanomassiliicoccales, 16S ribosomal RNA gene copies of methanogens, and order Methanobacteriales. In summary, the pulse of elevated dissolved H2 after feeding produced by elemental Mg inhibited rumen fermentation and feed digestibility by decreasing the abundance of carbohydrate-degrading bacteria, promoted H2 incorporation into succinate by increasing family Christensenellaceae and genus Bacteroidales_BS11, and increased H2 utilization for methanogenesis by favoring growth of methanogens.

Effects of rumen cannulation on dissolved gases and methanogen community in dairy cows.

Rumen cannulation is a widely employed technique in ruminant nutrition research. However, the gap between skin and rumen cannula can cause leakage of fermentation gases and influx of atmospheric air, which may adversely affect the anaerobic environment in the rumen. The present study was designed to investigate the effects of rumen cannulation on headspace gases, dissolved gases, fermentation end products, and methanogen community in the rumen of dairy cows. Eight Holstein cows were used in the experiment. Four cows were surgically fitted with rumen cannulas, whereas the other 4 intact cows were used as control. Rumen cannulation decreased gaseous hydrogen and methane concentrations, dissolved carbon dioxide concentration, and relative abundances of Methanosphaera, and increased the saturation factor of dissolved hydrogen and dissolved methane, dissolved methane concentration, volatile fatty acid concentration, 16S ribosomal RNA gene copies of methanogens, and Simpson index of methanogen community. In summary, rumen cannulation causes a reduction in headspace gaseous hydrogen and gaseous methane, which may not decrease dissolved gas concentrations due to an increase in saturation factors. Furthermore, rumen cannulation alters methanogen community with increased methanogen population and decreased relative abundances of Methanosphaera.

Technical note: Evaluation of interval between measurements and calculation method for the quantification of enteric methane emissions measured by respiration chamber.

Respiration chambers share one analyzer working in parallel, and methane (CH4) concentrations have to be measured at certain intervals. The maximum and minimum values in the kinetics of CH4 emissions can be missed during the interval between measurements, which may influence the quantification of CH4 emissions. Chambers must be opened for morning feeding and cleaning, which causes a loss of CH4 data. Calculation methods are needed to estimate the lost CH4 emission data, which may influence the estimated amount of daily CH4 emissions. In this study, we measured the CH4 emissions of 10 growing Chinese Holstein dairy heifers in respiration chambers. Methane concentrations were measured every 0.5 min to obtain the 23-h kinetics of CH4 emissions, which were further selected at different intervals between measurements (i.e., 5, 30, 60, 120, 180, and 240 min) to evaluate the effects of interval on quantification of CH4 emissions. The missing 1-h kinetics of CH4 emissions before feeding were not measured, and 2 calculation methods were used to estimate the missing 1-h kinetics of CH4 emissions: mean value of measuring period (the mean method) and the nearest value of measurement just before chamber opening (the nearest method). The results showed that the rates of CH4 emission from 10 heifers varied from 4.56 to 11.42 g/h. The increment of intervals decreased maximum rate of CH4 emission and increased minimum rate of CH4 emission. Interval caused less than 5% of the difference in measuring CH4 emissions. Although the mean method had greater estimated daily CH4 emission than the nearest method, the difference was within 3%. The interval between measurements (≤3 h) and calculation method had little influence on enteric CH4 emission measurements.

Nitrate improves ammonia incorporation into rumen microbial protein in lactating dairy cows fed a low-protein diet.

Generation of ammonia from nitrate reduction is slower compared with urea hydrolysis and may be more efficiently incorporated into ruminal microbial protein. We hypothesized that nitrate supplementation could increase ammonia incorporation into microbial protein in the rumen compared with urea supplementation of a low-protein diet fed to lactating dairy cows. Eight multiparous Chinese Holstein dairy cows were used in a crossover design to investigate the effect of nitrate or an isonitrogenous urea inclusion in the basal low-protein diet on rumen fermentation, milk yield, and ruminal microbial community in dairy cows fed a low-protein diet in comparison with an isonitrogenous urea control. Eight lactating cows were blocked in 4 pairs according to days in milk, parity, and milk yield and allocated to urea (7.0 g urea/kg of dry matter of basal diet) or nitrate (14.6 g of NO3-/kg of dry matter of basal diet, supplemented as sodium nitrate) treatments, which were formulated on 75% of metabolizable protein requirements. Nitrate supplementation decreased ammonia concentration in the rumen liquids (-33.1%) and plasma (-30.6%) as well as methane emissions (-15.0%) and increased dissolved hydrogen concentration (102%), microbial N (22.8%), propionate molar percentage, milk yield, and 16S rRNA gene copies of Selenomonas ruminantium. Ruminal dissolved hydrogen was positively correlated with the molar proportion of propionate (r = 0.57), and negatively correlated with acetate-to-propionate ratio (r = -0.57) and estimated net metabolic hydrogen production relative to total VFA produced (r = -0.58). Nitrate reduction to ammonia redirected metabolic hydrogen away from methanogenesis, enhanced ammonia incorporation into rumen microbial protein, and shifted fermentation from acetate to propionate, along with increasing S. ruminantium 16S rRNA gene copies, likely leading to the increased milk yield.

[Application of wireless neuronal recording system in fear conditioning of Alzheimer's disease mice - hippocampal Theta oscillation observation].

APP/PS1/tau triple transgenic (3xTg) mouse is a classical animal model of Alzheimer's disease (AD), which has abnormalities in recognition and electrophysiological properties at early 6-month-old age. However, few studies were performed by using simultaneously recording cognitive behavior and brain electrical activity in the conscious 3xTg mice. By using a new wireless recording system, we recorded hippocampal Theta oscillations in 3xTg mice during the process of fear conditioning test. The results showed that: (1) in training session, no significant difference in the fear behavior and hippocampal Theta activity was found between 3xTg mice and WT mice; (2) in test session, 3xTg mice showed a significant decrease in freezing ratio compared with WT mice when they were exposed to conditioning stimulus (CS); (3) the 3xTg mice showed lower peak power in Theta oscillation in both Pre-CS and CS duration compared with WT mice; (4) CS effectively induced an increase in the peak frequency of Theta oscillation in WT mice, but not in 3xTg mice. These results indicated that the impairment of cognition behavior in 3xTg mice was accompanied with the decreased peak power and peak frequency of Theta oscillation in the hippocampus, suggesting that a decline in Theta oscillation might be involved in the impairments of the fear conditioning, and the enhanced hippocampal Theta oscillation may be beneficial for improving AD cognitive function.

Puerarin inhibits angiotensin II-induced cardiac hypertrophy via the redox-sensitive ERK1/2, p38 and NF-κB pathways.

AIM: To investigate the effects of puerarin (Pue), an isoflavone derived from Kudzu roots, on angiotensin II (Ang II)-induced hypertrophy of cardiomyocytes in vivo and in vitro. METHODS: C57BL/6J mice were infused with Ang II and treated with Pue (100 mg·kg(-1)·d(-1), po) for 15 d. After the treatment, systolic blood pressure (SBP) and left ventricular wall thickness were assessed. The ratios of heart weight to body weight (HW/BW) and left ventricular weight to body weight (LVW/BW) were determined, and heart morphometry was assessed. Expression of fetal-type genes (ANP, BNP and ß-MHC) in left ventricles was measured using semi-quantitative RT-PCR. Mouse primary cardiomyocytes were treated with Pue (50, 100, 200 µmol/L), then exposed to Ang II (1 µmol/L). ROS level was examined with flow cytometry, the binding activity of NF-κB was determined using EMSA. Western blot was used to measure the levels of ERK1/2, p38 and NF-κB pathway proteins. [(3)H]leucine incorporation was used to measure the rate of protein synthesis. RESULTS: Oral administration of Pue significantly suppressed Ang II-induced increases in the myocyte surface area, HW/BW, LVW/BW, SBP and left ventricular wall thickness. Furthermore, Pue significantly suppressed Ang II-induced increases in ANP, BNP and ß-MHC expression in the left ventricles in vivo. Treatment of cardiomyocytes with Pue (50-500 µmol/L) did not affect the viability of cardiomyocytes in vitro. Pretreatment of cardiomyocytes with Pue dose-dependently inhibited Ang II-induced increases in ROS production, NF-κB binding activity, protein synthesis and cell breadth. Furthermore, pretreatment with Pue significantly suppressed Ang II-induced activation of ERK1/2, p38 and the NF-κB pathway proteins and the expression of ANP and ß-MHC in cardiomyocytes. The positive drug valsartan exerted similar effects on Ang II-induced cardiac hypertrophy in vivo and in vitro. CONCLUSION: Pue attenuates Ang II-induced cardiac hypertrophy by inhibiting activation of the redox-sensitive ERK1/2, p38 and the NF-κB pathways.

3-Nitrooxypropanol supplementation of a forage diet decreased enteric methane emissions from beef cattle without affecting feed intake and apparent total-tract digestibility.

Supplementation of ruminant diets with the methane (CH4) inhibitor 3-nitrooxypropanol (3-NOP; DSM Nutritional Products, Switzerland) is a promising greenhouse gas mitigation strategy. However, most studies have used high grain or mixed forage-concentrate diets. The objective of this study was to evaluate the effects of supplementing a high-forage diet (90% forage DM basis) with 3-NOP on dry matter (DM) intake, rumen fermentation and microbial community, salivary secretion, enteric gas emissions, and apparent total-tract nutrient digestibility. Eight ruminally cannulated beef heifers (average initial body weight (BW) ±â€…SD, 515 ±â€…40.5 kg) were randomly allocated to two treatments in a crossover design with 49-d periods. Dietary treatments were: 1) control (no 3-NOP supplementation); and 2) 3-NOP (control + 150 mg 3-NOP/kg DM). After a 16-d diet adaption, DM intake was recorded daily. Rumen contents were collected on days 17 and 28 for volatile fatty acid (VFA) analysis, whereas ruminal pH was continuously monitored from days 20 to 28. Eating and resting saliva production were measured on days 20 and 31, respectively. Diet digestibility was measured on days 38-42 by the total collection of feces, while enteric gas emissions were measured in chambers on days 46-49. Data were analyzed using the mixed procedure of SAS. Dry matter intake and apparent total-tract digestibility of nutrients (DM, neutral and acid detergent fiber, starch, and crude protein) were similar between treatments (P ≥ 0.15). No effect was observed on eating and resting saliva production. Relative abundance of the predominant bacterial taxa and rumen methanogen community was not affected by 3-NOP supplementation but rather by rumen digesta phase and sampling hour (P ≤ 0.01). Total VFA concentration was lower (P = 0.004) following 3-NOP supplementation. Furthermore, the reduction in acetate and increase in propionate molar proportions for 3-NOP lowered (P < 0.001) the acetate to propionate ratio by 18.9% as compared with control (4.1). Mean pH was 0.21 units lower (P < 0.001) for control than 3-NOP (6.43). Furthermore, CH4 emission (g/d) and yield (g/kg DMI) were 22.4 and 22.0% smaller (P < 0.001), respectively, for 3-NOP relative to control. Overall, the results indicate that enteric CH4 emissions were decreased by more than 20% with 3-NOP supplementation of a forage diet without affecting DM intake, predominant rumen microbial community, and apparent total-tract nutrients digestibility.

This study evaluated the effects of supplementing forage fed cattle with 3-nitrooxypropanol (150 mg/kg dry matter) on feed intake, rumen fermentation and microbial community composition, methane emissions, and nutrient digestibility. Eight ruminally cannulated beef heifers were used for the experiment. The results indicated that 3-nitrooxypropanol supplementation substantially reduced methane emissions without affecting feed intake and total-tract digestibility of nutrients.

[Optimal conditions of bone marrow mesenchymal stem cells on paraquat-induced acute lung injury in rats].

OBJECTIVE: To investigate the effect of bone marrow mesenchymal stem cell (BMSC) transplantation on acute lung injury induced by paraquat (PQ) poisoning in rats, and to identify the optimal transplantation conditions. METHODS: Two hundred female rats were randomly divided into six groups, i.e., PQ group (n = 15), BMSC treatment group 1 (n = 15 for each subgroup), BMSC treatment group 2 (n = 15 for each subgroup), BMSC control group 1 (n = 15 for each subgroup), BMSC control group 2 (n = 15), and normal control group (n = 5). In the PQ group, each rat was intraperitoneally injected with 20% PQ solution (15 mg/kg). In BMSC treatment group 1, each rat was injected via the caudal vein with 1×10(5), 10(6), 10(7)or 10(8) BMSCs (1 ml) after 6 hours of PQ exposure. In BMSC treatment group 2, each rat was injected via the caudal vein with 1×10(7) BMSCs (1 ml) after 1, 6, 12, or 24 hours of PQ exposure. In BMSC control group 1, each rat was injected via the caudal vein with 1×10(5), 10(6), 10(7), or 10(8) BMSCs (1 ml). In BMSC control group 2, each rat was injected via the caudal vein with 1×10(7) BMSCs (1 ml). In the normal control group, each rat was intraperitoneally injected with an equal volume of 0.9% saline. The lung wet/dry weight ratio and plasma tumor necrosis factor-α (TNF-α) and malondialdehyde (MDA) levels were measured at 1, 3, and 7 days after various treatments. RESULTS: Compared with the normal control group, the PQ group showed significantly higher lung wet/dry weight ratios at 3 and 7 days after PQ exposure and significantly higher plasma TNF-α and MDA levels at 1, 3, and 7 days after PQ exposure (P < 0.01). Compared with the PQ group, BMSC treatment group 1 showed significantly lower lung wet/dry weight ratios at 7 days after injection of 1×10(6) and 10(7) BMSCs, significantly lower plasma TNF-α levels at 3 and 7 days after injection of 1×10(5), 10(6), and 10(7) BMSCs, and significantly lower plasma MDA levels at 3 days after injection of 1×10(6) and 10(7) BMSCs and at 7 days after injection of 1×10(5), 10(6), and 10(7) BMSCs (P < 0.05 or P < 0.01). Compared with the PQ group, BMSC treatment group 2 showed significantly lower lung wet/dry weight ratios at 7 days after injection of BMSCs following 1, 6, and 12 hours of PQ exposure, significantly lower plasma TNF-α levels at 3 and 7 days after injection of BMSCs following 1, 6, and 12 hours of PQ exposure, and significantly lower plasma MDA levels at 3 days after injection of BMSCs following 6 hours of PQ exposure and at 7 days after injection of BMSCs following 1, 6, and 12 hours of PQ exposure (P < 0.01). CONCLUSION: BMSCs have a protective effect on the lung in rats with PQ poisoning, and the effect is closely related to the transplantation time and number of transplanted BMSCs. After 6 hours of PQ exposure, intravenous injection of 1×10(7) BMSCs can result in significant decreases in lung wet/dry weight ratio and plasma TNF-α and MDA levels.

Neuromuscular junction disorders: Experimental models and pathophysiological mechanisms.

Located between skeletal muscle fibers and motoneurons, the neuromuscular junction is a chemical synapse essential for the transmission of information from nervous system to skeletal muscle. There are many diseases related to neuromuscular junction dysfunction, including myasthenia gravis, Lambert­Eaton myasthenic syndrome, congenital myasthenic syndromes, amyotrophic lateral sclerosis, and spinal muscular atrophy. The pathophysiological mechanisms of these diseases have been investigated using many animal models. Among them, mouse models are the most commonly used and have provided the majority of current data. Moreover, advances in human induced pluripotent stem cell technology has resulted in new opportunities to study neuromuscular junction disorders from both patients and healthy individuals. Currently, patient­specific induced pluripotent stem cells derived from motor neurons have begun to be studied. These studies will help us achieve a more comprehensive understanding of diseases related to neuromuscular junction disorders. We will describe the research models of neuromuscular junction disorders and provide an overview of recent key findings.

Comparison of in situ ruminal straw fiber degradation and bacterial community between buffalo and Holstein fed with high-roughage diet.

Buffalo exhibits great efficiency in utilizing low-quality roughage, which can be due to the combined effect of host physiological feature and roughage diet fed. The present study was designed to compare the ruminal fiber degradation and the bacterial community attached to straws in buffalo and Holstein when fed with the same high-roughage diet using in situ ruminal incubation technique. Rice and wheat straws were selected as the incubation substrates and sampled at 0, 4, 12, 24, 48, 72, 120, and 216 h of incubation time to measure the kinetics of dry matter (DM) and neutral detergent fiber (NDF) disappearance. Additional two bags were incubated and sampled at 4 and 48 h of incubation time to evaluate the bacterial community attached to straws. The results showed that buffalo exhibited a greater (p ≤ 0.05) fraction of rapidly soluble and washout nutrients and effective ruminal disappearance for both DM and NDF of straw than Holstein, together with a greater (p ≤ 0.05) disappearance rate of potentially degradable nutrient fraction for NDF. Principal coordinate analysis indicated that both host and incubation time altered the bacterial communities attached to straws. Buffalo exhibited greater (p ≤ 0.05) 16S rRNA gene copies of bacteria and greater (p ≤ 0.05) relative abundance of Ruminococcus attached to straw than Holstein. Prolonging incubation time increased (p ≤ 0.05) the 16S rRNA gene copies of bacteria, and the relative abundance of phyla Proteobacteria and Fibrobacters by comparing 4 vs. 48 h of incubation time. In summary, buffalo exhibits greater ruminal fiber degradation than Holstein through increasing bacterial population and enriching Ruminococcus, while prolonging incubation time facilitates fiber degradation through enriching phyla Proteobacteria and Fibrobacteres.

Application of 3-nitrooxypropanol and canola oil to mitigate enteric methane emissions of beef cattle results in distinctly different effects on the rumen microbial community.

BACKGROUND: The major greenhouse gas from ruminants is enteric methane (CH4) which in 2010, was estimated at 2.1 Gt of CO2 equivalent, accounting for 4.3% of global anthropogenic greenhouse gas emissions. There are extensive efforts being made around the world to develop CH4 mitigating inhibitors that specifically target rumen methanogens with the ultimate goal of reducing the environmental footprint of ruminant livestock production. This study examined the individual and combined effects of supplementing a high-forage diet (90% barley silage) fed to beef cattle with the investigational CH4 inhibitor 3-nitrooxypropanol (3-NOP) and canola oil (OIL) on the rumen microbial community in relation to enteric CH4 emissions and ruminal fermentation. RESULTS: 3-NOP and OIL individually reduced enteric CH4 yield (g/kg dry matter intake) by 28.2% and 24.0%, respectively, and the effects were additive when used in combination (51.3% reduction). 3-NOP increased H2 emissions 37-fold, while co-administering 3-NOP and OIL increased H2 in the rumen 20-fold relative to the control diet. The inclusion of 3-NOP or OIL significantly reduced the diversity of the rumen microbiome. 3-NOP resulted in targeted changes in the microbiome decreasing the relative abundance of Methanobrevibacter and increasing the relative abundance of Bacteroidetes. The inclusion of OIL resulted in substantial changes to the microbial community that were associated with changes in ruminal volatile fatty acid concentration and gas production. OIL significantly reduced the abundance of protozoa and fiber-degrading microbes in the rumen but it did not selectively alter the abundance of rumen methanogens. CONCLUSIONS: Our data provide a mechanistic understanding of CH4 inhibition by 3-NOP and OIL when offered alone and in combination to cattle fed a high forage diet. 3-NOP specifically targeted rumen methanogens and partly inhibited the hydrogenotrophic methanogenesis pathway, which increased H2 emissions and propionate molar proportion in rumen fluid. In contrast, OIL caused substantial changes in the rumen microbial community by indiscriminately altering the abundance of a range of rumen microbes, reducing the abundance of fibrolytic bacteria and protozoa, resulting in altered rumen fermentation. Importantly, our data suggest that co-administering CH4 inhibitors with distinct mechanisms of action can both enhance CH4 inhibition and provide alternative sinks to prevent excessive accumulation of ruminal H2.

Dietary selection of metabolically distinct microorganisms drives hydrogen metabolism in ruminants.

Ruminants are important for global food security but emit the greenhouse gas methane. Rumen microorganisms break down complex carbohydrates to produce volatile fatty acids and molecular hydrogen. This hydrogen is mainly converted into methane by archaea, but can also be used by hydrogenotrophic acetogenic and respiratory bacteria to produce useful metabolites. A better mechanistic understanding is needed on how dietary carbohydrates influence hydrogen metabolism and methanogenesis. We profiled the composition, metabolic pathways, and activities of rumen microbiota in 24 beef cattle adapted to either fiber-rich or starch-rich diets. The fiber-rich diet selected for fibrolytic bacteria and methanogens resulting in increased fiber utilization, while the starch-rich diet selected for amylolytic bacteria and lactate utilizers, allowing the maintenance of a healthy rumen and decreasing methane production (p < 0.05). Furthermore, the fiber-rich diet enriched for hydrogenotrophic methanogens and acetogens leading to increased electron-bifurcating [FeFe]-hydrogenases, methanogenic [NiFe]- and [Fe]-hydrogenases and acetyl-CoA synthase, with lower dissolved hydrogen (42%, p < 0.001). In contrast, the starch-rich diet enriched for respiratory hydrogenotrophs with greater hydrogen-producing group B [FeFe]-hydrogenases and respiratory group 1d [NiFe]-hydrogenases. Parallel in vitro experiments showed that the fiber-rich selected microbiome enhanced acetate and butyrate production while decreasing methane production (p < 0.05), suggesting that the enriched hydrogenotrophic acetogens converted some hydrogen that would otherwise be used by methanogenesis. These insights into hydrogen metabolism and methanogenesis improve understanding of energy harvesting strategies, healthy rumen maintenance, and methane mitigation in ruminants.

Combined effects of 3-nitrooxypropanol and canola oil supplementation on methane emissions, rumen fermentation and biohydrogenation, and total tract digestibility in beef cattle.

The individual and combined effects of 3-nitrooxypropanol (3-NOP) and canola oil (OIL) supplementation on enteric methane (CH4) and hydrogen (H2) emissions, rumen fermentation and biohydrogenation, and total tract nutrient digestibility were investigated in beef cattle. Eight beef heifers (mean body weight ± SD, 732 ± 43 kg) with ruminal fistulas were used in a replicated 4 × 4 Latin square with a 2 (with and without 3-NOP) × 2 (with and without OIL) arrangement of treatments and 28-d periods (13 d adaption and 15 d measurements). The four treatments were: control (no 3-NOP, no OIL), 3-NOP (200 mg/kg dry matter [DM]), OIL (50 g/kg DM), and 3-NOP (200 mg/kg DM) plus OIL (50 g/kg DM). Animals were fed restrictively (7.6 kg DM/d) a basal diet of 900 g/kg DM barley silage and 100 g/kg DM supplement. 3-NOP and OIL decreased (P < 0.01) CH4 yield (g/kg DM intake) by 31.6% and 27.4%, respectively, with no 3-NOP × OIL interaction (P = 0.85). Feeding 3-NOP plus OIL decreased CH4 yield by 51% compared with control. There was a 3-NOP × OIL interaction (P = 0.02) for H2 yield (g/kg DM intake); the increase in H2 yield (P < 0.01) due to 3-NOP was less when it was combined with OIL. There were 3-NOP × OIL interactions for molar percentages of acetate and propionate (P < 0.01); individually, 3-NOP and OIL decreased acetate and increased propionate percentages with no further effect when supplemented together. 3-NOP slightly increased crude protein (P = 0.02) and starch (P = 0.01) digestibilities, while OIL decreased the digestibilities of DM (P < 0.01) and neutral detergent fiber (P < 0.01) with no interactions (P = 0.15 and 0.10, respectively). 3-NOP and OIL increased (P = 0.04 and P < 0.01, respectively) saturated fatty acid concentration in rumen fluid, with no interaction effect. Interactions for ruminal trans-monounsaturated fatty acids (t-MUFA) concentration and percentage were observed (P = 0.02 and P < 0.01); 3-NOP had no effect on t-MUFA concentration and percentage, while OIL increased the concentration (P < 0.01) and percentage (P < 0.01) of t-MUFA but to a lesser extent when combined with 3-NOP. In conclusion, the CH4-mitigating effects of 3-NOP and OIL were independent and incremental. Supplementing ruminant diets with a combination of 3-NOP and OIL may help mitigate CH4 emissions, but the decrease in total tract digestibility due to OIL may decrease animal performance and needs further investigation.

Structural characterization and functional properties of CNPP, a byproduct formed during CPP preparation.

Casein nonphosphopeptide (CNPP), a byproduct formed during the preparation of casein phosphopeptide (CPP), is often discarded on a large scale. Although our previous studies have demonstrated the ameliorative effect of CNPP on muscle wasting disorders, its structure-function mechanism is still unclear. Therefore, considering the great influence of structural characteristics on function, this study aims to explain the potential mechanism by characterizing the physicochemical and functional properties of CNPP. The results of structural characterization indicated that CNPP was of low molecular weight and composed of the complete range of amino acids; it was particularly rich in leucine. Compared with casein, CNPP had a lower molecular size and total/free sulfhydryl content (reduced 2.44 and 2.02 µmol/g in CNPP, respectively). Additionally, Fourier transform infrared spectroscopic analysis revealed that enzymatic hydrolysis caused protein unfolding, and the content of ß-turns and random coils reached 50.20% and 10.67%, respectively. Fluorescence-dependent detection of CNPP indicated a reduction of spectral intensity and the occurrence of a red shift. The changes in the structure of CNPP significantly affected its functional characteristics. CNPP has better solubility, foaming, and digestion properties than those of casein and whey protein. Specifically, the foam stability and emulsification properties decreased in the order of casein > CNPP > whey protein. The present study can provide a substantial basis for future application of CNPP as a functional ingredient against sarcopenia.

Hemp (Cannabis sativa L.) Seed Protein-EGCG Conjugates: Covalent Bonding and Functional Research.

In order to make HPI have a wide application prospect in the food industry, we used EGCG to modify HPI. In this study, we prepared different concentrations (1, 2, 3, 4, and 5 mM) of (-)-epigallocatechin gallate (EGCG) covalently linked to HPI and use methods such as particle size analysis, circular dichroism (CD), and three-dimensional fluorescence spectroscopy to study the changes in the structure and functional properties of HPI after being covalently combined with EGCG. The particle size data indicated that the covalent HPI-EGCG complex was larger than native HPI, and the particle size was mainly distributed at about 200 µm. CD and three-dimensional fluorescence spectroscopy analyses showed that the conformation of the protein was changed by conjugation with EGCG. The ß-sheet content decreased from 82.79% to 66.67% after EGCG bound to the protein, and the hydrophobic groups inside the protein were exposed, which increased the hydrophobicity of the protein and changed its conformation. After HPI and 1 mM of EGCG were covalently bonded, the solubility and emulsifying properties of the covalent complex were improved compared with native HPI. These results indicated that HPI-EGCG conjugates can be added in some foods.

A novel DNA vaccine constructed by heat shock protein 70 and melanoma antigen-encoding gene 3 against tumorigenesis.

Melanoma antigen-encoding gene 3 (MAGE-3) is an ideal candidate for a tumor vaccine although its potency need to be increased. Heat shock proteins (HSPs) represents a potential approach for increasing the potency of DNA vaccines. In the present study, a fusion DNA vaccine composed of Mycobacterium tuberculosis HSP70 and MAGE-3 was constructed and used to immunize C57BL/6 mice against B16 or B16-MAGE-3 tumor cells. The results show that the HSP70-MAGE-3 fusion DNA vaccine enhanced the frequency of MAGE-3-specific cytotoxic T-cells as compared to the MAGE-3 DNA vaccine or the HSP70/MAGE-3 cocktail DNA vaccine (P < 0.05). In conclusion, the results indicate that the HSP70-MAGE-3 fusion DNA vaccine can strongly activate MAGE-3 specific cellular immunological reactions and thus significantly inhibit the growth of B16-MAGE-3 tumors, improving the survival of tumor-bearing mice, and the HSP70-MAGE-3 fusion DNA vaccine has a significant therapeutic effect on the tumors that express MAGE-3 antigens.