Effects of short-term fasting on in vivo rumen microbiota and in vitro rumen fermentation characteristics.

OBJECTIVE: Fasting may lead to changes in the microbiota and activity in the rumen. In the present study, the effects of fasting on rumen microbiota and the impact of fasting on in vitro rumen fermentation were evaluated using molecular culture-independent methods. METHODS: Three ruminally cannulated Holstein steers were fed rice straw and concentrates. The ruminal fluids were obtained from the same steers 2 h after the morning feeding (control) and 24 h after fasting (fasting). The ruminal fluid was filtrated through four layers of muslin, collected for a culture-independent microbial analysis, and used to determine the in vitro rumen fermentation characteristics. Total DNA was extracted from both control and fasting ruminal fluids. The rumen microbiota was assessed using denaturing gradient gel electrophoresis (DGGE) and quantitative polymerase chain reaction. Microbial activity was evaluated in control and fasting steers at various intervals using in vitro batch culture with rice straw and concentrate at a ratio of 60:40. RESULTS: Fasting for 24 h slightly affected the microbiota structure in the rumen as determined by DGGE. Additionally, several microorganisms, including Anaerovibrio lipolytica, Eubacterium ruminantium, Prevotella albensis, Prevotella ruminicola, and Ruminobacter amylophilus, decreased in number after fasting. In addition, using the ruminal fluid as the inoculum after 24 h of fasting, the fermentation characteristics differed from those obtained using non-fasted ruminal fluid. Compared with the control, the fasting showed higher total gas production, ammonia, and microbial protein production (p<0.05). No significant differences, however, was observed in pH and dry matter digestibility. CONCLUSION: When in vitro techniques are used to evaluate feed, the use of the ruminal fluid from fasted animals should be used with caution.

Supplementing Rhodobacter sphaeroides in the diet of lactating Holstein cows may naturally produce coenzyme Q10-enriched milk.

OBJECTIVE: To examine the effects of Rhodobacter sphaeroides (R. sphaeroides) supplementation as a direct-fed microbial (DFM) on rumen fermentation in dairy cows and on coenzyme Q10 (CoQ10) transition into milk, an in vitro rumen simulation batch culture and an in vivo dairy cow experiment were conducted. METHODS: The characteristics of in vitro ruminal fermentation were investigated using rumen fluids from six cannulated Holstein dairy cows at 2 h post-afternoon feeding. A control treatment was included in the experiments based on a typified total mixed ration (TMR) for lactating dairy cows, which was identical to the one used in the in vivo study, plus R. sphaeroides at 0.1%, 0.3%, and 0.5% TMR dry matter. The in vivo study employed six ruminally cannulated lactating Holstein cows randomly allotted to either the control TMR (C-TMR) treatment or to a diet supplemented with a 0.5% R. sphaeroides culture (S-TMR, dry matter basis) ad libitum. The presence of R. sphaeroides was verified using denaturing gradient gel electrophoresis (DGGE) applied to the bacterial samples obtained from the in vivo study. The concentration of CoQ10 in milk and in the supernatant from the in vitro study was determined using high performance liquid chromatography. RESULTS: The results of the in vitro batch culture and DGGE showed that the concentration of CoQ10 significantly increased after 2 h of R. sphaeroides supplementation above 0.1%. When supplemented to the diet of lactating cows at the level of 0.5%, R. sphaeroides did not present any adverse effect on dry matter intake and milk yield. However, the concentration of CoQ10 in milk dramatically increased, with treated cows producing 70.9% more CoQ10 than control cows. CONCLUSION: The CoQ10 concentration in milk increased via the use of a novel DFM, and R. sphaeroides might be used for producing value-added milk and dairy products in the future.

Use of Lysozyme as a Feed Additive on In vitro Rumen Fermentation and Methane Emission.

This study was conducted to determine the effect of lysozyme addition on in vitro rumen fermentation and to identify the lysozyme inclusion rate for abating methane (CH4) production. An in vitro ruminal fermentation technique was done using a commercial concentrate to rice straw ratio of 8:2 as substrate. The following treatments were applied wherein lysozyme was added into 1 mg dry matter substrate at different levels of inclusion: Without lysozyme, 2,000, 4,000, and 8,000 U lysozyme. Results revealed that, lysozyme addition had a significant effect on pH after 24 h of incubation, with the highest pH (p<0.01) observed in 8,000 U lysozyme, followed by the 4,000 U, 2,000 U, and without lysozyme. The highest amounts of acetic acid, propionic acid (p<0.01) and total volatile fatty acid (TVFA) (p<0.05) were found in 8,000 U after 24 h of incubation. The CH4 concentration was the lowest in the 8,000 U and the highest in the without lysozyme addition after 24 h of incubation. There was no significant differences in general bacteria, methanogen, or protozoan DNA copy number. So far, addition of lysozyme increased the acetate, propionate, TVFA, and decreased CH4 concentration. These results suggest that lysozyme supplementation may improve in vitro rumen fermentation and reduce CH4 emission.

Effect of Lactobacillus mucosae on In vitro Rumen Fermentation Characteristics of Dried Brewers Grain, Methane Production and Bacterial Diversity.

The effects of Lactobacillus mucosae (L. mucosae), a potential direct fed microbial previously isolated from the rumen of Korean native goat, on the rumen fermentation profile of brewers grain were evaluated. Fermentation was conducted in serum bottles each containing 1% dry matter (DM) of the test substrate and either no L. mucosae (control), 1% 24 h broth culture of L. mucosae (T1), or 1% inoculation with the cell-free culture supernatant (T2). Each serum bottle was filled anaerobically with 100 mL of buffered rumen fluid and sealed prior to incubation for 0, 6, 12, 24, and 48 h from which fermentation parameters were monitored and the microbial diversity was evaluated. The results revealed that T1 had higher total gas production (65.00 mL) than the control (61.33 mL) and T2 (62.00 mL) (p<0.05) at 48 h. Consequently, T1 had significantly lower pH values (p<0.05) than the other groups at 48 h. Ammonia nitrogen (NH3-N), individual and total volatile fatty acids (VFA) concentration and acetate:propionate ratio were higher in T1 and T2 than the control, but T1 and T2 were comparable for these parameters. Total methane (CH4) production and carbon dioxide (CO2) were highest in T1. The percent DM and organic matter digestibilities were comparable between all groups at all times of incubation. The total bacterial population was significantly higher in T1 (p<0.05) at 24 h, but then decreased to levels comparable to the control and T2 at 48 h. The denaturing gradient gel electrophoresis profile of the total bacterial 16s rRNA showed higher similarity between T1 and T2 at 24 h and between the control and T1 at 48 h. Overall, these results suggest that addition of L. mucosae and cell-free supernatant during the in vitro fermentation of dried brewers grain increases the VFA production, but has no effect on digestibility. The addition of L. mucosae can also increase the total bacterial population, but has no significant effect on the total microbial diversity. However, inoculation of the bacterium may increase CH4 and CO2 in vitro.

Evaluation of umami taste in Hanwoo with different feed sources by chemical analysis, electronic tongue analysis, and sensory evaluation.

This study aimed to evaluate umami taste in Hanwoo with different feed by chemical analysis, sensory evaluation and an electronic tongue system. Hanwoo cattle were divided into three groups: control group (fed only total mixed ration [TMR]), T1 (fed soybean meal + TMR), and T2 (fed soybean meal + corn-dried distiller's grain with solubles [Corn DDGS] + TMR). The three most abundant fatty acids (C18:1n-9, C16:0, and C18:0) in the T1, T2, and control groups accounted for 83.63%, 86.07%, and 85.52% of the total fatty acid content, respectively. Umami taste-related glutamic acid levels were significantly high in T1 (109.89 mg/kg), followed by T2 (66.66 mg/kg) and control (47.27 mg/kg). Fatty acid levels showed a high correlation with umami taste. The results of this study showed that the amino acid and fatty acid levels had been affected by feed types and soybean- or Corn DDGS-based feed potentially enhanced Hanwoo's umami flavor.

Effects of supplementing limiting amino acids on milk production in dairy cows consuming a corn grain and soybean meal-based diet.

Limiting amino acids (AAs) for milk production in dairy cows fed on a concentrate diet of corn grain and soybean meal was evaluated in this study. Four lactating and multiparous Holstein cows (in third or fourth parities, with an average body weight of 633 ± 49.2 kg), 8 to 9 weeks into their lactation period, were used in a 4 × 4 Latin square design. The experiment comprised four dietary treatments: (1) no intravenous infusion (control); (2) control plus intravenous infusion of an AA mixture of 6 g/d methionine, 19.1 g/d lysine, 13.8 g/d isoleucine, and 15.4 g/d valine (4AA); (3) control plus intravenous infusion of the AA mixture without methionine (no-Met); and (4) control plus intravenous infusion of the AA mixture without lysine (no-Lys). All animals were fed on a controlled diet (1 kg/d alfalfa hay, 10 kg/d silage, 14 kg/d concentrate mixture, ad libitum timothy hay). The AA composition of the diet and blood were determined using an automatic AA analyzer. Milk composition (protein, fat, lactose, urea nitrogen, and somatic cell counts) was determined using a MilkoScan. The results showed that feed intake for milk production did not differ from that of intravenous infusion using a limiting AA mixture. The 4AA treatment numerically had the highest milk yield (32.4 kg/d), although there was no difference when compared with the control (31.2 kg/d), no-Met (31.3 kg/d), and no-Lys (31.7 kg/d) treatments. The concentration of AAs in blood plasma of cows in all treatments, mainly isoleucine and valine, increased significantly compared with that of control. The no-Met treatment increased (p < 0.05) the concentration of lysine in the blood relative to the control and no-Lys treatments, whereas the no-Lys treatment increased (p < 0.05) the concentration of methionine relative to the control and no-Met treatments. In conclusion, milk production increased when feeding 10 g/d methionine to the cows, together with their concentrate diet of corn grain and soybean meal.

Effects of illite supplementation on in vitro and in vivo rumen fermentation, microbial population and methane emission of Hanwoo steers fed high concentrate diets.

This study was conducted to evaluate the effects of feeding supplemental illite to Hanwoo steers on methane (CH4 ) emission and rumen fermentation parameters. An in vitro ruminal fermentation technique was conducted using a commercial concentrate as substrate and illite was added at different concentrations as treatments: 0%, 0.5%, 1.0%, and 2.0% illite. Total volatile fatty acids (VFA) were different (P < 0.05) at 24 h of incubation where the highest total VFA was observed at 1.0% of illite. Conversely, lowest CH4 production (P < 0.01) was found at 1.0% of illite. In the in vivo experiment, two diets were provided, without illite and with addition of 1% illite. An automated head chamber (GreenFeed) system was used to measure enteric CH4 production. Cattle received illite supplemented feed increased (P < 0.05) total VFA concentrations in the rumen compared with those fed control. Feeding illite numerically decreased CH4 production (g/day) and yield (g/kg dry matter intake). Rumen microbial population analysis indicated that the population of total bacteria, protozoa and methanogens were lower (P < 0.05) for illite compared with the control. Accordingly, overall results suggested that feeding a diet supplemented with 1% illite can have positive effects on feed fermentation in the rumen and enteric CH4 mitigation in beef cattle.

Antioxidant effects of cultured wild ginseng root extracts on the male reproductive function of boars and guinea pigs.

The main objective of this study was to investigate the effects of cultured wild ginseng root extracts (cWGRE) on the sperm of boars and the reproductive system of guinea pigs. Firstly, semen collected from boars (n=10) were incubated in 38°C for 1h with xanthine and xanthine oxidase to generate ROS. The cWGRE was added to the sperm culture system to test its antioxidant effect on the boar sperm. The amount of Reactive Oxygen Species (ROS) was measured by a chemiluminescence assay using luminol. The results indicated that the addition of cWGRE to boar sperm culture inhibited xanthine and xanthine oxidase-induced ROS concentrations. Treatment with cWGRE also had a positive effect on maintaining sperm motility. Effects of cWGRE administration on vitamin C-deficient guinea pigs were further investigated. Hartley guinea pigs (n=25) at 8 weeks of age were randomly divided into five groups. With the exception of the positive control group, each group was fed vitamin C-deficient feed for 21days (d). Respective groups were also orally administered cWGRE, ginseng extract, or mixed ginsenosides for 21 days. In comparison to the control group, oral administration of cWGRE reduced (P<0.05) amount of lipid peroxidation and increased (P<0.05) both glutathione peroxidase concentrations and the trolox equivalent antioxidant capacity. In addition, administration of cWGRE induced increases (P<0.05) in body weight, testosterone concentrations, and spermatid populations. The results of the present study support our hypothesis that cWGRE has positive effects on male reproductive functions via suppression of ROS production.

Production of conjugated linoleic acid (CLA) by Bifidobacterium breve LMC520 and its compatibility with CLA-producing rumen bacteria.

This study was performed to characterize the ability of an active Bifidobacterium strain to produce conjugated linoleic acid (CLA) and to test its possible utilization as a probiotic compatible to the ruminal condition. Bifidobacterium breve LMC520 can actively convert linoleic acid (LA) to cis-9,trans-11-CLA, which is a major isomer derived from microbial conversion. LMC520 showed reasonable tolerance under acidic conditions (pH 2.5 with 1% pepsin) and in the presence of oxgall (0-3%). The growth and CLA production of LMC520 were tested under ruminal conditions and compared with those of Butyrivibrio fibrisolvens A38, which is a major CLA producer in the rumen as an intermediate in the biohydrogenation (BH) process. LMC520 converted 15% of LA to CLA under ruminal conditions, which was 2 times higher activity than that of A38, and there was no decline in CLA level during prolonged incubation of 48 h. The BH activity of LMC520 was comparable to that of A38. When LMC520 was cocultured with A38, even with slight decrease of CLA due to high BH activity by A38, but the level of CLA was maintained by the high CLA-producing activity of LMC520. This comparative study shows the potential of this strain to be applied as a functional probiotic not only for humans but also for ruminants as well as to increase CLA production.

Dietary conjugated linoleic acid increases endurance capacity of mice during treadmill exercise.

This study was designed to evaluate the effect of dietary conjugated linoleic acid (CLA) on endurance exercise and availability of metabolic substrates in mice. Four-week-old male BALB/c mice were randomized to a control group (normal diet) and a CLA group (normal diet + 1% CLA). Each animal group received its respective diet for 10 weeks and was subjected to forced running on a treadmill system to estimate their maximum endurance capacity at the end of the experiment. All mice were immediately sacrificed after prolonged exercise, and the major metabolic substrates were measured in serum and liver. The body weights of mice in the CLA group were lower than those of the control group after the 10 weeks. The maximum running time in CLA-fed mice was significantly longer, by 26%, compared to that of the control mice. Dietary CLA decreased the serum concentrations of triglycerides, nonesterified fatty acids, and urea nitrogen and significantly reduced the consumption of liver glycogen. The present study demonstrated that dietary CLA enhances the endurance capacity of mice by increasing fat utilization and reducing the consumption of stored liver glycogen as substrates for energy metabolism.