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Abstract: A 2×4 factorial experiment was conducted to determine the effect of aflatoxin B1 (AFB1) at dose rates of 0, 320, 640, 960ng/ml on ruminal fermentation of substrates high in alfalfa hay (HA, alfalfa hay: maize meal=4:1) and ryegrass hay (HR, ryegrass hay: maize meal=4:1). In vitro dry matter disappearance was not affected by AFB1 dosage. The asymptotic gas production numerically declined by increase in AFB1 dosage for HA and HR. AFB1 dosage decreased gas production rate (P<0.05), and the rate for HR was lower for HA (P<0.05). Ammonia N concentration declined with the increase in AFB1 dosage for HA (P<0.05), and was higher for HA than HR (P<0.01). The highest AFB1 dosage decreased total VFA concentration by 12.7% for HA and 9.6% for HR (P<0.05), compared with that of the zero dosage. The molar proportions of propionate and butyrate did not differ between HA and HR, but molar proportions of isobutyrate, valerate and isovalerate were distinctly greater in HA than in HR (P<0.01). After 72h incubation, 0.831 (HA) and 0.842 (HR) of included AFB1 (960ng/ml) was degraded. The results implicate that rumen microbes have high ability to degrade AFB1. [Copyright &y& Elsevier]

Abstract: Hungate''s method is a well-accepted protocol for the isolation or incubation of anaerobes with a roll tube technique. The aim of this study was to stimulate fungal enzyme production by optimizing the components of Hungate''s medium for the growth of a rumen fungus Anaeromyces sp. YQ3. The organism was grown on corn stalks and incubated for 10 days in defined media with two glucose levels (G+, glucose in the Hungate''s medium as a glucose control; G−, glucose removed in a modified Hungate''s medium) and four N sources (N1: yeast extract+tryptone+(NH4)2SO4 in Hungate''s medium (control); N2: yeast extract+(NH4)2SO4; N3: tryptone+(NH4)2SO4; and N4: tryptone+yeast extract). In the G− media, the recovered activities of feruloyl esterase (FAE) (P<0.0001), acetyl esterase (AE) (P=0.0065) and xylanase (P<0.0001) were decreased, while the G+ media with N1 nitrogen stimulated the production of FAE and xylanase (P<0.0001). The G− medium with N2 nitrogen increased the recovered activities of carboxymethyl cellulase (P=0.0001) and avicelase (P<0.0001), while the N3 and N4 media increased the recovered activity of AE (P=0.0015). The N4 medium was comparable to the N1 medium in stimulating the amount of recovered xylanase activity. The activities of FAE (P<0.0001), AE (P<0.0001), and xylanase (P<0.0001) showed a time-dependent increase and reached their peaks at day 10, while the avicelase activity peaked at day 8 (P=0.0071). The esterase activities (FAE and AE) were positively correlated with the enzyme activities of xylanase and carboxymethyl cellulase (r >0.48, P<0.05). After a 10-day incubation, the glucose in the Hungate''s media contributed to an increase in organic matter disappearance (P<0.0001) and volatile fatty acid (VFA) concentration (P<0.0001), except for molar acetate proportions. The N4 treatment increased organic matter disappearance and total VFA concentration (P=0.0002). The change in N source did not alter molar proportions of acetate, propionate and valerate, while the N2 treatment increased molar butyrate proportion (P<0.0035), and both N2 and N3 increased the molar proportion of branched chain VFAs (P<0.0041). In summary, the glucose in the Hungate''s medium is beneficial for stimulating the production of esterases and xylanase, thereby promoting fungal growth. Amending the N source in Hungate''s medium brings about different yields of rumen fungal esterases and polysaccharide hydrolases that have important nutritional impacts on fibre degradation in ruminant animals. [Copyright &y& Elsevier]

Abstract: A 10-day pure culture of Neocallimastix sp. YAK11, isolated from rumen fluids of yak (Bos grunniens) was evaluated for fibrolytic activity, apparent dry matter disappearance (ADMD), ferulic acid, reducing sugars and volatile fatty acids (VFA). The fungus was grown on oat straw (OS), corn stalks (CS), rice straw (RS) and wheat straw (WS). Recovered activity of ferulic acid esterase peaked at day 5 in all roughages, and the lowest activity was found with low fibre OS throughout the incubation (P<0.0001). The recovered activity of acetyl esterase peaked at day 6 with WS and RS and at day 7 with CS and OS (P<0.0001). The highest acetyl esterase activity was found with high fibre WS throughout the incubation (P<0.0001). Xylanase activity increased with all roughages and was maintained at a high level throughout the incubation (P=0.0102). The lowest xylanase activity was found in WS (P<0.0001), but cultures grown on OS, CS and RS showed no differences. The highest ADMD and reducing sugar values occurred in OS and the lowest in WS (P<0.0001). Ferulic acid release did not differ between four roughages. ADMD was positively correlated with xylanase activity (r =0.51, P<0.01). Total VFA concentration was greatest in OS (P<0.0001) and highly correlated with fibrolytic enzyme activities (r >0.42, P<0.01). Molar proportions of acetate and valerate did not differ in the roughages. The lowest propionate and branch chained VFA levels occurred in WS (P=0.0115), and the highest butyrate levels occurred in OS throughout the 10-d incubation (P=0.0464). The highest ratio of non-glucogenic to glucogenic acids (NGR) occurred in WS (P=0.0481). NGR peaked for all roughages around day 3 and then slightly declined for the rest of the experiment. In general, the isolate was capable of a pronounced degradation that would depend on the fibre content of the roughages. Screening of fungi from rumen fluids of yak would appear to be a useful strategy to obtain highly active esterases and polysaccharide hydrolases. [Copyright &y& Elsevier]

Abstract: Fermentation characteristics of 5 combinations of fenugreek (Trigonella foenum-graecum L., FH) and alfalfa (Medicago sativa L., AH) hay in ratios (FH:AH) ranging from 100:0 to 0:100 were studied in response to inocula of either rumen fluids (RF) or rumen fluids plus a fungal culture of Neocallimastix sp. YAK11 (RFF). For the RFF treatment, the forage combinations were anaerobically incubated with fungal culture and Menke''s medium for the first 24h and then incubated with the stratified rumen fluids for 72h. The value of the in vitro dry matter disappearance did not differ between RF and RFF. Cumulative gas production (ml/g DM) was fitted to an exponential model: GP t =GPmax×(1− e −c×time); the maximum gas production (GPmax) was lower in RFF than in RF (P<0.01), but the rate constant c showed no difference. Molar proportions of CO2, CH4, and H2 were similar in both treatments. The pH values were lower for RFF than RF (P<0.01). Concentrations of ammonia N and total volatile fatty acids (VFA) did not differ between RF and RFF. In VFA pattern, molar proportions of propionate and valerate did not differ between RF and RFF. Molar proportion of acetate was greater in RFF than in RF (P<0.05), while molar butyrate and branch-chained volatile fatty acids proportions were lower in RFF than RF (P<0.01). Fibrolytic activities of carboxymethyl cellulase, avicelase, ferulic acid esterase, and acetyl esterase were similar between RF and RFF, but xylanase activity was greater in RFF than in RF (P<0.01). Regardless of the ratios of FH to AH in the forage combinations, the fungal inoculation at the inclusion level used did not alter the in vitro digestibility, but increased the molar acetate proportion and decreased the molar proportions of butyrate, branch-chained volatile fatty acids and maximum gas production. [Copyright &y& Elsevier]

Abstract: Sixteen combinations of 5 treatments at 4 levels were designed in a L16(45) orthogonal experimental design to evaluate associative effects of five methanogenesis inhibitors at four dose levels: nitroethane (NE, 0mM, 5mM, 10mM and 15mM), 2-nitroethanol (NEOH, 0mM, 5mM, 10mM and 15mM), 2-nitro-1-propanol (NPOH, 0mM, 5mM, 10mM and 15mM), pyromellitic diimide (PMDI, 0mM, 0.02mM, 0.05mM and 0.07mM) and 2-bromoethanesulphonate (BES, 0mM, 0.01mM, 0.03mM and 0.05mM) on in vitro ruminal methane production of the mixed substrate (Chinese wildrye hay:maize meal=4:1) using a cumulative gas production technique. After 48h incubation, in vitro dry matter disappearance (IVDMD), total gas production (GP48, ml/g DM) and total volatile fatty acids (VFA) production in various combinations of these inhibitors were decreased by 10.6–56.0, 26.5–44.5 and 20.3–47.6%, respectively (P<0.05). The molar proportion of acetate in the inhibitor combination groups was decreased by 6.6–12.5% while those of propionate and butyrate were increased by 7.0–19.2 and 21.9–56.5% (P<0.01), respectively. Methane proportion (MP) in total gas production was reduced by 79.4–98.5% (P<0.01), and the highest inhibition occurred in the combination of 10mM NE, 10mM NPOH, 0.07mM PMDI and 0.01mM BES in cultures. The partial correlation coefficients between NE, NEOH, NPOH, PMDI or BES and CH4 proportion were −0.465 (P<0.01), −0.417 (P<0.01), −0.355 (P<0.05), −0.408 (P<0.01) and −0.345 (P<0.05), respectively, indicating that NE was the most potent inhibitor, followed by NEOH and PMDI, and finally NPOH and BES. In general, VFA production in the inhibitor combinations was substantially shifted to produce much more butyrate and propionate and less acetate. The combination of 15mM NE, 10mM NEOH, 5mM NPOH, 0.07mM PMDI and 0.01mM BES in cultures, leading to >95% methane inhibition, may be the optimal application of these inhibitors with less depression of total VFA production. Further feeding trials to validate these combinations is still required on rumen function, methane production, growth performance and milk production. [Copyright &y& Elsevier]

Abstract: A comparison of caecal fermentation patterns of neutral detergent fibre prepared from alfalfa hay (aNDF) with glucose (GLU), cellobiose (CEB) and microcrystalline cellulose (MCC) was conducted with an in vitro batch culture. Caecal contents, freshly collected from adult rabbits, were taken as inoculum. After a 72-h incubation at 39°C, the highest mean activities of xylanase, acetyl and feruloyl esterases occurred in aNDF (P<0.05) while highest carboxymethyl cellulase activity occurred in GLU (P<0.05). Both GLU and CEB were rapidly fermented and characterized by much lower pH (P<0.01) and greater production of total volatile fatty acids (VFA). The lowest concentration of total VFA occurred in aNDF and MCC (P<0.05). The fermentation of aNDF and MCC resulted in higher molar proportions of propionate, valerate, branched chain VFAs than those of GLU and CEB (P<0.05); the molar proportions of acetate ranked as: GLU>MCC>CEB>aNDF (P<0.01); the molar proportion of butyrate ranked as: CEB>aNDF>GLU>MCC (P<0.01). The total gas production of GLU and CEB were greater than those of MCC and aNDF (P<0.05). Molar CH4 proportion ranked as: MCC>aNDF>CEB>GLU (P<0.01), and the molar H2 proportion in MCC was greater than those in other substrates (P<0.01). These results suggest that fermentation of MCC and aNDF produce more glucogenic acids, especially propionate. Low hydrogen recoveries for all substrates implicated that mechanisms of H2 disposal other than methanogenesis, such as reductive acetogenesis, may exist in the caecum and depend upon the nature of the carbohydrate. [ABSTRACT FROM AUTHOR]

Abstract: Fibrolytic activity distributions of 18 enzyme products, mostly obtained in China, were examined in terms of protein concentration and fibrolytic activities on model substrates. The hydrolytic capacities were determined by an in vitro ruminal degradation assessment and the enzymatic release of reducing sugars or ferulic acid from neutral detergent fibre fractions of corn stalks (NDFcs). Most enzyme products were characterized by xylanase, carboxymethyl cellulase (CMCase) and β-glucanase (EG), which had the potential to improve in vitro dry matter disappearance of NDFcs after a 16-h enzymatic pre-treatment followed by a 24-h ruminal degradation using a batch culture method (P<0.05). Quadratic effects of xylanase, CMCase, EG and ferulic acid esterase (FAE) occurred on the release of reducing sugars or ferulic acid from NDFcs (P<0.05). The effective fibrolytic activity combinations required to reach the highest release of reducing sugars were 418–1385U xylanase, 111–567U CMCase, 347–376U EG and 2.0U FAE per g NDFcs substrate. The addition of an enzyme product with a dominant FAE activity did not alter the total volatile fatty acid production at a low dose level of 1.0mg/g or 4140U/g substrate of corn stalks after a 48-h in vitro batch culture. However, a lower ratio of non-glucogenic to glucogenic acids occurred in the fermentation of corn stalks with inclusion of the FAE dominated enzyme product (P<0.05). These results suggest that all enzyme products in China were characterized or blended with enzyme extracts with xylanase, CMCase and EG. The rational combination of enzyme products necessary to reach a specific FAE activity with a specific fibrous forage, together with xylanase, CMCase and EG activities, should be considered in the design of a compound enzyme product for enhancing the cell wall degradation of fibrous crop residues, based on scientific understanding of the mode of enzymatic actions in the rumen. [Copyright &y& Elsevier]

Abstract: Neocallimastix sp. YQ1 and Anaeromyces sp. YQ3 were isolated from rumen fluids of Holstein steers using a modification of the roll-tube technique of . After a 9-day incubation for YQ1, and a 4-day incubation for YQ3, with optimal carbon and N supply in cultures, crude enzyme solutions of both fungi were prepared by centrifuging cultures for 10min at 1000× g at 4°C. Enzymological characteristics of ferulic acid esterase and acetyl esterase were measured in the crude enzyme solution. The Michaelis constants (K m ) and maximum velocities (V max ) of ferulic acid esterase against methyl ferulate at pH 6.0 and 39°C were 30μM and 8.05mU for YQ1 and 129μM and 3.15mU for YQ3, respectively. With substrate of p-nitrophenyl acetate, the K m and V max of AE at pH 6.0 and 39°C were 0.21mM and 336mU for YQ1 and 5.26mM and 854mU for YQ3. Enzymatic release of reducing sugar, ferulic acid, and p-coumaric acid from complex fibre-rich feedstuffs was evaluated, and YQ3 released more reducing sugars from wheat straw, but less from the other plant materials especially maize bran and corn stalks. YQ3 liberated much more hydroxycinnamic acids from corn stalks, and YQ1 liberated much more hydroxycinnamic acids from maize bran. Anaerobic rumen fungi showed species diversity of enzymological characteristics of ferulic acid esterase and acetyl ester esterase, and exhibited a strong capability to release ferulic acid and p-coumaric acid from complex fibre-rich feedstuffs. Therefore anaerobic fungi, especially their FAE, should not be neglected in studies on biodegradation of plant cell walls in the rumen as well as on industrial processes developed to release ferulic acid and p-coumaric acid from agricultural by-products with potential commercial industrial applications, such as precursors of natural vanillin, natural antioxidants, food preservative agents, anti-inflammatory agents and photoprotectants. [Copyright &y& Elsevier]

Abstract: To obtain an optimal feruloyl and acetyl esterase production of an anaerobic Neocallimastix sp. YQ2 grown on either Chinese wildrye grass hay (CW) or alfalfa hay (AH) or corn stalk (CS), three simultaneous 2×4 factorial experiments of 10-day pure cultures were applied with two levels of glucose (G+: glucose added at a level of 1.0g/L and G−: without glucose) and four levels of N source (N1: 1.0g/L yeast extract, 1.0g/L tryptone and 0.5g/L (NH4)2SO4; N2: 2.8g/L yeast extract and 0.5g/L (NH4)2SO4; N3: 1.6g/L tryptone and 0.5g/L (NH4)2SO4; N4: 1.4g/L yeast extract, 1.7g/L tryptone). Optimal combinations of glucose and N supplements for YQ2 to produce ferulic acid esterase, acetyl esterase, xylanase, carboxymethyl cellulase and avicelase were G+N2 for CS, G−N3 for CW, G−N3 for CW, G+N2 for CS, and G+N2 for CW, respectively. These five kinds of fibrolytic enzyme had different variation tendencies and maximum activity at different times of incubation. The fungus produced more total volatile fatty acids, and less propionic acids and iso-valeric acids, when CS was used as growth substrate, as well as more acetic acids when CW was used than when AH was used (P<0.05). As for the enzymological characteristics of feruloyl and acetyl esterase, Michaelis constants (K m) and maximum velocities (V max) of ferulic acid esterase against methyl ferulate at pH 6.0 and 39°C were 90μM and 1.15mU, and the Michaelis constants and maximum velocities of acetyl esterase at pH 7.0 and 39°C were 2.97mM and 294mU with specific substrate of p-nitrophenyl acetate. The crude enzyme preparation of the fungi grown on CS with the G+N1 combination had higher potential to release ferulic acid, and p-coumaric acid in CS up to 129.8g/kg the alkali-extractable ferulic acid and 88.1g/kg the alkali-extractable p-coumaric acid, respectively (P<0.05). The strong capability to hydrolyse plant materials by the fungus can be used to understand the degradation of fibre-rich feedstuffs in the rumen and also open prospects for development of direct fed feed enzyme products for ruminants. [Copyright &y& Elsevier]

Abstract: The aim of this study was to compare different methods to determine ferulic acid esterase (FAE) activity in commercial enzyme products and to measure the FAE activity in the rumen of steers fed different diets (A: 5kg forage and 4kg concentrate; B: 5kg forage and 5kg concentrate; C: 5kg forage and 6kg concentrate). FAE activities in seven commercial enzyme products were determined by high performance liquid chromatography (HPLC) at a wavelength of 320nm and by spectrophotometric assays at wavelengths of 335 and 340nm. FAE activities varied dramatically among the seven exogenous enzyme preparations. Correlation between the enzyme activities determined by HPLC and spectrophotometric assays was high (r >0.98), but consistency among the different methods was poor, especially with the spectrophotometrical method (SP) at 335nm. Therefore, the SP by microplate reader at 340nm with standard substrate of methyl ferulate (MFA) was recommended for rapid analysis of large numbers of samples. Rumen fluid of steers had low, and different, ferulic acid esterase activity at different times of the day. Furthermore, high diurnal variation occurred for the ferulic acid esterase and other fibrolytic enzymes that were measured. The activity of FAE and other fibrolytic enzymes differed along with the diet compositions and feed intake. Diet C had the highest mean fibrolytic enzyme activity in the rumen, and diet A had the lowest activity of FAE and birchwood xylanase (BX). High correlations occurred between activities of carboxymethyl cellulase (CMC), BX, acetyl esterase (AE) and those of FAE, both with all data and within each diet. As an accessory enzyme, ferulic acid esterase should not be neglected in studies on furthering understanding of biodegradation of plant cell wall in rumen, and impacts of exogenous enzyme products. [Copyright &y& Elsevier]

Abstract: In vitro fermentation characteristics of soluble, washout, and non-washout fractions separated from barley and maize and their technologically processed (i.e., pressure toasting, pelleting and their combination) products were measured. The aim was to establish whether the soluble equaled the washout fraction, whether these fractions are rapidly and completely fermented, and whether their fermentation characteristics are the same. A simple fractionation method was developed to separate the whole grain into a soluble fraction, a washout fraction, an insoluble washout fraction, and a non-washout fraction. Proximate analysis of the different fractions revealed in the soluble fraction a large (>400gkg-1DM) residual organic matter component of unknown origin, while the insoluble washout fraction contained between 800 and 900gstarch/kgDM. Fermentation characteristics of all fractions as well as the whole grain were measured for 72h using gas and volatile fatty acids (VFA) production in vitro. After fractionation, gas and VFA production profiles of the fractions with different chemical compositions were found to be different. Based on gas production profiles it was shown that the soluble fraction was easily fermentable and was fermented faster than the insoluble washout and the non-washout fraction. Differences were however much smaller than anticipated, which would suggest that the extent of fermentation of washout fractions in the rumen is incomplete. The maximum gas and VFA production from the soluble fraction was always lower than that of the insoluble washout, and the non-washout fraction. The combination of pressure toasting and pelleting reduced fermentability both in terms of gas and VFA production compared with the other treatments. [Copyright &y& Elsevier]

Butyric acid and its salt have been used as feed additives to improve feed efficiency, nutrient degradability and animal growth. In this study, an in vitro trial was conducted to evaluate the effects of tributyrin ( TB ) supplementation on fermentation, enzyme activity and gas production at dosages of 0, 2, 4, 6 and 8 g/kg dry matter ( DM ) basis in substrate, which was incubated for 36 h. In addition, an in vivo trial with the same concentrations of TB in diet as in vitro trial was conducted to assess the influence of TB addition on utilization of dietary nitrogen, calcium and phosphorus. Forty-five adult Small Tail ewes were randomly assigned to 5 treatments of 9 ewes each by initial body weight (55 ± 5 kg, mean ± SD). The in vivo trial lasted 18 days, and ewes had free access to water and the ration mixed with TB. The results showed that TB decreased in vitro molar proportion of acetate ( P  <  0.001), but increased propionate ( P  =  0.004), butyrate ( P  <  0.001) and valerate ( P  <  0.001). Tributyrin improved in vitro fermentation efficiency ( P  =  0.003) and enhanced activity of xylanase ( P  =  0.008), carboxymethyl cellulase ( P  =  0.062) and avicelase ( P  =  0.006). Tributyrin increased the in vitro apparent degradability of DM ( P  =  0.002), crude protein ( P  <  0.001), neutral detergent fiber ( P  <  0.001) and acid detergent fiber ( P  =  0.056). Tributyrin increased in vitro methanogenesis ( P  <  0.001), but decreased carbon dioxide production ( P  <  0.001). Besides, TB supplementation improved in vivo utilization of dietary nitrogen, calcium and phosphorus. The results indicated that TB addition had positive effects on not only in vitro fermentation but also in vivo dietary nutrient utilization, despite having an enhancing effect on methanogenesis. [ABSTRACT FROM AUTHOR]