Substrate DM Research Articles

Changes in microbial diversity, methanogenesis and fermentation characteristics in the rumen in response to medicinal plant extracts.

This study evaluated the in vitro effect of medicinal plant extracts on ruminal methanogenesis, four different groups of methanogens and ruminal fermentation characteristics. A fistulated Holstein cow was used as a donor of rumen fluid. Licorice and mugwort extracts (Glycyrrhiza uralensis and Artemisia capillaris, 0.5% and 1% of total substrate DM, respectively), previously used as folk remedies, were added to an in vitro fermentation incubated with buffered-rumen fluid. Total gas production in Glycyrrhiza uralensis extract treatment was not significantly different between treatments (p<0.05) while total gas production in the Artemisia capillaris extract treatment was lower than that of the control. Artemisia capillaris extract and Glycyrrhiza uralensis extract reduced CH4 emission by 14% (p<0.05) and 8% (p<0.05), respectively. Ciliate-associated methanogens population decreased by 18% in the medicinal plant extracts treatments. Medicinal plant extracts also affected the order Methanobacteriales community. Methanobacteriales diversity decreased by 35% in the Glycyrrhiza uralensis extract treatment and 30% in the Artemisia capillaris extract treatment. The order Methanomicrobiales population decreased by 50% in the 0.5% of Glycyrrhiza uralensis extract treatment. These findings demonstrate that medicinal plant extracts have the potential to inhibit in vitro ruminal methanogenesis.

In vitro production of methane with increasing levels of corn- or wheat-based dried distillers' grains with solubles in a barley silage-based diet

Methane production from wheat- or corn-based dried distillers' grains with solubles (CDDGS, WDDGS) was compared in vitro. Wheat DDGS (49 g fat/kg dry matter [DM]) or CDDGS (115 g fat/kg DM) partially or completely replaced whole-crop barley silage at 200, 400, 600, 800 or 1000 g/kg DM. Production of CH4 increased linearly and quadraticly (p<0.01) with increasing levels of CDDGS. Cumulative CH4 production at 24 h was higher (p<0.05) for WDDGS (12.0±0.5 mg/g DM) than CDDGS up to 800 g/kg DM. Molar proportions of propionate in incubation fluid were higher (p<0.05) for CDDG than for WDDGS at 200, 400 and 600 g/kg DM, respectively. In vitro CH4 production (mg CH4/g DM; mg CH4/g digested DM [DMD]) was lower for CDDGS than WDDGS up to 800 g/kg substrate DM. The higher residual oil content in CDDGS compared to WDDGS likely elicited this response.

Impacts of different spices on in vitro rumen dry matter disappearance, fermentation and methane of wheat or ryegrass hay based substrates

Two completely randomised experiments compared the impact of different spices, each at 30mg/g substrate DM, on the in vitro dry matter disappearance (IVDMD), methane, ammonia and volatile fatty acids (VFA) from either wheat flour (wheat) (experiment 1) as highly or hay based mixtures (experiment 2) as moderately fermentable substrates. Experiment 1 tested the effects of adding cinnamon, clove, coriander, cumin and turmeric, individually, on the rumen fermentation during their in vitro incubations with wheat for 24h. All spices reduced methane (P<0.05) from wheat as compared to the spice-free wheat (control). Coriander reduced methane more followed by turmeric, cumin and cinnamon than the control. The IVDMD of wheat did not differ for most spices except cinnamon where IVDMD was reduced (P<0.05) as compared to the control. Whilst, rumen ammonia was greatest for cumin (P<0.01) followed by coriander and turmeric, it was lowest for clove (P<0.01). The spices did not affect the pH and total VFA of rumen fluid for wheat. However, the presence of clove reduced acetic acid (P<0.05). Experiment 2 tested the effect of only three (coriander, cumin and turmeric) spices individually or together on the in vitro fermentation of hay based mixtures during 96h of incubation. Whilst all the individual spices or their mixture reduced methane from the hay based mixtures (P<0.05), the extent of this reduction was highest for turmeric, second highest for coriander and lowest for cumin. It appeared that most spices modified methane emission from wheat as a single ingredient or hay based mixtures without having detrimental effect on diet disappearance or fermentation in vitro. However, the extent of this modification depended on the type of a spice and substrate. Therefore, careful selection of a spice to suit specific feeds would be essential before their in vivo use to modify the fermentation efficiency of ruminant diets.

Corn or sorghum wet distillers grains with solubles in combination with steam-flaked corn: In vitro fermentation and hydrogen sulfide production1

The effects of wet distillers grains with solubles (WDG) on in vitro rate of gas production, IVDMD, H(2)S production, and VFA were evaluated. Five substrate treatments that were balanced for ether extract content were arranged in a 2 x 2 + 1 factorial. Factors were concentration (15 or 30%; DM basis) and source of WDG (corn or sorghum WDG; CDG and SDG, respectively) plus a 0% WDG control in substrates with steam-flaked corn as the basal grain. Control substrates had greater (P < 0.01) IVDMD and total gas production per gram of substrate DM than WDG-based substrates, and IVDMD was greater (P = 0.03) for CDG than for SDG substrates. Increasing WDG inclusion from 15 to 30% decreased IVDMD and total gas production (P < 0.05), but H(2)S production (micromol/g of fermentable DM) increased (P = 0.01) as inclusion of WDG increased. There were no differences (P > or = 0.10) among treatments in proportions of major VFA, acetate:propionate ratio, and total VFA concentration. These results suggest that including WDG in the substrate decreased IVDMD and gas production, which was particularly evident as WDG increased from 15 to 30% of substrate DM. In addition, CDG seemed to be more digestible than SDG. Hydrogen sulfide production increased with increasing WDG in the substrate, reflecting greater S concentrations in WDG, but in vitro VFA profiles were not affected by WDG concentration or source.

Effects of sulfur and monensin concentrations on in vitro dry matter disappearance, hydrogen sulfide production, and volatile fatty acid concentrations in batch culture ruminal fermentations1

Effects of monensin (MON) and S on in vitro fermentation and H(2)S production were evaluated in 2 experiments. In Exp. 1, 2 ruminally cannulated steers were adapted (>14 d) to a 75% concentrate diet [steam-flaked corn (SFC)-based], and ruminal fluid was collected approximately 4 h after feeding. Substrate composed (DM basis) of 85.2% SFC, 9% alfalfa hay, 5% cottonseed meal, and 0.8% urea was added with ruminal fluid and buffer to sealed 125-mL serum bottles to allow for gas collection. A Na(2)SO(4) solution was added to yield S equivalent to 0.2, 0.4, and 0.8% of substrate DM, and MON was included at 0, 2, 4, and 6 mg/L of culture volume. Bottle head-space gas was analyzed for H(2)S. No MON (P = 0.29) or MON x S interaction (P = 0.41) effects were detected for H(2)S production. Increasing S linearly increased (P < 0.01) H(2)S production (micromoles/g of fermented DM). The IVDMD (average 70.0%) was not affected by MON (P = 0.93), S (P = 0.18), or the MON x S interaction (P = 0.56). Total VFA concentrations were not affected by MON (P = 0.87), S (P = 0.14), or the MON x S interaction (P = 0.86), but increasing MON linearly decreased (P <or= 0.01) molar proportions of acetate, butyrate, and the acetate:propionate ratio (A:P), and linearly increased (P < 0.01) the proportion of propionate. In Exp. 2, 2 additional ruminally cannulated Jersey steers were adapted (>21 d) to a 75% concentrate diet (SFC base) that contained 15% (DM basis) wet corn distillers grains plus solubles (WDGS) and MON at 22 mg/kg of DM. In vitro substrate DM was composed of 75.4% SFC, 15% WDGS, 9% alfalfa hay, and 0.6% urea, and S and MON concentrations were the same as in Exp. 1. No effects of MON (P = 0.93) or the MON x S interaction (P = 0.99) were noted for H(2)S production; however, increasing S linearly increased (P < 0.01) H(2)S production. No effects of MON (P = 0.16), S (P = 0.43), or the MON x S interaction (P = 0.10) were noted for IVDMD (average 70.9%). Total VFA concentrations were not affected by MON (P = 0.40), S (P = 0.26), or the MON x S interaction (P = 0.59). As in Exp. 1, increasing MON linearly decreased (P < 0.05) molar proportions of acetate, butyrate, and A:P and linearly increased (P < 0.01) propionate. Increasing S concentration increased in vitro H(2)S production, but S did not affect VFA concentrations and proportions. Monensin did not affect in vitro H(2)S production, and changes in VFA molar proportions were evident with MON regardless of S concentration.

Effects of ionophores and antibiotics on in vitro hydrogen sulfide production, dry matter disappearance, and total gas production in cultures with a steam-flaked corn-based substrate with or without added sulfur1

Effects of 3 ionophores and 2 antibiotics on in vitro H(2)S production, IVDMD, total gas production, and VFA profile with or without added S were examined. In Exp. 1, ruminal fluid from 2 ruminally cannulated steers fed a steam-flaked corn-based diet (75% concentrate) without ionophore and antibiotics for 28 d before collection was used to inoculate in vitro cultures. Treatments were control (no ionophore or antibiotic), 3 ionophores (lasalocid sodium and monensin sodium at 5 mg/L or laidlomycin propionate at 1.65 mg/L), and 2 antibiotics (chlortetracycline hydrochloride at 5 mg/L and tylosin tartarate at 1.25 mg/L). Cultures also had 0 or 1.75 mg of S/L (from sodium sulfate). No S x ionophore-antibiotic treatment interactions were noted (P > 0.53) for IVDMD, total gas production, and H(2)S production. Hydrogen sulfide (mumol/g of fermentable DM) was increased (P < 0.001), and total gas production tended (P = 0.09) to be increased with additional S; however, IVDMD was not affected by added S (P = 0.90). Production of H(2)S was not affected by ionophores or antibiotics (P > 0.18). On average, IVDMD (P = 0.05) was greater for ionophores than for antibiotics, whereas total gas production was less for ionophores than for control (P < 0.001) and antibiotics (P < 0.001). Molar proportions of acetate (P < 0.01) and acetate:propionate (P < 0.01) were decreased and propionate was increased (P < 0.001) in ionophore treatments when no S was added, but when S was added there were no differences (P > 0.20) in acetate, propionate, or acetate:propionate between ionophores and control (S x treatment interaction, P = 0.03). In Exp. 2, the effects of ionophore-antibiotic combinations with added S were examined using the same procedures as in Exp. 1. Treatments were control, monensin plus tylosin (MT), and lasalocid plus chlortetracycline (LCTC), with concentrations of the ionophores and antibiotics as in Exp. 1. No differences were observed among treatments for H(2)S production (P > 0.55). Treatments MT and LCTC tended (P = 0.06) to increase IVDMD and decreased (P = 0.02) gas production vs. control. Proportion of acetate (P = 0.01) and acetate:propionate (P < 0.01) were decreased and propionate increased (P = 0.01) for both MT and LCTC compared with control. These data suggest that when S is approximately 0.42% of substrate DM, the 3 ionophores and 2 antibiotics we evaluated did not affect production of H(2)S gas in an in vitro rumen culture system.

Effects of an exogenous fibrolytic enzyme preparation on in vitro ruminal fermentation of three forages and their isolated cell walls

This study was completed to investigate whether a fibrolytic enzyme preparation with xylanase and cellulase activities (Fibrozyme™, Alltech Inc., Nicholasville, KY, USA), could stimulate in vitro rumen fermentation of alfalfa hay, grass hay and barley straw, and their isolated cell walls as neutral detergent fibre (NDF). Samples (500 mg) of each substrate were incubated in 120 ml bottles with 50 ml of buffered rumen fluid from sheep at 39 °C. The enzyme preparation was added directly to the bottles at the beginning of the incubation at levels of: 0 (control; CON), 50 mg/g substrate DM (LOWFIB) and 100 mg/g substrate DM (HIGHFIB). Effects on volatile fatty acid (VFA) production depended on substrate and incubation time. After incubation for 5 h, enzyme addition increased (P<0.05) production of VFA and acetate compared to CON for both alfalfa hay and its isolated cell wall, and increased (P<0.05) propionate and butyrate production in isolated cell wall, but reduced (P<0.05) them in alfalfa hay. After incubation for 10 h, addition of both doses of enzyme preparation increased (P<0.05) VFA and propionate production versus CON for both alfalfa hay and its isolated cell wall, and decreased (P<0.05) acetate to propionate ratios with both substrates, with intermediate values for LOWFIB and highest for HIGHFIB. After 24 h of incubation, there were no effects due to enzyme treatment or enzyme dose on rumen variables. Addition of both doses of supplemental enzyme increased (P<0.05) VFA, acetate and propionate production after 5 and 10 h incubation in both grass hay and its isolated cell wall, with increases being greater (P<0.05) for HIGHFIB versus LOWFIB in all variables. Both doses of enzyme addition reduced (P<0.05) the acetate to propionate ratio at 5 and 10 h of incubation. After 24 h of incubation, enzyme addition increased (P<0.05) VFA, acetate and butyrate production in both grass hay and its isolated cell wall versus CON, with some dose effects on both substrates. Enzyme supplementation also increased (P<0.05) VFA, acetate and propionate production versus CON in barley straw and its isolated cell wall at 5 and 10 h of incubation, with dose dependent effects after incubation for 5 h with higher (P<0.05) increases for HIGHFIB versus LOWFIB in both substrates. Both enzyme doses also reduced (P<0.05) the acetate to propionate ratio in both barley straw and its cell wall after incubation for 5 h. Production of total VFA and butyrate increased (P<0.05) with both doses of enzyme after incubation for 24 h. Final pH, ammonia-N and disappearance of aNDF and ADF did not differ among enzyme treatments after incubation of grass hay and barley straw or their isolated cell wall for 24 h. Results indicate that this fibrolytic enzyme preparation stimulated in vitro fermentation of substrates at short (5 and 10 h), but not at long (24 h) incubation times, and that effects depended on both the dose used and the presence of neutral detergent soluble components in the substrate.

Influence of exogenous fibrolytic enzymes and fumarate on methane production, microbial growth and fermentation in Rusitec fermenters

Two incubation runs were conducted with Rusitec fermenters to investigate the effects of three additive treatments (mixed fibrolytic enzymes from Trichoderma longibrachiatum (FE), disodium fumarate (FUM) and both additives (MIX)) on rumen microbial growth and fermentation of a grass hay:concentrate (600 : 400 g/kg DM) substrate. Each fermenter received daily 20 g substrate DM. Application rate (per g substrate DM) was 34.3 endoglucanase, 0.57 exoglucanase, 24.7 xylanase and 5.51 amylase units for FE and 30 mg fumarate for FUM. MIX fermenters received both additives. Both FE and MIX increased (P 0.05). Supplementing with FUM increased (P 0.05) any other variable, thus suggesting that observed effects were due to fermentation of FUM itself. The lack of effects of FUM and the absence of differences between FE and MIX on most of the measured variables would indicate that beneficial effects found in MIX fermenters were mainly due to the action of FE. Combining FE and FUM as feed additives under the conditions of the present experiment did not further improve rumen fermentation, compared to FE alone.

Effects of exogenous cellulase supplementation on microbial growth and ruminal fermentation of a high-forage diet in Rusitec fermenters1

Two incubation runs were carried out with a Rusitec system to investigate the effects of 2 exogenous pure cellulases on ruminal microbial growth and fermentation of a 70:30 grass hay:concentrate (DM basis) substrate. The substrate was sprayed with buffer (control; pH = 6.5), a cellulase from Trichoderma longibrachiatum (TRI), a cellulase from Aspergillus niger (ASP), or a 1:1 mixture of both cellulases (MIX) 24 h before being placed in the fermenters. Enzymes were applied at a rate of 30 endoglucanase units/g of substrate DM. Treating the substrate with enzymes reduced substrate NDF and ADF content (P < 0.001 to P = 0.002) and increased DM, NDF, and ADF disappearance after 6 and 24 h of incubation (P < 0.001 to P = 0.004) but not after 48 h of incubation. Daily VFA production was increased (P = 0.004) by 15, 9, and 15% for TRI, ASP, and MIX, respectively, with half of the increase being due to production of acetate. All enzyme treatments augmented (P = 0.009) methane production, but none of them altered the methane:VFA ratio (P = 0.70). There were no differences (P = 0.80) among treatments in the daily flow of solid-associated microorganisms, as measured using 15N as a microbial marker. Although the TRI and MIX treatments increased (P < 0.05) the daily flow of liquid-associated microorganisms and the proportion of microbial N in the solid residue after 48 h of incubation, no effects were observed (P = 0.92 and P = 0.95, respectively) for the ASP treatment. The results show that the TRI and MIX treatments enhanced in vitro fermentation by increasing substrate fiber degradation, VFA production, and ruminal microbial growth. The lack of differences between TRI and MIX in most of the measured variables indicates that treating the substrate with a mixture of both cellulases did not further improve the effects of the TRI treatment.