Effect of rare earth elements on in vitro rumen microbial fermentation and feed digestion

The objectives of this study were to investigate the effects of rare earth elements (REEs) on in vitro rumen fermentation, gas production, microbial protein synthesis and nutrient digestion using in vitro batch culture and continuous culture technique. A mixture of REE containing (g/kg) 380 g of LaCI₃·6H₂O, 521 g of CeCI₃·6H₂O, 30 g of PrCI₃·6H₂O and 69 g chlorides of other light REEs. The experimental diet consisted of 885 g/kg barley grain, 84 g/kg barley silage and 31 g/kg supplement (dry matter (DM) basis). Diet supplemented with different dosages of REE (control, no additional REE; low, 400 mg/kg REE; and high, 800 mg/kg REE, DM basis) were incubated for 4, 8, 14 and 24 h in diluted rumen fluid. At the end of 24 h of incubation, gas production and concentration of volatile fatty acid (VFA) linearly increased with increasing REE supplementation; whereas, influence of REE supplementation on VFA profile was marginal. Dry matter disappearance was not affected (P>0.10). Six dual-flow continuous culture fermenters were used in a replicated 3 × 3 Latin square with same treatments and same diet used in the batch culture. Mean ruminal pH (5.71) and total VFA (93.6 mM) concentration were not affected by supplementation of REE. The molar proportion (mol/100 mol) of acetate (39.1) and propionate (50.5) was similar among the treatments. However, the proportion (mol/100 mol) of butyrate was higher with the high REE (6.6) than with low REE (5.3) or the control (5.8). Ruminal true digestibilities of organic matter (OM) (0.785, 0.811 and 0.828), acid detergent fibre (0.360, 0.431 and 0.432) and crude protein (0.496, 0.590 and 0.589) for control, low and high REE, respectively, linearly increased with increasing REE supplementation, whereas, the increase in ruminal digestibility from low to high dosage of REE was minimal. Microbial nitrogen (N) production (g/day) and microbial efficiency (g N/kg of truly fermented OM) were not affected by treatments. Improvement of ruminal digestibility of OM due to REE supplementation was attributed to the increase in digestibility of fibre and degradability of protein. The results suggest that REE supplementation improved ruminal fibrolytic and proteolytic activities.     

Effects of preservation procedures of rumen inoculum on in vitro microbial diversity and fermentation

Sheep rumen contents were used as inoculum for an in vitro semi-continuous incubation system to study whether preservation method affects microbial fermentation pattern. Rumen fluid was filtered and either used immediately as inoculum (CTL) or dispensed into 110 mm × 16 mm tubes, that were stored refrigerated at 6 °C for 4 h (REF) or frozen at ?20 °C (FRZ), frozen in liquid N (FLN) or added with 0.04 glycerol and frozen in liquid N (FGL) for 48 h. Frozen inocula were thawed at 39 °C for 2 min before use (16 ml per bottle). Two 24 h incubations with four bottles per treatment were completed. The microbial utilisation of added glycerol after thawing in FGL increased total gas production (P<0.05) and 24 h volatile fatty acid (VFA) production (P<0.05), and also increased propionate and butyrate proportions at the expense of acetate. The other freezing inocula (i.e., FLN and FRZ) reduced the rate of gas production (as ml/g dry matter per hour), compared with CTL in the first 2 and 4 h of incubation (P<0.05), but this was compensated by increased fermentation at 8 and 12 h, respectively. Differences in gas production did not manifest a different VFA pattern at either 6 or 24 h incubation. Bacterial diversity was slightly affected by the preservation process, and the similarity index between untreated inocula and the 24 h incubated CTL samples was 0.690–0.724. Similarity between bacterial communities in FRZ and FLN with that in CTL after incubation was 0.678. The freezing preservation method of rumen inocula for subsequent in vitro gas production studies does not affect microbial fermentation pattern or bacterial biodiversity, provided that processing is rapid enough by using a high surface to volume ratio. Freezing in liquid N is more appropriate than at ?20 °C.     

Effect of the supplementation of a high-concentrate diet with sunflower and fish oils on ruminal fermentation in sheep

This study was conducted to test the hypothesis that the supplementation of a high-concentrate diet with lipids, reportedly a good strategy for improving the nutritional value of ruminant-derived products, may not necessarily be associated with detrimental effects on ruminal fermentation in sheep. Four ruminally cannulated adult ewes were fed a high-concentrate diet, with no oil (Control diet), for a 14-day adaptation period. Afterwards, they were fed the same basal diet but supplemented with sunflower oil [20 g/kg fresh matter (FM)] and fish oil (10 g/kg FM) (SOFO diet) for a further 11 days, to investigate the impact of the addition of oils on the ruminal fermentation of the diet. On days 0 (Control), 3 and 10 of the experimental period rumen fluid was sampled at 0, 1.5, 3, 6 and 9 h after the morning feeding, for analysis of pH, and ammonia, lactate and total volatile fatty acid (VFA) concentrations. Alfalfa hay was incubated in situ, using the nylon bag technique, for 12 and 24 h to examine the effect of oil supplementation on ruminal disappearance of dry matter (DM), crude protein (CP) and neutral-detergent fibre (NDF). On days 0 and 11, rumen fluid was collected just before the morning feeding and used to incubate alfalfa hay and the Control and SOFO diets by means of the in vitro gas production technique. The mean concentrations of acetate (87.8 mmol/L vs. 73.7 mmol/L) and butyrate (21.2 mmol/L vs. 17.7 mmol/L) were reduced by oil supplementation (P < 0.05) and the total VFA showed a tendency (P = 0.098) to be lower with the SOFO diet (139.0 mmol/L vs. 122.1 mmol/L). However, none of the other in vivo ruminal fermentation parameters were affected by the treatment (P > 0.10). The oil supplementation affected neither in situ rumen disappearance of DM, CP and NDF of alfalfa hay, nor rates of gas production (P > 0.10). On the other hand, a little, but significant reduction in cumulative gas production was observed when the experimental diets were incubated with rumen fluid derived from animals fed the oil-rich diet (P < 0.05).Overall, the results suggest that the supplementation of high-concentrate diets with sunflower oil (20 g/kg FM) plus fish oil (10 g/kg FM) had little effect on ruminal fermentation and therefore its use to improve the nutritional value of ruminant-derived products cannot be precluded.     

Batch culture evaluation of carbohydrase inhibitors to moderate rumen fermentation

While carbohydrase inhibitors have been widely investigated for regulating human carbohydrate assimilation, their potential application to animal nutrition has been largely ignored. Batch culture fermentations were conducted to determine how commercially available α-amylase and α-glucosidase inhibitors affect rumen fermentation. Fermentations were with 0.5 g of ground corn grain, and 40 mL of buffered rumen fluid inoculum. Rumen fluid donors were fed a 0.5 concentrate 0.5 forage diet. Incubations were conducted in duplicate and replicated on consecutive days with pH and volatile fatty acid (VFA) concentrations measured. The microbial-derived inhibitors, acarbose (ACB) and trestatin (TRE), prevented the decreases in pH and VFA production observed in the control tubes at doses of 1.2–9.5 and 0.1–1.1 mg, respectively. Miglitol, glipizide, and the plant-derived amylase inhibitors failed to affect pH or reduce VFA concentrations with the same apparent potency as did ACB and TRE, which both show the potential to reduce the amount of starch fermented in the rumen. These compounds may be potentially useful for reducing ruminal acid production and allowing more starch to pass to the small intestine from the forestomachs.     

Effect of elemental nano-selenium on feed digestibility,rumen fermentation,and purine derivatives in sheep

The objective of this experiment was to study the effect of elemental nano-selenium (NS) on feed digestibility, rumen fermentation, and urinary purine derivatives in sheep. Eight male ruminally cannulated sheep (42.5 ± 3.2 kg of body weight, BW) were used in a replicated 4×4 Latin square experiment in four 20 day periods. Depending on treatment designation, sheep were fed the basal diet supplemented with 0 (control), 0.3, 3 and 6 g of nano-Se/kg dry matter (DM). Ruminal pH (range of 6.68–6.80) and ammonia N concentration (range of 9.95–12.49 mg/100 mL) was decreased (P<0.01), and total VFA concentration (range of 73.63–77.72 mM) was increased linearly (P<0.01) and quadratically (P<0.01) with increasing nano-Se supplementation. The ratio of acetate to propionate was linearly (P<0.01) and quadratically (P<0.01) decreased due to the increasing of propionate concentration. In situ ruminal neutral detergent fiber (aNDF) degradation of Leymus chinensis and crude protein (CP) of soybean meal were linearly (P<0.01) and quadratically (P<0.01) improved by feeding nano-Se. Similarly, nutrients digestibility in the total tract and urinary excretion of purine derivatives were also quadratically (P<0.01) changed by increasing nano-Se supplementation. The present results indicated that nano-Se supplementation in basal diet improved rumen fermentation and feed utilization. Nano-Se could also stimulate rumen microbial activity, digestive microorganisms or enzyme activity. The optimum dose of nano-Se was about 3.0 g/kg dietary DM in sheep.     

Influences of flavomycin,ropadiar, and saponin on nutrient digestibility,rumen fermentation,and methane emission from sheep

This study focused on the effects of three additives given together with a hay/concentrate-based diet on nutrient digestibility, rumen fermentation, and methane emission from sheep. The basal diet consisted of 1.29 kg mixed hay and 0.43 kg concentrate mixture based on dry matter (DM). Treatments consisted of control (no additive), flavomycin⁴⁰ (250 mg/d), ropadiar from an oregano extract (250 mg/d), and saponin in the form of a yucca schidigera extract (170 mg/d). Results indicated that intake and digestibility were unaffected by treatments (P>0.05). The NH₃-N concentration of rumen liquor was lower (P<0.05) for additive treatments versus the control treatment. Higher concentrations of volatile fatty acid (VFA) were observed in the saponin (75.8 mmol/L) and ropadiar (73.1 mmol/L) treatments. The proportion of individual fatty acid of rumen liquor was unchanged, whereas lower ratio of acetate to propionate in the saponin treatment was observed (P<0.05). The average methane production expressed on digested organic matter (OM) and neutral detergent fiber (aNDFom) basis were decreased by approximately 3.3 and 12.0 g/kg, respectively in saponin, and 4.2 and 11.9 g/kg in ropadiar treatment compared to the control. Methane production was positively correlated with the concentrations of NH₃-N, and negatively correlated with total VFA and the proportion of propionate of rumen liquor (P<0.05). The study found that saponin and ropadiar could have the potential to reduce rumen methanogenesis in sheep.     

Effects of dose and adaptation time of a specific blend of essential oil compounds on rumen fermentation

Essential oils can be used as natural additives in animal feeds. The present study evaluated the effects of three different doses and different adaptation times of a specific blend of essential oils (BEO) on rumen microbial fermentation. Eight dual flow continuous culture fermenters (1320 ml) were used in two periods of 9 days each to study the effects of increasing doses of BEO. Treatments were: CTR (no BEO), D5 (5 mg/l of BEO), D50 (50 mg/l of BEO) and D500 (500 mg/l of BEO). During the last 3 days, samples were taken at 0, 2, 4 and 6 h after the morning feeding and analyzed for large peptide (LPep), small peptides plus amino acid (SPep + AA) and ammonia N concentrations, and at 2 h after feeding for volatile fatty acids (VFA) concentration and profile. The D5 increased total VFA concentration, acetate proportion and acetate to propionate ratio, and decreased propionate and valerate proportion, compared with CTR. The concentration of LPep N tended (P=0.08) to be lower for D5 compared with CTR. In the second experiment, eight sheep were used to study the effects of long-term adaptation of rumen fluid to BEO on ruminal fermentation. Four sheep were assigned at random to the CTR treatment (no BEO) and four sheep were adapted to BEO (110 mg/day of BEO) for 4 weeks (ADBEO). After 4 weeks samples of ruminal fluid were obtained at 0 and 3 h after the morning feeding and in 2 consecutive days using an oro-ruminal probe. Samples were analyzed for LPep, SPep + AA and ammonia N concentrations, total and individual VFA, and pH. Treatment ADBEO tended (P<0.10) to increase acetate proportion and decrease valerate proportion, compared with CTR. Ruminal fluid collected from each of CTR and ADBEO sheep was used to study in vitro fermentation profile of soybean meal, corn meal, alfalfa hay and ryegrass hay. Treatments were: Control fluid (CTR without BEO), CTR fluid plus a single dose of BEO (11 mg/l; CTR + BEO) and ADBEO fluid plus a single dose of BEO (11 mg/l; ADBEO + BEO). Acetate proportion and acetate to propionate ratio was higher, and propionate and isovalerate proportion, and BCVFA and ammonia N concentration were lower in ADBEO + BEO fluid compared with CTR fluid. The addition of essential oils can shift the microbial fermentation in the rumen by increasing the acetate to propionate ratio and inhibiting deamination.     

Influence of natural magnesium sources on the in vitro fermentation and protozoan population in the rumen fluid collected from sheep

The objective of this experiment was to determine the effect of two types of caustic calcinated magnesite (caustic magnesite (CM) and Agromag (AG)) upon the end products of in vitro fermentation (total gas, methane, total and individual fatty acids, and VFA) and protozoan population in the rumen fluid collected from sheep. Both magnesium additives (CM and AG) as natural products in the dose of 0.01 g were added to the fermentation bottles containing rumen inoculum from sheep and different substrates. Meadow hay (MH), wheat straw (WS), amorphous cellulose (AC) and barley grain (BG) were used as substrates and incubated with the buffered rumen fluid using an in vitro gas measuring technique during 72 h of incubation. The rumen protozoa, Entodinium spp., Trichostomatids and large Entodiniomorphids and the total protozoan concentration were counted after 24 h of incubation. The methane production was significantly decreased with CM or AG, respectively, by 58 or 62% (MH), by 65% (WS), by 52% (AC) and by 58% (BG). The total VFA concentration was significantly lower compared to control for CM plus MH, WS, AC, BG and AG plus WS. The total VFA concentration was significantly higher compared to control for AG plus AC. The effect of the both additives on ciliate population was not uniform and depended on the substrates used and protozoan type. Ciliate population was significantly increased in Entodinium spp. (AG plus BG) and Diploplastron affinae (CM or AG plus BG) compared to control. Tested additives significantly decreased population of Entodinium spp. (AG plus MH or AC), Dasytricha ruminantium (AG plus AC), Ophryoscolex c. tricoronatus, Eremoplastron dilobum and Polyplastron multivesiculatum (CM or AG plus BG). It can be concluded that both natural magnesium sources influenced rumen fermentation patterns and protozoan population in vitro depending on the type of the substrate used; therefore, the relative efficacy of individual tested additive cannot be determined from these experiments. In vivo experiments are required in future.     

Lactobacillus plantarum effects on silage fermentation and in vitro microbial yield

In four parallel experiments, herbage [three harvests of alfalfa (308 to 379 g dry matter (DM)/kg), one of whole-plant corn (331 g DM/kg)] was ensiled with three different treatments: no inoculant (control), Lactobacillus plantarum (LP) or formic acid (FA), in 1-L mini-silos and fermented for 60 d at room temperature (22 °C). Mini-silos were opened and analyzed for fermentation characteristics and soluble N fractions. A subsample of wet silage from each mini-silo was ground to 4 mm and stored at ?20 °C. Silages were thawed and subjected to 9 h ruminal in vitro incubations to measure gas production and volatile fatty acid (VFA) production as well as microbial biomass yield (MBY) and microbial non-ammonia N (MNAN) formation using ¹⁵N as a marker. In all four experiments, silage fermentation products and pH indicated good preservation across all treatments. Analysis of data showed that FA- and LP-treated silages had lower concentrations of ammonia-N and free amino acids N than control. The FA treatment was lower in soluble N, but higher in peptide-N, than control. Silage pH was lowest in FA (4.25), followed by LP (4.28), and control (4.38). Ruminal in vitro gas production and VFA concentrations were not different among treatments (P>0.05). Compared to control, FA- and LP-treated silage yielded greater MNAN and MBY. These findings suggested that L. plantarum preserved more true protein during silage fermentation than control, which in turn increased in vitro ruminal microbial growth.     

Effect of by-product gypsum application rate to grass pasture,grass hay,and corn silage on fermentation by rumen microorganisms in continuous culture

A dual-flow continuous culture fermenter system was used to investigate ruminal fermentation in response to increased by-product gypsum application rate of three forages. The treatments included 0, 22, 45, and 90 tonnes/ha by-product gypsum applied to grass plots and 0, 22, and 45 tonnes/ha by-product gypsum applied to corn plots. Forage was harvested to represent grass pasture (GP), grass hay (GH), and corn silage (CS), dried, ground, and fed to fermenters at a rate of 60 g dry matter (DM)/day. Organic matter (OM) and neutral detergent fiber (aNDF) digestibilities, rumen pH, total volatile fatty acid (VFA) production, and N metabolism were not affected by gypsum application rate for all forage types. The GH had greater sulfur content than recommended as the maximum tolerable level by the National Research Council (NRC). The results of this study indicate that ruminal fermentation was not compromised when by-product gypsum was applied to GP, GH, or CS at rates up to 90 tonnes/ha. By-product gypsum application to pastures and crops shows promise as an economical soil amendment to reduce dissolved phosphorus loss in runoff, although potential animal health issues should be further evaluated.     

Effect of chitosans on in vitro rumen digestion and fermentation of maize silage

The gas in vitro technique was used to study the effects of six types of chitosans, each having different molecular weights and acetylation degrees, on rumen microbial fermentation. In a first trial, a separate concentration of 750 mg/l of culture fluid for each of the six chitosans (CHI1, CHI2, CHI3, CHI4, CHI5, and CHI6) was incubated for 24 h in diluted ruminal fluid with maize silage as the substrate. The ionophore antibiotic monensin (MON) was used as a positive control, and a negative control with no chitosan (CTR) was also included. Each treatment was tested in triplicate for three different periods. At the end of the trial, samples were collected to determine volatile fatty acid (VFA) and ammonia N concentrations, and pH and gas production values were recorded. Methane concentration was estimated stoichiometrically. In vitro true organic matter digestibility (IVOMD) and partitioning factor (PF, mg OM truly degraded/ml gas produced) were also calculated. In a second trial, a separate concentration of 750 mg/l of each of the six chitosans was incubated for 144 h in diluted ruminal fluid with maize silage as the substrate, to study the effects of these compounds on fermentation kinetics.All six chitosans decreased the IVOMD and PF values. Chitosan inclusion did not affect the fermentation of the substrate's soluble fraction, but did reduce the fermentation kinetics of the insoluble but fermentable fraction. However, only CHI5 and CHI6 decreased total VFA concentration. CHI3 and CHI6 decreased the molar proportion of acetate and increased the molar proportion of propionate, thus increasing the propionate-to-acetate ratio. Chitosan inclusion did not affect molar proportions of butyrate. With the exception of CHI2, the molar proportion of branch-chained VFA was lowered by all of the chitosan treatments. Most of the treatments also decreased methane production, also with the exception of CHI2.In conclusion, chitosan extracts may enable the manipulation of rumen microbial fermentation, but further research is required to elucidate the effect of chitosans on ruminal fermentation parameters in commercial diets as well as the adaptability of rumen microflora to these additives.     

Debaryomyces hansenii fermentation for arabitol production

The fermentation process for arabitol production from glycerol was developed using a Debaryomyces hansenii strain recently selected from a broad screening. The high-producing strain produced arabitol as the only detectable polyol from glycerol. In this work, the pH, dissolved oxygen concentration (DO), inoculum size and magnesium concentration, and the nitrogen-to-phosphorus (N/P) ratio were systematically evaluated for effects on cell growth rate and arabitol productivity. Among those evaluated, the medium with N/P = 9, DO of 5% air saturation and pH 3.5 supported the highest arabitol production. Under these optimal conditions, arabitol production of 40 g/L was achieved in 5 days compared to earlier studies with 15 g/L arabitol in 5 days. Volumetric productivity and specific productivity were successfully improved from 0.13 to 0.33 g/L-h and 0.007 to 0.02 g/g-h respectively with arabitol yield of 55% from glycerol.     

Mycophenolic acid production in solid-state fermentation using a packed-bed bioreactor

Mycophenolic acid (MPA) was produced from Penicillium brevicompactum by solid-state fermentation (SSF) using pearl barley, and submerged fermentation (SmF) using mannitol. It was found that SSF was superior to SmF in terms of MPA concentration (1219 mg/L vs. 60 mg/L after 144 h fermentation), and the product yields were 6.1 mg/g pearl barley for SSF and 1.2 mg/g mannitol for SmF. The volumetric productivities were 8.5 and 0.42 mg/L h for SSF and SmF, respectively.The optimum solid substrate of SSF for MPA production was pearl barley, producing 5470 mg/kg compared with wheat bran (1601 mg/kg), oat (3717 mg/kg) and rice (2597 mg/kg). The optimum moisture content, incubation time and inoculum concentrations were 70%, 144 h and 6%, respectively. Neither the addition of mannitol or (NH4)₂HPO₄ nor adjustment of media pH within the range of 3–7 significantly enhanced MPA production.MPA production by SSF using a packed-bed bioreactor was performed and an increased maximum production of MPA 6.9 mg/g was achieved at 168 h incubation time. The higher volumetric productivity and concentrations makes SSF an attractive alternative to SmF for MPA production.     

The relationship between the volatile fatty acids supply and the nitrogen retention in growing sheep nourished by total intragastric infusions

Twelve four-month old Suffolk × Small-tail-Han male sheep (live weight 21–26 kg), fitted with rumen and abomasum fistulas and nourished by total intragastric infusions, were used to study the relationship between the volatile fatty acids (VFA) supply and the nitrogen (N) retention in sheep. The animals were randomly divided into four groups and four levels of mixed VFA energy (the molar proportion of acetic acid, propionic acid and butyric acid was 65:25:10), i.e. 333, 378, 423 and 468 kJ kg^?1 W^0.75 d^?1, were infused into the rumen, as treatments I, II, III and IV, respectively. The results showed that the N retention was significantly increased (P < 0.05) with the VFA infusion level. Significant regression relationship was found between the VFA supply (x, g d^?1) and the N retention (y, mg d^?1): y = 2.75x ? 403, r² = 0.86, n = 12, P < 0.01. It was concluded that to get efficient utilization of dietary N and high N retention in sheep, it is necessary to supply enough dietary energy.     

A novel medium for the production of cephamycin C by Nocardia lactamdurans using solid-state fermentation

In this study, Nocardia lactamdurans NRRL 3802 was explored for the first time for production of cephamycin C by using solid-state fermentation. The effects of various substrates, moisture content, inoculum size, initial pH of culture medium, additional nitrogen source and amino acids were investigated for the maximum production of cephamycin C by N. lactamdurans NRRL 3802 in solid-state fermentation. Subsequently, selected fermentation parameters were further optimized by response surface methodology (RSM). The soybean flour as a substrate with moisture content of 65%, initial pH of culture medium of 6.5 and inoculum size of 10⁹ CFU/ml (2 × 10⁸ CFU/gds) at 28 ± 2 °C after 4 days gave maximum production of 15.75 ± 0.27 mg/gds of cephamycin C as compared to 8.37 ± 0.23 mg/gds before optimization. Effect of 1,3-diaminopropane on cephamycin C production was further studied, which further increased the yield to 27.64 ± 0.33 mg/gds.     

Effect of natural dolomites on the in vitro fermentation and rumen protozoan population using rumen fluid and fresh faeces inoculum from sheep

The objective of the study was to determine the effect of dolomites from five different sources upon the end products of in vitro fermentation (total gas, methane, total and individual fatty acids, hydrogen recovery) and protozoan population. Dolomites as natural products in the dose of 0.1 g were added to the fermentation bottles containing inoculum from sheep and substrates. Both rumen fluid (RF) and fresh faeces (FF) from sheep as the sources of inocula for in vitro fermentation were used. Meadow hay (MH) and barley grain (BG) were used as fermentation substrates and incubated with the buffered rumen fluid using an in vitro gas measuring technique in separate incubation during 72 h. Both inocula (RF and FF) and dolomites impact in vitro fermentation characteristics. The gas volume was significantly increased with dolomites with RF or FF, respectively, by 20% or 20–40% (MH) and by 10% or 10–30% (BG). The methane production was significantly decreased with dolomite additives with RF inocula by 15–32% (MH) and by 50–70% (BG). A significant effect of the dolomite additives on the rumen protozoan population was observed during fermentation of MH; the total protozoan concentration and the number of Entodinium spp. was decreased (P < 0.05). Populations of Isotrichids and large Entodiniomorphids were not influenced by experimental incubations. More studies are needed to optimize the combination of different diets with dolomite additives for practical feeding conditions.     

Effects of feed intake on enteric methane emissions from sheep fed fresh white clover (Trifolium repens) and perennial ryegrass (Lolium perenne) forages

Published analyses of enteric methane (CH₄) emissions from sheep and cattle show an inverse relationship between feed intake and CH₄ yield (g CH₄/kg dry matter (DM) intake), which suggests opportunities for reducing CH₄ emissions from feed eaten and per unit of animal production. Most relationships between feed intake and CH₄ yield have been based on animals fed conserved feeds, especially silages and grains. Our research is a series of experiments with fresh white clover (Trifolium repens) and perennial ryegrass (Lolium perenne; ryegrass) forages fed to sheep at a range of feed intake levels. This study was comprised of four experiments where good quality freshly harvested white clover or ryegrass were fed to sheep over a three-fold range in DM intake, and CH₄ emissions were measured in respiration chambers for two consecutive days in each experiment. Measurements were made from 16 sheep in Experiment 1 (fed at 1.6 × metabolizable energy requirements for maintenance; ME_m), 28 sheep in Experiment 2 (at 0.8 and 2.0 × ME_m), eight sheep and two measurement periods in Experiment 3 (at 1.6 × ME_m), and 30 sheep in Experiment 4 (fed at 0.8, 1.2, 1.6, 2.0 and 2.5 × ME_m). Prior to each experiment, sheep had a 10 d acclimatization period to diets. Apparent digestibility was measured over 7 d from sheep in Experiments 1, 3 and 4, along with collection of rumen digesta for volatile fatty acid (VFA) determination. Although CH₄ yields differed when sheep were fed white clover or ryegrass at similar intakes, the differences were inconsistent and mean values similar across all experiments. This, and a similar structure of all experiments, enabled combined analysis of data from all four experiments using the restricted maximum likelihood (REML) procedure to estimate effects of feed intake level on digestibility, digestible nutrient intake, gas emissions, and VFA concentrations in the rumen. The REML analysis showed that when DM intake increased from 0.40 to 1.60 kg/d, the predicted responses were an increase in CH₄ production (g/d) of 187% (12.4–35.6 g/d; P<0.001), and a decline in CH₄ yield of 21% (25.6–20.2 g/kg DM intake; P<0.001). High feed intake levels were associated with increased molar proportions (mM of total VFA) of propionate from 0.17 to 0.21 (P=0.038). Single and multiple regressions were completed on the data from all experiments, with organic matter (OM) intake predicting 0.87 of the variation in CH₄ production, and molar proportion of propionate predicting 0.60 of the variation in CH₄ yield. Increasing feed intakes by 1 kg/d of DM reduced CH₄ yield by 4.5 g/kg DM intake. Plant chemical composition was weakly related to CH₄ yield. High intakes of fresh forages will lower CH₄ yield from fermentation, but effects of feed composition on CH₄ emissions were minor. The interaction between effects of feed intake and rumen function requires further investigation to understand relationships with CH₄ emissions.     

Varying endophyte status and energy supplementation of fresh tall fescue in continuous culture

Eight dual-flow continuous culture vessels (700 ml) were used to compare in vitro effects of toxic, endophyte-infected (E+), endophyte-free (E−), and non-toxic, endophyte-infected (EN) Jesup tall fescue (vegetative stage) on ruminal fermentation at 4 levels (0, 150, 300, and 450 g kg⁻¹ DM) of concentrate supplementation (ground corn) for a total of 12 experimental diets in a randomized incomplete block design with 2 replicates. Each culture vessel was offered a total of 15 g DM d⁻¹. Forage was fed in four equal portions (fed at 03:00, 09:00, 15:00, and 21:00 h); and corn was fed in two equal portions (fed at 09:00 and 21:00 h). Headspace gas and liquid samples were analyzed for methane, ruminal culture pH, ammonia–N, and volatile fatty acid production. Ammonia–N output (g d⁻¹) varied by grass; EN had lower values compared to those of E+ and E−. Increasing the level of grain linearly decreased ruminal culture pH, ammonia–N, acetate production, and the acetate-to-propionate ratio, whereas propionate and butyrate production increased with higher grain supplementation. Ruminal fermentation was minimally altered by the presence of the endophyte; however, for the highest level of grain fed (450 g kg⁻¹ DM fed) the methane production pattern for all three grasses was altered. In addition to having the lowest ruminal ammonia–N accumulation, the non-toxic, endophyte-infected fescue resulted in the lowest methane production measured.     

Specific activity and viability of Nitrosomonas europaea during discontinuous and continuous fermentation

The energy conservation and number of viable cells of Nitrosomonas europaea fluctuate dramatically during cultivation. In discontinuous culture the specific activity (SA) reaches its maximum after 9 h with about 2700 nmol O₂ (mg protein)^?1 min^?1, where the highest number of viable N. europaea cells is detectable after 21 h with 2 × 10⁸ cell ml^?1. Afterwards, both SA and viable cell number immediately start to decrease. Accordingly, the exponential growth turns into a linear growth, whereby the number of viable cells permanently decreases. The exponential growth phase can be extended from about 21 to 38 h by increasing the concentration of CO₂ or trace elements. In continuous fermentation of N. europaea, SA of about 2500 nmol O₂ (mg protein)^?1 min^?1 and viable cell number of 2.5 × 10⁸ cell ml^?1 is detectable at dilution rates between 1 and 1.8 day^?1. At dilution rates below 1 day^?1, SA and number of viable cells are reduced. The minimal doubling time is 13 and 15 h during continuous and discontinuous fermentation, respectively. Consequently, cell production of N. europaea should be performed in continuous fermentation. When bacteria are grown in discontinuous systems, they should be harvested in the early exponential growth phase.     

Rumen enzyme profile and fermentation characteristics in sheep as affected by treatment with sodium lauryl sulfate as defaunating agent and presence of ciliate protozoa

The efficiency of sodium lauryl sulfate as a defaunating agent and effect of rumen protozoa on nutrient utilization, fermentation characteristics and enzyme profile were evaluated in adult sheep maintained on a mixed ration containing 65:35% Pala (Ziziphus numularia) leaf: concentrate. Twenty-one adult Malpura sheep divided into three equal groups (DF, RF and F) were either defaunated by oral administration of sodium lauryl sulfate at the rate of 8 g/100 kg body weight (DF), or defaunated and again refaunated (RF), or maintained faunated (F). Daily dry matter intake was similar in defaunated, refaunated and faunated sheep. However, digestibility of cell wall and cell wall contents (NDF, ADF and cellulose) were lower (P < 0.01) in defaunated than refaunated and faunated sheep. Irrespective of the presence or absence of rumen protozoa, daily intake of DCP and DE were similar in the three experimental groups. Even with similar DM, DCP and DE intake, N-retention, blood glucose level, ruminal concentration of total VFA and total-N were higher (P < 0.01), while rumen pH and NH₃-N concentration were lower (P < 0.01) in defaunated sheep. Ruminal activity of amylase, xylanase, protease and urease enzymes were not influenced by presence or absence of ciliate protozoa. However, carboxymethyl cellulase enzyme activity was lower (P < 0.01) in the rumen of defaunated sheep. The total and differential counts of rumen protozoa were similar in refaunated and faunated sheep indicating lack of residual toxic effect of sodium lauryl sulfate. It is concluded that absence of ciliate protozoa increased ruminal TVFA, total-N with lower NH₃-N concentration and fibre digestibility in sheep. Moreover, sodium lauryl sulfate was fully effective for complete removal of rumen ciliate protozoa and successfully defaunated the sheep.