E. Owen

A semi-automated in vitro gas production technique for ruminant feedstuff evaluation

This technical note describes the Reading Pressure Technique (RPT) ‐ an in vitro feed evaluation system based on a semi-automated gas production technique. Substrates are incubated together with buffered rumen fluid in sealed fermentation flasks. A pressure transducer, interfaced with a PC allows accumulated head-space gas pressure values to be directly entered into a spreadsheet. These pressure measurements are then used to generate gas volume estimates using a quadratic function derived from simultaneous pressure and volume measurements. Earlier attempts to derive volume from pressure resulted in predicted volume being under estimated. This discrepancy was most likely due to diffusion of head-space gases into the liquid phase, a factor not considered in Boyle’s Law, the initial relationship used. In comparison with the syringe technique (Theodorou et al., 1994), the modifications improved both the accuracy (reduced operator error) and rate at which measurements could be taken (5‐6 s per flask) greatly increasing the analytical capacity (to 336 flasks or 75 substrates per incubation series). The ability of this system to provide information not just on both rate and extent of degradation, but also fermentation efficiency and degradation kinetics offers a wide variety of applications, for example the systematic evaluation of tropical feedstuffs. In conclusion it is suggested that the simplicity, low cost and high capacity of the RPT system make it ideal for those situations where either budget constraints or the level of technical expertise required render the more complex systems inappropriate. # 1999 Published by Elsevier Science B.V. All rights reserved.

Biochemical characterization and mode of action of a thermostable endoglucanase purified from Thermoascus aurantiacus

A major extracellular endoglucanase purified to homogeneity from Thermoascus aurantiacus had a M(r) of 34 kDa and a pI of 3.7 and was optimally active at 70-80 degrees C and pH 4.0-4.4. It was stable at pH 2.8-6.8 at 50 degrees C for 48 h and maintained its secondary structure and folded conformation up to 70 degrees C at pH 5.0 and 2.8, respectively. A 33-amino acid sequence at the N terminus showed considerable homology with 14 microbial endoglucanases having highly conserved 8 amino acids (positions 10-17) and Gly, Pro, Gly, and Pro at positions 8, 22, 23, and 32, respectively. The enzyme is rich in Asp (15%) and Glu (10%) with a carbohydrate content of 2.7%. Polyclonal antibodies of endoglucanase cross-reacted with their own antigen and with other purified cellulases from T. aurantiacus. The endoglucanase was specific for polymeric substrates with highest activity toward carboxymethyl cellulose followed by barley beta-glucan and lichenan. It preferentially cleaved the internal glycosidic bonds of Glc(n) and MeUmbGlc(n) and possessed an extended substrate-binding site with five subsites. The data indicate that the endoglucanase from T. aurantiacus is a member of glycoside hydrolase family 5.

Prediction of in situ rumen dry matter disappearance of Ethiopian forages from an in vitro gas production technique using a pressure transducer, chemical analyses or in vitro digestibility

In each of four experiments, forages (Experiment 1, 20 hays from Napier and Pigeon grass cut at different intervals; Experiment 2, eight leaves and branches from tree lucerne harvested at different intervals; Experiment 3, six hays from Reed Canary grass cut at different intervals; Experiment 4, 15 crop residues from sorghum, tef, wheat and barley) were assessed for in situ rumen dry matter (DM) disappearance, in vitro gas production using a pressure transducer technique (PTT), chemical composition (nitrogen and fibre fractions) and in vitro digestibility (DMD). In situ DM disappearance data were fitted to the model of Orskov and McDonald, i.e. P = ao + bo(1 − exp(− cot)), where P is the DM disappearance at time t, ao and bo are fitted constants and co (h−1) is the fractional rate of DM disappearance. PTT data were fitted to the model of France et al., i.e. Y=A(1 − e−b(1−T)−c(t−T)) where Y is gas production after time t, b and c are rate constants, A is the asymptote and T is lag time. In situ DM disappearance parameters were linearly regressed against in vitro gas production parameters, chemical composition and in vitro DMD. Overall co of forages was correlated (P < 0.01) with fractional rate of gas production μ (μ = b + c2√t). co was also correlated with nitrogen, fibre fractions and in vitro DMD. In all experiments, effective in situ DM disappearance (EDMD: DM degraded in the rumen at a given rate of passage) was significantly (P < 0.01) correlated with extent of gas production (at the assumed passage rate from the rumen). EDMD was also correlated (P < 0.01) with in vitro DMD in all experiments, and with chemical composition (N, NDF, ADF, KMnO4 lignin). It is concluded that in situ DM disappearance parameters of forages such as used in the present study, may be predicted from in vitro gas production parameters, and to some extent, also from chemical composition and in vitro digestibility. However, the accuracy of prediction is likely to vary, depending on the type of forages used.

Botanical and nutritional composition of maize stover, intakes and feed selection by dairy cattle

Maize stover is the most abundant crop residue in the central Kenya highlands and it is often the sole forage offered to dairy cattle during the dry seasons. In recent years increasing offer rates of crop residues (excess feeding) to promote selective feeding by confined animals has been shown to improve utilization of the residues, but no studies have been conducted with dairy animals offered maize stover. This study was conducted to determine the effects of increasing the ad libitum amounts of maize stover offered, on intake, selection and milk production by dairy cattle, when supplemented with cottonseed cake. The effects of post harvest handling on the quality of maize stover were also studied. Hybrid 511 maize was grown under similar agronomic conditions in 2 consecutive years, 1994 and 1995. Maize harvested under dry weather conditions in 1994 produced stover with a higher leaf+sheath+husk:stem ratio (1.48), than maize harvested in 1995 under rainy weather conditions (0.99). In both years, leaves recorded the highest crude protein (CP) contents among the botanical components. Acid detergent lignin (ADL) levels were highest in stem (85 g/kg DM) and lowest in husk (29 g/kg DM). Stem had the lowest digestible organic matter (DOMD) values (405 g/kg DM) while husk had the highest (707 g/kg DM). Stover intake by dairy cows increased significantly from 19 to 26 and then to 30 g DM/kg live weight per day, as the amount of stover offered increased from 31 to 59 and then to 86 g DM/kg live weight per day, respectively. The cows selected for more leaves and husks as the amount of stover offered increased. All animals were supplemented with 3.2 kg DM/day of cottonseed cake. Milk yields increased significantly (P<0.05) from 10.0 to 11.2 and then to 12.2 kg/cow/day. It was concluded that offering excess can be an effective strategy to improve the intake of maize stover and increase milk production although adoption of the technology would depend on other factors such as availability of large amounts of stover and an economical use of the refusals.

Feeding straw to small ruminants: effect of amount offered on intake and selection of barley straw by goats and sheep

The hypothesis that increasing the amount of straw offered and allowing animals to refuse proportionately more than 0·1 to 0·2 would increase intake and quality of the straw consumed was tested in two experiments with castrated goats (aged 6 to 31 months) and one with wether sheep (aged 6 to 18 months). Each trial (over 21 to 42 days following 14 to 35 days preliminary feeding) involved individually feeding long barley straw and concentrate supplement (15 g dry matter (DM) per kg live weight (M075) daily) and monitoring the quantity and quality of straw offered and straw refused. The results supported the hypothesis. In experiment 1, with 18 goats per treatment, those allowed to refuse 500 rather than 200 g straw per kg DM offered consumed more (18·9 and 14·4 g DM per kg M75 per day, s.e.d. 0·70) and their straw refusals contained more digestible organic matter (DOM) in vitro (347 and 320 g/kg DM, s.e.d. 7·7). For both treatments, refusals were less digestible in vitro than the straw offered (412 g DOM per kg DM). Thirty-six goats in experiment 2 (over 42 days) offered increasing amounts of straw (18, 54 and 90 g DM per kg M075 per day) consumed more (15·5, 22·8 and 26·2 g DM per kg M per day, s.e.d. 0·74), refused more (125, 566 and 703 g/kg DM offered) and the refusals were of increasing digestibility in vitro (354, 370 and 403 g DOM per kg DM, s.e.d. 14·5). All refused straw was inferior to that offered (443 g DOM per kg DM). The estimated intake of straw DOM was markedly improved by offering more straw (7·2, 12·8 and 14·5 g per kg M75 per day). Experiment 3, using 30 wethers over 21 days fed as in experiment 2, showed similar treatment responses, although absolute intakes of straw were lower. The responses observed are comparable to improvements in intake following treatment of straw with alkali. Further research is required to determine optimum feeding rates as affected by straw quality and animal productivity level. Practical feeding strategies will also need to consider utilizing refused straw.

Sodium hydroxide treatment of barley straw; effect of volume and concentration of solution on digestibility and intake by sheep

1. Four experiments have been carried out to determine the effect of treatment of spring-sown barley straw (var. Deba Abed) with sodium hydroxide (NaOH) solution and subsequent neutralization with hydrochloric acid (HCl) on its digestibility and intake by sheep. 2. In Experiment 1, chopped straw was mixed with 4·5 or 9·0 g NaOH in 200 or 800 ml water/100 g, straw, and after 24 hr neutralized with HCl and left for a further 24 hr before being offered to castrated male sheep (wethers) in a maintenance diet containing 35% concentrates. Organic-matter digestibility of straw significantly increased, by 8 and 11 percentage units respectively, after treatment with 200 ml solution containing 4·5 and 9·0 g NaOH. Volume of solution did not affect digestibility. 3. In Experiment 2, straw treated as in Experiment 1 was given ad libitum . The highest intake was for straw treated with 200 ml solution containing 4·5 g NaOH/100 g straw, treatment with 9·0 g NaOH giving a significantly lower intake but higher than that of untreated straw. 4. In Experiment 3, the in vitro digestibility of milled straw, treated as in Experiments 1 and 4, increased with increasing volumes of solution up to 120 ml/100 g straw, but the response to successive increments of NaOH declined progressively. 5. In Experiment 4 chopped straw was mixed with 4·5, 6·75 or 9·0 g NaOH in 30, 60 or 120 ml water/100 g straw and offered as in Experiment 1. Treatments significantly increased straw digestibility, by 8 to 16 percentage units. Increasing the volume of water from 30 to 60 ml significantly improved digestibility, by 5 percentage units at the two lower levels of NaOH. The response to an increase in the level of NaOH was less, and inconsistent. In vitro and in vivo digestibilities were significantly correlated, but it is concluded that the in vitro technique used overestimates the digestibility of treated straw.

Comparison of sheep and goats under stall-feeding conditions: roughage intake and selection

Three experiments measured intake and assessed quality of roughage refused by individually-fed castrated Saanen goats and Suffolk × Mule wethers, aged 21 months. In experiment 1, in which long, lucerne hay was offered (10 animals per species; 14 days) goats ate more than sheep (33·2 v. 28·3 g dry matter (DM) per kg M daily; P v. 16·4 g DM per kg M daily; P In each experiment, food refusals (0·2 of food offered) were of lower nutritive value (lower nitrogen, higher acid detergent fibre, lower digestibility in vitro) than food offered. Throughout, food refusals by goats were of slightly higher nutritive value than food refusals by sheep. It is concluded that both sheep and goats are selective feeders under stall-feeding conditions, but the greater intake of roughage by goats cannot be attributed to their selection of more nutritive components.

Isolation of four major subunits from Clostridium thermocellum cellulosome and their synergism in the hydrolysis of crystalline cellulose.

The cellulosome of Clostridium thermocellum, purified by affinity chromatography, was dissociated under mild conditions and separated by SDS-PAGE. Two major p-nitrophenylcellobiosidases (PNPCases I and II) corresponding to the S5 (103 kDa) and S8 (78 kDa) subunits and one major carboxymethylcellulase (CMCase) coinciding with the S11 (60.5 kDa) subunit were isolated and characterized using carboxymethylcellulose (CMC), H3PO4-swollen cellulose and cello-oligosaccharides. Both PNPCases showed little effect on the viscosity of CMC and released twice as much total sugar as reducing sugar from H3PO4-swollen cellulose. The CMCase released ten times more total sugar than reducing sugar from H3PO4-swollen cellulose and reduced the viscosity of CMC rapidly. None of these enzymes was active on cellotriose. Both PNPCases released cellobiose from cellotetraose, and cellobiose and cellotriose from cellopentaose. In contrast, CMCase was active only on cellopentaose and released mainly glucose. Use of MeUmb(Glc)n revealed that both PNPCases cleaved preferentially either the second or fourth linkage from the non-reducing end while the CMCase was specific for the internal glycosidic bonds. Thus, the PNPCases and CMCase behaved as typical exo- and endoglucanases, respectively. When tested individually, all three enzymes degraded Avicel only to a small extent. A 1.5-2.0-fold increase in sugar release was observed when CMCase was combined with either PNPCase I, II or both. Combining S1 with either PNPCase II or CMCase resulted in fourfold synergism in the hydrolysis of Avicel. Synergism was sevenfold when all three enzymes were combined with S1.

Ensiling of whole-crop wheat with cellulase-hemicellulase based enzymes. 1. Effect of crop growth stage and enzyme on silage composition and stability

Whole-crop wheat was harvested at the heading, mealy-ripe, medium-dough and hard-dough stages of grain development corresponding to crop dry matter (DM) contents of 310 g DM kg−1, 405 g DM kg−1, 520 g DM kg−1 and 680 g DM kg−1, respectively. Crop from each harvest was either untreated or treated with one of two enzyme additives (Clampzyme or Deezyme) at three application rates of 333, 667 and 1000 ml t−1 DM and 1000, 1334 and 1667 ml t−1 DM (low, medium and high level) for Clampzyme and Deezyme, respectively. Crop from each harvest was ensiled in 5.0 kg quantities in polythene-bag silos, and sealed either immediately or after 24 h. Contents of DM, acid detergent fibre, neutral detergent fibre and in vitro digestible organic matter increased with advancing maturity, but contents of nitrogen, water-soluble carbohydrates and fermentation products decreased. Silage stability, as measured by silo DM loss when exposed, also increased as crops matured. Compared with untreated crop, enzyme treatment increased lactic and acetic acid contents and decreased pH, but these effects were more pronounced in less mature crops. Neither fibre content nor in vitro digestible organic matter content were affected by enzyme treatment; increasing levels of enzyme did not affect silage composition. An interesting finding was that delaying silo sealing by 24 h improved fermentation and improved the aerobic stability of silages. Albeit using small-scale silos, it is concluded that whole-crop wheat may be harvested at DM contents of up to 700 g DM kg−1 (hard-dough stage) and be effectively preserved by ensiling. Addition of cellulase-hemicellulase-based enzymes would enhance fermentation when ensiling less mature (less than 500 g DM kg−1 wheat.

Ensiling of whole crop wheat with cellulase–hemicellulase based enzymes: 3. Comparing effects of urea or enzyme treatment on forage composition and stability

This work compared the effect of ensiling technique and application of urea or a cellulase‐ hemicellulase enzyme mixture (FSO2) on the conservation of whole crop wheat. Awheat crop harvested at 600 g dry matter (DM) kg ˇ1 was conserved with or without the application of urea (40 g kg ˇ1 DM) and four levels of FSO2 (0, 1750, 3500 and 15 503 ml t ˇ1 DM). Each forage (5 kg DM) was conserved in polythene-bag silos that were evacuated and sealed immediately, sealed immediately but not evacuated or evacuated and sealed after 24 h. After at least 42 days of conservation, the silos were opened and analysed for chemical composition, rumen fluid-pepsin in vitro digestibility and aerobic stability. In non-urea-treated forages, increasing enzyme application rate did not affect in vitro digestibility, but increased water soluble carbohydrate and lactic acid contents, and reduced pH, neutral detergent fibre, acid detergent fibre and cellulose contents. Compared to the FSO2 only treatments, urea treatment increased pH and N content and reduced ensiling DM loss, neutral detergent fibre, acid detergent fibre, acid detergent lignin and cellulose contents. Application of FSO2 and urea (FSO2a U) produced forages with higher in vitro digestibility and lower cell wall contents than in FSO2 only forages. NDF and ADF contents were also 5‐10% lower in FSO2a U forages than in those conserved with only urea. Immediate evacuation of silos did not enhance fermentation quality. Delaying silo sealing by 24 h increased lactic acid content and aerobic stability relative to either of the immediate seal treatments. Urea treatment alone and the high enzyme level alone also enhanced aerobic stability. However, increasing the enzyme application rate in the FSO2a U treatments did not enhance