δ13C as a marker to study digesta passage kinetics in ruminants: a combined in vivo and in vitro study

The aim of the current study was to explore the use of the tracer ¹³C as an internal marker to assess feed fraction-specific digesta passage kinetics through the digestive tract of dairy cows. Knowledge on feed-specific fractional passage rates is essential to improve estimations on the extent of rumen degradation and microbial protein efficiency; however, this information is largely lacking. An in vivo and in vitro experiment was conducted with grass silages (Lolium perenne L.) that were enriched with ¹³C by growing the grass under elevated ¹³CO₂ conditions. In a crossover design, two dairy cows received pulse doses of two ¹³C-enriched grass silages and chromium-mordanted neutral detergent fibre (Cr-NDF) into the rumen. The two ¹³C-enriched grass silages used differed in digestibility and were grown under identical field conditions as the bulk silages fed to the animals. Faecal excretion patterns of ¹³C-enriched dry matter (¹³C-DM), neutral detergent fibre (¹³C-NDF) and Cr-NDF were established, and a nonlinear multicompartmental model was used to determine their rumen passage kinetics. In addition, the ¹³C-enriched silages were incubated in rumen liquid in an in vitro batch culture system at different time intervals to determine the effect of fermentation on ¹³C-enrichment in the residue. The in vitro study showed that the ¹³C : ¹²C ratios in DM and NDF residues remained stable from 24 h of incubation onwards. In addition, in vitro fractional degradation rates for ¹²C in the DM and NDF did not differ from those of ¹³C, indicating that fermentative degradation does not affect the ¹³C : ¹²C ratio in the DM nor in the NDF fraction of the residue. Model fits to the faecal excretion curves showed a significant difference in fractional rumen passage rates between Cr-NDF, ¹³C-DM and ¹³C-NDF (P ⩽ 0.025). Silage type had no clear effect on rumen passage kinetics (P ⩾ 0.081). Moreover, it showed that peak enrichments for ¹³C-DM and ¹³C-NDF in faeces were reached at 30.7 and 41.7 h post dosing, respectively. This is well after the time (24 h) when the ¹³C : ¹²C ratios of the in vitro unfermented residues have reached stable enrichment level. Fractional rate constants for particle passage from the rumen are estimated from the descending slope of faecal excretion curves. The present study shows that the decline in ¹³C : ¹²C ratio after peak enrichment is not affected by fermentative degradation and therefore can be used to assess feed component-specific fractional passage rates.     

Stable Isotope Labeled n-Alkanes to Assess Digesta Passage Kinetics through the Digestive Tract of Ruminants

We describe the use of carbon stable isotope (¹³C) labeled n-alkanes as a potential internal tracer to assess passage kinetics of ingested nutrients in ruminants. Plant cuticular n-alkanes originating from intrinsically ¹³C labeled ryegrass plants were pulse dosed intraruminally in four rumen-cannulated lactating dairy cows receiving four contrasting ryegrass silage treatments that differed in nitrogen fertilization level (45 or 90 kg nitrogen ha⁻¹) and maturity (early or late). Passage kinetics through the gastrointestinal tract were derived from the δ¹³C (i.e. the ratio ¹³C:¹²C) in apparently undigested fecal material. Isotopic enrichment was observed in a wide range of long-chain n-alkanes (C₂₇–C₃₆) and passage kinetics were determined for the most abundant C₂₉, C₃₁ and C₃₃ n-alkanes, for which a sufficiently high response signal was detected by combustion isotope ratio mass spectrometry. Basal diet treatment and carbon chain length of n-alkanes did not affect fractional passage rates from the rumen (K ₁) among individual n-alkanes (3.71–3.95%/h). Peak concentration time and transit time showed a quantitatively small, significant (p≤0.002) increase with carbon chain length. K ₁ estimates were comparable to those of the ¹³C labeled digestible dry matter fraction (3.38%/h; r = 0.61 to 0.71; p≤0.012). A literature review has shown that n-alkanes are not fermented by microorganisms in the rumen and affirms no preferential depletion of ¹³C versus ¹²C. Our results suggest that ¹³C labeled n-alkanes can be used as nutrient passage tracers and support the reliability of the δ¹³C signature of digestible feed nutrients as a tool to measure nutrient-specific passage kinetics.     

Grass maturity effects on cattle fed silage-based diets. 2. Cell wall digestibility,digestion and passage kinetics

Four silages were harvested at approximately 1-week intervals from the same timothy-meadow fescue sward and studied in a 4 × 4 Latin square experiment with four ruminally and duodenally cannulated young cattle. The diets comprised silage and concentrate (7:3 dry matter (DM) basis) and were fed at a rate of 70 g DM kg^−0.75 liveweight in two equal meals per day.Neutral detergent fibre (NDF) digestibility was 0.757, 0.765, 0.692 and 0.686 on diets based on the four silages in order of harvest date. Increasing maturity of grass ensiled showed linear (P_L < 0.001) and cubic (P_C < 0.01) trends. NDF was separated into digestible (DNDF) and indigestible (INDF) fractions, which differed clearly in their rate of passage from the rumen (on average 0.0141 vs. 0.0258 h⁻¹). The rate of digestion (k_d) of DNDF was on average 0.076 h⁻¹ when derived from the rumen evacuations but only 0.036 h⁻¹ when calculated from the disappearance from nylon bags incubated in the rumen. Both methods detected decreased k_d of NDF with increasing maturity of grass ensiled.Rate of passage from the rumen increased with increasing maturity of grass both when determined for NDF with rumen evacuation technique and from the faecal excretion of ytterbium calculated with a two-pool model. Mean retention time (MRT) in the non-escapable pool of particles increased (P_L < 0.01) with increasing grass maturity, the opposite being true for the escapable pool (P_L < 0.05), resulting in no change in the total ruminal MRT. Pool sizes of ruminal DM P_L < 0.01) and NDF (P_L < 0.001) increased with increasing maturity of grass. Ruminal NDF digestibility was calculated by different methods. When digestion kinetic parameters were derived from rumen evacuations and two-pool models used for passage kinetics, estimated digestibilities were very close to the observed ones.     

Synthetic peptides derived from the nonmuscle myosin light chains are highly specific substrates for protein kinase C

The phosphorylation of synthetic peptides derived from the NH₂-terminal sequence of smooth-muscle myosin was studied with purified protein kinase C. The protein kinase C phosphorylation domain included both serine residues and threonine residues in the sequence SSKRAKAKTTKKR(G), denoted myosin light chain (1–13) (MLC(1–13)). Kinetic analysis of MLC(1–13) and truncated peptides derived from the parent peptide established that removal of the serine residues had little effect on protein kinase C reactivity. MLC(1–13) had a V/K of 2.4 min⁻¹·mg⁻¹, whereas the V/K of MLC(3–13) was 3.0 min⁻¹·mg⁻¹. Removal of Lys-3 resulted in a 50% decrease in V/K which was attributable to a 50% decrease in apparent V_max. Arg-4 was established as a significant protein kinase C specificity determinant, since the apparent K_m increased 7-fold and the V_max decreased 3-fold when the parent peptide was truncated at that residue. All peptides studied required calcium and lipid effectors for full activity with protein kinase C, indicating that they are Class C substrates as defined by Bazzi and Nelsestuen (Biochemistry 26 (1987) 5002) for protein kinase C. Other protein kinases, including cyclic AMP- and cyclic GMP-dependent protein kinase, S6/H4 kinase, myosin light-chain kinase and calcium/calmodulin-dependent kinase II, had little or no activity with these peptides. In studies on the purification of lymphosarcoma protein kinase C by several chromatographic procedures, the results showed that the myosin light-chain can provide convenient and well-characterized substrates for purification and mechanistic studies of protein kinase C biochemistry.     

Effect of fibrolytic enzymes added to a Andropogon gayanus grass silage-concentrate diet on rumen fermentation in batch cultures and the artificial rumen (Rusitec)

In vitro batch cultures were used to screen four fibrolytic enzyme mixtures at two dosages added to a 60 : 40 silage : concentrate diet containing the C₄ tropical grass Andropogon gayanus grass ensiled at two maturities – vegetative stage (VS) and flowering stage (FS). Based on these studies, one enzyme mixture was selected to treat the same diets and evaluate its impact on fermentation using an artificial rumen (Rusitec). In vitro batch cultures were conducted as a completely randomized design with two runs, four replicates per run and 12 treatments in a factorial arrangement (four enzyme mixtures×three doses). Enzyme additives (E1, E2, E3 and E4) were commercial products and contained a range of endoglucanase, exoglucanase and xylanase activities. Enzymes were added to the complete diet 2 h before incubation at 0, 2 and 4 μl/g of dry matter (DM). Gas production (GP) was measured after 3, 6, 12, 24 and 48 h of incubation. Disappearance of DM (DMD), NDF (NDFD) and ADF (ADFD) were determined after 24 and 48 h. For all four enzyme mixtures, a dosage effect (P<0.05) was observed for NDFD and ADFD after 24 h and for DMD, NDFD and ADFD after 48 h of incubation of the VS diet. For the FS diet, a dosage effect was observed for GP and NDFD after 24 h and for GP, DMD, NDFD and ADFD after 48 h of incubation. There was no difference among enzyme mixtures nor was there an enzyme×dose interaction for the studied parameters. Because of the greatest numerical effect on NDF disappearance and the least cost price, enzyme mixture E2 at 4 µl/g of diet DM was selected for the Rusitec experiment. The enzyme did not impact (P>0.05) DM, N, NDF or ADF disappearance after 48 h of incubation nor daily ammonia-N, volatile fatty acids or CH₄ production. However, enzyme application increased (P<0.05) microbial N production in feed particle-associated (loosely-associated) and silage feed particle-bound (firmly associated) fractions. With A. gayanus silage diets, degradation may not be limited by microbial colonization, but rather by the ability of fibrolytic enzymes to degrade plant cell walls within this recalcitrant forage.     

Prediction of rumen degradability parameters of a wide range of forages and non-forages by NIRS

Kinetics of nutrient degradation in the rumen is an important component of feed evaluation systems for ruminants. The in situ technique is commonly used to obtain such dynamic parameters, but it requires cannulated animals and incubations last several days limiting its application in practice. On the other hand, feed industry relies strongly on NIRS to predict chemical composition of feeds and it has been used to predict nutrient degradability parameters. However, most of these studies were feedstuff specific, predicting degradability parameters of a particular feedstuff or category of feedstuffs, mainly forages or compound feeds and not grains and byproducts. Our objective was to evaluate the potential of NIRS to predict degradability parameters and effective degradation utilizing a wide range of feedstuffs commonly used in ruminant nutrition. A database of 809 feedstuffs was created. Feedstuffs were grouped as forages (FF; n=256), non-forages (NF; n=539) and of animal origin (n=14). In situ degradability data for dry matter (DM; n=665), CP (n=682) and NDF (n=100) were collected. Degradability was described in terms of washable fraction (a), slowly degradable fraction (b) and its rate of degradation (c). All samples were scanned from 1100 to 2500 nm using an NIRSystems 5000 scanning in reflectance mode. Calibrations were developed for all samples (ALL), FF and NF. Equations were validated with an external validation set of 20% of total samples. NIRS equations to predict the effective degradability and fractions a and b of DM, CP and NDF could be evaluated from being adequate for screening (r²>0.77; ratio of performance to deviation (RPD)=2.0 to 2.9) to suitable for quantitative purposes (r²>0.84; RPD=3.1 to 4.7), and some predictions were improved by group separation reducing the standard error of prediction. Similarly, the rate of degradation of CP (CP_c) and DM (DM_c) was predicted for screening purposes (RPD⩾2 and 2.5 for CP_c and DM_c, respectively). However, the rate of degradation of NDF was not predicted accurately (NDF_c: r²<0.75; RDP<2).     

Multinuclear NMR of cis-dicarbonylbis(dithiocarbamato)iron(II) complexes in chloroform solution

The ¹³C and ¹⁵SN NMR spectra of eleven cis-Fe(S₂CNRR′)₂(CO)₂ complexes, where R and R′ are organic substituents, have been measured at ambient temperature in CDCl₃ (0.08–0.16 M). The ¹³C absorptions for the carbonyl ligands correlate well with the force constants for the CO stretching vibrations in CHCl₃ solution. Each of the parameters (¹³CO absorption and k_cis for CO) correlate well with the aqueous solution pK_a for⁺H₂NRR′, corrected for the phenyl-containing substituents, high pK_a values corresponding to high ¹³CO absorptions and low k_cis CO force constants. [p ]Evidence was found in the ¹³C NMR spectra for hindered rotation about the CN bond in S₂CNC₂ in complexes with higher pK_a(corr) values and in the ¹³C spectra of the corresponding thiuram disulfides. [p ]The ¹⁵N (natural abundance) NMR spectra for each of the complexes was determined. Each revealed a single sharp absorption in a region of the ¹⁵N NMR spectrum which indicates substantial CN double bond character, as one would expect for coordinated dithiocarbamate ligands.     

Grass silage pulp as a dietary component for high-yielding dairy cows

Green biorefineries provide novel opportunities to use the green biomass efficiently and utilize the ecosystem services provided by grasslands more widely. The effects of the inclusion of fractionated grass silage solid fraction (pulp) on feed intake, rumen fermentation, diet digestion and milk production in dairy cows were investigated. Pulp was separated from grass silage using a screw press simulating a green biorefinery. Partial removal of liquid from forage increased DM concentration from 220 to 432 g/kg and NDF from 589 to 709 g/kg DM while CP decreased from 144 to 107 g/kg DM. A feeding trial using an incomplete changeover design with 24 Nordic Red cows and two 3-week periods was conducted. The pulp replaced grass silage in the diet at 0 (P0), 25 (P25) and 50 (P50) percentage of total forage, which was fed ad libitum with 13 kg of concentrate for all treatments. The forage DM intake was highest on P25 (14.1 kg/day) while P0 and P50 did not differ from each other (13.2 and 13.0 kg/day, respectively). There were no differences between the treatments in rumen pH or ammonia N, but the proportion of acetate increased with increasing pulp inclusion. The digestibility was measured using acid insoluble ash and indigestible NDF (iNDF) as internal markers. Neither of the markers detected differences in NDF digestibility, but according to iNDF, apparent total tract organic matter digestibility decreased with increasing pulp inclusion. The cows maintained milk production at P25, but it showed some decline at P50 (energy-corrected milk at P0 and P25 was 39.8 kg/day while for P50, it was 38.5 kg/day, P = 0.056) and the milk protein yield significantly declined with higher pulp inclusion. Simultaneously, the nitrogen use efficiency in milk production increased. It seems that the fibrous grass-based fraction from a biorefinery process has potential to be used as a feed for ruminants.     

Relation between feeding behaviour and energy metabolism in pigs fed diets enriched in dietary fibre and wheat aleurone

Feed intake and its daily pattern are regulated both at a short and a long term by several control pathways, including energy balance regulation. This trial aimed to determine the effect of dietary fibre (DB) (mix of wheat, soy and sugar beet pulp fibres) and aleurone supplementation and their interaction on energy and nitrogen balances in growing pigs with ad libitum access to feed. Forty pigs (BW: 35 kg) were fed diets differing by fibre concentration (NDF concentration: 10% or 14% DM) and aleurone supplementation (0, 2 or 4 g/kg) during 3 weeks. Pigs were housed individually in a respiration chamber during the last week to record feeding behaviour and measure energy and nitrogen balances (n = 36). Glucose oxidation was studied on the 6th day with an injection of [U-¹³C] glucose and measurement of ¹³CO₂ production. There was no significant interaction between DB inclusion and aleurone supplementation on any variables characterizing feeding behaviour. Pigs had less but longer meals with high level of DB, with an increased interval between two meals without effect on daily feed intake. The meal frequency significantly decreased when aleurone supplementation increased. Total tract apparent digestibility coefficient of DM, organic matter, ash, nitrogen and gross energy decreased when pigs received high DB level. Dietary fibre level increased significantly faecal excreted nitrogen. Aleurone supplementation decreased nitrogen retention. Free access to the feed induced a great individual variability not only in feed intake level (from 784 to 2290 g/day) but also in feeding behaviour (from 5.5 to 21.5 meals per day). This variability can be linked with the importance of underlying feed intake regulation pathways and difference in energy balance and metabolism efficiency. Several profiles of metabolism efficiency can be discriminate, thanks to a clustering based on feeding behaviour and pre-prandial concentrations of metabolites and hormones. In conclusion, DB inclusion decreased meal frequency, increased average meal size, decreased total tract apparent faecal digestibility coefficient of nitrogen and gross energy. Supplementation of aleurone decreased average daily feed intake with a reduction of the meal number per day, without modification of average meal size. Aleurone supplementation decreased nitrogen retention and nutrient deposition. Independently of experimental diets, the high individual variability permitted discriminating different profiles with different metabolic strategies. Efficient pigs with a high energy retention as protein and lipid seem to be able to adapt their metabolism according to energy sources.     

Partial purification and characterization of cyclic AMP phosphodiesterases from Funaria hygrometrica

Natural-abundance ¹³C NMR spectroscopy at 15.04 MHz has been used to examine the effects of pH, calcium, and lanthanide ions on the polypeptide cardiac stimulant Anthopleurin-A in aqueous solution. The carboxyl resonance from the aspartic acid residue not observed in a previous study (R. S. Norton and T. R. Norton, 1979, J. Biol. Chem.254, 10220–10226) has been identified and an apparent pK_a of 3.4 obtained. More accurate estimates have been derived for the apparent pK_a values of the two histidine residues. Binding of Ca²⁺ ions has been found by equilibrium dialysis and ¹³C NMR to be weak (K_d > 0.1 M). The interaction with lanthanide ions is slightly stronger, but binding occurs at the C terminus as well as at a site involving one or both of the aspartate carboxylate groups. These results suggest that possible Anthopleurin-A-induced calcium translocation in the myocardial cell is a secondary effect. The interaction of Anthopleurin-A with lipid monolayers has also been examined. Binding occurs to neutral and zwitterionic lipids, but is stronger with negatively charged lipids, particularly cardiolipin. This interaction is also influenced by the presence of Ca²⁺ ions. The implications of these results for the mechanism of action of this polypeptide are discussed.     

Effect of glyphosate contaminated feed on rumen fermentation parameters and in sacco degradation of grass hay and corn grain

Four rumen fistulated wethers were used to investigate the effect of glyphosate contaminated feed on rumen fermentation. The rations were based on corn silage, urea and a vitamin-mineral premix, either in the absence or presence of 0.77?g glyphosate per kg DM. Furthermore, rations were fed either with or without aromatic amino acid supplementation. During four periods of 28 days, sheep received each of the four dietary treatments according to a Latin square. After 14 days of adaptation rumen fermentation parameters (pH, ammonia, volatile fatty acids) were measured on day 15 over a five-hour period after the morning feeding. The remaining 13 days served for in sacco degradation studies with grass hay and corn grain. Ammonia (NH₃) and pH of rumen fluid were within the normal range for all dietary treatments (NH₃: 9.1 – 32.3?mmol·l^???1, pH: 6.2 – 6.7). Neither rumen fermentation parameters nor in sacco DM and NDF degradation of incubated feedstuffs were significantly affected by glyphosate, with or without aromatic amino acid supplementation. Kinetic profiles of the in sacco dry matter and NDF degradation of grass hay were almost identical for the dietary treatments.     

Enhancing the Secretion Efficiency and Thermostability of a Bacillus deramificans Pullulanase Mutant (D437H/D503Y) by N-Terminal Domain Truncation

Pullulanase (EC 3.2.1.41), an important enzyme in the production of starch syrup, catalyzes the hydrolysis of α-1,6 glycosidic bonds in complex carbohydrates. A double mutant (DM; D437H/D503Y) form of Bacillus deramificans pullulanase was recently constructed to enhance the thermostability and catalytic efficiency of the enzyme (X. Duan, J. Chen, and J. Wu, Appl Environ Microbiol 79:4072–4077, 2013, http://dx.doi.org/10.1128/AEM.00457-13). In the present study, three N-terminally truncated variants of this DM that lack the CBM41 domain (DM-T1), the CBM41 and X25 domains (DM-T2), or the CBM41, X25, and X45 domains (DM-T3) were constructed. Upon expression, DM-T3 existed as inclusion bodies, while 72.8 and 74.8% of the total pullulanase activities of DM-T1 and DM-T2, respectively, were secreted into the medium. These activities are 2.8- and 2.9-fold that of the DM enzyme, respectively. The specific activities of DM-T1 and DM-T2 were 380.0 × 10⁸ and 449.3 × 10⁸ U · mol⁻¹, respectively, which are 0.94- and 1.11-fold that of the DM enzyme. DM-T1 and DM-T2 retained 50% of their activity after incubation at 60°C for 203 and 160 h, respectively, which are 1.7- and 1.3-fold that of the DM enzyme. Kinetic studies showed that the K_m values of DM-T1 and DM-T2 were 1.5- and 2.7-fold higher and the K_cat/K_m values were 11 and 50% lower, respectively, than those of the DM enzyme. Furthermore, DM-T1 and DM-T2 produced d-glucose contents of 95.0 and 94.1%, respectively, in a starch saccharification reaction, which are essentially identical to that produced by the DM enzyme (95%). The enhanced secretion and improved thermostability of the truncation mutant enzymes make them more suitable than the DM enzyme for industrial processes.     

Potential use of radiography for measuring feed intake of southern rock lobster (Jasus edwardsii)

The potential of using radiography for measuring feed intake in southern rock lobster Jasus edwardsii (Hutton) was evaluated. Lobsters (14-40 g) readily consumed feeds containing radio-opaque marker (400—450 μm). Marker particles were clearly identified in the stomach 90 min after feeding enabling feed intake to be estimated. Initially low estimates (15±17%; mean±S.D.) of feed intake (expressed as percentage of gravimetric estimates) were recorded due to loss of markers prior to ingestion. Radiographic estimates improved (100±23%; mean±S.D.) when changes to pellet diameter (1.5 mm), moisture content (11%) and binding agent (alginate) were made. Thus, estimates were dependent on pellet diameter and resistance to fragmentation prior to ingestion. To test for the presence of feeding hierarchies in groups of lobsters an experiment was conducted to examine the effect of ration level (high ration: 2.0% BW day⁻¹ and low ration: 0.2% BW day⁻¹) on intra-individual (measured as coefficient of variation for feed intake: CV_C) and inter-individual variation (measured as mean share of the group meal: MSM%). There was a significant (P<0.05) negative correlation between CV_C and MSM% for the low ration treatments which indicated that small lobsters (CV_C=114±19% and MSM%=22±10%: mean±S.D.) were less successful than large lobsters (CV_C 44±11% and MSM%=41±13%: mean±S.D.) at obtaining feed. This may be indicative of the formation of feeding hierarchies.     

Effects of feeding level and NDF content of grass-clover silages on chewing activity,fecal particle size and NDF digestibility in dairy heifers

The objective of this study was to assess effects of feed intake and NDF content of highly digestible grass-clover silage on chewing behavior, fecal particle size distribution and apparent digestibility in restrictively fed heifers. Four grass-clover silages (Lolium perenne, Trifolium pratense and Trifolium repens) were harvested in 2009 at different regrowth stages, resulting in silages with NDF contents of 312, 360, 371 and 446 g/kg dry matter (DM), respectively. Four rumen-fistulated Jersey heifers (343±32 kg BW) were fed silage at 90% of ad libitum levels in a 4×4 Latin square design, replicated with further restricted feeding levels (50%, 60%, 70% or 80% of ad libitum) in a balanced 4×4×4 Greco-Latin square design. Eating activity was estimated from test meal observations, while rumination activity was estimated from jaw movements logged by a jaw recorder system. Total tract digestibility was estimated from chromic oxide marker and fecal spot sampling, and fecal particle size distribution in washed and freeze-dried particulate DM was determined by dry sieving (2.36, 1.0, 0.5, 0.212 and 0.106 mm, and bottom bowl). Higher NDF content of silage stimulated longer eating time per kg DM intake (P<0.001), while reduced feeding level caused a reduction in eating time per kg DM intake (P<0.001) and NDF (P<0.001). Rumination time per kg DM intake (P<0.01) increased with reduced feeding level, with less effect of feeding level at lower NDF contents (P<0.01) and more rumination with greater NDF content (P<0.01). Relative to NDF intake, rumination time increased with greater NDF content (P<0.01), at a higher rate with reduced feeding level (P<0.05). Digestibility of potentially digestible NDF (DNDF) decreased with greater NDF content (P<0.001) and increased with reduced feeding level (P<0.05). Increasing NDF content resulted in more particulate DM in feces (g/kg DM; P<0.05) and larger mean particle size (P<0.001). In conclusion, feeding heifers with grass-clover silages of decreasing NDF content increased chewing time relative to NDF intake, reduced mean fecal particle size, and increased DNDF digestibility. Restricting feeding level made heifers eat for a shorter time period while rumination and total chewing was increased, causing the ratio between eating and rumination time to decrease with lower intake of forage fiber. Particle size reduction and digestibility depended mostly on changes in NDF content, especially the indigestible NDF content.     

The effect of dietary inclusion of starch,sucrose and xylose on the utilization of dietary energy in sheep

A calorimetric experiment of 4 × 4 Latin square design was undertaken to study the effect of sugar-beet pulp (SBP), maize starch, sucrose and xylose on energy metabolism in sheep. The four diets comprised a diet (A) of dried grass, soya-bean meal and SBP (450, 50 and 500 g kg⁻¹ on dry matter (DM) basis) and corresponding diets in which 400 g kg⁻¹ of SBP was replaced by maize starch (B), sucrose (C) or xylose (D); all diets were offered at a level of 600 g DM day⁻¹. After the Latin square was completed, energy value of the basal diet of dried grass and soya-bean meal (900 and 100 g kg⁻¹ DM; 600 g day⁻¹) was determined in the same four sheep.Treatment differences in organic matter, gross energy, nitrogen (N) and neutral detergent fibre (NDF) digestibility were non-significant. Differences in N retention were not significant.Digestible energy (DE) contents (MJ kg⁻¹ DM) were 13.27, 13.22, 13.21 and 13.21 MJ kg⁻¹ for diets A, B, C and D, respectively. Energy loss in methane was higher (P < 0.05) for Diet A than for other diets. Metabolizable energy (ME) contents for the diets A-D were 11.25, 11.22, 11.32 and 11.40 MJ kg⁻¹ DM, respectively. Metabolizability (q) of the diets averaged 0.642 and was not significantly affected by the diet given. There was a trend for heat production to be higher in sheep given the sucrose-containing diet (C) than in those given other diets (6.34 versus 6.04 MJ day⁻¹) and as a result, energy retention was lower (0.38 versus 0.64 MJ day⁻¹), but the difference did not reach statistical difference. Efficiencies of utilization of ME for maintenance and fattening (k_mf) averaged 0.67 and were in good agreement with those predicted from equations of the Agricultural Research Council (1980) excepting the lower k_mf (0.63) for Diet C.The mean ME content of SBP calculated by difference was 13.05 MJ kg⁻¹ DM and the corresponding values for mixtures of SBP + starch, SBP + sucrose and SBP + xylose (600 and 400 g kg⁻¹ DM) were 12.98, 13.16 and 13.36 MJ kg⁻¹ DM, respectively.     

The effect of rabbit age on in vitro caecal fermentation of starch, pectin, xylan, cellulose, compound feed and its fibre

In vitro gas production kinetics of six different substrates, pectin (PEC), xylan (XYL), starch (STA), cellulose (CEL), commercial compound feed (FEED; 201 g crude protein per kg, 155 g crude fibre per kg, 334 g neutral-detergent fibre (NDF) per kg and 190 g acid-detergent fibre (ADF) per kg) and an NDF prepared from commercial compound feed (NDF_FEED) were determined using the caecum contents of weaned rabbits (36 days of age) and of rabbits at slaughter age (78 days of age) as inoculums. The cumulated gas production over 96 h of incubation was modelled with Gompertz model, and the kinetic parameters compared. The total potential gas production (parameter ‘B’ of the Gompertz model) was not affected (P>0.05) by the inoculum source, except with STA, where rabbits at slaughter weight had significantly higher total potential fermentability (314 ml/g dry matter (DM)) than those at weaning age (189 ml/g DM). Intensities of fermentation (maximum fermentation rate; MFR) of PEC (32.2 ml/h) and XYL (24.4 ml/h) were significantly greater in rabbits at weaning, while that of STA (45 ml/h) was significantly lower than at slaughter age (23.0, 14.3 and 14.0 ml/h for PEC, XYL and STA, respectively). The MFRs of CEL and NDF_FEED were very similar between inoculum sources. In the first 10 h of fermentation which correspond to the normal retention time of the substrates in the caecum, the highest amount of gas was produced from PEC, followed by FEED and XYL. These substrates had a time of maximum fermentation rate (TMFR) at both rabbit ages short enough (8.0 and 9.5 h for PEC, 9.5 and 6.6 h for FEED, 13.7 and 14.2 h for XYL at weaning and at slaughter age, respectively) to be almost completely fermented in vivo.     

Evaluation of physical structure value in spring-harvested grass/clover silage and hay fed to heifers

The physical structure value of conserved grass/clover forages of spring harvest was evaluated by assessing effects of harvest time, conservation method, iNDF/NDF ratio and NDF intake (NDFI) per kg BW on chewing activity and fecal particle size in dairy heifers. A mixed sward consisting of ryegrass (Lolium perenne), red clover (Trifolium pratense) and white clover (Trifolium repens) was harvested in 2009 on May 9 (early) and 25 (late), and both cuts were conserved as silage and hay. The early silage, early hay, late silage and late hay contained dry matter (DM) of 454, 842, 250 and 828 g/kg, and NDF of 315, 436, 414 and 503 g/kg DM, respectively. Forages were fed as sole feed to four Jersey heifers of 435±30 kg BW in a 4×4 Latin square experiment. Feeding level was 90% of individual ad libitum intake, divided equally across two daily meals offered at 0800 and 1530 h. Chewing activity was estimated from recorded jaw movements (JM) oscillations continuously logged for 96 h and summarized per 24 h as mean effective rumination time and eating time. Eating behavior was further observed during four 20-min test meals. Weight proportion of large feces particles (>1.0 mm) and geometric mean fecal particle size (GPS) were calculated. Potentially indigestible NDF (iNDF) was estimated by incubation for 288 h in situ. The daily DM intake (DMI) decreased with progressing maturity at harvest (P<0.001) while daily NDFI was unaffected by harvest time (P>0.05). Earlier harvest led to less rumination per kg NDFI (P<0.01), similar eating time per kg NDFI (P>0.05) and similar proportion of large particles (P>0.01) compared with later harvest. Rumination time per kg NDFI decreased with higher NDFI per kg BW (P<0.001) and with lower iNDF/NDF ratio (P<0.01). Content and potential digestibility of NDF was greater in hay than in silage from the same harvest probably due to field loss and therefore confounded effects of conservation method. This study of high digestibility grass/clover silage and hay showed that NDF content and NDFI per kg BW affect fecal particle size and rumination time per kg NDF, and suggests implementation of NDFI per kg BW in systems evaluating physical structure in diets.     

Effect of feeding long or short wheat hay v. wheat silage in the ration of lactating cows on intake,milk production and digestibility

The objective of this study was to evaluate in lactating cows the effect of either chopping or ensiling of wheat roughage on: intake, digestibility, lactation performance and animal behavior. Three groups of 14 lactating cows each, were fed total mixed rations (TMRs) based on either long wheat hay (HL), short wheat hay (HS) or wheat silage (SI), as the sole roughage source (30% of TMR dry matter (DM)). Parameters examined: sorting behavior, DM intake, milk yield and composition, rumination, recumbence, average daily rumen pH, digesta passage rate, and in-vivo digestibility. Performance data was summarized by day and analyzed using a proc-mixed model. The content of physically effective neutral detergent fiber (peNDF) was similar in the HL and SI and lower in the HS, resulting in similar differences among the three corresponding TMRs. In vitro DM digestibility of wheat silage was higher than that of the two hays (65.6% v. 62.8%) resulting in higher in vitro DM digestibility of the SI-TMR compared with the hay-based TMRs (79.3 v. 77.0%). HS-TMR was better than HL- or SI-TMRs at preventing feed sorting by cows after 12 or 24 h eating of the diets. Cows fed HS-TMR consumed more DM and NDF but less peNDF than the other two groups. Average daily rumen pH was similar in the three groups, but daily rumination time was highest in the cows fed HS-TMR. Rumen retention time was longest in cows fed HL-TMR. DM digestibility in cows fed SI-TMR was higher than that of HS and HL groups (65.2%, 61.8% and 62.4%, respectively), but NDF digestibility was similar in the three treatments. The highest intake of digestible DM was observed in cows fed SI-TMR, HS cows were intermediate and HL cows were the lowest. Consequently, cows fed SI-TMR had higher yields of milk, 4% fat corrected milk and energy-corrected milk (47.1, 42.9 and 43.2 kg/day, respectively) than cows fed HS-TMR (45.7, 41.0 and 41.0 kg/day, respectively) or HL-TMR (44.1, 40.3 and 40.3 kg/day, respectively). Net energy production (NE_L+M+gain) per kg DM intake was highest in the SI-TMR, lowest in the HS-TMR and intermediate in the HL-TMR (1.52, 1.40 and 1.45, respectively). Animal welfare, as expressed in daily recumbence time and BW gain was similar in the SI and HS groups and higher than the HL cows.     

Ionisations within a subtilisin–glyoxal inhibitor complex

Z-Ala-Pro-Phe-glyoxal (where Z is benzyloxycarbonyl) has been shown to be a competitive inhibitor of subtilisin with a K_i=2.3±0.2 μM at pH 7.0 and 25 °C. Using Z-Ala-Pro-[2-¹³C]Phe-glyoxal we have detected a signal at 107.3 ppm by ¹³C NMR, which we assign to the tetrahedral adduct formed between the hydroxy group of serine-195 and the ¹³C-enriched keto-carbon of the inhibitor. The chemical shift of this signal is pH independent from pH 4.2 to 7.0 and we conclude that the oxyanion pK_a<3. This is the first observation of oxyanion formation in a reversible subtilisin–inhibitor complex. The inhibitor is bound as a hemiketal which is in slow exchange with the free inhibitor. Inhibitor binding depends on a pK_a of ～6.5 in the free enzyme and on a pK_a<3.0 when the inhibitor is bound to subtilisin. Protonation of the oxyanion promotes the disassociation of the inhibitor. We show that oxyanion formation cannot be rate limiting during catalysis and that subtilisin stabilises the oxyanion by at least 45.1 kJ mol^?1. We conclude that if the energy required for oxyanion stabilisation is utilised as binding energy in drug design it should make a significant contribution to inhibitor potency.     

An alternative oven method combined with different detergent strengths in the analysis of neutral detergent fibre

Two methods for preparing neutral detergent fibre (NDF) – the standard method of Van Soest, P.J., Robertson, G.B., Lewis, B.A., `Symposium: Carbohydrate methodology, metabolism, and nutritional implications in dairy cattle.' J. Dairy Sci. 74 3583–3597 (1991), and an alternative 16 h oven method at 90°C. Neutral detergent solution (100%) was compared with solutions diluted to 50, 25 and 0% of normal strength. Sodium sulfite and a heat-stable amylase were used in all treatments. Forty-four samples consisting of 11 dried and ensiled forages, three straws, nine starchy concentrates, 10 high-protein concentrate feeds, three samples of tropical browse leaves and eight manures from both ruminants and non-ruminants were used. The 0% treatment was the only treatment giving significantly higher NDF values compared to the others (p<0.05). Sensitivity to detergent strength seemed to be sample-specific in the high protein group as meat meal and maize gluten had higher NDF values at reduced detergent strengths. We recommend using the oven method and a detergent strength of 25% of the current standard in combination with sodium sulfite for low protein feeds. For starchy feeds a heat-stable amylase should be used and high lipid samples should be acetone-extracted before isolating NDF. For high protein feed samples only the standard method can yet be recommended. The oven method is labour-saving and more convenient and arguments for reduced detergent strength suggest lower cost and reduced waste water pollution.