W.F. Pellikaan

Comparative in vitro fermentation activity in the canine distal gastrointestinal tract and fermentation kinetics of fiber sources

The current study aimed to evaluate the variation in fermentation activity along the distal canine gastrointestinal tract (GIT, Exp. 1). It also aimed to assess fermentation kinetics and end product profiles of 16 dietary fibers for dog foods using canine fecal inoculum (Exp. 2). For Exp. 1, digesta were collected from the distal ileum, proximal colon, transverse colon, and rectum of 3 adult dogs. Digesta per part of the GIT were pooled for 3 dogs, diluted (1:25, wt/vol), mixed, and filtered for the preparation of inoculum. A fructan, ground soy hulls, and native potato starch were used as substrates and incubated for cumulative gas production measurement as an indicator of the kinetics of fermentation. In addition, fermentation bottles with similar contents were incubated but were allowed to release their gas throughout incubation. Fermentation fluid was sampled at 4, 8, 12, 24, 48, and 72 h after initiation of incubation, and short-chain fatty acids and ammonia were measured. Results showed comparable maximal fermentation rates for rectal and proximal colonic inocula (P > 0.05). Production of short-chain fatty acids was least for the ileal and greatest for the rectal inoculum (P < 0.05). Therefore, for in vitro studies, fecal microbiota can be used as an inoculum source but may slightly overestimate in vivo fermentation. Experiment 2 evaluated the gas production, fermentation kinetics, and end product profiles at 8 and 72 h of incubation for citrus pectin, 3 fructans, gum arabic, 3 guar gums, pea fiber, peanut hulls, soy fiber, sugar beet fiber, sugar beet pectin, sugar beet pulp, wheat fiber, and wheat middlings. Feces of 4 adult dogs were used as an inoculum source. Similar techniques were used as in Exp. 1 except for the dilution factor used (1:10, wt/vol). Among substrates, large variations in fermentation kinetics and end product profiles were noted. Sugar beet pectin, the fructans, and the gums were rapidly fermentable, indicated by a greater maximal rate of gas production (R(max)) compared with all other substrates (P < 0.05), whereas peanut hulls and wheat fiber were poorly fermentable, indicated by the least amount of gas produced (P < 0.05). Sugar beet fiber, sugar beet pulp, soy fiber, and wheat middlings were moderately fermentable with a low R(max). Citrus pectin and pea fiber showed a similar low R(max), but time at which this occurred was later compared with sugar beet fiber, sugar beet pulp, soy fiber, and wheat middlings (P < 0.05). Results of this study can be used to formulate canine diets that stimulate dietary fiber fermentation along the distal GIT that may optimize GIT health and stimulate the level of satiety in dogs.

The production of intrinsically labeled milk and meat protein is feasible and provides functional tools for human nutrition research

Administration of labeled, free amino acids does not allow direct assessment of in vivo dietary protein digestion and absorption kinetics. Consequently, dietary protein sources with labeled amino acids incorporated within their protein matrix are required. The aim of the present study was to produce intrinsically L-[1-(13)C]phenylalanine-labeled milk and meat protein that would permit in vivo assessment of postprandial protein digestion and absorption kinetics in humans. One lactating dairy cow was continuously infused with 420 μmol of L-[1-(13)C]phenylalanine/min for 96 h, with plasma and milk being collected before, during, and after isotope infusion. Twenty-four hours after infusion, the cow was slaughtered to produce intrinsically labeled meat. Levels of L-[1-(13)C]phenylalanine enrichment as high as 40 mole percent excess (MPE) in milk and 1.5 MPE in meat protein were achieved. In a subsequent human proof-of-principle experiment, 2 healthy young males (25±1 yr; 66.2±5.2 kg) each ingested 135 g of L-[1-(13)C]phenylalanine intrinsically labeled minced beef, after which plasma samples were collected at regular time intervals. Plasma L-[1-(13)C]phenylalanine enrichments increased during the first 90 min following beef ingestion, reaching peak plasma enrichment levels of 0.61±0.04 MPE. Whole-body net protein balance, assessed by continuous infusion of L-[ring-(2)H(5)]phenylalanine and L-[ring-(2)H(2)]tyrosine, was higher in the postprandial period compared with basal values (6.4±0.1 vs. -4.5±0.1 μmol/kg per h). In conclusion, the production of intrinsically L-[1-(13)C]phenylalanine-labeled milk and meat protein is feasible and provides functional tools to investigate in vivo protein digestion and absorption kinetics in humans.

Effects of dietary starch content and rate of fermentation on methane production in lactating dairy cows

The objective of this study was to investigate the effects of starch varying in rate of fermentation and level of inclusion in the diet in exchange for fiber on methane (CH4) production of dairy cows. Forty Holstein-Friesian lactating dairy cows of which 16 were rumen cannulated were grouped in 10 blocks of 4 cows each. Cows received diets consisting of 60% grass silage and 40% concentrate (dry matter basis). Cows within block were randomly assigned to 1 of 4 different diets composed of concentrates that varied in rate of starch fermentation [slowly (S) vs. rapidly (R) rumen fermentable; native vs. gelatinized corn grain] and level of starch (low vs. high; 270 vs. 530g/kg of concentrate dry matter). Results of rumen in situ incubations confirmed that the fractional rate of degradation of starch was higher for R than S starch. Effective rumen degradability of organic matter was higher for high than low starch and also higher for R than S starch. Increased level of starch, but not starch fermentability, decreased dry matter intake and daily CH4 production. Milk yield (mean 24.0±1.02kg/d), milk fat content (mean 5.05±0.16%), and milk protein content (mean 3.64±0.05%) did not differ between diets. Methane expressed per kilogram of fat- and protein-corrected milk, per kilogram of dry matter intake, or as a fraction of gross energy intake did not differ between diets. Methane expressed per kilogram of estimated rumen-fermentable organic matter (eRFOM) was higher for S than R starch-based diets (47.4 vs. 42.6g/kg of eRFOM) and for low than high starch-based diets (46.9 vs. 43.1g/kg of eRFOM). Apparent total-tract digestibility of neutral detergent fiber and crude protein were not affected by diets, but starch digestibility was higher for diets based on R starch (97.2%) compared with S starch (95.5%). Both total volatile fatty acid concentration (109.2 vs. 97.5mM) and propionate proportion (16.5 vs. 15.8mol/100mol) were higher for R starch- compared with S starch-based diets but unaffected by the level of starch. Total N excretion in feces plus urine and N retained were unaffected by dietary treatments, and similarly energy intake and output of energy in milk expressed per unit of metabolic body weight were not affected by treatments. In conclusion, an increased rate of starch fermentation and increased level of starch in the diet of dairy cattle reduced CH4 produced per unit of eRFOM but did not affect CH4 production per unit of feed dry matter intake or per unit of milk produced.

Substantial Differences between Organ and Muscle Specific Tracer Incorporation Rates in a Lactating Dairy Cow

We aimed to produce intrinsically L-[1-13C]phenylalanine labeled milk and beef for subsequent use in human nutrition research. The collection of the various organ tissues after slaughter allowed for us to gain insight into the dynamics of tissue protein turnover in vivo in a lactating dairy cow. One lactating dairy cow received a constant infusion of L-[1-13C]phenylalanine (450 µmol/min) for 96 h. Plasma and milk were collected prior to, during, and after the stable isotope infusion. Twenty-four hours after cessation of the infusion the cow was slaughtered. The meat and samples of the various organ tissues (liver, heart, lung, udder, kidney, rumen, small intestine, and colon) were collected and stored. Approximately 210 kg of intrinsically labeled beef (bone and fat free) with an average L-[1-13C]phenylalanine enrichment of 1.8±0.1 mole percent excess (MPE) was obtained. The various organ tissues differed substantially in L-[1-13C]phenylalanine enrichments in the tissue protein bound pool, the highest enrichment levels were achieved in the kidney (11.7 MPE) and the lowest enrichment levels in the skeletal muscle tissue protein of the cow (between 1.5–2.4 MPE). The estimated protein synthesis rates of the various organ tissues should be regarded as underestimates, particularly for the organs with the higher turnover rates and high secretory activity, due to the lengthened (96 h) measurement period necessary for the production of the intrinsically labeled beef. Our data demonstrates that there are relatively small differences in L-[1-13C]phenylalanine enrichments between the various meat cuts, but substantial higher enrichment values are observed in the various organ tissues. We conclude that protein turnover rates of various organs are much higher when compared to skeletal muscle protein turnover rates in large lactating ruminants.

Evaluation of n-alkanes and their carbon isotope enrichments (δ 13 C) as diet composition markers

Plant cuticular n-alkanes have been successfully used as markers to estimate diet composition and intake of grazing herbivores. However, additional markers may be required under grazing conditions in botanically diverse vegetation. This study was conducted to describe the n-alkane profiles and the carbon isotope enrichment of n-alkanes of common plant species from the Mid Rift Valley rangelands of Ethiopia, and evaluate their potential use as nutritional markers. A total of 23 plant species were collected and analysed for long-chain n-alkanes ranging from heptacosane to hexatriacontane (C(27) to C(36)), as well as their carbon isotopic ratio ((13)C/(12)C). The analysis was conducted by gas chromatography/combustion isotope ratio mass spectrometry following saponification, extraction and purification. The isotopic composition of the n-alkanes is reported in the delta notation (δ(13)C) relative to the Vienna Pee Dee Belemnite standard. The dominant n-alkanes in the species were C(31) (mean ± s.d., 283 ± 246 mg/kg dry matter) and C(33) (149 ± 98 mg/kg dry matter). The carbon isotopic enrichment of the n-alkanes ranged from -19.37‰ to -37.40‰. Principal component analysis was used to examine interspecies differences based on n-alkane profiles and the carbon isotopic enrichments of individual n-alkanes. Large variability among the pasture species was observed. The first three principal components explained most of the interspecies variances. Comparison of the principal component scores using orthogonal procrustes rotation indicated that about 0.84 of the interspecies variances explained by the two types of data sets were independent of each other, suggesting that the use of a combination of the two markers can improve diet composition estimations. It was concluded that, while the n-alkane profile of the pasture species remains a useful marker for use in the study region, the δ(13)C values of n-alkanes can provide additional information in discriminating diet components of grazing animals.

δ 13C 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.

Passage kinetics of 13C-labeled corn silage components through the gastrointestinal tract of dairy cows

Fractional passage rates form a fundamental element within modern feed evaluation systems for ruminants but knowledge on feed type and feed component specific passage rates are largely lacking. This study describes the use of carbon stable isotopes ((13)C) to assess component-specific passage kinetics of 6 intrinsically (13)C-labeled corn silages varying in quality (2 cultivars×3 maturity stages) in a 6×6 Latin square design using 6 rumen-fistulated lactating dairy cows. An increase in maturity increased starch and decreased neutral detergent fiber and acid detergent fiber contents of corn silages. Passage kinetics were assessed for an external (chromium mordanted fiber; Cr-NDF) and an internal marker ((13)C isotopes) collected in feces and omasal digesta. The best fit was obtained with a deterministic multicompartmental model compared with stochastic Gn and GnG1 models with increasing order of age dependency (n=1 to 5) for both sampling sites. The Cr-NDF marker yielded higher rumen fractional passage rates (K1) than did (13)C in the dry matter ((13)CDM) in feces (0.042/h vs. 0.023/h). Omasal marker excretion patterns support the conclusions based on conventional fecal marker excretions. Component-specific passage was assessed for acid detergent fiber ((13)CADF) in feces and for starch ((13)CST) in omasal digesta. The fractional passage rate based on fecal (13)CDM and (13)CADF did not differ. Omasal (13)CST provided higher K1 values (0.042/h) than omasal (13)CDM (0.034/h) but lower values than omasal Cr-NDF (0.051/h). Fractional passage rates from the proximal colon-cecum (K2) based on fecal marker concentrations showed trends similar to K1, with Cr-NDF providing a value (0.425/h) more than twice as high as that of (13)CDM (0.179/h) and (13)CADF (0.128/h). Total mean retention time in the gastrointestinal tract was approximately double for (13)CDM (64.1h) and (13)CADF (77.6h) compared with Cr-NDF (36.4h). Corn silage quality did not affect any of the estimated passage kinetic parameters. In situ fractional degradation rates did not differ among corn silages, except for a decreased fractional degradation rate of starch with advancing maturity. Results indicate that isotope labeling allows assessment of component-specific passage kinetics of carbohydrate fractions in corn silage.

Effects of preservation conditions of canine feces on in vitro gas production kinetics and fermentation end products

This study investigated the effect of chilling and freezing (for 24 h) canine feces on in vitro gas production kinetics and fermentation end product profiles from carbohydrate-rich (in vitro run 1) and protein-rich (in vitro run 2) substrates. Feces were collected from 3 adult retriever-type dogs fed a canned diet for at least 2 wk. Each fecal sample was divided into 3 portions: 1 portion was used immediately as an inoculum (fresh) and the other 2 portions were used after either chilling to 5°C for 30 min and storage in crushed ice for 23.5 h (chilling) or freezing to -20°C for 30 min and storage in a prefrozen (-20°C) container for 23.5 h (freezing). The medium solution for run 1 contained N whereas that for run 2 was N free. Substrates included fructooligosaccharide (FOS), sugar beet pulp, and wheat middlings in run 1 and soybean meal, poultry meat meal, and feather meal in run 2. Gas production kinetics were calculated from cumulative gas production data measured for 72 h. After incubation, fermentation liquids were analyzed for short-chain fatty acids, NH3, and aromatic compounds. For both in vitro runs, chilling feces did not affect gas production kinetics and end product profiles of substrates compared with inocula from fresh feces. Freezing feces decreased the maximum rate of gas production in phase 2 for FOS (P<0.001) and across substrates increased gas produced (P≤0.005) and time of maximum gas production in phase 2 (P<0.001). Furthermore, compared with fresh fecal inocula, inocula from frozen feces resulted in increased overall indole concentrations in run 1 (P=0.006) and indole concentrations from soybean meal and poultry meat meal in run 2 (P<0.001). In run 2, phenol concentrations were greater (P=0.015) for frozen feces than for fresh feces (P=0.015). In conclusion, freezing canine feces for 24 h slightly altered fermentative characteristics of fecal inoculum whereas chilling feces in crushed ice for 24 h maintained fermentative characteristics. Chilling feces in crushed ice is a practical method to preserve feces during transport between laboratories within 24 h for in vitro fermentation studies evaluating dietary ingredients.

Changes in fatty acid content and composition in silage maize during grain filling.

BACKGROUND The stage of maturity at harvest has a major effect on the fatty acid (FA) content and composition of forage plants consumed by dairy cows. The present study investigated the dynamics of FA content and composition in stover (leaves and stem) and ears (cob, shank and husks) of two maize genotypes (G2 and G6) grown on sandy and clay soils and harvested at 14, 42, 56, 70 and 84 days after flowering (DAF). In addition, the FA content and composition of six maize genotypes (G1-G6) grown on the two soil types were compared at the normal harvest time of early genotypes in the Netherlands (70 DAF). RESULTS The contents of total FAs and major individual FAs in both stover and ears changed significantly (P < 0.001) during the grain-filling period (14-84 DAF). In stover the contents of C16:0, C18:2, C18:3 and total FAs declined (P < 0.001) while those of C18:0 and C18:1 increased (P < 0.001) with progressive grain filling. The rate of decline in C18:3 and total FA contents was slower during 14-56 DAF as compared with 56-84 DAF. In ears, the contents of C16:0, C18:1, C18:2 and total FAs increased up to 56 DAF and then remained more or less constant until 84 DAF. At 70 DAF the content of polyunsaturated fatty acids (PUFAs) in both stover and ears did not differ among the six genotypes. However, the average contents of C16:0, C18:3 and total FAs in stover were higher (P < 0.05) on clay soil, whereas those of C18:0 and C18:1 were higher on sandy soil. CONCLUSION The results demonstrate that the maximum PUFA content in silage maize is harvested around 56 DAF, in the present study at a T(sum) of 927 °C.d or at an ear dry matter content of 440 g kg(-1) , which is before the onset of rapid senescence. Any further delay in harvesting will cause a rapid decline in C18:3 content in maize silages.

Structural features of condensed tannins affect in vitro ruminal methane production and fermentation characteristics

An in vitro study was conducted to investigate the effects of condensed tannin (CT) structural properties, i.e. average polymer size (or mean degree of polymerization), percentage of cis flavan-3-ols and percentage of prodelphinidins in CT extracts on methane (CH 4 ) production and fermentation characteristics. Condensed tannins were extracted from eight plants in order to obtain different CT types: blackcurrant leaves, goat willow leaves, goat willow twigs, pine bark, redcurrant leaves, sainfoin plants, weeping willow catkins and white clover flowers. They were analysed for CT content and CT composition by thiolytic degradation, followed by high performance liquid chromatography (HPLC) analysis. Grass silage was used as a control substrate. Condensed tannins were added to the substrate at a concentration of 40 g/kg, with or without polyethylene glycol (+ or −PEG 6000 treatment) to inactivate tannins, then incubated for 72 h in mixed buffered rumen fluid from three different lactating dairy cows per run. Total cumulative gas production (GP) was measured by an automated GP system. During the incubation, 12 gas samples (10 µ l) were collected from each bottle headspace at 0, 2, 4, 6, 8, 12, 24, 30, 36, 48, 56 and 72 h of incubation and analysed for CH 4 . A modified Michaelis-Menten model was fitted to the CH 4 concentration patterns and model estimates were used to calculate total cumulative CH 4 production (GP CH4 ). Total cumulative GP and GP CH4 curves were fitted using biphasic and monophasic modified Michaelis-Menten models, respectively. Addition of PEG increased GP, GP CH4 , and CH 4 concentration compared with the −PEG treatment. All CT types reduced GP CH4 and CH 4 concentration. All CT increased the half time of GP and GP CH4 . Moreover, all CT decreased the maximum rate of fermentation for GP CH4 and rate of substrate degradation. The correlation between CT structure and GP CH4 and fermentation characteristics showed that the proportion of prodelphinidins within CT had the largest effect on fermentation characteristics, followed by average polymer size and percentage of cis flavan-3-ols.