M. Rodehutscord

Linear regression approach to study amino acid digestibility in broiler chickens

1. An experiment was conducted to investigate whether a linear regression approach is a suitable tool for determining the amino acid (AA) digestibility up to the terminal ileum of broiler chickens. Solvent-extracted rapeseed meal (RSM) was used as the model ingredient. 2. Ten diets with 5 different inclusion rates of RSM (60, 120, 180, 240 and 300 g/kg, corresponding to crude protein concentrations from 170 to 250 g/kg in the diet), each without or with a supplementation of phytase (500 U/kg), were fed ad libitum to broiler chickens between 14 and 21 d of age. Seven pens of 12 chickens were allocated to each treatment. Digesta were sampled on a pen basis from the section of the gastrointestinal tract between Meckels diverticulum and 2 cm anterior to the ileo-caeco-colonic junction. Titanium dioxide was included as an indigestible marker. 3. The amounts of crude protein and AAs digested up to the terminal ileum constantly increased with increasing AA intake over the entire range of intakes. When the amount of an AA digested at the terminal ileum is linearly regressed against its intake, the deviation of the slope from 1 is caused by both the unabsorbed AA from RSM and from specific endogenous losses related to RSM. These slopes varied between 0·68 and 0·88 for individual AAs, and the slopes were unaffected by phytase supplementation. 4. It is suggested that a linear regression approach be adopted to study the AA digestibility of raw materials in chickens. Digestibility determined this way does not need any correction for basal endogenous loss.

Studies on the intestine section to be sampled in broiler studies on precaecal amino acid digestibility

Abstract Measurements of precaecal amino acid digestibility with digesta sampled from slaughtered animals may be affected by the chosen length of the sampled section. The length needs standardization, therefore, when digestibility is understood to be a measure of feedstuff potential. It was our objective to study the change in the net disappearance of amino acids from the lower small intestine of broiler chicken. The section between Meckels diverticulum and 2 cm anterior of the ileo-caeco-colonic junction was cut into three subsections of equal length: proximal, medial, and terminal. The contents of each subsection were pooled within the birds of each pen (12 in Experiment 1 and 10 in Experiment 2). TiO2 was used as an indigestible marker. Prior to digesta sampling, broilers had been fed the experimental diets for seven days. In Experiment 1, two diets with either soybean meal or a mix of soybean meal and peas as the main protein sources were used. Each diet was allocated to eight pens and feeding commenced on day 14 of age. Net disappearance was significantly affected by diet only in regard to aspartic acid and methionine. No significant interaction between diet and subsection occurred. Net disappearance was significantly affected by subsection for all amino acids. It ranged from 74 – 92% for individual amino acids without significant differences in the medial and terminal subsections. Net disappearance was, however, between 3% and 9% lower in the proximal subsection. In Experiment 2, diets contained soybean meal as the main protein source and were given to 18 pens from day 22 of age. Again, the effect of subsection on net disappearance was significant for all amino acids. Net disappearance was significantly lower in the proximal than in the middle subsection, and differences ranged from 5 – 10%. Significant differences in the net disappearance were also found for most of the amino acids between the middle and the terminal subsection ranging from 2 – 4%. In conclusion, when precaecal amino acid digestibility should be used as a measure for a protein sources potential, digesta sampling should not consider the proximal third of the section between Meckels diverticulum and the end of the ileum.

Effect of variable intake of alfalfa and wheat on faecal alkane recoveries and estimates of roughage intake in sheep

The alkane technique allows estimates of intake and diet composition. For the latter a correction for incomplete recovery of faecal alkane concentrations has to be made. Feeding level and diet proportions have been discussed as factors possibly influencing recovery. A balance trial was conducted to study these effects on faecal alkane recovery rates and their consequences on estimates of roughage intake in sheep. The diets consisted of force-dried pelleted alfalfa meal (A) and wheat whole meal (W) in three levels each (360, 480, 600 and 270, 360, 450 g/day, respectively); the wheat was partly labelled with beeswax to provide a distinctive alkane pattern. Each diet was fed to four wethers. Despite significant effects (P<0.05) of A and W on organic matter digestibility, no such effects were observed for alkane recovery rates. Estimates of alfalfa intake were better if alkanes with a higher coefficient of variation of recovery were not included in diet composition estimates. Furthermore, in spite of the lack of significance of the dietary factors A, W and A x W, faecal alkane concentration correction based upon diet-specific faecal recoveries gave better estimates of alfalfa intake than based on overall recoveries. The estimate of alfalfa intake differed from known intake by -1.7 to -2.6 %, depending on the method of estimation. The relative mean discrepancy was regarded as a tool to assess estimated intakes of individual animals. It ranged from 4.4 to 14.2% for dietary treatments and was 7.6 % for all animals. The poor quality of estimates for individual animals may result from the variation of faecal recovery rates and needs further investigation.

Variation in chemical composition and physical characteristics of cereal grains from different genotypes

ABSTRACT Genotypes of cereal grains, including winter barley (n = 21), maize (n = 27), oats (n = 14), winter rye (n = 22), winter triticale (n = 21) and winter wheat (n = 29), were assayed for their chemical composition and physical characteristics as part of the collaborative research project referred to as GrainUp. Genotypes of one grain species were grown on the same site, except maize. In general, concentrations of proximate nutrients were not largely different from feed tables. The coefficient of variation (CV) for the ether extract concentration of maize was high because the data pool comprised speciality maize bred for its high oil content. A subset of 8 barley, 20 rye, 20 triticale and 20 wheat samples was analysed to differ significantly in several carbohydrate fractions. Gross energy concentration of cereal grains could be predicted from proximate nutrient concentration with good accuracy. The mean lysine concentration of protein was the highest in oats (4.2 g/16 g N) and the lowest in wheat (2.7 g/16 g N). Significant differences were also detected in the concentrations of macro elements as well as iron, manganese, zinc and copper. Concentrations of arsenic, cadmium and lead were below the limit of detection. The concentration of lower inositol phosphates was low, but some inositol pentaphosphates were detected in all grains. In barley, relatively high inositol tetraphosphate concentration also was found. Intrinsic phytase activity was the highest in rye, followed by triticale, wheat, barley and maize, and it was not detectable in oats. Substantial differences were seen in the thousand seed weight, test weight, falling number and extract viscoelasticity characteristics. The study is a comprehensive overview of the composition of different cereal grain genotypes when grown on the same location. The relevance of the variation in composition for digestibility in different animal species will be subject of other communications.

Hydrolysis of phytate and formation of inositol phosphate isomers without or with supplemented phytases in different segments of the digestive tract of broilers.

The objective was to characterise degradation of myo-inositol 1,2,3,4,5,6-hexakis (dihydrogen phosphate) (InsP6) and formation of inositol phosphate (InsP) isomers in different segments of the broiler digestive tract. Influence of an Aspergillus niger (PhyA) and two Escherichia coli-derived (PhyE1 and PhyE2) phytases was also investigated. A total of 600 16-d-old broilers were allocated to forty floor pens (ten pens per treatment). Low-P (5·2 g/kg DM) maize–soyabean meal-based diets were fed without (basal diet; BD) or with a phytase added. On day 25, digesta from different digestive tract segments were pooled per segment on a pen-basis, freeze-dried and analysed for P, InsP isomers and the marker TiO2. InsP6 degradation until the lower ileum (74 %) in BD-fed birds showed a high potential of broilers and their gut microbiota to hydrolyse InsP6 in low-P diets. Different InsP patterns in different gut segments suggested the involvement of phosphatases of different origin. Supplemented phytases increased InsP6 hydrolysis in the crop (P < 0·01) but not in the lower ileum. Measurements in the crop and proventriculus/gizzard confirmed published in vitro degradation pathways of 3- and 6-phytases for the first time. In the intestinal segments, specifically formed InsP4–5 isomers of supplemented phytases were still present, indicating further activity of these enzymes. Myo-inositol tetrakisphosphate (InsP4) accumulation differed between PhyE1 and PhyE2 compared with PhyA in the anterior segments of the gut (P < 0·01). Thus, the hydrolytic cleavage of the first phosphate group is not the only limiting step in phytate degradation in broilers.

A note on sampling digesta from the ileum of broilers in phosphorus digestibility studies

The responses of broilers to increments of dietary P concentration in P retention and P prececal digestibility were studied in 2 experiments using Ross 308 broilers in their fourth week of age. The low-P basal diet was mainly based on corn, potato protein, and corn starch. Titanium dioxide was used as the indigestible marker. Monobasic calcium phosphate was added in 6 (experiment 1) or 5 (experiment 2) graded levels up to a maximum of 8.1 g of P/kg of the diet. Five (experiment 1) and 6 (experiment 2) replicated pens of 10 birds were used. Excreta were collected from trays underneath the pens. Birds were asphyxiated by carbon dioxide exposure and the ileum (from Meckels diverticulum to 2-cm anterior to the ileo-ceca-colonic junction) was dissected. Digesta was collected from the entire section (experiment 1) or from 3 subsections of equal lengths (experiment 2). Excretion of P increased linearly with increasing dietary P concentration up to a level of about 5.2 g of P/kg of the diet and increased nonlinearly with further increase in dietary P. In contrast, P flow in the ileum increased linearly over the entire range of P intake. Up to the level of 5.2 g of P/kg of the diet, P excretion was slightly lower than P flow in the ileum, but responses in P excretion and P flow in the ileum were similar, suggesting that P excretion with urine is very low and unaffected by P intake when the birds are supplied with P below their requirement. Between the 3 subsections of the ileum, calculated digestibility was significantly different for the basal diet and the diet with the 3 lowest levels of P supplementation. We concluded that the response in P prececal digestibility to increments in dietary P concentration is linear over a wider range of dietary P than the response in P retention. When digesta is collected from the ileum for determination of P prececal digestibility, the first third of the ileum should not be considered.

Ileal microbiota of growing pigs fed different dietary calcium phosphate levels and phytase content and subjected to ileal pectin infusion.

Two experiments with growing pigs were conducted to determine the effects of dietary P and Ca levels, phytase supplementation, and ileal pectin infusion on changes in bacterial populations in the ileum and on ileal and fecal fermentation patterns. Growing pigs (BW 30.1 +/- 1.3 kg) were fitted with simple T-cannulas at the distal ileum and were fed a low-P corn-soybean meal control diet (3 g of P/kg), or the control diet supplemented with either 15 g of monocalcium phosphate (MCP)/kg (Exp. 1) or 1,000 phytase units of phytase/kg (Exp. 2). Daily infusion treatments consisted of either 60 g of pectin dissolved in 1.8 L of demineralized water or 1.8 L of demineralized water as a control infusion, infused via the ileal cannula. In each experiment, 8 barrows were assigned to 4 dietary treatments according to a double incomplete 4 x 2 Latin square design. The dietary treatments in Exp. 1 were the control diet with water infusion, the control diet with pectin infusion, the MCP diet with water infusion, or the MCP diet with pectin infusion. In Exp. 2, the pigs received the same control treatments as in Exp. 1 and the phytase diet in combination with water or pectin infusion. Gene copy numbers of total bacteria, Lactobacillus spp., Lactobacillus reuteri, Lactobacillus amylovorus/Lactobacillus sobrius, Lactobacillus mucosae, Enterococcus spp., Enterococcus faecium, Enterococcus faecalis, bifidobacteria, the Clostridium coccoides cluster, the Clostridium leptum cluster, the Bacteroides-Prevotella-Porphyrmonas group, and Enterobacteriaceae were determined by quantitative PCR in DNA extracts of ileal digesta. In Exp. 1, addition of MCP reduced ileal gene copy numbers of Enterococcus spp. (P = 0.048), E. faecium (P = 0.015), and the C. leptum cluster (P = 0.028), whereas pectin infusion enhanced (P = 0.008) ileal d-lactate concentration. In Exp. 2, supplemental phytase led to greater ileal gene copy numbers of the C. coccoides (P = 0.041) and C. leptum (P = 0.048) clusters and the Bacteroides-Prevotella-Porphyrmonas group (P = 0.033), whereas it reduced (P = 0.027) fecal n-butyrate concentration. Pectin infusion reduced (P = 0.005) ileal gene copy number of the C. leptum cluster. In conclusion, ileal bacterial populations and fermentation patterns are susceptible to changes in the intestinal availability of Ca and P as well as to the supply of pectin as a fermentable substrate. Greater intestinal Ca availability decreased the numbers of some gram-positive bacteria, whereas greater P availability in the small intestine caused by phytase activity enhanced the growth of strictly anaerobic bacteria.

Determination of titanium dioxide supplements in different matrices using two methods involving photometer and inductively coupled plasma optical emission spectrometer measurements

Titanium dioxide (TiO2) is a viable marker in digestibility studies using different animal species. The photometrical analysis is based on an intense orange colour following the addition of hydrogen peroxide to an acid solution. The measurement using inductively coupled plasma optical emission spectrometry (ICP-OES) is a method for analysing more than one element from the same sample preparation. The present study was conducted to investigate whether an established ICP-OES element analysis following acid-based hydrolysis is appropriate for titanium analysis. Defined amounts of TiO2 were added to samples obtained in studies with cows, pigs and turkeys and recoveries were determined. It was shown that supplemented TiO2 can be determined in samples of feeds, faeces or excreta, and digesta using a method based on photometric or ICP-OES measurement. The differences between the true and measured titanium concentrations indicate that using the ICP-OES method leads to a higher accuracy of determination.

Faecal alkane recoveries from multi-component diets and effects on estimates of diet composition in sheep.

In a feeding trial with sheep, four animals each were fed one of the three roughages (perennial ryegrass (PR), meadow fescue (MF) and red clover (RC)) or one of the four different mixtures (g/day 250 : 250 : 250, 375 : 75 : 300, 525 : 187.5 : 37.5 and 75 : 525 : 150, respectively) of those three roughages, both with and without the inclusion of 100 g of beeswax-labelled barley. Further, four sheep received a pure lucerne diet to enlarge available data on single species faecal recoveries. All sheep except those fed single-component diets and the 250 : 250 : 250 roughage mix were administered intra-ruminal alkane controlled-release devices (CRD). The aim of the study was to investigate the effect of diet composition on faecal alkane recoveries, and to assess the accuracy of the alkane-based estimate of diet composition and intake based either upon a general set of faecal recoveries across diets or upon dietary recoveries and dietary samples more specifically attributable to individual dietary treatments. For each roughage component independently, the accuracy of diet composition estimates was assessed using linear regression across all diets. The estimates of the proportion of barley were analysed further using mean differences and mean prediction errors. Faecal alkane recovery increased with increasing chain length and was affected by diet composition. RC had a significantly higher faecal recovery for alkanes C25, C27, C29 and C31 than at least one of the other single-roughage diets. When considering mixed diets consisting of PR, MF, RC and barley, the composition of the roughage component significantly affected faecal recovery of all alkanes except C30 and C33. The inclusion of beeswax-labelled barley caused a decrease in faecal recovery of alkanes up to C29. This effect was attributable to the beeswax rather than the barley itself. By contrast, the decrease of faecal recovery of synthetic dosed alkanes from the CRD in diets containing barley, compared with the corresponding diets without barley, was attributable to the supplement itself. It was concluded that synthetic dosed alkanes behaved differently during gut transit from natural alkanes. The proportions of individual dietary components were estimated well over a wide range of proportions. Generally, the more information available, the more accurate the estimates achieved. However, a general set of faecal alkane recoveries and bulked samples of dietary components yielded estimates of diet composition sufficiently accurate for a large number of studies, especially in situations where groups of animals are of concern and not the individual animal.

Variation in precaecal amino acid and energy digestibility between pea (Pisum sativum) cultivars determined using a linear regression approach

1. An experiment was conducted to study the variation in the precaecal amino acid (AA) and energy digestibility of 4 cultivars of white-flowering peas (Pisum sativum) for broiler chickens. The 4 cultivars were grown and harvested under the same agronomic and environmental conditions. 2. One basal diet and 8 diets including each pea cultivar at inclusion rates of either 150 or 300 g/kg were used. Peas were included at the expense of starch. Hence, the differences in dietary crude protein and AA concentrations resulted only from the inclusion of peas. Titanium dioxide was included as an indigestible marker. 3. Crude protein concentrations in the complete diets ranged from 164 to 244 g/kg. All diets were fed ad libitum to broiler chickens between 14 and 21 d of age. Seven pens of 12 chickens were allocated to each treatment. Digesta were sampled on a pen basis from the section of the gastrointestinal tract between Meckels diverticulum and 2 cm anterior to the ileo-caeco-colonic junction. 4. The proportions of crude protein and AAs digested responded linearly to increased intake and the relationships between quantitative intake and digested amounts of AAs were described by multiple linear regressions. The slope determined for each pea cultivar was taken as a measure of AA digestibility without the need for consideration of basal endogenous crude protein and AA secretions. 5. Digestibility of AAs in peas ranged from 0·60 to 0·91. One cultivar had significantly lower AA digestibilities than the other three cultivars. This could be explained neither by the trypsin inhibitor activity nor by tannin levels. The AAs with the highest digestibilities in all cultivars were arginine > glutamic acid > lysine > methionine > phenylalanine > leucine. The AAs with the consistently lowest digestibilities were cystine < threonine. The ranking of the remaining AAs varied slightly between cultivars. 6. The multiple linear regression approach is suitable to study differences in AA digestibilities without the need for consideration of basal endogenous AA losses. 7. Diets containing 300 g/kg of peas had significantly lower energy digestibilities than the pea-free basal diet. The pea cultivar with the lowest AA digestibility caused the lowest energy digestibility at both levels of pea inclusion.