Rumen digestibility of starch and nitrogen
in near-isogenic lines of wheat
P.C. Garnsworthy
*, J. Wiseman
School of Biological Sciences, University of Nottingham, Sutton Bonington Campus, Loughborough, Leics LE12 5RD, UK
Received 15 November 1999; received in revised form 2 March 2000; accepted 5 April 2000
Abstract
Comparisons between cereal species have shown that wheat starch is fermented more rapidly in the rumen than starch from maize, barley, potato or sorghum, probably due to the protein matrix surrounding the starch granules, but there are few comparisons of starch digestion from different cultivars of wheat. Although wheat varieties are classi®ed as being hard or soft, genetic factors affecting nutritional value cannot be evaluated using name alone because any two named varieties may be distantly or closely related. Near-isogenic lines allow the nutritional implications of speci®c characteristics to be investigated against a comparatively uniform genetic background. The purpose of this study was to investigate genetic effects on starch and nitrogen degradation kinetics in sacco for four pairs of near-isogenic wheat lines. Pair A comprised one hard and one soft line; Pair B comprised two hard lines, one of which contained the 1B/1R rye translocation; Pair C comprised a hard line with the 1B/1R rye translocation and a soft wheat without the 1B/1R translocation; Pair D comprised two lines differing in the visco-elastic properties of dough. Minor differences in the soluble and degradable fractions of starch and nitrogen were found within pairs. Only in Pair C, where the 1B/1R translocation was accompanied by a change in hardness, was there a signi®cant effect on effective rumen starch and nitrogen degradability (soft non-1B/1R versus hard 1B/1R, starch digestibility0.86 versus 0.88, standard error of the difference (S.E.D.) 0.001; nitrogen degradability0.80 versus 0.76, S.E.D. 0.008). Rumen by-pass starch was in¯uenced mainly by the starch content of the wheat rather than differences in rumen digestion. It is concluded that in ruminants, unlike poultry, genetic differences in wheats have very little impact on their overall nutritive value, which is determined mainly by total starch content.#2000 Elsevier Science B.V. All rights reserved.
Keywords:Wheat; Starch; Nitrogen; Rumen; Ruminants; Digestion; Degradation; Genetics 85 (2000) 33±40
*Corresponding author. Tel:44-0115-951-6065; fax:44-0115-951-6060.
E-mail address: phil.garnsworthy@nottingham.ac.uk (P.C. Garnsworthy)
1. Introduction
Wheat is potentially an excellent source of fermentable carbohydrates for ruminants, but there is a shortage of information on the kinetics of wheat digestion in the rumen. The major carbohydrate of wheat is starch, which is fermented by amylolytic bacteria and protozoa (Kotarski et al., 1992). Comparisons between cereal species have shown that wheat starch is fermented more rapidly in the rumen than starch from maize, barley, potato or sorghum (Axe et al., 1987; Cone et al., 1989). An in vitro study of gas production from cereals fermented in rumen ¯uid found that the rate of gas production was faster in wheat than in triticale, oats, barley, maize rice and sorghum (Opatpatanakit et al., 1994). There are few comparisons of starch digestion from different cultivars of wheat, although Opatpatanakit et al. (1994) found signi®cant effects of varieties and growing conditions on gas production from wheat using an in vitro system.
Wheats are generally classi®ed as hard or soft according to their endosperm texture, which re¯ects the protein±starch interaction and is genetically determined (Barlow et al., 1974). In soft wheats the starch and protein granules are embedded in a friable matrix that is readily crushed to release the starch and protein with little damage. However, in hard wheats a continuous protein matrix physically entraps the starch granules making it dif®cult to separate the protein and starch. In a comparison of wheat, maize and barley, McAllister et al. (1993) concluded that the protein matrix seemed to be the major factor responsible for differences in ruminal digestion of starch.
Although wheat varieties are described as being hard or soft, genetic factors affecting nutritional value cannot be evaluated by simply comparing named varieties. This is because any two named varieties may be distantly or closely related in a generally unpredictable way and name alone would give no indication of the genetic relationship. Thus, programmes based on varietal names are of little value in assessing reasons for variability in nutritional value of wheat. The emergence of near-isogenic lines, developed through conventional plant breeding techniques, represents a major development as nutritional implications of speci®c characteristics may now be investigated against a comparatively uniform genetic background. For example, the speci®c effect of hardness on rumen digestion of wheat can be examined without possible confounding effects of other factors.
A characteristic present in most feed wheats is the 1B/1R translocation, in which the short arm of chromosome 1B has been replaced with the short arm of the 1R chromosome of rye. The translocation has several reported agronomic advantages, such as resistance to powdery mildew, stripe rust, leaf rust and stem rust (Mettin et al., 1973; Zeller, 1973). The 1B/1R translocation does, however, have detrimental effects on the quality of hard wheats for bread-making (Dhaliwal et al., 1987). Plant breeders are currently trying to remove disadvantageous traits whilst maintaining the bene®ts. Although the agronomic implications of the presence of the 1B/1R translocation have been extensively studied, and in¯uence on bread-making investigated, there appears to have been only one report on the nutritional implications for animals. Short et al. (2000), in a study of near-isogenic lines of wheat, found a detrimental effect of the 1B/1R translocation on amino acid digestibility in young chickens and also concluded that hard endosperm texture was associated with lower nutritional value.
The objective of the current study was to determine the rumen digestion characteristics of starch and nitrogen in near-isogenic wheat lines differing in only one or two characteristics, so that nutritional consequences of speci®c genetic characteristics can be determined.
2. Materials and methods
Four pairs of near-isogenic wheat lines differing in only one or two characteristics were selected for study. Pair A comprised one hard and one soft line; Pair B comprised two hard lines, one of which contained the 1B/1R rye translocation; Pair C comprised a hard line with the 1B/1R rye translocation and a soft wheat without the 1B/1R rye translocation; Pair D comprised two lines differing in the visco-elastic properties of dough.
Approximately 2 kg of each wheat line was milled through a hammer mill ®tted with a 4 mm screen and passed through a 2 mm sieve to exclude large particles (<1% of the sample weight, principally comprising chaff). A 50mm sieve was used to remove ®ne particles that would pass through the pores of the incubation bags (<1% of the sample weight). Bags were made from polyester with a pore size of 50mm and measured 20 cm10 cm (Ankom Technology, Fairport NY). Approximately 10 g of milled wheat was weighed into each bag and a metal ring was attached using an elastic band, which also sealed the opening of the bag. A total of 24 bags were prepared for each wheat line and attached to stainless steel D-shaped bolts, via their metal rings, in groups of six.
Four non-lactating Holstein±Friesian dairy cows, each ®tted with a rumen ®stula, were given a daily ration of 11 kg ®rst-cut grass silage, 10 kg maize silage, 8 kg brewers grains, 5.6 kg dairy concentrates (based on wheat, barley, soya bean meal and rapeseed meal) and 0.75 kg molassed sugar beet pulp nuts. Cows received this ration for at least 2 weeks before the start of the experiment.
Six samples from each wheat line were placed in the rumen of each cow and incubated for 0, 4, 8, 12, 24 or 48 h. After removal from the rumen, bags were rinsed gently in cold water to remove any coarse digesta. Bags were then washed in a domestic washing machine at 308C for 60 min to remove soluble material and dried in an oven at 708C for 48 h. Starch and nitrogen contents of residues from each bag were determined using a near-infrared scanning monochromator (NIRS6500, Perstorp UK). Starch content of the calibration samples and the original wheat samples was determined using an enzymatic method developed at Nottingham University (Nicol, 1999). Nitrogen content was determined by ¯ash combustion in oxygen using a NA2000 nitrogen analyser (Fisons, UK). Disappearance of starch and nitrogen from bags was regressed against incubation time using the non-linear model dab(1ÿeÿct), where d (g/g) is the proportional
All statistical analyses were performed using Genstat 5, Release 4.1, 4th Edition (Lawes Agricultural Trust). Regression and effective digestibility coef®cients were determined separately for each wheat line tested with each individual cow. Coef®cients for each pair of lines were compared by analysis of variance, with cows as blocks.
3. Results
Considerable differences in starch content were found between lines within pairs of near-isogenic wheats (Table 1); nitrogen contents were similar within pairs (Table 2).
Within the pair of lines that were near-isogenic except for hardness (Pair A), no signi®cant difference (p>0.05) was found in starch digestion and rumen escape contents were identical (Table 1). The rapidly soluble (a), nitrogen fraction was greater (p<0.05) in the hard wheat. The potentially degradable (b) nitrogen fraction was greater (p<0.05) in the soft wheat. No signi®cant difference was found in rate of nitrogen degradation (c), so that effective rumen degradability of nitrogen was similar (Table 2).
For wheats in Pair B, which were near-isogenic except that one line possessed the 1B/ 1R translocation, similar effects were found on starch and nitrogen degradation. The rapidly soluble fraction (a) was increased (p<0.05) and the potentially degradable fraction (b) was decreased (p<0.05) by the translocation. No effect was found on the rate of degradation (c) of starch or nitrogen. Rumen digestibility of starch and rumen escape
Table 1
Starch content and rumen digestibility characteristics of four pairs of near-isogenic wheat lines (4 replicates per wheat line) Pair Characteristic a(g/g) b(g/g) c(g/g/h)
A Hard 645 0.43 0.56 0.44 0.90 64
Soft 582 0.39 0.60 0.41 0.89 64
S.E.D.b 0.019 0.018 0.029 0.004 2.0
B Hard non-1B/1R 576 0.31 0.68 0.49 0.89 64
Hard 1B/1R 637 0.37 0.62 0.45 0.89 70
S.E.D. 0.022* 0.021* 0.038 0.004 3.2
C Soft non-1B/1R 622 0.23 0.75 0.42 0.86 87
Hard 1B/1R 502 0.30 0.69 0.42 0.88 60
S.E.D. 0.023* 0.015* 0.059 0.001* 2.2*
D Low elasticity 580 0.12 0.87 0.36 0.83 99
High elasticity 565 0.14 0.85 0.46 0.85 85
S.E.D. 0.012 0.010 0.067 0.016 6.8
aais the rapidly soluble fraction,bthe potentially digestible fraction,cthe rate of digestion ofbfraction. Rumen digestibility was calculated asabc/(ck), wherekis the rumen out¯ow rate (0.08/h). Rumen escape starch was calculated as (1ÿrumen digestibility)starch content.
bStandard error of the difference.
*Signi®cant (p<0.05) difference between wheat lines within a near-isogenic pair.
starch content were similar in the two wheats, but the rumen degradability of nitrogen was greater (p<0.05) in the wheat with the 1B/1R translocation.
Wheats in Pair C showed opposite effects on starch and nitrogen degradation. The soft non-1B/1R wheat had a lower (p<0.05) soluble starch fraction, but a higher (p<0.05) soluble nitrogen fraction; it also had a higher (p<0.05) potentially digestible starch fraction, but a lower (p<0.05) potentially degradable nitrogen fraction. No difference was found in the rate of starch or nitrogen degradation. The effective digestibility of starch was lower (p<0.05) in the soft non-1B/1R wheat and the effective degradability of nitrogen was higher (p<0.05). The rumen escape starch content of the soft non-1B/1R wheat was signi®cantly greater (p<0.05) than the hard 1B/1R wheat.
No signi®cant difference in starch or nitrogen kinetics was found between the wheats with low or high visco-elastic properties of dough (Pair D).
4. Discussion
It has been suggested that the protein matrix and structural carbohydrates within the endosperm of cereal grains, rather than the properties of the starch granule itself, have a greater effect on starch digestion by rumen microorganisms (McAllister et al., 1993). This conclusion was reached after studying the digestion of barley, wheat and maize grains in vitro, and the major difference in starch digestion was between maize and other cereals. The protein matrix in maize endosperm is extremely resistant to digestion by Table 2
Nitrogen content and rumen degradability characteristics of four pairs of near-isogenic wheat lines (4 replicates per wheat line)
Wheat Nitrogen
(g/kg DM)
Regression coefficientsa Rumen degradability (g/g) Pair Characteristic a(g/g) b(g/g) c(g/g/h)
A Hard 18.9 0.36 0.57 0.26 0.79
Soft 18.7 0.33 0.59 0.25 0.78
S.E.D.b 0.005* 0.005* 0.015 0.005
B Hard non-1B/1R 17.9 0.32 0.61 0.26 0.79
Hard 1B/1R 17.4 0.35 0.58 0.27 0.80
S.E.D. 0.007* 0.008* 0.015 0.003*
C Soft non-1B/1R 19.0 0.39 0.54 0.26 0.80
Hard 1B/1R 18.2 0.25 0.69 0.25 0.76
S.E.D. 0.005* 0.011* 0.028 0.008*
D Low elasticity 19.4 0.16 0.76 0.22 0.72
High elasticity 21.8 0.18 0.74 0.29 0.75
S.E.D. 0.005 0.005 0.035 0.010
aais the rapidly soluble fraction,bthe potentially digestible fraction,cthe rate of digestion ofbfraction.
Rumen digestibility was calculated asabc/(ck), wherekis the rumen out¯ow rate (0.08/h). bStandard error of the difference.
rumen microorganisms (McAllister et al., 1990). Studies with near-isogenic lines of sorghum have shown that the waxy gene increases protein and starch digestibility in the rumen (Lichtenwalner et al., 1978; Kotarski et al., 1992). If the protein matrix limits starch digestion, a high correlation between rates and extent of digestion would be expected. The correlation between rate of starch digestion and rate of nitrogen degradation was 0.953 (p<0.001) and the correlation between extent (ab) of starch digestion and nitrogen degradation was 0.837 (p<0.001). This supports the hypothesis that the protein matrix within the endosperm of wheat affects starch digestion by rumen microorganisms.
Wheat is inherently more digestible in the rumen than maize or sorghum (Axe et al., 1987; Cone et al., 1989) and it is usually not desirable to further increase its rate of fermentation. Rapid fermentation of starch can cause rumen acidosis due to a build up of volatile fatty acids and drop in rumen pH. In dairy cattle, it is usually preferable to slow the rate of fermentation of starch in the rumen since this allows more starch to ¯ow through to the small intestine (De Visser, 1993). In the current study, rate of starch digestion in the rumen was not in¯uenced by any of the genetic modi®cations. Effective digestibility of starch in the rumen was only affected signi®cantly when both hardness and the 1B/1R translocation were altered simultaneously. However, the magnitude of the change in effective digestibility was small and the main determinant of rumen escape starch was the original starch content of the wheat line.
Microbial protein yield in the rumen can be optimised by synchronising carbohydrate and nitrogen fermentation in the rumen (Sinclair et al., 1993, 1995). The optimum ratio of nitrogen to carbohydrate degradation in the rumen has been suggested to be 30 g/kg (AFRC, 1984). If fed as a single meal, the wheats used in this study would have been below this optimum for the ®rst 4 h after feeding and above the optimum thereafter. In practical diets for ruminants, wheat would not be used as a single ingredient; the faster rate of starch digestion, in relation to nitrogen degradation, makes wheat a useful supplement for forages with a rapidly degraded nitrogen fraction, such as grass silage.
Near-isogenic lines of wheat, differing in only one or two characteristics, showed only minor differences in the kinetics of rumen fermentation. Differences that were signi®cant were mainly concerned with the relative proportions of soluble and potentially degradable starch or nitrogen. In no comparison was the rate of degradation signi®cantly different within pairs.
Effective rumen degradability of nitrogen was signi®cantly increased by the 1B/1R translocation in Pair B, but it was decreased in Pair C where hardness was also changed. In contrast, effective rumen digestibility of starch was not affected by the 1B/1R translocation in Pair B, but was increased in Pair C. This contradicts the general observation of a high correlation between nitrogen degradability and starch digestion. It is possible, therefore, that speci®c components of the protein matrix are critical in allowing bacterial access to starch granules.
Controlled genetic changes in endosperm structure, together with the 1B/1R translocation, have been shown to have major implications in the nutrition of poultry (Short et al., 2000), but poultry are generally considered to be particularly sensitive to changes in the quality of dietary inputs. This is because the endogenous enzyme secretions of poultry cannot digest non-starch polysaccharides. Whilst there is modest
bacterial activity in the caecum of poultry, this is poorly developed in young broilers of the age studied by Short et al. (2000) and even in adult birds would be considerably less effective than that found in ruminants. For ruminants, changes in endosperm structure may be important in other cereals, such as maize and sorghum, but the changes in rumen fermentation characteristics observed in the current study would have very little impact on the overall supply of nutrients to a ruminant animal. The major determinant of variability in the quantity of starch digested in the rumen is total starch content of the wheat. This suggests that future programmes designed to examine variability in nutritional value of wheat for ruminants need to be concerned essentially with the total starch content only. In addition, it is evident from the current study, together with that of Short et al. (2000), that the development of any system to predict the nutritive value of wheat must treat ruminants and non-ruminants separately.
Acknowledgements
We would like to thank the John Innes Centre and Nickersons Seeds for supplying samples of the wheat lines, P. Gorton, M. Dickin and R. Allison for laboratory analysis, and staff of the Animal research Unit for care of the cows. This trial forms part of a wider study investigating the nutritional value of near-isogenic wheat lines that is substantially funded by the UK Home-Grown Cereals Authority.
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