Alteration of absorption of d -xylose by parasitism and feeding of grain legumes to sheep

ANIMAL FEED SCIENCE AND TECHNOLOGY

ELSEVIER

Animal Feed Science and Technology 49 (1994) 325-333

Alteration of absorption of D-xylose by parasitism and feeding of grain legumes to sheep F. Woldetsadick "'*'~, R.M. Dixon a'2, H.P. Yuen b, J.H.G.

Holmes a

aAnimal ProductionSection, School of Agriculture and Forestry, Universityof Melbourne, Park-ville, Vic. 3052, Australia bStatistical Consulting Centre, Universityof Melbourne, Parkville, Vic. 3052, Australia Received 6 October 1993; accepted 9 February 1994

Abs~a~

To assess the effect of anti-nutritional factors on nutrient absorption, D-xylose was used as an index of absorption in sheep fed grain legumes. The kinetics of absorption and excretion of o-xylose were studied in six Merino wethers fed oat hay/lucerne hay ( 1 : 1 ). DXylose disappeared from plasma 6 h after i.v. injection and 63.7 + 11.5% of the dose was recovered in urine in 48 h, indicating metabolism of about 36% of circulating D-xylose. After abomasal injection, ~xylose in plasma reached a maximum in 2 h and disappeared in about 12 h. In 48 h, 17.5 + 11.5% was recovered in urine, indicating that about 27% had been absorbed. Six wethers received either lucerne (Medicago sativa), chickpeas (Cicer aritenum ), navy beans ( Phaseolus vulgaris ), Lablab ( Lablab purpureus ), cowpeas ( Vigna unguiculata) or lupins (Lupinus angustifolius) at 15 g per kg of live weight (LW) plus oaten hay at 5 g per kg of LW. After abomasal injection, peak levels of O-xylose in plasma did not differ significantly but excretion in urine was greater for lupins (34% of the dose) than for other legumes ( 16-22%, P < 0.05). Compared with lucerne, no grain legumes reduced absorption from the intestines, while lupins apparently enhanced uptake of D-xylose (P < 0.03 ). Absorption was strongly reduced by level of infestation with internal parasites (P< 0.001 ).

~" Part of the findings described in this paper were presented at the 16th Annual Scientific Meeting of the Nutrition Society of Australia, held in Canberra, A.C.T., on 9-11 December 1991. * Corresponding author. Current address: Western Zone Agricultural Development Office, PO Box 194, Jimma, Ethiopia. 2 Current address: Dept. of Primary Industry, "Swans Lagoon" MiUaroo, via Ayr, Qld. 4807, Australia. 0377-8401/94/$07.00 © 1994 Elsevier Science B.V. All rights reserved SSD10377-8401 ( 94 )00628-M

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1. Introduction

Grain legumes may contain a wide range of anti-nutritional factors (ANFs) which interfere with digestion, the amounts present varying not only with species and variety, but also with growing conditions. The most important ANFs are protease inhibitors and lectins. These must be destroyed by heating if such grain legumes are to be used for food for humans or monogastric livestock. It might be expected that these soluble proteins would be detoxified by microbial breakdown in the rumen. Holmes et al. ( 1991 ) reported that trypsin inhibitors and lectins disappeared from nylon cloth bags containing ground lupins (Lupinus angustifolius), lablab (Lablab purpureus), cowpeas (Vigna unguiculata) and navy beans (Phaseolus vulgaris) suspended in the rumen of sheep but subsequently demonstrated that trypsin inhibitors retained their activity after more than 12 h incubation with rumen fluid in vitro ( Holmes et al., 1993). Addison et al. (1984) with beef cattle and Garcia et al. (1990) with sheep and goats reported unsatisfactory growth responses when roughages were supplemented with lablab. With navy beans as a supplement, poor growth, diarrhoea and the development of antibodies to lectins occurred in cattle (Williams et al., 1984/1985 ) and poor growth and diarrhoea in sheep (Paduano et al., 1990). These data suggest that ANFs which disappear from nylon bags have merely dissolved but have not been detoxified and continue to exert deleterious effects upon digestion. Lectins reduce the absorptive efficiency of the intestinal mucosa, while trypsin inhibitors block proteolysis prior to absorption; other anti-nutritional factors, such as tannins and hypersensitivity factors, interfere with other components of digestion. The effect of lectins of five grain legumes on absorptive efficiency of the intestine of sheep was compared with absorption by sheep fed a forage legume, using D-xylose as an index. D-Xylose is used as an orally administered index of absorptive capacity in humans and other monogastric mammals because it shares a common carrier with glucose and other hexoses (Csaky and Ho, 1965 ). Since D-xylose is partially degraded by ruminal micro-organisms when administered orally to ruminants (Pausur et al., 1958) it was injected per abomasum in this study. Internal parasitism, which is ubiquitous in grazing sheep, has significant effects on absorptive capacity, so faecal egg counts were used in covariance analysis as an index of parasitic infestation. 2. Materials and methods

2.1. Experiment 1 2.1.1. Design Two treatments, intra-jugular injection of 8 g of D-xylose and intra-abomasal injection of 20 g of D-xylose, were administered to six sheep in a switch-over design, with three sheep receiving each treatment on each occasion, separated by a 3 week recovery period. Serial sampling of venous blood was used to study the

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kinetics of O-xylose absorption and clearance from blood. Excretion in urine was measured by total collection over 36 h.

2.1.2. Animals and treatments Six Merino wethers, about 17 months old, weighing 38-46 kg, were prepared with abomasal fistulas, treated with anthelmintic and housed in metabolism crates. They were fed a diet of chopped oaten hay and lucerne ( 1 : 1 ) to maintain live weight (LW), using an automatic belt feeder to provide a constant feed supply throughout the 24 h. A multi-mineral block was provided. The two treatments were (i) intrajugular administration of 8 g of D-xylose in 20 ml saline, warmed to 37 °C; blood samples were obtained from the opposite jugular vein at 0, 5, 15, 30, 60, 120, 180, 240, 300, 360 and 720 rain; (ii) intra-abomasal administration of 20 g D-xylose in 50 ml of saline by injection through the plug of the abomasal cannula, with blood samples collected at 30-min intervals from 0 to 4 h, then hourly for the next 4 h. In both treatments, total urine collection was carded out for 48 h following injection Of D-xylose.

2.1.3. Analysis Blood was centrifuged for 15 rain at 3000 rev. rain- 1 and the plasma stored at 10 ° C. D-xylose in plasma and urine was measured by the method of Deckert et al. (1967). -

2.1.4. Calculation of absorption IfA g of D-xylose were injected via the jugular vein and B g were recovered in the urine, the proportion of circulating D-xylose not metabolised by the sheep's tissues but excreted equals B/A. If C g of D-xylose were injected into the abomasum and D g were recovered in the urine, the m o u n t absorbed by the intestine and entering the circulation is D~ (B/A) and the proportion absorbed is D×A

B×C 2.2. Experiment 2 2. 2.1. Design Six diets containing different legumes were fed to nine sheep in four periods of 17 days, using a non-orthogonal modified Latin Square design with a total of six replications of each treatment. In each period, three of the legumes were fed to single sheep and three to two sheep. Each sheep received only four of the legumes during the experiment. D-Xylose was infused per abomasal fistula; absorption and clearance of ~xylose were measured in plasma and total excretion in urine was determined. Faecal parasitic egg counts were performed on all sheep in each period as an index of internal parasitism and used as a covariate in analysis.

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2.2.2. Animals and treatments Nine abomasally fistulated Merino wethers used, including five used in Experiment 1, weighing 45.3 _+3.2 kg, were treated once with anthelmintic immediately before experimental feeding began. In each period, they were penned individually and fed experimental rations once daily for a week, then were placed in metabolism crates and fed continuously by a belt feeder. All sheep received chopped oaten hay at the rate of 0.5% LW (air-dry feed) day -1, plus one of six legumes at 1.5% of LW (air-dry feed) day- 1. The legumes were lucerne (Medicago sativa ), chickpeas ( Cicer aritenum ), navy beans ( P. vulgaris ), lablab beans ( Lablab purpureus ) , cowpeas ( V. unguiculata ) and lupins ( Lupinus angustifolius). All sheep consumed their entire rations. Lucerne and lupins were obtained locally in Victoria, the other grain legumes from Central Queensland. Feed allowances were calculated at the beginning of each period. A multi-mineral block was provided. In each period, on Day 16, 20 g of D-xylose were administered per abomasal fistula and serial blood samples were collected via jugular catheter at intervals of 30 rain for 6 h and hourly for another 4 h. Urine was collected at 12-h intervals for 48 h after administration of D-xylose. Grab samples of faeces were collected on Day 17. 2.2.3. Analyses D-Xylose was estimated in plasma and urine as before. Faecal egg counts were performed by the McMaster flotation technique. 2.2.4. Statistical analyses In Experiment 1, peak concentrations (mg 1-1), areas under the curves (mg 1-1X h ) and total excretion of D-xylose were subjected to ANOVA. Experiment 2 was analysed as a non-orthogonal modified Latin square with partial balancing and six replicates using faecal egg count as a covaxiate. 3. Results

3. I. Experiment I In sheep dosed intravenously, the maximum concentration of D-xylose, 0.71 + 0.34 mg m l - t of plasma, occurred at the first sampling, at 5 rain (Fig. 1 ). Subsequently, concentrations declined according to first-order kinetics at 0.0095 m i n - i, according to the equation y ( m g m l - l ) = - 0 . 4 5 4 8 - 9 . 5 8 7 2 e -3x,

R2=0.997

to almost pre-injection levels in 8 h. In the 48 h following administration, 63.7 +_11.5% of the injected D-xylose was recovered in the urine. In sheep dosed via the abomasum, D-xylose was present in the blood at 0.03 nag m l - 1 in the first sample, collected after 30 min. D-Xylose reached maximum

F. Woldetsadick et al. ~Animal Feed Science and Technology 49 (1994) 325-333

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0.8

I.V. injection 0.6

°r a.o%.s7

~0.4

q

x, "

0.2

V1

100

200

300

400

I

500

MINUTES

Fig. 1. Concentrations of D-xylose in plasma of sheep after injection via jugular vein or abomasum. Table 1 Relative absorption (area under the curve, mg ml-~ × min) and peak plasma levels of a 20 g dose of D-xylose injected into the abomasum of sheep fed six different legumes at 15 g per kg of LW per day, using faecal parasitic egg count as a covariate

Lucerne Chick pea Navy bean Lablab Cowpea Lupins SEM

Relative absorption (mg 1-~ ×rain)

Peak plasma level (mg ml -~ )

2.44 3.12 2.53 2.63 2.91 2.91 0.263

0.215 0.229 0.163 0.181 0.220 0.240 0.029

concentration, 0.136_+0.029 nag ml -~, between 90 and 150 min and declined from then according to first-order kinetics, to almost pre-injection values in 8 h (Fig. 1 ). In the 48 h after administration, 17.5 +_5.5% of the injected D-xylose was recovered in the urine, more than half in the first 12 h. As urinary D-xylose represents 63.7% of the D-xylose entering the bloodstream, the amount of D-xylose absorbed from the abomasum was estimated as 27.5% of the dose administered.

3.2. Experiment 2 The peak concentrations ofl~xylose in plasma, reached after 90-120 min, were highest for lupins, lowest for navy beans but the difference was not statistically significant when faecal egg count was included as a covariate (P--0.1 ). Relative absorptions did not differ significantly (Table 1 ). Recovery of D-xylose in the urine (Table 2) was greatest with the lupin diet, least with navy beans (P> 0.03 ). The recovery of D-xylose from wethers fed lucerne, 18.5% (3.7 g of 20 g administered), was similar to the recovery in Experiment 1. Faecal egg counts (Table 3) were highly variable, with a mean of

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Table 2 Measured urinary excretion and calculated intestinal absorption of a dose of 20 g of D-xylose injected into the abomasum of sheep fed six different grain legumes at 15 g per kg LW per day, using faecal egg count as a covariate

Lucerne Chick pea Navy bean Lablab Cowpea Lupins SEM Significance, P, for legumes

Urinary excretion (g day- i )

Intestinal absorption (g day- l )

Systemic metabolism (g day- i )

3.70 b 4.32 b 3.73 b 3.10 b 3.38 b 6.8(P 0.67

5.81 6.78 5.86 4.87 5.31 10.68

2.11 2.46 2.13 1.77 1.93 3.88

0.28

~bMeans with different superscripts differ at P < 0.05. Table 3 Faecal egg counts (eggs per g) for nine sheep fed six different legumes at 15 g per kg of LW, in four consecutive periods, for 17 days prior to faecal collection Period 1

2

Lucerne

60

Chick pea

60

Navy bean

30

Lablab

60

3

0 30 270 840 3330 870 240

-

Cowpea Lupins

180 60 30 -

30 750

4

90 360 240 210 510 60 150 240 1110

0 2490 120 660 450 90 30 0 330

411 _+704. With the exception of one sheep, egg counts were below 900 at all times, indicating a mild degree of infestation. Faecal egg counts were highly significant as a covariate ( P < 0.001 ) with an increase in egg count of 100 associated with a reduction in recovery of 0.6 _+0.1 g D-xylose. 4. Discussion In Experiment 1, the peak D-xylose level after abomasal infusion was 0.136_+ 0.029 mg ml-~ of plasma at 120 min which agrees closely with the value of0.138 _+0.03 mg m l - 1, 90 min after administration to cows of 0.5 g of D-xylose

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per kg LW via abomasal fistula (Pearson and Baldwin, 1980). For both routes of administration, the clearance of D-xylose by first-order kinetics represents the normal nutrient absorption pattern (Seegraber and Morill, 1979), indicating normal absorption and clearance of D-xylose under our experimental conditions. In Experiment 2, the lack of statistical significance in the relationship between diet, animal or parasitic burden and the area under the curve may have been due, in part, to variation between sheep in the rate at which D-xylose entered the duodenum after infusion, since abomasal contractions and emptying are intermittent events even when food is supplied at a constant rate. This may have obscured the relationship to diet, animal or parasitic burden. Alternatively, the feeding regime employed may have supplied the anti-nutritional factors to the small intestine at a steady slow rate so that toxic levels were never achieved. Under practical feeding situations, animals fed once per day may experience rapid emptying of the forestomachs and high concentrations of anti-nutritional factors for short periods. Significant differences between grain legumes in peak plasma levels were in the same direction as the differences in urinary excretion but exhibited no greater differentiation. We conclude that the serial measurement of D-xylose in plasma contributed no additional information to that obtained by total urine collection. Lupins resulted in the highest plasma peak concentration of D-xylose and double the D-xylose excretion of the other legumes. Urinary recoveries of D-xylose with other grain legumes were not statistically lower than that found with lucerne (which is not considered to contain any factors causing malabsorption). However, the ranking of the legumes was in accordance with their known anti-nutritional effects, with the lowest peak values and recoveries found with navy beans and lablab, which have been associated with significantly poorer performance and symptoms of severe gut dysfunction in monogastric animals (Lasheras et al., 1980; Santidrian and Marzo, 1989) and milder symptoms in ruminants (Addison et al., 1984; Ismartoyo, 1989; Garcia et al., 1990). Ismartoyo (1989)found that goats fed lablab at 12 g per kg of LW per day and dosed orally with D-xylose had lower D-xylose levels in plasma than goats receiving a control diet (0.47 mg m l - 1 versus 1.24 nag m l - 1) although in later experiments in our laboratory with sheep fed lablab at up to 15 g per kg of LW per day, Domingo (1990) found no reduction in I>-xylose uptake. This may have been due to differences in digestion between goats and sheep or to different batches of lablab. With intakes of navy beans of 20 g per kg of LW per day, there was a small reduction in plasma Dxylose. These values were still above 'normal' values reported by Pearson and Baldwin (1980). Intakes of grain legumes of 15-20 g per kg of LW per day are above the amounts likely to be fed as supplements in Australian animal production systems. Infestation with internal parasites has long been known to reduce absorption from the intestine (Blood and Henderson, 1960). In the current experiment, the relationship between faecal egg count and D-xylose excretion in the urine was the most significant factor in the statistical analysis, demonstrating the relatively

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greater effect on absorption of the damage to the intestine caused by parasitic infestation. Lectins are the anti-nutritional factor most likely to cause reduced efficiency of absorption of the end-products of digestion by blocking the binding sites on mucosal cells, which would be detected by the D-xylose absorption test. Other antinutritional factors affect digestion in other ways, e.g. by blocking digestive enzyme action, specifically as in trypsin inhibition and non-specifically as in the binding of proteins by tannins, or by increasing gut motility. The deleterious effects of navy beans, for example, are likely to be due to the action of several antinutritional factors acting in concert. We conclude that there are only small differences in the effects of the grain legumes studied on absorptive capacity of sheep but that these are much less than the effect of a minor degree of internal parasitism. Acknowledgements One of us (F.W.) was supported by a fellowship from the Australian International Development Assistance Bureau. Faecal egg counts were performed at the Attwood laboratory of the Victorian Department of Agriculture Institute of Animal Science. Refereaces Addison, ICB., Cameron, D.G. and Blight, G.W., 1984. Highworth lablab grain as a supplement for beef cattle on native pastures. Proc. Aust. Sof. Anita. Prod., 15: 227-230. Blood, D.C. and Henderson, J.A., 1960. Veterinary Medicine. BaiUiere, Tindall and Cox, London. Csaky, C.Z. and Ho, P.M., 1965. Intestinal transport of V-xylose. Prof. Sof. Exp. Biol. Med., 120: 403--408. Deckert, T., Hvidt, S. and Kjeldsen, IC, 1967. Normal values of v-xylose excretion in urine after oral loading dose in children and adults. A modification of the method of Roe and Rice. Scand. J. Clin. Lab. Invest., 19: 263-268. Domingo, J., 1990. Antinutritional factors in legume seeds. M.AgSci. Thesis, University of Melbourne. Gareia, E., Ismartoyo, Sloenmbe, R.F., Dixon, R.M. and Holmes, J.H.G., 1990. Nutritive value of Lablab purpureus grain for sheep and goats. Prof. Aust. Sof. Anim. Prod., 18: 478. Holmes, J.H.G., Dixon, R.M., Domingo, J.A., Garcia, E., Ismartoyo, Lodebo, B., Paduano, D.C., Pomares, C. and Woldetsadick, F., 1991. Grain legumes (lupins, lablab, cowpeas and navy beans) as supplements for sheep and goats. In: D.J. Farrell (Ed.), Recent Advances in Animal Nutrition in Australia, Univ. New Engl. Publishing Unit, Armidale, Australia, pp. 62-71. Holmes, J.H.G., Dixon, R.M., Smith, C., Entonu, S., Chau, H., Sanpote, S. and Acharyya, B., 1993. Resistance of trypsin inhibitors to fermentation by rumen microflora. In: A.F.B. van der Poel, J. Huisman and H. Saini (Editors), Recent Advances of Research in Antinutritional Factors in Legume Seeds. EAAP Publication No. 70. Wageningen Pers, Wageningen, pp. 183-186. Ismartoyo, 1989. Lablab purpureus as a supplement for goats fed roughage diets. M.Ag.Sci. Thesis, University of Melbourne. Lasheras, B., Boulfer, J., Cenaruzabeitia, E.M.N., Lluch, M. and Larralde, J., 1980. Effect of raw legume diets upon intestinal absorption of D-galactose by chick. Rev. Esp. Fisiol., 36: 89-92. Paduano, D.C., Slofombe, R.F., Holmes, J.H.G. and Dixon, R.M., 1990. Cowpeas and navy beans as supplements for roughage diets for sheep. Prof. Aust. Sof. Anita. Prod., 18: 536.

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Pausur, J.H., Shuey, E.W. and Georgi, C.E., 1958. The conversion of D=xylose into volatile organic acids by rumen bacteria. Arch. Biochem. Biophys., 77: 387-402. Pearson, E.G. and Baldwin, B.H., 1980. D-xylose absorption test in the adult bovine. Cornell Vet., 71: 288-296. Santidrian, S. and Marzo, F., 1989. Effect of feeding tannic acid and kidney bean (Phaseolus vulgaris) on the intestinal absorption of D-galactose and L-leucine in chickens. J. Sci. Food Agric., 47: 435442. Seegraber, F.J. and Morill, J.L., 1979. Effect of soy protein on intestinal absorptive capacity of calves by the xylose absorption test. J. Dairy Sei., 62: 972-977. Williams, P.E.V., Pusztai, A.J., MacDearmid, A. and Innes, G.M., 1984/1985. The use of kidney beans (Phaseolus vulgaris) as protein supplements for young, rapidly growing beef steers. Anim. Feed Sci. Technol., 12: 1-10.