Relationships between in vitro gas production characteristics, chemical composition and in vivo quality measures in goats fed tree fodder supplements

Relationships between in vitro gas production characteristics, chemical composition and in vivo quality measures in goats fed tree fodder supplements

Small Ruminant Research 31 (1999) 117±126 Relationships between in vitro gas production characteristics, chemical composition and in vivo quality mea...

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Small Ruminant Research 31 (1999) 117±126

Relationships between in vitro gas production characteristics, chemical composition and in vivo quality measures in goats fed tree fodder supplements Florence V. Nhereraa, L.R. Ndlovua,*, B.H. Dzowelab a

Department of Animal Science, University of Zimbabwe, P.O. Box MP167, Mount Pleasant, Harare, Zimbabwe b SADC-ICRAF Agroforestry Project, P.O. Box CY594, Causeway, Harare, Zimbabwe Accepted 7 April 1998

Abstract Methods currently used to estimate the nutritive value of tropical forages fail to predict nutrient content and availability. The in vitro gas production technique has been proposed as a potential tool to evaluate tropical forages. Four tree fodder legume/ maize stover (1:3, w/w) diets of known chemical composition, digestible OM intake (26.9±29.1 g DMÿ1 W0.75 dayÿ1), OM digestibility (520 to 540 g kgÿ1 DM) and microbial nitrogen supply (0.43±2.68 g kgÿ1 W0.75 dayÿ1) were used in in vitro gas production tests to evaluate the relationship between feed intake, digestibility and microbial nitrogen (MN) supply and the in vitro gas production characteristics and chemical composition. The tree fodder legumes used were Leucaena esculenta, L. diversifolia, L. pallida and Calliandra calothyrsus. Two rumen ®stulated goats fed on L. leucocephala/maize stover diet (1:3, w/w) were used as donor animals for rumen liquor. Gas production from the fermentation of tree fodder legume/maize stover diets was measured in 24 and 96 h in vitro gas tests adapted to describe kinetics of fermentation based on the modi®ed exponential model P ˆ b…1 ÿ eÿct †. Correlation and simple and multiple regression analysis were used to test relationships between gas production parameters and chemical composition and in vivo data. Potential gas production (b), the rate constant (c) and gas volumes were negatively correlated to digestible organic matter intake (DOMI) and organic matter digestibility. There was, however a positive relationship between microbial nitrogen yield and intake (r2ˆ0.60, p<0.001) and truly degraded NDF (r2ˆ0.61, p<0.001). The rate constant, c, and gas volume at 24 h, V24, had a signi®cant impact on DOMI (r2ˆ0.42, p<0.01; r2ˆ0.52, p<0.001, respectively). Gas volume at 6 h and truly degraded NDF were useful in predicting microbial nitrogen yield (r2ˆ65, p<0.001). The effect of polyphenols on intake was low (r2ˆ0.31, p<0.001) but not signi®cant with OMD. The polyphenols accounted for 90% of the variation in gas production after 24 h, 80% for 96 h and 75% of the variation in the rate constant. # 1999 Elsevier Science B.V. All rights reserved. Keywords: Gas production; Tree fodder; Intake; Digestion; Microbial nitrogen

1. Introduction

*Corresponding author. Tel.: +263 4303211 x:1402; fax: +263 4333407; e-mail: [email protected]

Evaluation of the nutritional characteristics of tree fodder legumes is important because of the recent increase in the use of this material in ruminant live-

0921-4488/99/$ ± see front matter # 1999 Elsevier Science B.V. All rights reserved. PII: S0921-4488(98)00128-X

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stock diets (Reed, 1995). Tree fodder legumes are known to contain several types of polyphenols that affect intake and DM digestibility (Khazaal and Orskov, 1994). Rapid and accurate assessment methods are therefore needed to ensure that all material is evaluated before it is made available to livestock. One such tool is the in vitro gas production technique modi®ed by Menke and Steingass (1988). The technique describes the kinetics of fermentation based on the exponential model: P ˆ a ‡ b…1 ÿ eÿct † of Orskov and McDonald (1979), where P, describes gas production at time t, a, the gas produced (ml) by instantaneous fermentation of the soluble and readily available fraction of feed, b, the gas produced (ml) by the fermentation of the insoluble, but slowly fermentable fraction and c, the fractional rate (rate constant) at which gas is produced per hour (ml hÿ1). The technique is based on the assumption that gas volumes re¯ect substrate fermentation to short chain volatile fatty acids (SCFA), carbon dioxide (CO2) and methane (CH4) (Blummel and Becker, 1997). The constant, a, is often ignored as it is assumed that there is no instantaneous substrate fermentation (Nsahlai et al., 1994). Blummel and Orskov (1993) enhanced the precision of this technique by terminating fermentation at 24 h and digesting the residue in neutral detergent solution. This modi®cation accounts for the soluble but not fermentable fractions that cause overestimation of dry matter loss in other in vitro and in situ systems (Khazaal et al., 1995; Blummel and Becker, 1997). The accuracy with which this method predicts feed intake, digestibility, microbial nitrogen supply and animal performance has been reported by Blummel and Orskov (1993, 1994) and Blummel and Becker (1997) using hay-based diets. However, Khazaal et al. (1995) reported that the gas production method was slightly inferior to the nylon bag technique in determining the nutritive value of hay but suggested that the technique was a better method for determining the nutritive value of tree fodder containing anti-nutritional factors such as polyphenols. It must be noted that the in vitro method derives its value from the accuracy with which it predicts in vivo measurements. The objective of this experiment was to test the accuracy of the in vitro gas production technique in evaluating the nutritive value of three Leucaena species and Calliandra calothyrsus as indi-

cated by the chemical composition of the feeds and in vivo goat responses. 2. Materials and methods 2.1. Samples Four diets consisting of three Leucaena accessions resistant to the pest Heteropsylla cubana (Crawford) and Calliandra calothyrsus (Oxford Forestry Institute (OFI) accession number 9/89) were used. The three Leucaenas were (i) Leucaena esculenta sub-species paniculata (OFI 52/87), (ii) L. diversifolia sub-species stenorcarpa (OFI 53/88) and (iii) L. pallida (Commonwealth Plant Introduction (CPI) 85890). The leguminous fodders were obtained from the Southern African Development Community-International Centre for Research in Agroforestry (SADC-ICRAF) plots (Latitude 17.58S and Longitude 31.58S), with an altitude of 1530 m above sea level, mean annual rainfall of 895 mm and mean annual temperature range of 15±208C. The fodders were each mixed with maize stover at 1:3 (w/w) in order to achieve a crude protein content of not less than 90 g kgÿ1 on as fed basis. Samples of the diet mixture were used as substrate in the gas production tests. The diets had been used in a previous intake and digestibility trial using 16 goats (Nherera et al., in press). Dry matter and organic matter were analysed using the method of Goering and Van Soest (1975). Nitrogen and neutral detergent ®bre were determined as described by the Association of Of®cial Analytical Chemists, 1990 and Licitra et al. (1996), respectively. Soluble phenolics and soluble and insoluble proanthocyanidins were analysed according to the methods of Giner-Chavez et al. (1997). Protein precipitating capacity was determined using the method of Hagerman (1987) and expressed as the diameter of the protein±tannin band. In vivo organic matter digestibility (OMD), digestible organic matter intake (DOMI) and microbial nitrogen (MN) supply determined from allantoin excretion (Chen and Gomes, 1992) are shown in Tables 1 and 2. 2.2. 96 Hour in vitro gas production test The 96 h in vitro gas production test was carried out to determine the extent and rate of gas production as

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Table 1 The content of dry matter (DM, g/kg), organic matter (OM), nitrogen (N), neutral detergent fibre (NDF) in g kgÿ1 DM, soluble phenolics (SP, g kgÿ1 DM), insoluble proanthocyanidins (IPA, AU550 nm gÿ1 NDF), soluble proanthocyanidins (SPA, AU550 nm 100 mgÿ1 DM) and protein precipitating capacity (PPC, mm) of forages fed to goats Component

Forage

SEM

Leucaena esculenta

Leucaena diversifolia

Leucaena pallida

Calliandra calothyrsus

Maize stovers

DM OM N NDF

913b 953c 38.8a 437a

921a 947d 36.3b 398b

911b 948bc 34.8c 378c

920a 956b 34.0c 385c

918a 966a 6.1d 802a

SP IPA SPA PPC

178.6b 19.6b 0.76a 7.8b

181.8b 19.7b 0.79a 9.1a

156.8c 18.7c 0.39c 7.8b

234.4a 29.9a 0.47b 9.6a

ND ND ND ND

dfˆ10 1.2 1.0 0.42 5.4 dfˆ8 3.04 0.22 0.008 0.27

p value

0.001 0.001 0.001 0.001 0.001 0.001 0.001 0.001

AU, absorbance units at 550 nm; ND, not determined. LS means in a row with the same superscript are not significantly different (p>0.05).

a,b,c,d

in¯uenced by the different substrates. Two rumen cannulated indigenous bucks of the small East African type, 3 years of age and weighing about 30 kg provided rumen liquor for the gas production technique. The bucks were penned individually and maintained on maize stover supplemented with dried L. leucocephala fodder (3:1, w/w). The legume (200 g) was offered at 0800±1200 hours and the stover (600 g) was offered from 1200±0800 hours of the following morning. Mineral licks and water were available ad libitum. Rumen liqour and digesta were collected from both bucks and mixed in a prewarmed CO2-®lled thermos ¯ask in the morning before the goats were offered the legume supplement. The rumen digesta was transferred to a domestic blender and homogenised for 15 s, strained through a double layer of

cheese cloth and ®ltered through glass wool into a prewarmed ¯ask. All laboratory handling of rumen liquor was done under continuous ¯ushing with CO2 so as to maintain anaerobic conditions. About 300 mg of each test diet was weighed into twelve 100 ml calibrated glass syringes, each syringe was ®tted with a plunger as described by Menke et al. (1979). The syringes were pre-warmed (398C) prior to injection of 30 ml of incubation media consisting of 10 ml of rumen liquor and 20 ml of buffer, prepared as described by Menke and Steingass (1988), in each syringe. The syringes were incubated in an upright position in a water bath at 398C as described by Blummel and Orskov (1993). Parallel incubations for the measurement of gas production without substrate (blank) and gas produced from 300 mg of solulta

Table 2 Digestible organic matter intake per metabolic liveweight (DOMI, g kgÿ1 W0.75 dayÿ1 ), apparent digestibility coefficients of organic matter (OMD) and daily microbial nitrogen supply (g dayÿ1) of goats used in the digestibility trial Parameter

OMD DOMI MN supply

Forage Leucaena esculenta

Leucaena diversifolia

Leucaena pallida

Calliandra calothyrsus

0.52 27.56 ÿ0.43b

0.53 26.92 1.85a

0.54 29.05 1.91a

0.54 28.08 2.68a

nsˆnot significant. dfˆ40. a,b LS means in a row with the same superscript are not significantly different (p>0.05). d

SEMd

p value

0.005 0.840 0.400

ns ns 0.001

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hay (standard) of known gas production parameters, were included at the beginning of the incubation period. The syringes were shaken by hand, twice in the ®rst hour of incubation and afterwards at every gas measurement to prevent the plunger from picking up substrate as it rose. Readings of gas volumes were recorded after 2, 4, 6, 8, 12, 24, 48, 72 and 96 h of incubation. After 96 h the contents of the syringes were rinsed into 50 ml centrifuge tubes and centrifuged twice at 3000g for 30 m. The supernatant was decanted and the residue dried and ashed to estimate in vitro organic matter degradability (IVOMD). 2.3. 24 Hour in vitro gas production test A shorter gas production test was done to determine truly degradable neutral detergent ®bre (TNDF) and to estimate the ratio of degraded NDF-to-gas produced, RA (Mgomezulu and Blummel, 1996). Rumen digesta collection, sample and buffer preparations and incubation techniques were as described above. The gas volumes were recorded at 2, 4, 6, 8, 12 and 24 h of incubation. Fermentation was terminated at 24 h and the entire residue was transferred into 600 ml spoutless beakers. The syringes were rinsed with 70 ml of neutral detergent solution. The method of Van Soest and Robertson (1985) for determination of true digestibility was then applied. A ratio of truly degraded substrate to gas produced (mg mlÿ1) was computed to give an estimate of variation in SCFA per microbial cell yield per unit substrate truly degraded. 2.4. Statistical analyses The time series measurements of gas volumes from the 96 h gas production test were ®tted into the modi®ed Orskov and McDonald (1979) non-linear exponential model P ˆ b…1 ÿ eÿct † to describe the rate and extent of fermentation where: P is gas production at time t, b is the potential extent of gas production and c, the fractional rate of gas production and the constant a, is equal to zero because unfermented feed does not produce gas. All gas volumes were adjusted to a common sample weight of 300 mg. Data from the two experiments was subjected to a one-way analysis of variance (ANOVA) for a completely randomised design (CRD). The model accounted for diet effects. Pearson's correlation coef-

®cients were used to determine the relationship between the chemical composition of feed and animal responses (digestible organic matter intake, digestibility and microbial nitrogen supply) and gas production characteristics. Simple and multiple linear regression procedures (selection/stepwise) were used in the validation of the importance of the gas production parameters in the prediction of animal responses and the importance of chemical composition in predicting gas production parameters. Only those parameters significant at p<0.05 for at least one variable were retained in the correlation and regression analyses. All analyses were done using the General Linear Model (GLM) procedures of the Statistical Analysis Systems (SAS, 1994). 3. Results 3.1. The 96 hour in vitro gas production test Potential gas production, b, (ml 300 mg DM) varied signi®cantly (p<0.001) between diets, ranging from 54.9 ml (L. pallida) to 65.2 ml (L. esculenta) (Table 3). The fractional rate, c, (ml hÿ1) also varied signi®cantly (p<0.001) between diets. The rates ranged from 0.024 ml hÿ1 for C. calothyrsus/maize stover diet to 0.034 ml hÿ1 in the L. esculenta/maize stover diet. The L. esculenta diet had the highest (p<0.001) absolute gas volume production at all sampling times, followed by L. diversifolia, C. calothyrsus diets and the L. pallida diet had the least. In vitro organic matter degradability (IVOMD) ranged from 494 to 520 g kgÿ1 DM and showed signi®cant (p<0.001) variation between diets. 3.1.1. Correlations between gas production parameters and feed chemical components Correlations between gas production components and selected feed chemical properties of the diets are given in Table 4. The correlations of dietary N and NDF with gas production components were positive and highly signi®cant (p<0.001). However, none of the gas production components were signi®cantly (p>0.05) correlated to soluble proanthocyanidins (SPA) and the protein precipitating capacity of tannins. Insoluble proanthocyanidins (IPA) were negatively correlated to c (rˆÿ0.46; p<0.01) and gas volume after 24 h (rˆÿ0.31; p<0.05). Potential gas

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Table 3 The potential extent of gas production (b, ml), fractional rate of gas production (c, ml hÿ1), gas volumes measured at 24, 48, 72 and 96 h (V24, V48, V72 and V96, ml) and in vitro organic matter digestibility coefficient (IVOMD) of the diets used Parameter

Forage

b c V24 V48 V72 V96 IVOMD d

Leucaena esculenta

Leucaena diversifolia

Leucaena pallida

Calliandra calothyrsus

65.2a 0.034a 38.7a 51.3a 58.2a 63.7a 0.49c

55.9c 0.030b 30.6b 41.6b 48.3b 53.7b 0.51b

54.9c 0.025c 26.2c 37.2c 44.6c 50.5c 0.52a

59.0b 0.024c 27.3c 39.8b 47.3b 53.1b 0.51b

SEMd

p value

1.00 0.0006 0.53 0.66 0.81 0.83 0.004

0.001 0.001 0.001 0.001 0.001 0.001 0.001

dfˆ40. LS means in a row with the same superscript are not significantly different (p>0.05).

a,b,c

production, b, and gas volume after 96 h (V96), however, were not correlated (p>0.05) to PAs and soluble phenolics (SP). Correlations between in vitro gas production components and dietary chemical components to IVOMD, OMD and DOMI are shown in Table 5. All gas production parameters were signi®cantly (p<0.05) and negatively correlated to both in vitro (IVOMD) and in vivo (OMD) digestibilities and DOMI. The correlations between volume of gas produced per unit of DM incubated and in vivo variables was highest (p<0.001) at V24. Neutral detergent ®bre was highly negatively correlated to OMD and DOMI (rˆÿ0.67; p<0.001) but weakly correlated to IVOMD (rˆÿ0.32; p<0.01). 3.1.2. Prediction of gas production parameters from feed constituents Nitrogen, NDF and polyphenolic components accounted for the between diet variation in the gas volumes at 24 and 96 h (V24 and V96) as well as the fractional rate of gas production, c (Table 6). Table 4 Correlations (r) between the extent of gas production (b), rate of gas production (c), gas volumes at 24 and 96 h (V24 and V96), neutral detergent fibre (NDF), nitrogen (N) and insoluble proanthocyanidins (IPA)

NDF N IPA ns

b

c

V24

V96

0.68c 0.45b 0.02ns

0.66c 0.76c ÿ0.46b

0.81c 0.75c ÿ0.31a

0.79c 0.63c 0.19ns

Not significant; ap<0.05; bp<0.01; cp<0.001.

Although polyphenolics could account for 80±90% of the variation in gas volumes (V96 and V24, respectively) and predict the rate constant, c, with high accuracy (r2ˆ0.75; p<0.001), they could not predict b (p>0.05). The latter component could only be predicted with some accuracy by including both N and NDF in a multiple linear regression equation (r2ˆ0.60; p<0.001). 3.1.3. Prediction of OMI and OMD from in vitro components and feed constituents Equations for the prediction of OMD and DOMI from chemical constituents and in vitro gas parameters Table 5 Correlations (r) between gas production components, chemical composition and in vitro organic matter digestibility (IVOMD), in vivo organic matter digestibility (OMD) and digestible organic matter intake (DOMI) IVOMD Gas production components b ÿ0.31a c ÿ0.50b V24 ÿ0.47b V96 ÿ0.42b Feed chemical components NDF ÿ0.32b N 0.51b IPA ÿ0.34a

MD

DOMI

ÿ0.51b ÿ0.68b ÿ0.72c ÿ0.68c

ÿ0.36a ÿ0.65c ÿ0.72c ÿ0.52c

ÿ0.67c 0.46b ÿ0.33a

ÿ0.67c 0.77c ÿ0.22a

ns, Not significant. ap<0.05. bp<0.01. cp<0.001. b, c, gas production constants; V24 and V96, gas volumes after 24 and 96 h; NDF, neutral detergent fibre; N, nitrogen; IPA, insoluble proanthocyanidins.

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Table 6 Predicting gas production components from feed chemical components (standard errors are indicated in parenthesis) Y variate

Equation

R2

b

ÿ86(23.9)‡2.5(0.42) NDF 25.5(0.2)‡22.(6.7) N ÿ40.2(21.2)‡3.1(0.4) NDFÿ2.2(6.4) N

0.46b 0.20a 0.60c

ÿ0.1(0.02)‡0.002(0.0004) NDF ÿ0.02(0.007)‡0.03(0.004) N ÿ0.07(0.02)‡0.001(0.0004)NDF ‡0.03(0.005) N 0.06(0.003) ‡0.03(0.002) SPA ÿ0.01(0.001) PPC ÿ145.4(19.7)‡3.0(0.34) NDF ÿ26.4(7.7)‡37.5(5.1) N ÿ124(16.8)‡2(0.34) NDF‡ 21.3(4.5) N 69.9(2.6)‡0.05(0.003) IPA‡ 31.6(2.3) SPAÿ14.1(0.74) PPC ÿ134(22.2)‡3.2 (0.38) NDF 2.6(9.8)‡34.6(6.4) N ÿ119(21.8)‡2.6(0.4) NDF‡ 14.5(5.9) N 93.8(4.1)‡0.06(0.005) IPA‡ 27.7(3.59) SPAÿ14.3(1.1)PPC

0.44b 0.58c 0.66c

c

V24

V96

0.75c c

0.65 0.56c 0.77c 0.90c 0.63c 0.40b 0.67c 0.80c

a p<0.05. bp<0.01. cp<0.001. b, c, gas production constants; V24 and V96, gas volumes at 24 and 96 h; NDF, neutral detergent fibre; N, nitrogen; IPA and SPA, insoluble and soluble proanthocyanidins; PPC, protein precipitating capacity.

are presented in Table 7. Neutral detergent ®bre was the only chemical component that signi®cantly but weakly predicted OMD (r2ˆ0.45; p<0.01). However, NDF could only account for 7% of the variation in DOMI whilst N accounted for 60%. Prediction of DOMI in a multiple linear regression based on polyphenols (IPA, SPA and PPC) was low but signi®cant (r2ˆ0.31; p<0.01). Predictions of intake and digestibility from gas production components were only signi®cant (p<0.05) for c and V24. 3.2. 24 Hour in vitro gas production test Total gas production after 24 h (V24), varied signi®cantly (p<0.001) between diets, ranging from 33.1 ml (C. calothyrsus diet) to 42.2 ml (L. esculenta diet) (Table 8). Degraded NDF (mg 300 mgÿ1) also varied from 96 mg to 106 mg and differed (p<0.01) between diets (Table 8). Truly degradable NDF (TNDF, g kgÿ1 DM) was signi®cantly lower

Table 7 Predicting in vivo organic matter digestibility (OMD) and digestible organic matter intake (DOMI) from chemical composition and gas production components and in vitro organic matter digestibility (IVOMD) (standard error is indicated in parenthesis) Y variate

Equation

R2

OMD DOMI

133.3(13.4)ÿ1.4(0.23) NDF 40.2(2.23)ÿ4.34.5(78.8)c 66.9(1.83)ÿ0.4(0.06)V24 62.6(4.34)ÿ22.6(2.84) N 97(11.8)ÿ0.74(0.24) NDFÿ 16.9(3.2) N 14(4.0)ÿ0.02(0.005) IPAÿ11(3.5) SPA‡4.9(1.1) PPC

0.45b 0.42b 0.52c 0.60c 0.67c 0.31b

a p<0.05. bpP<0.01. cp<0.001. c, fractional rate of gas production; V24, gas volume at 24 h; NDF, neutral detergent fibre; N, nitrogen; IPA and SPA insoluble and soluble proanthocyanidins; PPC, protein precipitating capacity.

(p<0.01) in the L. esculenta diet compared to other diets. The ratio relating truly degraded NDF (mg) to gas production (ml) (RA) also varied signi®cantly (p<0.001) between diets with the RA for L. esculenta being approximately 20% lower (p<0.001) than the rest. Relationships between the 24 h gas test parameters and DOMI and MN supply are shown in Table 9. Gas volume at 6 h (V6) was strongly and signi®cantly (rˆ0.94; p<0.001) correlated to gas production after 24 h. Gas volumes were signi®cantly (p<0.01) and inversely correlated to DOMI, TNDF, MN supply and RA. DOMI was positively and signi®cantly correlated with TNDF (rˆ0.59; p<0.001), MN (rˆ0.61; p<0.001) and RA (rˆ0.46; p<0.01). Truly degradable NDF was also signi®cantly correlated to MN supply (rˆ0.60; p<0.001) and RA (rˆ0.74; p<0.001). The correlation between MN supply and RA was positive and highly signi®cant (rˆ0.73; p<0.001). 3.2.1. Prediction of DOMI and MN supply Predictions of DOMI and MN yield using the 24 h in vitro parameters are shown in Table 10. Total gas volume after 24 h incubation and the amount of NDF degraded (mg) were non-signi®cant (p>0.05) in the prediction of these two components. Truly degradable NDF (TNDF) and RA poorly predicted DOMI (r2ˆ0.35; p<0.05 and r2ˆ0.20; p<0.01, respectively). Gas volume at 6 h and RA was better than TNDF in

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Table 8 Least squares mean estimates of volume of gas produced at 24 h (V24), neutral detergent fibre degraded (degrad. NDF), ratio of NDF degraded/ml of gas produced (RA) and content of truly degradable neutraldetergent fibre (TNDF), derived from the 24 h in vitro fermentation of 300 mg of the browse/maize stover mix Parameter

Forage

V24 (ml) Degrad. NDF(mg) RA (mg/ml gas) TNDF (g/kg DM) d

Leucaena esculenta

Leucaena diversifolia

Leucaena pallida

Calliandra calothyrsus

42.2a 96.2b 2.28b 601c

36.2b 105.5a 2.91a 663a

35.2b 100.3ab 2.84a 650ab

33.1c 96.3b 2.9a 628b

SEMd

p value

0.73 1.61 0.06 8.4

0.001 0.001 0.001 0.001

dfˆ40. LS means in a row with the same superscript are not significantly different (p>0.05).

a,b,c

predicting MN yield (r2ˆ0.50; p<0.001, r2ˆ0.53; p<0.01, respectively). Ef®ciency of predicting MN supply was improved by including both V6 and TNDF in a multiple linear regression equation (r2ˆ0.65; p<0.001) but was not improved (p>0.05) by inclusion of both RA and TNDF. 4. Discussion 4.1. 96 Hour in vitro gas production test The diet with the greatest potential b, fastest rate c, and highest gas volume per given time was L. esculenta, a shrub of moderate PA concentration, but it had the lowest degradability (494 gÿ1 kg DM). The reverse trend was true for L. pallida, low in PA content. A greater proportion of this variation can be attributed to compositional differences of the tree Table 9 Correlations (r) between in vitro gas production parameters (volumes at 6 and 24 h V6 and V24), mg truly degraded mlÿ1 of gas produced (RA) and truly degradable neutral detergent fibre (TNDF)) and digestible organic matter intake (DOMI) and microbial nitrogen output per day (MN) Parameter

V6

V 24

V6 V24 DOMI TNDF MN RA

0.94b ÿ0.48a ÿ0.42a ÿ0.71b ÿ0.86b

ÿ0.38b ÿ0.38a ÿ0.70b ÿ0.89b

a

b

p<0.01. p<0.001.

DOMI

TNDF

MN

fodder legumes tested, particularly their ®bre, nitrogen, nature of polyphenolic concentrations and possibly other anti-nutritional factors. These two forage legumes had similar protein precipitating capacities but different concentrations of SP, SPA and IPA. Insoluble proanthocyanidins (IPAs) were inversely correlated to the fractional rate, c, and gas volume after 24 h (V24). Haslam (1989) stated that IPA are strongly bonded to ®bre and therefore affect the rate and extent of ®bre degradation. No signi®cant (p>0.05) relationship was established between the extent of gas production, b, and proanthocyanidin concentrations. However, Nsahlai et al. (1994) found a negative correlation between b and IPA (rˆÿ0.71; p<0.01) but none with SPA. Correlations between SPAs and PPC and gas production characteristics in the present study were not signi®cant (p>0.05). Khazaal and Orskov (1994) also reported non-signi®cant negative correlations between polyphenols and the Table 10 Prediction of digestible organic matter intake (DOMI) and microbial nitrogen output (MN) from truly degradable neutral detergent fibre (TNDF), volume at 6 h and the ratio of degraded NDF mlÿ1 of gas produced (RA), derived from the 24 h gas production technique (standard error is indicated in parenthesis) Y variate

Equation

R2

DOMI

ÿ126.8(12)‡8(3.2) TNDF 40.6(12.6)ÿ10(4.6)RA ÿ16.8(5.7)‡0.29(0.09)TNDF ÿ8.4(1.34)‡3.6(0.52)RA 519(30)ÿ24.1(5.2)V6 ÿ3.6(5.4)‡15.5(7.4)TNDFÿ 0.63(0.16)V6

0.35b 0.20a 0.34a 0.53c 0.50b 0.65c

MN/day 0.59b 0.61b 0.46a

0.60b 0.74b

0.73b

p<0.05. bP<0.01. cP<0.001.

a

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volume of gas production and attributed their ®ndings to the large number and diversity of the browse species they used. The weak relationships reported in this study could also have been due to differences in fermentation patterns of the different browses as affected by the different chemical and functional structures of polyphenols. Correlating gas production parameters, b, c, V24 and V96, to IVOMD, OMD and DOMI gave inverse relationships in all cases. However, Blummel and Orskov (1993) using hay, obtained positive and highly signi®cant correlations between the gas parameters and true organic matter fermentation after 24 and 48 h of incubation (rˆ0.95, p<0.001; rˆ0.89, p<0.001). Blummel and Orskov (1993) stated that true fermentable organic matter is partitioned between fermentation gases (volatile short chain fatty acids (SCFA), CO2 and CH4) and microbial biomass. The relationship between microbial biomass and fermentation gases has been reported to be negative (Blummel et al., 1994) when both are related to a given unit of truly fermented substrate. It is therefore postulated that since there was an inverse relationship between digestibility and gas production parameters, degradation of tree fodder legume-supplemented diets favoured microbial protein synthesis, though to varying extents, to SCFA production and hence the negative correlations. Khazaal et al. (1995) reported that protein fermentation does not lead to much gas production. This difference between hay and tree fodder could be due to the high content of N and its slow release in tree fodder which better matches energy release in ®bre fermentation (Van Soest, 1982) thus ensuring synchrony between nitrogen and energy supply for microbial cell synthesis. This hypothesis needs to be tested both in vitro and in vivo using highly sensitive microbial cell markers. IPAs were negatively correlated to intake and digestibility (Table 5). This is in agreement with the results of Rittner and Reed (1992), who found that insoluble tannins had greater impact on digestibility and intake through binding of carbohydrate and protein. The browse species used by Rittner and Reed (1992) had lower NDF and N content than those used in this study. However, in this study the association between IPA intake and digestibility was low. This suggests a possible interaction between N, NDF and IPAs in modulating the impact of IPAs on ruminant

digestion. No signi®cant correlations were found between PPC of tannins and degradability. Makkar et al. (1989, 1993) and Khazaal et al. (1994) did not ®nd signi®cant (p>0.05) correlations between these two parameters and explained that PPC is determined under conditions (pH 5.0) that are different from those in the rumen. In the protein precipitation assay, bovine serum albumin is used whereas the rumen contains different proteins. Of all gas parameters, only V24 and c could predict intake with some precision while for chemical components N and NDF gave the best equation. Information about the IVOMD proved to be of less importance since it could only account for 22% (p>0.05) of the variation in DOMI. Khazaal et al. (1993) found similarly poor prediction equations using nylon bag data. The low precision with which degradability predicts intake in browses can be due to the presence of (poly)phenolics which interfere with palatability and hence the intake is without necessarily affecting microbial degradation of the feed (digestibility). Khazaal and Orskov (1994) suggested that the poor predictions may be due to the fact that the constants b and c are arithmetic derivatives. These authors also suggested that another possible cause of the poor predictions could be the low concentration of substrate (300 mg 30 mlÿ1) compared to that in the rumen of sheep (2.5 g 30 mlÿ1). However, other researchers using the same substrate concentration have found excellent predictions, casting serious doubts as to the validity of the above suggestion. Blummel and Orskov (1993) found that the asymptote, b, could account for 88% and total gas production 90% of the variance in intake but the rate constant did not signi®cantly (p>0.05) account for the variation. Nsahlai and Umunna (1996) also reported that gas production could predict in vivo dry matter digestibility of roughages (r2ˆ0.64, p<0.001) and legumes (r2ˆ0.82, p<0.001) and intake of roughages (r2ˆ0.41, p<0.05) but not intake of legumes. An additional possible contributory factor to the low predictions in our study could be the narrow ranges of DOMI and ODM used. 4.2. 24 Hour in vitro gas production test Blummel et al. (1994) and Mgomezulu and Blummel (1996) de®ned RA as the proportion of truly

F.V. Nherera et al. / Small Ruminant Research 31 (1999) 117±126

degraded substrate per unit of gas produced (mg mlÿ1 gas) which indicates variation in volatile SCFA production to microbial cell yield per unit of truly fermented substrate. Calliandra calothyrsus, L. diversifolia and L. pallida-based substrates produced less gas per unit of fermented material as was estimated in the 96 h experiment, pointing to the proportionately higher microbial protein yield. This was con®rmed by the high MN yield (Table 2). Proportionately less microbial yield and higher production of SCFA and other gases from fermented substrate would be a suitable explanation for the higher gas volumes in L. esculenta. Gas production was inversely correlated to the in vivo MN yield estimate and its in vitro equivalent (RA) (rˆÿ0.71; p<0.001; rˆÿ0.88; p<0.001; respectively). The positive correlation between MN and TNDF (rˆ0.60; p<0.001) and DOMI (rˆ0.61; p<0.001) also con®rms that degradation of feeds with a legume component promotes microbial protein synthesis. This could be due to the high N content of the legume. The ratio of SCFA-to-MN yield per unit of substrate fermented (RA) was highly correlated to MN yield. The coef®cient of determination for predicting MN supply from RA was relatively high in agreement with the results reported by Mgomezulu and Blummel (1996). These results show that prediction of MN supply and, to a lesser extent, intake can be achieved by investigating the relative proportion of SCFA-to-microbial cell yield per unit substrate of forages using a simple 24 h in vitro technique. 5. Conclusion The in vitro gas production technique has a potential to be an important tool for assessing degradation (rate and extent of fermentation over 96 h) and estimating in vivo microbial nitrogen supply (24 h gas test) of tree fodder legume-supplemented diets when fed to goats. The 24 h gas production test was able to re¯ect trends in gas production and degradation similar to those established in the 96 h gas production test and also offered the advantage of being able to predict MN yield. It is therefore recommended that the shorter incubation be used as this will enable more feeds to be evaluated over a short period of time. There is a need, though, for more studies to con®rm the relationship

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between the 24 h gas production estimates and microbial nitrogen supply using tree fodder. Further research is also necessary to determine the relationship between gas production and in vivo responses on the one hand and speci®c phenolics as well as research that incorporates a more diverse array of browse on the other. Acknowledgements The authors are grateful to the African Network for Agroforestry Education (ANAFE) of the International Centre for Research in Agroforestry (ICRAF) for ®nancing this work and to the department of Animal Science, University of Zimbabwe for the technical and logistical support

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