Directory UMM :Data Elmu:jurnal:A:Animal Feed Science and Technology:Vol85.Issue3-4.Jun2000:

The effect of PEG addition in vitro on dry matter and

nitrogen digestibility of

Calliandra calothyrsus

and

Leucaena leucocephala

leaf

B. Palmer, R.J. Jones

*

CSIRO Tropical Agriculture, Davies Laboratory, PMB Post Of®ce, Aitkenvale, Qld 4814, Australia

Received 8 October 1999; received in revised form 24 January 2000; accepted 15 February 2000

Abstract

A modi®ed two stage in vitro digestion method was used to study the effects of rate of PEG addition (0±1100 mg/g DM) on dry matter (IVDMD) and nitrogen digestibility (IVND) of freeze dried leaf materialfrom actively growingshootsofCalliandra calothyrsus(Calliandra)andLeucaena leucocephala

cv. Cunningham (Leucaena). For both species, IVDMD and IVND increased asymptotically with PEG rate; the IVND response being markedly greater for Calliandra. There was a three-fold greater amount of PEG bound to the residue after Stage 1 (rumen ¯uid/buffer) digestion with Calliandra than with Leucaena (105 versus 33 mg/g DM). The presence of PEG in the residue, which was not reduced after Stage 2 (acid±pepsin) digestion, resulted in a higher residue weight and therefore an underestimated IVDMD. Quanti®cation of the PEG in the residue enabled a corrected IVDMD (CIVDMD) to be esti-mated. PEG did not bias estimates of nitrogen digestibility in the same way. In a second experiment, estimates of CIVDMD and IVND were made after Stage 1 and after Stage 2 in the presence and absence of PEG at 160 mg/g sample. For Leucaena, there were small, but signi®cant, effects of PEG, whereas with Calliandra there were large responses to PEG and to timing of addition. When measured after Stage 1, IVND in the absence of PEG was not signi®cantly different to zero (compared with 57% for Leucaena), whereas with PEG, IVND was 75% (compared with 68% for Leucaena). The low IVND for Calliandra was associated with low NH4N levels in the rumen ¯uid/buffer after Stage 1.

In summary, about 160 mg PEG/g sample is appropriate for most studies with tropical tanniniferous shrub legumes to estimate any deleterious tannin effect. The use of PEG to estimate this effect on IVDMD is not valid without accounting for the PEG bound to the residue. For estimation of the adverse effect of tannins on IVND the use of PEG may be more appropriate.#2000 Published by Elsevier Science B.V.

Keywords:In vitro digestibility; Tannin containing shrub legumes; PEG binding; DMD; N digestibility 85 (2000) 259±268

*_{Corresponding author. Tel.:‡61-7-47538520; fax:‡61-7-47538600.}

E-mail address: raymond.jones@tag.csiro.au (R.J. Jones)

1. Introduction

The ability of PEG to bind with condensed tannins (CT) has been used in ®eld and laboratory studies to measure and to reduce the adverse effects of CT in ruminant diets (Pritchard et al., 1988; Barry, 1989; Silanikove et al., 1994; McSweeney et al., 1999; Jones et al., 2000). Most of these laboratory studies have measured in vitro dry matter digestibility (IVDMD) or gas production. The improvement in IVDMD in some of these studies has been smaller than expected or even negative for tanniniferous feeds (Jones et al., 2000). If PEG binds with some component of the feed to produce an insoluble complex then it would remain in the residue and cause an underestimation of IVDMD. However, this would not be the case if any complexes formed at rumen pH were dissociated in the subsequent acid±pepsin stage (Makkar et al., 1995). Estimation of the PEG in the residue after Stage 1 and release from the residue in Stage 2 would allow an assessment of such changes. Use of14C-PEG offers an accurate method for estimating the PEG bound to the residue after Stage 2, thus allowing a correction to be made to the IVDMD measurement (CIVDMD).

We examined the effect of a range of PEG rates on IVDMD with leaf material from two tropical browse species known to differ in tannin content and IVDMD. We also measured in vitro nitrogen digestibility (IVND) since although PEG may increase IVND, the presence of PEG in the residue would not cause bias in this estimate. We also explored the effect of PEG added at Stages 1 and 2 on digestibility during each stage.

2. Materials and methods

2.1. Plant samples

Samples were the terminal ®ve fully expanded leaves from actively growing shoots of the tropical shrub legumes Calliandra calothyrsus (CPI1 115690) (Calliandra) and

Leucaena leucocephala cv. Cunningham (Leucaena) growing on an alluvial soil at the CSIRO Lansdown Research Station, 50 km south of Townsville, north Queensland, Australia (198400S, 1468500E). The plant material was frozen and transported from the ®eld in insulated containers and kept frozen in an anaerobic atmosphere prior to freeze-drying. After drying the material was ground to pass a 1 mm screen and analysed for nitrogen.

2.2. In vitro digestibility Ð PEG rate

PEG 4000 solutions, prepared to give various rates in the range of 0±1000 mg PEG/g sample, were spiked with14C-PEG 4000 (Amersham, UK) in 2 ml water and added to 0.4 g DW (dry weight) of sample in glass centrifuge tubes. To duplicate samples 40 ml rumen ¯uid/buffer (1:4) was then added. There were 10 levels of PEG for Calliandra and ®ve levels for Leucaena. Tubes were then incubated in an anaerobic chamber at 398C using a modi®ed Tilley and Terry (1963) in vitro technique (Jones et al., 1998). After

1_{Commonwealth Plant Introduction Number (Australia).}

Stage 1 (72 h in rumen ¯uid/buffer), tubes were centrifuged for 20 min at 2500g. Two samples, each of 1 ml were withdrawn from the supernatant and added to 10 ml of scintillant (OptiPhase `HiSafe' 31

; Fisher Chemicals, UK). Tubes were subsequently counted for 10 min in a scintillation counter (Wallac 1410 Pharmacia, Finland). Residues in the tubes were washed with water on a vortex mixer, then centrifuged for 10 min, the supernatant discarded and the process repeated before adding 40 ml of acid pepsin (2 g 1:10,000 pepsin in 1 l 0.1 M HCl). Each tube was then thoroughly mixed on the vortex mixer and incubated at 398C for 24 h. Tubes were centrifuged, the supernatant sub-sampled for counting as before, the remaining supernatant discarded and the residues washed, dried at 808C for 48 h and weighed. Residues were analysed for nitrogen.

2.3. In vitro digestibility Ð timing of PEG addition

In the second experiment, the same plant samples were used and the same digestion protocol was followed. Two rates of PEG only were used; 0 (Water) and 160 mg PEG/g sample. Five treatment sequences were compared (Table 1). IVDMD and IVND were measured after Stage 1 for the ®rst two treatments and after Stage 2 for the remainder.

2.4. Statistical analyses

Data for IVDMD, CIVDMD and IVND were analysed using ANOVA to test for differences between individual levels of PEG rate and speciesPEG rate interactions (SPSS, 1995). For the ®rst experiment, curves were also ®tted to treatment means using an iterative non-linear regression model of the form

Yˆb0‡b1(1ÿexpb2X) (SigmaPlot, 1997).

3. Results

3.1. PEG rate

Labelled PEG released during Stage 2 was not signi®cantly different to zero, indicating that any PEG bound to the residue in Stage 1 was not released in the acid±pepsin. PEG bound to the residue was subsequently only measured using the14C-PEG activity in the supernatant after Stage 1 digestion.

Table 1

Treatment sequences for digestibility measurements

Treatment No. Code Stage 1 (rumen fluid/buffer) Stage 2 (acid±pepsin)

1 w1 Water

2 p1 PEG

3 w1‡w2 Water Water

4 w1‡p2 Water PEG

For Calliandra, increasing rates of PEG increased IVDMD, CIVDMD and IVND (Fig. 1) with an asymptotic value of 56.4, 65.1 and 88.1%, respectively. These levels were all achieved at 160 mg PEG/g DM or less. For Leucaena, there was no signi®cant response to PEG in terms of IVDMD (69.5%). However, the asymptotic responses for CIVDMD and the IVND were 78.5 and 86.3% at 80 mg PEG/g DM or less (Fig. 2). Leucaena had signi®cantly higher IVDMD and CIVDMD than Calliandra at all rates of

Fig. 1. Effect of rate of PEG addition on IVDMD, CIVDMD and IVND forC. calothyrsusleaf.

Fig. 2. Effect of rate of PEG addition on IVDMD, CIVDMD and IVND forL. leucocephalaleaf.

PEG addition (Figs. 1 and 2). At zero PEG, IVND of Leucaena was higher than for Calliandra, but above 80 mg PEG/g DM, there were no differences between species.

The relationships between IVDMD, CIVDMD and IVND and PEG are shown in the following equations:2

Calliandra

IVDMDˆ53:2…1:40†‡3:23…0:250†…1ÿexp…ÿ0:100…0:0318†PEG rate††; R

2_ˆ0 :96

CIVDMDˆ51:6…0

:30†‡13:5…0:32†…1ÿexp…ÿ0:0195…0:00110†PEG rate††; R2ˆ0:99

IVNDˆ53:2…1:40†‡34:9…1:48†…1ÿexp…ÿ0:0227…0:00230†PEG rate††; R

2_ˆ0 :99

Leucaena

IVDMDˆ69:5…0:27†‡0:0008…0:00060†PEG rate; R

2_ˆ0 :42

CIVDMDˆ69:8…1

:27†‡8:74…1:520†…1ÿexp…ÿ0:0299…0:01450†PEG rate††; R2ˆ0:94

IVNDˆ74:4…0:90†‡11:9…1:05†…1ÿexp…ÿ0:0526…0:02040†PEG rate††; R

2_ˆ0 :98

The amount of PEG bound by Calliandra was approximately three times that bound by Leucaena (105 and 33 mg/g, respectively) (Fig. 3). This is in line with the levels to support maximum IVND for the two species (Figs. 1 and 2).

2_{Value in subscript (within parentheses) is standard error of the coef®cient.}

Fig. 3. Effect of rate of PEG addition on the level of PEG bound in the residue after Stage 1 in vitro digestion for

The relationship between PEG binding and rate of PEG addition to Calliandra and Leucaena are shown in the following equations:

Calliandra

PEG bindingˆ105:2…2:17†…1ÿexp…ÿ0:0142…0:00110†PEG rate††; R

2_ˆ0 :99

Leucaena

PEG bindingˆ33:2…0:74†…1ÿexp…ÿ0:0280…0:00280†PEG rate††; R

2_ˆ0 :99

Fig. 4. In vitro digestibility ofC. calothyrsusandL. leucocephalaleaf following Stage 1 and following Stage 2 digestion in the presence and absence of PEG added at Stage 1 or Stage 2 (see Table 1): (a) dry matter digestibility; (b) corrected dry matter digestibility, and (c) nitrogen digestibility.

3.2. Timing of PEG addition

For all treatments, IVDMD and CIVDMD values for Leucaena were higher than for Calliandra (Fig. 4). With Leucaena, only treatment 5 gave a signi®cantly higher IVDMD, whereas with Calliandra the addition of PEG at Stage 1 (treatments 2 and 5) resulted in higher IVDMD; treatment 1 gave a markedly lower value than the other treatments. Similar results were obtained for CIVDMD except that with Calliandra treatment 4 (PEG applied at Stage 2) signi®cantly higher values were obtained than for treatment 3 where no PEG was added (Fig. 4). For Stage 1, PEG increased IVND (p<0.01) from 57 to 68% for Leucaena and very strongly for Calliandra from 0 to 75%. After Stage 2 with Leucaena, there was little difference in IVND whether PEG was applied at Stage 1 or at Stage 2 but both treatments were higher than the control with no PEG (treatment 3). In contrast, with Calliandra there was a large response to PEG applied at Stage 1 and at Stage 2 with IVND being higher, but not signi®cantly different from Leucaena, when PEG was added in Stage 1 (Fig. 4).

In the absence of PEG, NH4±N in the supernatant after Stage 1 was vastly different

between the two species, with levels for Calliandra not signi®cantly different from zero. In the presence of PEG, values were higher but not signi®cantly different between species (Fig. 5).

4. Discussion

4.1. In vitro digestibility Ð PEG rate

1980; Ahn et al., 1989; Norton, 1994; Jones et al., 1998). The range of PEG rates used in this experiment was wide enough to measure a full response curve for both species and showed that there was little response in terms of digestibility to rates above 160 mg/g sample. In the light of these results, it is surprising to see recommendations for rates up to 1 g/g sample for studies using PEG to bind tannins in gas production PEG binding studies (Makkar et al., 1995; Silanikove et al., 1996).

With IVND, the initial response to PEG addition was extremely high with major increases from small additions in PEG, e.g., a 50% response to the addition of only 30 mg PEG/g sample. It is quite clear from these results that the major cause of low N digestibility in Calliandra is the high CT content, since nullifying the effects of tannin has resulted in IVND as high as with Leucaena. However, it should be noted that CIVDMD for Leucaena (78%) is signi®cantly higher than for Calliandra (65%). This may well re¯ect the intractable nature of the ®bre fraction in Calliandra.

The presence of PEG in the residue strongly suggests that insoluble tannin-PEG complexes have been formed during digestion. It has been suggested (Makkar et al., 1995) that these complexes may also be insoluble in acid±pepsin. Our results clearly show that such complexes are not soluble and therefore introduce serious bias in the estimation of IVDMD. For this reason, very little response in IVDMD to PEG has accompanied the use of PEG with some tanniniferous species (Salawu et al., 1997; Jones et al., 2000).

The three-fold difference in PEG binding between the two species has been shown in tannin measurements using the butanol±HCl and vanillin methods (Jackson et al., 1996) but was not reported by Norton (1994). Such variation is probably due to fact that inappropriate standards (not those from the plants concerned) were used (Hagerman and Butler, 1989; McNeill et al., 1998). The method of PEG binding to assess tannin activity better re¯ects the IVND response to PEG between the two species (11.9 and 34.9% units for Leucaena and Calliandra, respectively).

4.2. In vitro digestibility Ð timing of PEG addition

With Calliandra and Leucaena, there were increases in CIVDMD with PEG addition both during Stages 1 and 2, but with Calliandra, the increases were far more pronounced suggesting a greater tannin activity. These differences are somewhat masked with IVDMD because of the PEG bound to the residue. Without PEG addition, there was no increase in dry matter digestibility in the second stage with Leucaena whereas with Calliandra there was an increase from 36 to 45%, again suggesting greater tannin activity with Calliandra. In terms of nitrogen digestibility, the effect of this greater tannin activity has been totally eliminated by the addition of PEG to Stage 1 of a two-stage digestion. Without the addition of PEG in Stage 1, there was no digestibility of the protein from Calliandra. In fact, as shown in Fig. 5, the ammonium in the digestion medium was dramatically reduced, any ammonium present from the rumen ¯uid had been utilised by the microorganisms.

In conclusion, about 160 mg PEG/g sample is appropriate for most studies with tropical tanniniferous shrub legumes to estimate any deleterious tannin effects. The use of PEG to estimate this effect on IVDMD is not valid without accounting for the PEG bound

to the residue. Furthermore, estimates of IVDMD on the residues in gas production systems are also overestimated although the rate of gas production per se may be a useful parameter. Nevertheless, the effect of bound PEG on the underestimation of IVDMD is important and the use of nitrogen digestibility as an estimate of nutritional value is strongly recommended.

Acknowledgements

We thank the Australian Centre for International Agricultural Research (ACIAR) for funding the studies, and the School of Biomedical and Molecular Sciences, James Cook University of north Queensland, Australia, for use of their scintillation counter.

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