Effect of freeze drying on in vitro ruminal fermentation dynamics of three tropical shrub legumes with and without condensed tannins

T T Tiemann*,**, P Avila**, C E Lascano**, M Kreuzer*and H D Hess***

* ETH Zurich, Department of Agricultural and Food Science, Universitaetstrasse 2, CH-8092 Zurich, Switzerland
** Tropical Grass and Legume Project, CIAT, Cali, Colombia
*** Agroscope Liebefeld-Posieux, Research Station ALP, P.O. Box 64, CH-1725 Posieux, Switzerland
dieter.hess@alp.admin.ch

Abstract

Extensive comparisons of the effects of tropical shrub legumes rich in condensed tannins (CT) require well-conserved material. It is, however, unclear if the application of even gentle methods like freeze drying (lyophilization) affects the results in comparison to fresh material. Therefore, an experiment with the gas-pressure transducer technique, simulating ruminal fermentation dynamics in vitro, was conducted to investigate the effect of freeze drying on the ruminal nutrient degradability of three tropical multipurpose shrub legumes. Leaves of the CT shrubs Calliandra calothyrsus and Flemingia macrophylla and of the CT-free shrub Cratylia argentea were tested either in fresh form or lyophilized. In order to simulate practical feeding conditions, the legume leaves were incubated together with Brachiaria humidicola (1:2) for 144 h. Additionally, incubations were carried out either with or without polyethylene glycol (PEG) to be able to separate effects either dependent or independent of the CT.

Only few differences were found between fresh and lyophilized leaves. These included that the proportion of apparently undegraded nitrogenous compounds was higher in fresh than in lyophilized leaves of Flemingia macrophylla, and freeze drying had a limited influence on volatile fatty acid production in Calliandra calothyrsus. The variables related to degradation dynamics (i.e., total gas production, the time until the point of inflection, apparent dry matter degradability), however, were not influenced. There was also no difference between CT and non-CT plants in that respect, as is also obvious from the lack of interactions of state of the plant material and PEG addition.

This indicates that effects of freeze drying of shrub leaves on overall ruminal nutrient degradation in mixed grass-legume diets were minor.

Keywords: Calliandra, condensed tannins, Flemingia, lyophilization, rumen degradation dynamics

Introduction

In vitro experiments are rarely conducted with fresh plant material due to its limited shelf life and difficult handling. Hence the use of oven-dried, lyophilized or even sun-dried forage plants for laboratory analyses and experiments is common. There is little information on how drying affects the nutritional properties of tanniniferous species like the non-herbaceous legumes from the tropics, and these scarce findings are contradictory. There are several authors who report or assume that freeze drying does not significantly alter condensed tannin (CT) concentrations compared to fresh leaves (Terril et al 1990; Ahn et al 1997; Norton and Ahn 1997; Dalzell et al 1998) which, however, does not exclude that the drying process could change CT properties. Hove et al. (2003) reported for different sun and oven drying procedures a significant effect on the degree of polymerisation of CT. Palmer and Ibrahim (1996) described that using lyophilized instead of fresh (frozen) material of Calliandra calothyrsus leaves markedly reduced in situ ruminal degradability. Mahyuddin et al (1988), comparing various drying treatments, noted that the apparent reduction in digestibility of leaves from Calliandra calothyrsus upon drying may be less marked in vitro than in situ. Additionally, they found that lyophilized samples were more similar to oven dried than to fresh samples. Concerned by these results, Stewart et al (2000) more recently investigated the effect of various post-harvest treatments on Calliandra calothyrsus. They described a significantly higher in vitro digestibility of air-dried and lyophilized leaves compared to leaves frozen in liquid nitrogen, ground and later thawed when using one of two accessions (provenances) of Calliandra calothyrsus. Nevertheless, Stewart et al (2000) found no significant differences between air-dried and lyophilized samples in contents of crude protein (CP), neutral detergent fiber (NDF) and acid detergent fiber (ADF) as well as astringency and procyanidin:prodelphinidin ratio. From their results Stewart et al (2000) concluded that drying effects might depend on provenance. Such a phenomenon might be even more pronounced between plant species. The purpose of the present study was, therefore, to quantify the effects of freeze drying on ruminal nutrient degradation and gas formation as simulated with the well-established gas-pressure transducer technique (GTT) for three major tropical shrub legume species. The emphasis was put on the comparison of the effects of CT in typical mixed grass-legume diets using either fresh (never even frozen) or lyophilized legume samples. Polyethylene glycol (PEG) was applied to inactivate CT as there might be an interaction between CT properties and ruminal nutrient degradation and because effects even of gentle drying such as freeze drying cannot be totally excluded.

Materials and methods 

Plant material

In line with farm practice in many tropical countries, the diet included a tropical grass. In the present study, the low-quality (<40 g/kg of crude protein) Brachiaria humidicola (formerly called Brachiaria dictyoneura, CIAT 6133 of the germplasm collection held at the Centro Internacional de Agricultura Tropical (CIAT)) was selected. The legumes tested included two CT-shrubs, Calliandra calothyrsus (CIAT 22310) and Flemingia macrophylla (CIAT 17403) as well as the non-CT shrub Cratylia argentea (CIAT 8516). The grass was harvested after 12 weeks of re-growth from a 4-years old pasture and was sun dried. The legumes were cultivated in pure stands on a Mollisol (pH about 4.2) at CIAT’s Headquarters in Palmira, Colombia (3° 30’ N, 76° 21’ W; 965 m a.s.l). Foliage of the legumes was harvested from mature plants.

Simultaneously harvesting material for the comparison of fresh (i.e., never even frozen) and lyophilized material was not possible due to the time needed for freeze drying. Comparing the materials at different times with different rumen fluid was considered as the more serious bias. Therefore, it was decided to keep conditions for both harvests the same (always 9 weeks after the respective last cut; harvest from the same pure stands of the shrubs) and to schedule the experiment to take place in the middle of the rainy season where not only plant growth was high but also climatic conditions were relatively constant. At the first cut the harvested material was immediately put on ice. Subsequently, samples were stored at –20°C until being lyophilized (Edwards Freeze Dryer, Edwards, West Sussex, UK). The lyophilized plant material was then ground to pass a 1-mm screen using a Wiley laboratory mill. For CT extraction and analysis, the plant material was finely ground employing a piston-action ball mill. Fresh material of all experimental legumes was harvested 9 weeks later, put on ice for transport and used immediately in the experiment.

Experiment with the gas-pressure transducer technique

Fermentation dynamics and ruminal nutrient degradation were measured in vitro by the GTT as described by Theodorou et al (1994) and following the protocol as described in detail in Tiemann et al (2008). The six feed mixtures (three legume species, two drying methods) were tested both with and without PEG to determine the CT dependent part of the effects. Each of the resulting 12 treatments was evaluated in three replicates. Rumen fluid was collected from two rumen fistulated Zebu steers grazing a pasture of star grass (Cynodon plectostachyus, medium feed quality). The pH of the fresh rumen fluid used was 6.6 and the redox potential was –290 mV. Directly prior to incubation, DM contents of the fresh and the already prepared lyophilized materials were analyzed. The results of the fresh samples were subsequently used for the determination of the amount of fresh plant material required to (approximately) achieve the intended ratio of Brachiaria humidicola and legume dry matter (DM) of 2:1. Two days later fresh legume leaves were harvested again, stored immediately on ice and ground with a meat grinder under permanent ice cooling. This material was kept on ice in small aluminum dishes, and the amounts determined as described above were weighed rapidly onto pre-cooled pieces of aluminium foil. Simultaneously, a second sample was weighed to determine the precise DM content for later data adjustment. The forage was incubated in 160 ml serum bottles in a digestion medium containing micro- and macro-elements, a reducing agent and a reduction indicator (Menke and Steingass 1988). A total of 90 ml of this solution was complemented with 10 ml of rumen fluid. An amount of 100 g PEG/l was added to the incubation buffer for the respective samples. One gram of DM of the experimental diets was added to this mixture.

Laboratory analyses

The individual forages were analyzed in sun-dried form (grass) or in fresh and lyophilized form (legumes) for DM (24 h at 105 °C), total ash (3 h at 500 °C), N (San+ Autoanalyzer, Skalar Analytical, Breda, The Netherlands), NDF and ADF (Van Soest et al 1991). The NDF was determined without the use of α-amylase, as no starch was expected in the plants used, but with the addition of sodium sulphite as recommended by Robbins et al (1987) for CT plants. Sulphuric acid detergent lignin was analyzed by solubilisation of cellulose with sulphuric acid (Robertson and Van Soest 1981). Detergent fiber data were not corrected for ash content. Contents of extractable and bound CT of the two CT-plants, Calliandra calothyrsus and Flemingia macrophylla, were analyzed by photospectrometric measurement (absorbance at 550 nm) of plant extracts prepared according to Terrill et al (1992). Purified tannins, extracted from each material by the procedure of Hagerman and Butler (1980), were used as standards.

In fresh rumen fluid and fermentation fluid after incubation pH, redox potential and VFA profile (HPLC) were determined (cf. Tiemann et al 2008). Fermentation residues were analyzed for DM and N as described above for the forages. This data was used to calculate in vitro dry matter degradability (DMD) and the proportion of total dietary N incorporated in CP apparently remaining undegraded (UDN). Gas production data was fitted to a modified sigmoid model of Gompertz which was recommended for gas production systems (cf. Tiemann et al 2008)
 

Where:

y is the cumulative total gas production (ml) at time t (h),
A is the maximal (asymptotic) gas production (ml/g),
B is the latency period before exponential gas production starts (lag period) (h), and
C is the specific gas production rate (ml/h) (representing the microbial activity).

Additionally, the point of inflection of the curves was calculated as lnB/C, where gas production rate is maximal and the cumulative gas volume is equivalent to 36.8 % of A. Calculations of gas production parameters were performed using the PROC NLIN procedure of SAS (version 8.2 for Linux, SAS Institute, Cary, NC, USA).

Statistical analysis

Analysis of variance was performed separately for each species with the GLM procedure of SAS (version 8.2, SAS Institute, Cary, NC) applying the following model:
 

y_ijk = m + S_i + P_j + SxP_ij + e_ijk 

Where:

S=plant state (fresh or lyophilized),
P=PEG level (–/+), and
SxP=interaction among S and P, and
ε_ijk =residual error.

Multiple comparisons among means within plant species were performed with the Ryan-Einot-Gabriel-Welsh method (REGWQ). Table 2 gives means, standard errors of the mean (SEM) and P values for the effects.

Results and discussion 

The results on the chemical composition of the forages (Table 1) were consistent with findings from other studies (e.g. Tiemann et al 2008) and may therefore be considered representative for the species and their accessions investigated.

To get data relevant to practical feeding systems, quite a high percentage of low quality grass hay had been supplemented with either fresh or lyophilized plant material with or without CT. As the basal diet was not the focus of this experiment it was kept equal for all treatments to guarantee that potential differences in the fermentation process exclusively resulted from variations in the supplemented legumes when applied in dietary proportions reflecting the upper limit used in feeding practice.

There were only very few statistically significant differences in fermentation characteristics between diets containing fresh and lyophilized legumes (Table 2).

None of these differences was occurring in the fermentation dynamics variables, suggesting that overall microbial degradation processes were not substantially influenced by the state of the forage. However, residues from diets with fresh Flemingia macrophylla contained more (P<0.05) UDN than those with lyophilized Flemingia macrophylla. In diets containing Calliandra calothyrsus, total VFA concentration and propionate proportion of total VFA were higher and butyrate proportion was lower when using lyophilized material. In Flemingia macrophylla, the effect disappeared when PEG was added, indicating that the difference was due to modifications related to CT. Based on the lack of substantial effects of lyophilisation on fermentation dynamics, it was also assumed that harvest times had only minor influences, if any, since an exact compensation of effects of harvest batch and lyophilisation is rather unlikely. Additionally, contents and composition of CT and of most nutrients was similar (Table 1). As expected, the addition of PEG to the incubation buffer clearly increased the degradation of nitrogenous compounds (reduced proportion of UDN) in diets containing Calliandra calothyrsus or Flemingia macrophylla, and had no effect in the diet with the CT-free C. argentea. On the other hand, DMD of Flemingia macrophylla was lower (P<0.001) and DMD of Calliandra calothyrsus also tended to be lower (P<0.1) when PEG was added. This apparent reduction of DMD could be an artefact due to the formation of complexes between CT, fibre and PEG as described by Makkar et al (1995). Furthermore there were PEG effects on certain fermentation variables of the diet with C. argentea, which did not contain any detectable amounts of CT. Although similar observations have been made in previous studies (Stürm et al 2007), the reasons for these unexpected effects remain unclear.

Stewart et al (2000) investigated the effect of air-drying and freeze drying in comparison to frozen and later thawed leaves in two accessions of Calliandra calothyrsus. They found that, in accession CIAT 22310, the same as investigated here, in vitro DMD (applying the two stage method of Tilley and Terry 1963) was lower in fresh leaves compared to dried samples, while this was not the case for accession CIAT 22316. The lower in vitro DMD in accession CIAT 22310 occurred even though protein-bound CT had made up a smaller proportion in the fresh samples. Our data do not confirm the presence of such alterations when using GTT-derived DM degradation in Calliandra calothyrsus CIAT 22310. It cannot be excluded that the longer incubation period applied with GTT, as compared to the Tilley and Terry (1963) method, might have masked such a difference. However, 37% of the total gas production (h-infl) was reached within 34 h and 26 h with fresh material from Calliandra calothyrsus and Flemingia macrophylla, respectively, and 9 h (P<0.05) and 3 h later with lyophilized material, while the opposite would have been expected from the results of Stewart et al (2000). This would indicate that data obtained with lyophilized forage would, if at all, rather lead to an underestimation of the fermentability than to an overestimation. The present observation in Calliandra calothyrsus supports also the findings of Ben Salem et al (1999) who reported that drying Acacia cyanophylla had no major effect on the nutritive value in response to its high CT content. Norton and Ahn (1997) reported that feed utilisation was lower when feeding previously frozen leaves of Calliandra calothyrsus to sheep compared to feeding oven-dried leaves. However it is most likely that this effect was due to modifications of CT as drying was done using a forced draught oven at 60 °C for 48 h and properties of CT may be modified at this temperature. By contrast, in the present study the wide lack of significant interactions between plant state and PEG illustrates that changes in CT-properties due to freeze drying were mostly negligible.

In conclusion, potential changes in composition and structure of plant compounds in tropical shrub legumes with or without CT by freeze drying appear to have no substantial effect on ruminal degradation dynamics and fermentation end products. Therefore samples may be gently conserved by this method without causing a serious bias in the estimation of the nutritional value of such forages in vitro.

Acknowledgements 

The authors are grateful to the staff of the laboratory of ‘Forrajes tropicales’ at CIAT in Cali, Colombia. This study was supported by the North-South Centre of ETH Zurich (formerly ‘ZIL’), Switzerland.

References 

Ahn J H, Elliott R and Norton B W 1997 Oven drying improves the nutritional value of Calliandra calothyrsus and Gliricidia sepium as supplements for sheep given low-quality straw. Journal of the Science of Food and Agriculture 75: 503-510.

Ben Salem H, Nefzaoui A, Ben Salem L and Tisserand J L 1999 Intake, digestibility, urinary excretion of purine derivatives and growth by sheep given fresh, air-dried or polyethylene glycol-treated foliage of Acacia cyanophylla Lindl. Animal Feed Science and Technology 78: 297-311.

Dalzell S A, Stewart J L, Tolera A and McNeill D M 1998 Chemical composition of Leucaena and implications for forage quality. In H M Shelton, R C Gutteridge, B F Mullen and R A Bray editors. Leucaena – adaptation, quality and farming systems, Volume 86 of ACIAR Proceedings, Australian Centre for International Agricultural Research, Canberra, Australia, pp. 227-246.

Hagerman A E and Butler L 1980 Condensed tannin purification and characterization of tannin-associated proteins. Journal of Agriculture and Food Chemistry 28: 947-952.

Hove L, Ndlovu L R and Sibanda S 2003 The effects of drying temperature on chemical composition and nutritive value of some tropical fodder shrubs. Agroforestry Systems 59: 231-241.

Mahyuddin P, Little D A and Lowry J B 1988 Drying treatment drastically affects feed evaluation and feed quality with certain tropical forage species. Animal Feed Science and Technology 22: 69-78.

Makkar H P S, Blummel M, Becker K, 1995 Formation of complexes between polyvinyl pyrrolidones or polyethylene glycols and tannins, and their implication in gas production and true digestibility in in vitro techniques. British Journal of Nutrition 73: 897–913

Menke K H and Steingass H 1988 Estimation of the energetic feed value obtained from chemical analyses and in vitro gas production using rumen fluid. Animal Research and Development 28: 7-55.

Norton B W and Ahn J H 1997 A comparison of fresh and dried Calliandra calothyrsus supplements for sheep given a basal diet of barley straw. Journal of Agricultural Science 129: 485-494.

Palmer B and Ibrahim T M 1996 Calliandra calothyrsus forage for the tropics – a current assessment. In D O Evans editor. International workshop on the genus Calliandra, Bogor, Indonesia, Winrock International, Morrilton, AR, pp. 183-194.

Robbins C T, Mole S, Hagerman A E and Hanley T A 1987 Role of tannins in defending plants against ruminants: Reduction in dry matter digestion. Ecology 68: 1606-1615.

Robertson J B and Van Soest P J 1981 The detergent system of analysis. In W P T James and O Theander editors. The analysis of dietary fibre in food. Marcel Dekker, New York, pp. 123-158.

Stewart J L, Mould F and Mueller-Harvey I 2000 The effect of drying treatment on the fodder quality and tannin content of two provenances of Calliandra calothyrsus Meissner. Journal of the Science of Food and Agriculture 80: 1461-1468.

Stürm C, Tiemann T T, Lascano C E, Kreuzer M and Hess H D 2001 Nutrient composition and in vitro ruminal fermentation of tropical legume mixtures with contrasting tannin contents. Animal Feed Science and Technology 138: 29-46.

Terrill T H, Rowan A M, Douglas G B and Barry T N 1992 Determination of extractable and bound condensed tannin concentrations in forage plants, protein concentrate meals and cereal grains. Journal of the Science of Food and Agriculture 58: 321-329.

Terrill T H, Windham W R, Evans J J and Hoveland C S 1990 Condensed tannin concentration in Sericea lespedeza as influenced by preservation method. Crop Science 30: 219-224.

Theodorou M K, Williams B A, Dhanoa M S, McAllan A B and France J. 1994 A simple gas production method using a pressure transducer to determine the fermentation kinetics of ruminant feeds. Animal Feed Science and Technology 48: 185-197.

Tiemann T T, Avila P, Ramírez G, Lascano C E, Kreuzer M and Hess H D 2008 In vitro ruminal fermentation of tanniniferous tropical plants: Plant-specific tannin effects and counteracting efficiency of PEG. Animal Feed Science and Technology 146: 222-241.

Tilley J M A and Terry R A 1963 A two-stage technique for the in vitro digestion of forage crops. Journal of the British Grassland Society 18: 104-111.

Van Soest P J, Robertson J B and Lewis B A 1991 Methods for dietary fiber, neutral detergent fiber, and nonstarch polysaccharides in relation to animal nutrition. Journal of Dairy Science 74: 3583-3597 http://jds.fass.org/cgi/reprint/74/10/3583

Received 8 August 2009; Accepted 21 October 2009; Published 3 December 2009

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