Content of phenolics and tannins in leaves and pods of some Acacia and Dichrostachys species and effects on in vitro digestibility

Four browse legume foliages (leaves and pod fruits) from three species of Acacia (A. polyacantha, A. tortilis, A. nilotica) and Dichrostachys sp. native to Tanzania were evaluated for nutritive potential to establish levels and effect of normal phenolics and tannins antinutritive factors (ANFs) on in vitro feed digestibility (IVD). Total extractable phenolics (TP), tannins (TT) were estimated by Folin-Ciocalteu assay, and condensed tannins (CT) by butanol/HCl assay. In leaves, total proanthocyanidins were assayed into constituent anthocyanidins’ flavonoids by high performance liquid chromatography (HPLC). Organic matter degradability (OMD) was estimated in vitro by gas production technique. Adverse effects of tannin’s ANF on IVD were assessed by polyethylene glycol (PEG) tannin bioassay. Crude protein (CP) varied (P<0.05) between fodder species and foliages. Leaves had (P<0.05) higher CP (141-194 g/kg DM) in Dichrostachys sp. and A polyacantha, respectively, compared to pods (133-142 g/kg DM) in A. tortilis and Dichrostachys sp., respectively. Neutral detergent fibre (NDF), acid detergent fibre (ADF) and acid detergent lignin (ADL) va* Part of the data were presented to the 4th Korea-Japan Rumen Metabolism Joint Symposium, May 21st-24th, 2002, Cheju, Republic of Korea 6 Corresponding author: e-mail: fujihara@life.shimane-u.ac.jp 646 PHENOLICS AND TANNINS IN BROWSE LEGUMES 647 RUBANZA C.D.K. ET AL. ried (P<0.05) between fodder species and foliages. A. nilotica had lowest (P<0.05) NDF, ADF and ADL (182, 68 and 44 g/kg DM, respectively). A. polyacantha and Dichrostachys sp. had highest (P<0.05) NDF and ADF (416 and 146 g/kg DM, respectively). Pods had higher (P<0.05) fibre values than leaves. Leaves had higher (P<0.05) TP, TT, and CT (162, 138, and 55 mg/g DM), than pods (112, 105 and 35 mg/g DM), respectively. Proanthocyanidin flavonoids (mg/g DM) varied (P<0.05) from 0.062 (A. nilotica) to 5.288 (A. tortilis), 0.188 (A. tortilis) to 4.179 (A. polyacantha), and 0.009 (A. nilotica) to 4.392 (Dichrostachys sp.) for delphinidins, cyanidins and pelargonidin, respectively. The browse foliages had relatively high tannin content greater than 5% DM, a beneficial level in animal feeding and nutrition, and thus could impair feed digestibility. OMD varied significantly (P<0.05) both between fodder species and foliage components. Pods had higher (P<0.05) gas production (GP) and OMD than leaves. Addition of PEG improved (P<0.05) gas production, IVD and metabolizable energy (ME) in leaves and pods by binding tannins. Leaves had higher response on gas production and IVD due to PEG treatment compared to pods except A. nilotica. Improved gas production and digestibility due to PEG treatment indicate nutritive potential in browse fodder previously depressed by tannin ANFs. Therefore, phenolics and tannins ANFs could limit utilization of browse fodder nutritive potential as supplements to ruminants consuming low quality roughages. Utilization of browse legume fodder could be optimized through reduction of tannin antinutritive activity. Under farmers’ conditions, optimal utilization of browse could be achieved through feeding a mixture of tanniferous browse with other feeds especially high in nitrogen to dilute tannin antinutritive activity.


INTRODUCTION
Browse tree legume fodder represents cheap protein supplement for livestock in tropical countries where both feed quality and quantity pose a great constraint on animal production.In most tropical region ruminant production depends on low quality forages that are deficient in essential nutrients especially protein and minerals that fluctuate with seasons (Crowder and Chheda, 1982).In the dry season forage quality declines tremendously, CP drops to as low as 3.0 to 5.0% of DM (Kakengi et al., 2001), a level that is too low to meet minimum normal nitrogen requirements of 8% CP for optimal rumen microorganisms function (Hungate, 1966;Annison and Bryden, 1998).In turn, basal roughages alone could not meet animal requirements for maintenance and production unless supplemented with nitrogen (protein) sources (Leng, 1990).Conventional protein and energy supplements (concentrates, fish meal) are expensive and unaffordable by most low capital traditional farmers.Browse fodder could improve animal nutrition through increased CP content (110 to 250 g/kg DM) (Le Houérou, 1980).However, utilization of nutritive potential from browse legume fodder is limited by presence of anti-nutritive factors (ANFs) such as phenolics and tannins (Mangan, 1988;Kumar and D'Mello, 1995).Tannins refer to naturally occurring high molecular weight soluble polyphenolic secondary plant compounds that bind to, and form complex with proteins, carbohydrates, minerals, and other dietary nutrients during feed digestion (Mangan, 1988), rendering them unavailable to the animal.Phenolics and tannins cause reduced digestibility through inhibition of digestive enzymes, complex formation with rumen microorganisms, and toxic effects to animal body (Mangan, 1988;Makkar et al., 1995).Subject to establishment of level of phenolics and tannins ANFs in browse foliages, the fodder could be used as protein supplements to ruminants fed on low quality basal roughages.Acacia spp.and other browsable tree leaves, fruits (fresh and dry pods) and seeds represent an important protein supplements for livestock fed on low quality roughages (standing hay, straw and stover) basal feeds in most arid and semiarid regions, or even in the tropics especially during dry seasons.However, utilization of browse as protein supplement has not been optimized following presence of polyphenolics antinutritive factors.
The current work aimed to develop a low cost and sustainable dry season protein feed supplements from promising tree legume fodder species native to semi-arid western Tanzania.A study was therefore conducted to determine essential chemical composition (feed protein and dietary fibres), phenolics and tannin levels in three species of Acacia and one species of Dichrostachys leaves and pods.Also, to estimate extent and rate of organic matter (OM) degradation, and to assess adverse effects of tannin on feed digestibility in vitro.

Study area
Browse leaves and pod fruits from three species of Acacia (A. nilotica, A. tortilis and A. polyacantha), and Dichrostachys sp. were harvested from five administrative districts (Bariadi, Kahama, Meatu, Shinyanga Urban and Shinyanga Rural) in Shinyanga region, north-western Tanzania (2 -3 o S; 31-31.5 o E).Shinyanga region forms a semiarid agro-ecological zone (SAEZ) located at 1100-1300 m above sea level.Minimum and maximum temperatures vary from 15 to 18.3 o C and 27.6 to 30.2 o C, respectively.This SAEZ receives low annual rainfall of 600 to 800 mm raining from November to mid May.Dry season is from June to October.Common vegetations include short grasses and scattered shrubs and trees mostly dominated by Acacia spp.

Forage sample collection and processing
For each four species, leaves and soft twigs samples were hand plucked from three positions of a tree canopy: on the lower, middle and upper parts of the tree trunk.The foliage samples were harvested from 8 to 10 trees selected at random in each of four sub-plots (70 x 70 m) in four rangelands in the five districts in the study area.Collected browsable fodder samples constituted a mixture of leaves and thin twigs (midrib and twigs less than 5 mm), there after referred to as "leaves and soft twigs", and pod fruits.Pod fruits were collected while still intact to the plants just before drying when had turned to yellowish and were separated from pods with inflorescence as well as from dry pods either still attached on the tree or already dropped on the ground.The leaves and pod foliages were harvested at advanced maturity stage in late rainy season between mid April and mid May in 2001.Harvested browse samples were pooled for each individual tree species and foliage components (leaves and soft twigs separated from pod fruits).The samples were dried at 50 o C in a forced air oven for 48 h to constant weight, then ground to pass through a 2.00 mm sieve and sub-sampled into 24 bulk samples (three samples for each species and foliage components) for further laboratory analyses.Samples for phenolics, tannins and for in vitro degradability assays were ground to pass through 1 mm-sieve.
In vitro organic matter degradability study Animals and management.Rumen fluid for in vitro gas production, digestibility and tannin bioassay studies was obtained from three healthy mature Japanese Corriedale female sheep fitted with permanent rumen cannulae (70 mm).The fistulated animals were kept on standard daily ration of 800 g timothy hay and 200 g concentrates (2-parts wheat bran and 1-part rolled barley) divided into two equal meals fed at 8.00 and 16.00 h daily.The animals were supplemented with minerals to meet body requirements according to ARC (1990), and had free access to water throughout the experimental duration.
In vitro gas production.Rumen fluid was obtained from the three sheep early in the morning before feeding.Preparation of an in vitro mineral buffer media for gas test was conducted as described by Menke and Steingass (1988).Rumen fluid was filtered through a cheese cloth and mixed (1:2 v/v) with an anaerobic buffermineral solution containing, per litre, g: NaHCO 6H 2 O, 0.002 and sodium resazurin, 0.125.The mixture was kept stirred under CO 2 flushing at 39 o C using a magnetic stirrer fitted with a hot plate.
Browse legume fodder test feed samples were incubated in vitro with buffered rumen fluid in 100 ml graduated glass syringes following the procedure described by Menke and Steingass (1988).Approximately 200 mg DM (1 mm milled feed samples) were weighed into 100 ml syringes in triplicate, and in each series a blank, buffered rumen fluid without test feed sample, was run in triplicate.Vaseline oil was applied to the piston to ease movement and prevent escape of gas.The syringes were pre-warmed (39 o C) for 1 h, before addition of 30 ± 0.5 ml of rumen buffer mixture into each syringe.All the syringes were incubated in a water bath maintained at 39 ± 0.1 o C. The syringes were gently shaken every hour during the first 8 h of incubation.Gas production readings (ml) were recorded after 3, 6, 12, 24, 48, 72 and 96 h.The OM degradability characteristics, extent and rate of feed degradation were estimated by fitting gas production readings (ml) into degradability curve (Ørskov and McDonald, 1979) based on mathematical model: (McDonald, 1981), where y is the potential gas production (ml/g OM), and a is gas production (ml/g OM) from immediately soluble OM fraction.Constant b is gas production (ml/g OM) from the slowly but potentially degradable feed fraction; and c is gas production rate constant (ml/h) from slowly fermentable feed fraction, b.

Effect of polyethylene glycol (PEG) treatment on digestibility.
Adverse effects of tannins ANFs on in vitro OM digestibility (IVD) were assessed by incubation of approximately 500 mg DM test feed samples with or without 1.0 g polyethylene glycol (PEG) molecular weight (MW, 4 000) (Wako Pure Chemicals Industries Ltd., Japan).The feed samples were incubated in 100 ml glass syringes essentially by the procedure of Menke and Steingass (1988).The PEG tannin bioassay was conducted according to Makkar et al. (1995) as detailed by Makkar (2000).Rumen fluid for tannin bioassay experiment was obtained from similar sheep handled and treated in a similar procedure as for in vitro gas production test above except composition of the buffer medium.As the amount of feed incubated was 500 mg, composition of the rumen buffer medium was according to Tilley and Terry (1963).Reduced buffer medium composition, per litre, were, g: NaHCO 3 , 35.0; NH 4 HCO 3 , 4.00; Na 2 HPO 4 , 5.7; KH 2 PO 4 , 6.2; NaCl, 2.22; MgSO 4 .7H 2 O, 0.6; Na 2 S, 0.52; CaCl 2 .H 2 O, 13.2; MnCl 2 .4H 2 O, 10.0; CoCl 2 .6H 2 O, 1.0 and sodium resazurin, 0.01 and, 60 ml freshly prepared reduction solution containing 580 mg Na 2 S.9H 2 O and 3.7 ml 1 M-NaoH.A portion (40.0 ± 0.5 ml) of the rumen fluid medium was transferred into each syringe and incubated into water bath at 39 ± 0.1 o C as described by Blümmel and Ørskov (1993).The syringes were pre-warmed (39 o C) for 1 h, before addition of 40 ± 0.5 ml of rumen buffer mixture into each syringe, and incubated in a water bath maintained at 39 ± 0.1 o C. The syringes were gently shaken every hour during the first 8 h of incubation.Gas production readings (ml) were recorded after 2, 4, 6, 8, 12, 16 and 24 h for both PEG treated samples and blank samples.Feed organic matter digestibility (OMD) (%) and metabolizable energy (ME) (MJ/kg DM) were estimated from equation of Menke and Steingass (1988) and Makkar and Becker (1996) based on 24 h gas production, (GAS, ml) and crude protein content (CP, % DM): OMD (%) = 14.88 + 0.889GAS + 0.45CP, and ME (MJ/kg DM) = 2.20 + 0.136Gv + 0.057CP.

Statistical analyses
Data on chemical compositions, in vitro gas production, degradability characteristics and digestibility estimates were subjected to analysis of variance (ANOVA) using the General Linear Model Procedure (GLMP) (SAS/ Statview, 1999).Data were analyzed based on statistical model: where, Y ij is the general observation on chemical composition, gas production (GP), degradability characteristics, and in vitro digestibility parameters.µ ij is the general mean common for each parameter under investigation; S i is the i th effect of browse fodder species on the observable parameters; F j is the j th effect of foliage components (leaves or pods), (S+F) ij represent i th and j th interaction effects of fodder species and foliage component on the observed parameters, and e is the standard error term.

Chemical compositions
Results on chemical compositions are presented in Table 1.Feed protein (CP), ash content, fibre components (NDF, ADF, and ADL) were significantly (P<0.05)different between both fodder species and between leaves and pods.Leaves harvested from A. polyacantha and A. tortilis had higher (P<0.05)CP (194 and 188 g/kg DM, respectively), compared to A. nilotica and Dichrostachys sp. that had lower (P<0.05)CP (144 and 141 g/kg DM, respectively).There was no significant (P>0.05)difference in CP between leaves and pods except in A. polyacantha (194 vs 133 g/kg DM) and A. tortilis (188 vs 133 g/kg DM) (Table 1).Ash varied (P<0.05) between both leaves and pod foliages (Table 1), and was influenced (P<0.001) by fodder species and foliages interaction.There was no difference (P>0.05) in ash content (g/kg DM) between leaves and pod foliages except A. polyacantha (121 vs 85) and Dichrostachys sp.(96 vs 67), respectively.Leaves harvested from A. polyacantha and Dichrostachys sp. had higher (P<0.05)ash content than pod fruits.In leaves, ash varied (P<0.05) from 70 (A.nilotica) to 121 g/kg DM (A. polyacantha).A. nilotica pods had the lowest ash content (63 g/kg DM), and was not different (P>0.05) from pods harvested from other fodder species except A. polyacantha (85 g/kg DM).Neutral detergent fibre (NDF), acid detergent fibre (ADF) and acid detergent lignin (ADL) varied (P<0.05) both between fodder species and between leaves and pod foliages (Table 1).In both leaves and pod foliages, A. nilotica had lowest (P<0.05)NDF (182 vs 187 g/kg DM, respectively) compared to A. polyacantha (416 vs 488 g/kg DM, respectively).Leaves and soft twigs had lower NDF fraction though not statistically different from pod fodder except Dichrostachys sp.(Table 1).ADF ranged from 68 (A.nilotica) to 150 g/kg DM (A. tortilis) in leaves, and from 86 to 214 g/kg DM in A. nilotica and A. polyacantha pods, respectively.ADL ranged from 44 to 123 g/kg DM in A. nilotica and Dichrostachys sp.leaves, respectively, and from 37 to 162 g/kg DM in A. nilotica and A. polyacantha pods, respectively.There was no difference (P>0.05) in ADL contents between leaves and pods except A. polyacantha (162 vs 107 g/kg DM) and Dichrostachys sp.(74 vs 123 g/kg DM) (Table 1).

Effect polyethylene glycol (PEG) treatment on in vitro digestibility
Addition of PEG resulted in significant (P<0.05)improvement in gas production (Table 4), OM digestibility and metabolizable energy (ME) estimates (Table 5).

TABLE 3
Cumulative in vitro gas production potential (ml gas/g OM) in browse legume foliage leaves and pod fruits at different incubations, h, OM fermentation characteristics, a: b and (a+b) (ml gas/g OM); OM degradation rate constant, c (ml gas/h)

DISCUSSION
Browse leaves and pod foliages from Acacia spp.and Dichrostachys sp. could form potential feed resources mainly as protein supplements to ruminants fed on low quality basal forages such as standing hay and crop by-products (straw, stover) that have low CP values (30-70 g/kg DM) especially during dry seasons.High CP values in browse (133-194 g/kg DM) could correct deficient nitrogen in basal roughages.Presented results on browse chemical compositions (Table 1) were comparable to other workers (Le Houérou, 1980;Topps, 1997;Abdulrazak et al., 2000).The latter authors reported high CP contents (103 to 336 g/kg DM) in browse.However, some variations in CP could be due to proportion of foliage sampled for analyses as well as stage of maturity.For example, Topps (1997) reported low CP contents of 153, 153 and 219 g/kg DM in A. tortilis, A. hockii and A. senegal old leaves compared to high CP values (210, 194 and 319 g/kg DM) in newly emerged leaves, respectively.Low CP contents in pod fruits for example those harvested from A. polyacantha and A. tortilis compared to the respective species' leaves, is partly explained by genotype factors that control differential accumulation of nutrients in leaves and fruits (pods) as related to stage of maturity.Pods were harvested before drying (when had started turning to yellow) at their advanced maturity stage.Therefore, browse legume fodder represent a potential feed resource that could be utilized as a protein supplement to correct deficient feed nitrogen to animals fed on poor quality basal roughages in tropical regions especially during dry seasons.
High ash contents in browse to a large extent could be related to browse fodder nutritive potential.
Lower fibre compositions (NDF, ADF and ADL or lignin) indicate promising nutritive potential in browse legume fodder.Low NDF content suggests feed of high cell contents (CC) that could be related to high feed digestibility.Other workers (Reed, 1986) had similarly reported low fibre compositions in East African Acacia spp.However, low fibre fraction in tanniferous browse should be interpreted with precaution.Most browse had low NDF, for example A. nilotica leaves and pods (182 vs 187 g/kg DM) that would suggest high cell contents (CC).High CC in some Acacia spp.foliages for example A. nilotica could be due to soluble polyphenolic compounds, that would lead to underestimation of NDF, ADF, and therefore over estimation of digestibility. A. nilotica had high total phenolics and tannins (Table 1) that possibly could had interfered with fibre determination.
High phenolics and tannins compositions in browse would depress feed nutritive values.The browsable fodder (leaves and pods) had tannins greater than lower beneficial level (5 % DM) in animal feeding and nutrition (Mangan, 1988).High levels of phenolics and tannins ANFs could have adverse effects (depressed feed intake, impaired feed digestibility and toxic effect on rumen microbes) especially when fed in high proportions in ruminants' diets depending on nature and tannin activity.Relatively low polyphenolics composition in pod foliages suggests its superiority as a protein supplement to animals fed on low quality basal forages compared to browse leaves and soft tender twigs.Accumulation of polyphenolics secondary plant metabolites represents a self-defence and adaptation mechanism in plants against foraging herbivores and insects, and even moisture stress.High polyphenolics in Acacia spp.has been reported elsewhere (Reed, 1986;Abdulrazak et al., 2000), or even Dichrostachys sp.(Shayo and Udén, 1999).
Delphinidin, cyanidin and pelargonidin are products of leucodelphinidins, leucocyanidins and leucopelargonidins flavonoids, respectively, following cleavage of polymerized (condensed) tannin flavan in hot mineral acid-alcohol mixture.Delphinidins, cyanidins and pelargonidins represent flavan-3-diol and flavan-3,4-diols flavonoids or a mixture of the two, are the depolymerized constituents of leucoanthocyanidins (condensed tannins).Detected delphinidin, cyanidin and pelargonidin flavonoids in A. polyacantha, A. tortilis, A. nilotica; and Dichrostachys sp.leaves elucidate the structure, type and nature of tannin as related to tannin biological activity.The flavan-3-diol and flavan-3,4-diols molecules elucidate the tannin molecule steriochemistry, and suggest presence of several isomers in the polymerized tannin molecule.For example, a flavan-3,4-diols flavonoids possess asymmetric carbon at C-2, C-3 and C-4, a structure that indicate presence of eight isomers.Tannin molecules with different isomeric forms in their flavan have different tannin structure and possibly different biological activity, and would have variable tannin antinutritive activity whether in vitro or in vivo.
Gas production is a result of feed fermentation in vitro that simulates the rumen degradability phenomenon.Gas production originates from feed fermentation and indirectly from CO 2 released from buffer mixture by volatile fatty acids (acetates, butyrates and propionates).The extent of gas production partly reflects efficiency of fermentation or extent of degradability of feed OM.Feed OM is fermented to microbial mass (microbial protein) and gases (CO 2 , methane, CH 4 ) and short chain fatty acids (acetates, butyrates and propionates).Proportion of fermentation products depends on nature of feed, especially CP and fibre contents.Most of CP is degraded to microbial mass and gives less gas.
High gas production potential in A. nilotica, A. tortilis leaves and in pods suggest high extent and rate of feed fermentation.On the other hand, variable gas production within fodder species' foliages could be due to nature and proportion of fibre in the feed sample.Decreased degradability could mostly explained by phenolics and tannins binding feed nutrients in the process of fermentation.Makkar et al. (1995), Makkar and Becker (1996) and Getachew et al. (2000) reported similar observations.High feed in vitro degradability estimates indicated by gas production in A. nilotica different from expectations due to high phenolics and tannins could be attributed to specific nature of tannin activity as related to biological anti-nutritive activity.Some of variations could be due to plant genotypic characteristics in relation to type of phenolics and tannins' activity on digestibility.Makkar and Becker (1993) reported a variable nature of tannins between and within plant species as related to their nature and biological activity.
Similarly, observed differences on degradability both between fodder species and foliage components could be due to fibre type and extent of lignification (Van Soest, 1994;Fonseca et al., 1998).High degradability values in pod foliages could be due to lower phenolics and tannin contents and that could be related to decreased anti-nutritive activity.High gas production from A. polyacantha at initial incubations (3 and 6 h) could be explained by high CP.CP represents a large proportion of an immediately fermentable OM feed fraction at initial incubations, and decreases at subsequent incubations.
The reflected high gas production from A. nilotica could be explained by low fibre composition (Table 1), though had high total phenolics and tannins (Table 2).Results suggest that polyphenolics in A. nilotica had less depressive effect on gas production.Also low fibre could probably had less effect on depressed OM degradability in vitro.Based on gas production potential in leaves, A. polyacantha ranked lowest followed by Dichrostachys sp., A. tortilis and A. nilotica (the highest).This trend could be due to amount of lignin and extent of lignification and possibly due to level of tannin and related biological activity.The constant a, represent the immediately fermentable OM fraction.The b constant denotes a slowly fermentable feed fraction with time.The constants a and b are mainly related to type of fibre and extent of fibre lignification, and plant genotype as related to factors that control the a and b constants.gonidyny, była podobnie zróżnicowana.Udział tanin w liściach był większy niż 5% w s.m., który to poziom uważany jest za korzystny z punktu widzenia żywienia zwierząt, i dlatego mógł wpływać ujemnie na strawność paszy.OMD różniła się istotnie (P<0,05) zarówno między gatunkami roślin, jak między liśćmi i strąkami.Produkcja gazu i OMD strąków były większe (P<0,05) niż liści.Dodatek PEG zwiększał (P<0,05) produkcję gazu, a tym samym IVD i zawartość energii metabolicznej w liściach i strąkach, dzięki wiązaniu tanin.

TABLE 1
Chemical compositions in selected browse tree leaves and pod fruits, g/kg DM

TABLE 2
Phenolics and tannins compositions in selected browse leaves and pod fruits, mg/g DM Addition of PEG had no effect on gas production, OMD and ME values in blank samples.In both leaves and pod foliages, A. nilotica showed the highest relative response to PEG treatment on gas production, OMD and ME estimates.Increase in gas production at 24 h due to PEG treatment ranged from 28.7 to 46.6% in A. polyacantha and A. nilotica in leaves, respectively; and from 14.4 (A.polyacantha) to 74.9% (A.nilotica) in pods.In leaves, increase in OMD due to PEG treatment ranged from 14.7 (A.polyacantha) to 23.4% (A.nilotica).Pod foliages had lower response (8.2 -22.2%) on OMD due to addition of PEG except in A. nilotica pods (40.6%).Response on ME due to addition of PEG varied (P<0.05) between both leaves and pod foliages in three species of Acacia and Dichrostachys sp.. Increase in ME due to PEG treatment varied from 15.3 to 24.3% in A. polyacantha and A. nilotica leaves, respectively.A. polyacantha pods had the lowest response on ME (8.5 %) due to addition of PEG compared to A. nilotica (42.0%).

TABLE 4
Effect of polyethylene glycol (PEG) treatment on in vitro gas production (ml/g OM) at different incubation intervals, h