Chemical Composition , Degradation Characteristics and Effect of Tannin on Digestibility of Some Browse Species from Kenya Harvested during the Wet Season

A study was conducted with the objective of evaluating the nutritive value of some browse species from Kenya. The species evaluated included: Bauhinia alba, Bauhinia variegata, Bridelia micrantha, Calliandra calothyrsus, Carisa edulis, Cratylia argentea, Gliricidia sepium, Lantana camara, Maerua angolensis, Sesbania micrantha and S. sesban. The browses were evaluated by their chemical composition including phenolics, in vitro gas production and tannin activity (tannin bioassay). All the species had high crude protein content (149-268 g/kg DM) and low NDF content (239-549 g/kg DM). The feeds had varying contents of total extractable tannins (TET) ranging from low (3-22 mg/g DM), moderate (42-58 mg/g DM) and high (77-152 mg/g DM). Calliandra calothyrsus had the highest tannin content. Significant (p<0.05) variation in gas production was recorded among the species. Sesbania micrantha had the highest (p<0.05) potential gas production while Gliricidia sepium had the highest (p<0.05) rate of gas production. Use of polyethylene glycol (PEG 6000), to assess the adverse affect of tannins, indicated that tannins in browse species with high tannin content had inhibitory effects on rumen microbial fermentation as indicated by the gas production. Estimated organic matter digestibility and metabolizable energy also increased with PEG addition. The results of this study indicate that such Kenyan browse species have the potential to be used as feed supplements for ruminant animals. (Asian-Aust. J. Anim. Sci. 2005. Vol 18, No. 1 : 54-60)


INTRODUCTION
Ruminant production in tropical regions such as Kenya is constrained by inadequate supply of feeds in terms of quality and quantity.The native pastures and crop residues are the major feed sources available in these areas for the ruminants.With the increase in human population, more land is being dedicated to crop production and hence unavailable for pasture production.This has resulted in animals increasingly being fed on crop residues.The intake and digestibility of such feeds is low for productive animal production.The cause the low intake and digestibility of such feeds has been suggested to be due to their low content of essential nutrients such as proteins, minerals and vitamins and coupled with high lignifications of their structural carbohydrates (Nair et al., 2002), which slows rumen fermentation.It has been suggested that supplementing these feed resources with nitrogenous feeds such as multipurpose trees and shrubs (MPTS) is one way of alleviating the nitrogen (McSweeny et al., 1999) and mineral deficiencies (Abdulrazak et al., 2000).
There are many trees and shrubs in the tropics and subtropics that can supply fodder.Most of them have been shown to be of high nutritive value (Abdulrazak et al., 2001;Viengsavanh et al., 2002).The MPTS is rich in most nutrients such as proteins and are more digestible.However, use of the MPTS is limited by the presence of antinutritional compounds such as tannins, which are toxic to rumen microbes or the animal.The phenolic compounds also reduce digestibility and availability of protein, contribute to unpalatability and hence, reduce intake (Kibon and Ørskov, 1993).Rubanza et al. (2003) showed that the in vitro digestibility of browse leaves declines with increase in tannin content.Abdulrazak et al. (2000) reported a negative correlation between concentration of phenolics and in vitro gas production of some Kenyan browse species.This negative influence of phenolics on digestibility has been attributed to their toxic effects on rumen microbes (Tolera et al., 1997).However, when they occur in low quantities (less the 5% DM), they tend to increase the rumen by-pass of nutrients such as proteins, which increases nutrient flow to the intestines (Abdulrazak et al., 2001).In Kenya, there is generally scanty information available on the nutritive value of the trees and shrubs and especially on indigenous species.The objective of this study was to investigate the nutritive value of multipurpose trees and shrubs as feed for ruminants and the effect of tannins on estimated in vitro organic matter digestibility and metabolizable energy.study.They include: Bauhinia alba, Bauhinia variegata, Bridelia micrantha, Calliandra calothyrsus, Carisa edulis, Cratylia argentea, Gliricidia sepium, Lantana camara, Maerua angolensis, Sesbania micrantha and Sesbania sesban.All the samples except Maerua angolensis were harvested from around World Agroforestry centre (ICRAF)-Maseno (0° 0'; 34° 35'E) in western Kenya.The area is located at an altitude of 1600m above sea level with average annual rainfall and temperature of 1,500-2,000 mm and 20°-23°C respectively.Maerua angolensis was harvested from Marigat area (0° 31'S; 35° 78'E) Baringo district, Kenya.The area is located at an altitude of 1,066 m above sea level with average annual rainfall and temperature of 700 mm and 24°C respectively.
The browse forages were harvested during the wet season (April/May).They were then oven dried at 50°C for 48 h to constant weight and were ground to pass through a 2.0 mm sieve for the laboratory analysis.However, for the analysis of phenolics and in vitro gas production experiments, the forages were further ground to pass through a 1.0 mm sieve.

Chemical analysis
Dry matter (DM), organic matter (OM) and crude protein (CP) contents were measured according to the official methods of the Association of Official Analytical Chemists (AOAC, 1990).Neutral detergent fibre (NDF), acid detergent fibre (ADF) and acid detergent lignin (ADL) were determined according to the methods of Van Soest et al. (1991).

Phenolic compounds
The extraction of phenolics was done using 70% aqueous acetone.Total extractable phenolics (TEPH) were determined using Folin Ciocalteu as described by Makkar (2000).The concentration of TEPH was calculated using the regression equation of tannic acid standard.Total extractable tannins (TET) were estimated indirectly after being absorbed to insoluble polyvinyl pyrrolidine (PVP).The concentration of TET was calculated by subtracting the TEPH remaining after PVP treatment from the TEPH.For both the TEPH and TET, the absorbance was measured using a spectrophotometer (Quantification-2 program pack, Shimadzu Corporation, Kyoto, Japan).

In vitro gas production study
Animals and rumen fluid extraction : Rumen liquor for in vitro degradability (in vitro gas production and tannin bioassays) was obtained from three mature healthy Japanese Corriedale sheep fitted with permanent rumen fistula.The animals were fed on standard diet of 800 g DM timothy hay and 200 g DM concentrate twice daily at 0900 and 1600 h.The animals had free access to water and mineral lick throughout the experiment period.The rumen liquor was withdrawn prior to morning feeding, mixed, strained through various layers of cheesecloth and kept at 39°C under a CO 2 atmosphere.

In vitro gas production
Samples were incubated in vitro with rumen fluid in calibrated glass syringes following the procedure of Menke and Steingass (1988).About 200 mg of sample (milled through 1.0 mm sieve) were weighed into 100 ml calibrated glass syringes in duplicate.Vaseline oil was applied to the pistons to ease movement and to prevent escape of gas.The syringes were pre-warmed at 39°C before addition of 30±1 ml of rumen liquor-buffer mixture (ratio 1:2) into each syringe.Blanks with buffered rumen fluid without feed sample were also included in duplicate.All the syringes were incubated in a water bath maintained at 39±0.1°C.The syringes were gently shaken every hour during the first 8 hours of incubation and readings were recorded after 3, 6, 12, 24, 48, 72 and 96 h of incubation.The degradability characteristics were estimated by fitting the mean gas volumes in the exponential equation p=a+b(1-e -ct ) (Ørskov and McDonald, 1979) using 'Neway' computer program (Chen X. B., Rowett Research Institute, Aberdeen), where p is the gas production (ml/g OM) at time t, a is the gas production (ml/g OM) from immediately soluble OM fraction, b is the gas production (ml/g OM) from the slowly but potentially degradable fraction and c is the gas production (ml/g OM) rate constant from b.

Effect of Polyethylene glycol (PEG) addition on digestibility
Incubation was carried out using the method of Menke and Steingass (1988) as modified by Makkar et al. (1995).Makkar et al. (1995) increased the sample size from 200 mg to 500 mg and consequently increased the amount of buffer two-fold.The incubation volume was also increased from 30 ml to 40 ml of the rumen-buffer mixture.About 500 mg DM of the feed samples were incubated with or without 1.0 g Polyethylene glycol (PEG) (MW 6000).
The syringes were pre-warmed at 39°C for 1 h before addition of 40±0.5 ml rumen liquor-buffer mixture into the syringes and incubated in water bath maintained at 39±0.1°C.Blanks were also included in the incubation.The syringes were gently shaken every hour during the first 4 h and thereafter, every 2 h up to 8 h of incubation.The gas production readings (ml) were recorded after 2, 4, 6, 8, 12, 16 and 24 h of incubation.

Statistical analysis
Analysis of variance (ANOVA) was carried out on chemical composition, phenolics, in vitro gas production, degradability characteristics and digestibility estimates data using the General Linear Model (GML) procedure (SAS/StatView, 1999).The in vitro gas production data was analysed by two factorial design (species and time).Significance between means was tested using the least significance difference (LSD).

RESULTS AND DISCUSSION
The chemical composition and the phenolics content of the browse species are presented in Table 1.Maerua angolensis had the highest ash content, at 176 g/kg DM while Bauhinia variegata had the lowest ash content, at 55 g/kg DM.The CP contents varied among the species, ranging from 268 g/kg DM (Calliandra calothyrsus) to 149 g/kg DM (Bridelia micrantha).The concentrations of fibre varied significantly (p<0.05)among the species.The NDF, ADF and ADL were highest (p<0.05) in Bridelia micrantha and lowest (p<0.05) in Maerua angolensis except Sesbania sesban, which had lowest ADL.The TEPH concentrations ranged between 223 mg/g DM to 9 mg/g DM.The TET concentration ranged from 152 mg/g DM to 3 mg/g DM.Calliandra calothyrsus had the highest TEPH and TET concentration, which was significantly (p<0.05)different from all the other browse species.
The chemical composition did not vary much from values published in the literature for the browse species (Teguia et al., 1999;El hassan et al., 2000).The browses had high CP content, which would indicate their high nutritive value, since the browses are intended to be used as protein supplements for low quality tropical pastures and crop by-products, which are low in protein.Norton (1994) reported that feeds with less than 6% CP levels are unlikely to provide the minimum ammonia levels required for maximum microbial growth in the rumen.Therefore, all the browse species evaluated in this study would be good protein supplements if they were adequately degraded and were non-toxic to the rumen microbes and host animal.
The NDF content of the browse species was low to moderate.This indicates the browses have high cell contents, which is related with high digestibility.The fibre of browses has also been shown to be digestible (El hassan et al., 2000).Hence, digestibility of the fibre may not be a problem.
High phenolics and tannins in browse species have been shown to be the major drawback in their use as protein supplements.This is because the tannins depress feed intake, impair feed digestibility and are toxic to rumen microbes (Mangan, 1988).The high tannin content of C. calothyrsus has also been reported (Salawu et al., 1997).The other species had low to moderate content of the phenolics and tannins, which would indicate high nutritive value.
Table 2 summarises the cumulative gas production (ml/g OM) and the fermentation characteristics of the tested feeds.There were significant (p<0.05)variations among the browse species.The highest gas production values at 24 h of incubation were obtained with Sesbania sesban, Sesbania micrantha, Gliricidia sepium and Lantana camara.At 96 hour, Sesbania micrantha, Sesbania sesban, Maerua angolensis, Lantana camara, and Gliricidia sepium had higher gas production in that order.Calliandra calothyrsus produced signicantly (p<0.05) the least gas volumes in all the hours.For all the browse species except Calliandra calothyrsus and Sesbania sesban, there was no significant  (p<0.05)increase in gas volume after 72 h of incubation.The potential gas production (a+b) was significantly (p<0.05)highest in Sesbaina micrantha and lowest in Bridelia micrantha while the rate constant (c) was highest in Gliricidia sepium and lowest in Calliandra calothyrsus.
Gas production basically results from fermentation of carbohydrates to short chain fatty acids (SCFA)-mainly acetate, propionate and butyrate (Blümmel and Ørskov, 1993) and from the buffering of the SCFA (CO 2 released from the bicarbonate buffer).Hence, the extent of gas production reflects the efficiency and/or extent of degradability of the feed OM.Besides SCFA and gas (mainly CO 2 and CH 4 ) the feed OM is also fermented to microbial biomass (microbial protein) (Getachew et al., 1998).In vitro fermentation of protein produces ammonium carbonate and the ammonia.The ammonia produced from protein breakdown may be directly used by bacteria for protein synthesis.Hence, low amount of gas is recorded from protein synthesis (González et al., 1998).
In vitro gas production (ml/g OM) was significantly (p<0.05)higher in Sesbania micrantha, Sesbania sesban, Maerua angolensis, Lantana camara and Gliricidia sepium.This would indicate high rate and extent of fermentation of the feed OM of these species.This could be due to their high CP content (253-267 g/kg DM) and comparatively low fibre and tannin content.Variations in gas production among the species could also be due to the nature and proportion of the forage fibre such as proportion of pectin in the cell walls (Marinas et al., 2003).Most of the fermentation of the OM of forages was achieved up to 48 hours of incubation, indicating rapid degradation by the rumen microbes (Marinas et al., 2003).The decrease in degradability of some of the species could be explained by their tannin contents.For instance, the poor performance of Calliandra calothyrsus could be related to its high content of soluble phenolics especially tannins (Salawu et al., 1997;Teguia et al., 1999).The tannin activity differences among the plant species is also known to affect the degradability of a, b, c constants in the equation p = a+b (1-e -ct ) (Ørskov and McDonald, 1979).the browse species (Makkar and Becker, 1998).Bridelia micrantha had the lowest potential gas production (a+b), probably due to its high NDF, ADF and ADL contents.Data on effect of PEG addition on gas production, OMD and ME estimates are presented in Table 3 and 4. At 24 h of incubation, the increase in gas production ranged from 0.2% (Lantana camara) to 148.6% (Carisa edulis).The OMD increase ranged from 0.1% (Lantana camara) to 50.9% (Carisa edulis) while the range for ME was 0.1% (Lantana camara) to 53.6% (Carisa edulis).Carisa edulis and Calliandra calothyrsus had relatively the highest response to PEG addition on gas production, OMD and ME estimates.
The gas method has been used to assess the actions of anti-nutritive factors on rumen fermentation of browse species (Nsahlai et al., 1994;Makkar et al., 1995;Getachew et al., 2002).PEG binds to the tannins forming inert PEGtannin complexes, which results in increase in gas production.The higher the biological activity of the tannins on rumen microbes, the higher the increase in gas production in the presence of PEG (Makkar and Becker, 1998).The increase in gas production due to incubation with PEG could be due to increase in SCFA production and/or changes in molar proportions of the acids (Blümmel and Ørskov, 1993).This suggests that removal of adverse effects of tannins by PEG increases the digestibility and energy content of the browse feeds (Makkar et al., 1995).In the current study, Carisa edulis and Calliandra calothyrsus had the highest (p<0.05)response to PEG addition.This could be because of the high tannin content of the two species.However, the tannin in Carisa edulis seems of high activity owing to their smaller concentration than Calliandra calothyrsus but high response in gas production.Getachew et al. (2002) concluded that tropical browses with less than 45 and 20 g/kg DM of total phenols and tannin respectively are unlikely to produce significant adverse effects on ruminant livestock.The same trend was observed in this study.The species, which had low tannin content, had minimal response to PEG addition on gas production, OMD and ME.
Table 5 presents the correlations between fibre fractions and phenolic compounds with in vitro gas production parameters, OMD and ME.The NDF, ADF and ADL were negatively correlated with the gas production parameters and the OMD and ME.The TEPH and TET concentration were strongly and negatively correlated with the gas production parameters and the OMD and ME ranging from r=-0.583 (a+b) to r=-0.838 (24 h incubation) except a, which was positively correlated.These findings were consistent with the findings of other studies reported in the literature (Abdulrazak et al., 2000;Fadel Elseed et al., 2002;Rubanza et al., 2003b).While Rubanza et al. (2003b) and Abdulrazak et al. (2000) found a negative but poor relationship between the TET content and the digestibility of the browses, our results found a strong negative correlation.This could be attributed to the different nature and activity of the tannins among the browse species (Abdulrazak et al., 2000).The negative correlation between  the tannins content and the degradability characteristics indicates the adverse effects of the tannins on the use of such browse species as animal feeds.

CONCLUSION AND IMPLICATIONS
It is concluded that the browse species used in this study could be good protein supplements to low quality basal diets.However, the high phenolics content and activity in some of the browse species could limit their nutritive value.Use of tannin binding agents such as PEG would increase their nutritive value.Further studies to evaluate more browse species especially the locally available species are recommended.Animal feeding experiments are also necessary to ascertain the nutritive value of the promising species in terms of palatability, intake, digestion and effect on production performance of the animals.

Table 1 .
Chemical composition of the browse species

Table 2 .
Cumulative in vitro gas production (ml gas/g OM) and degradation characteristics (a, b, a+b and c) of the browse species SEM: Standard error of the means; LSD: Least significant difference.

Table 3 .
Effect of PEG addition on gas production (ml gas/g OM)

Table 4 .
Effect of PEG addition on estimated in vitro OMD (%) and ME (MJ/kg DM) SEM: Standard error of the means.LSD: Least significant difference.

Table 5 .
Correlation coefficient (r) of the relationship between the concentration of NDF, ADF, ADL, TEPH, TET and gas production parameters, OMD and ME