Potential nutritive value of selected browse species from Kenya using in vitro gas production technique and polyethylene glycol

I M Osuga^*,^**, S A Abdulrazak***, N Nishino****, T Ichinohe** and T Fujihara**

^*The United Graduate School of Agricultural Sciences, Tottori University, Tottori-shi, Japan
**Laboratory of Animal Science, Faculty of Life and Environmental Science, Shimane University, Matsue-shi, Japan
fujihara@life.shimane-u.ac.jp
***Division of Research and Extension, Egerton University, Njoro, Kenya
****Department of Animal Science, Okayama University, Okayama-shi, Japan

Abstract

Keywords: Browse species, gas production, In vitro degradability, PEG

Introduction

Tree and shrub legumes have gained interest and importance in the recent past as feeding resources in ruminant livestock diets in the tropics. This is especially under the harsh conditions in arid and semi-arid areas where livestock farming is the major economic activity. The tree and shrub legume forages are rich in most essential nutrients such as proteins and minerals and tend to be more digestible than the grasses and crop residues. This necessitates the evaluation of the nutritional characteristics of the forages in order to maximise their use in the ruminant diets.

The in vitro gas production technique as modified by Menke and Steingass (1988) is one of the useful tools for determining the potential nutritional value of feed resources consumed by ruminants, especially tree and shrub legume forages containing anti-nutritional factors such as polyphenols. The technique measures the volume of gas produced over time, which reflects the end result of the fermentation of the feed substrate to volatile fatty acids (VFAs), microbial biomass and the neutralization of the VFAs produced. Application of models such as that of Ørskov and McDonald (1979), allows the rumen microbial fermentation characteristics of the feeds to be described. Blümmel and Ørskov (1993) enhanced the precision of this technique by terminating fermentation at 24 h and digesting the residue in neutral detergent solution. Using hay-based diets, Blümmel and Ørskov (1993) and Blümmel and Becker (1997) reported the accuracy with which this method predicts feed intake, digestibility, microbial nitrogen supply and animal performance. The technique has also been used to demonstrate and quantify the adverse effect of some chemical constituents such as tannins in the browse forages (Makkar et al 1995). Therefore the objective of this study was to assess the degradability and adverse effect of tannins on the degradability of the tree and shrub forages using chemical composition and in vitro gas production technique.

Materials and methods

Forage samples

Browsable tree leaves from twelve species which are commonly consumed by animals were used in this study. The species used included: Balanites aegyptiaca, Boscia angustifolia, Berchemia discolor, Calliandra calothyrsus, Grewia bicolor, Maerua angolensis, Olea europaea, Persia americana, Pappea capensis, Terminalia brownii, Tamarindus indica and Zizyphus mucronata. All forages except Calliandra calothyrsus and Persia americana were harvested from Egerton University's Chemeron Field Station in Marigat, Baringo District, Kenya. The area is located at an altitude of 1066 m above sea level with average annual rainfall and temperature of 700 mm and 24 ^oC, respectively. Calliandra calothyrsus and Persia americana were harvested from Egerton University, Njoro campus, Nakuru, Kenya, which is located at an altitude of 2,238 m above sea level with mean day temperatures of 21 ^oC, and a mean annual rainfall of 900 - 1020 mm. The browse forages were harvested from at least 10 trees for each species selected at random in four locations within the study area at the end of the rain season. The harvested samples were then pooled for each individual tree species and then oven dried at 50 ^oC for 48 h to constant weight and ground to pass through a 2.0 mm sieve. The samples were then sub-sampled to obtain three samples for each tree species and used 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

Organic matter (OM) and crude protein (CP) contents were measured according to the official methods of the Association of Official Analytical Chemists (AOAC 1984). 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).

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. The various leucoanthocyanidin flavonoids were measured by high performance liquid chromatography (HPLC) as described by Steward et al (2000) with slight modifications. One gram sample was extracted with 4 ml of acetone/water (70/30, v/v) in water bath by ultrasonication for 30 min followed by centrifugation for 15 min at 3000 rpm. A 0.5 ml of supernatant was vortexed with 0.25 ml dichloromethane and centrifuged again at 3000 rpm for 15 min to remove the pigments. A 0.05 ml of the aqueous lower layer was combined with 3.0 ml of butanol/HCl (95/5, v/v) and heated for 1 h at 95 ^oC. Butanol/HCl was evaporated at 50 ^oC under a stream of nitrogen and the residue redissolved in 0.5 ml of methanol/HCl (99/1, v/v) followed by filtration through a 0.02 µm polytetraflouroethylene membrane. 10 µL of the aliquot was then injected to the HPLC (Shimadzu co. Japan) column (Inertsil ODS-80 A, 150 x 4.6 mm). Gradient elution rate was 0.9 ml/min using water/acetic acid (sol. A, 95/5 v/v) and methanol (sol. B). The gradient profile was: 80% A/20 % B (0 min), 50% A/50% B (0-15 min), 0% A/100% B (15-20 min), 0%A/100%B (20-25 min) and 80%A/20%B (25-30 min). The absorbance at 525 nm was recorded using the HPLC (Shimadzu co. Japan). The peaks were identified using cyanidin chloride, delphinidin chloride and pelargonidin chloride which had retention times of 9.77, 12.26 and 14.26 min respectively. The total condensed tannins were taken as the sum of the various flavonoids measured.

In vitro study

Animals

Three mature sheep fitted with permanent rumen fistula were used in this study. The animals were fed on standard diet made up of 800 g DM timothy hay and 200 g DM concentrate twice daily at 09.00 and 16.00 h in equal sized meals. The animals had free access to water and mineral licks throughout the experiment period. The animals provided the rumen liquor for the in vitro gas production and tannin bioassay experiments. The rumen liquor was withdrawn at 14 h post feeding, mixed, strained through four layers of cheesecloth and kept at 39 ^oC under a CO₂ atmosphere.

In vitro gas production

About 200 mg of sample (milled through a 1.0 mm sieve) were incubated in vitro with rumen fluid in calibrated glass syringes following the procedure of Menke and Steingass (1988). The syringes were pre-warmed at 39 ^oC before addition of 30±1 ml of rumen liquor-buffer mixture (ratio 1:2) into each syringe and incubated in a water bath maintained at 39 ^oC. Blanks with buffered rumen fluid only were also included. The gas production readings were recorded after 3, 6, 12, 24, 48, 72 and 96 h of incubation. The gas production characteristics were estimated by fitting the mean gas volumes to the exponential equation of Ørskov and McDonald (1979):

G = a + b(1 - e ^{-c t}) ,

where

G is the gas production (ml) at time t,
a is the gas production from the immediately soluble fraction (ml),
b is the gas production from the insoluble but degradable fraction (ml),
a + b is the potential gas production (ml),
c is the rate constant of gas production (fraction/h).

Tannin bioassay

Incubation was carried out as described by Makkar et al (1995). 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 ^oC for 1 h before addition of 40±0.5 ml rumen liquor-buffer mixture (1:3) into the syringes and incubated in triplicate in a water bath maintained at 39 ^oC. Blanks were also included in the incubation. The gas production readings were recorded after 2, 4, 6, 8, 12, 16 and 24 h of incubation.

In vitro true DM and OM degradability and partitioning factor

After termination of the incubation at 24 h, the syringe contents were quantitatively transferred into a 600 ml beaker by rinsing the syringes with about 70 ml of neutral detergent solution ( Blümmel and Becker 1997) and refluxed for 1 h. Residual DM and ash were then determined. The ratio of DM truly degraded (mg) to gas volume (ml) produced at 24 h of incubation was used as the partitioning factor (index of microbial protein synthesis) (Blümmel and Becker 1997)

Statistical analysis

In vitro gas production data were fitted to the asymptote exponential model using Neway-Excel computer program (Macaulay Institute, Aberdeen, UK). All the data was subjected to analysis of variance (ANOVA) using the General Linear Model procedure (SAS/StatView 1999) and significance between means tested using least significant difference (LSD).

Results and discussion

The chemical composition of the browse species forages are shown in Table 1. The OM, CP, fibre and lignin contents were highly variable, with significant (P<0.05) differences detected among the various species. The OM (g/kg DM) ranged from 822.8 (Maerua angolensis) to 936.0 (T. indica). The CP content (g/kg DM) range was 98.4 (Olea europea) to 302.3 (Calliandra calothyrsus). Boscia angustifolia had the highest NDF and ADF contents while Maerua angolensis had the lowest NDF and ADF contents. The ADL content ranged from 48.6 in Terminalia brownii to 138.3 g/kg DM in Persia americana.

The chemical compositions of the browse forages were within the range reported in the literature for browse forages from Kenya (Osuga et al 2006; Osuga et al 2005; Abdulrazak et al 2000). The role of browse forages as nitrogen sources for ruminants, especially during lean periods, is the major contribution of the browses in many parts of the tropics where other nitrogen sources may not be readily available and/or are expensive. Except for Olea europaea, which had CP content of less than 100 g/kg DM, all the other species had high CP contents of more than 100 g/kg DM. The high CP content, which is above the minimum required level for optimal rumen microbial activity (80 g/kg DM) (Annison and Bryden 1998), justifies the use of the browse forages in small quantities in order to supplement poor quality pastures and crop residues. The browse forages had low to moderate content of fibre. This is a positive attribute of the browse forages since the voluntary DM intake and DM digestibility are dependent of the cell wall constituents (fibre) especially the NDF and lignin (Bakshi and Wadhwa 2004). In addition, the fibre of browse species forages has been shown to be more digestible (El Hassan et al 2000) than that of grasses and crop residues. The variation in the various chemical compositions evaluated may be due to several factors such as species, soil, stage of maturity and harvesting (Singh et al 2005).

Table 2 presents the phenolics and tannin compositions of the browse forages. The forages varied widely in the phenolics composition. The TEPH ranged from 10.0 (Maerua angolensis) to 165.8 mg/g DM (Terminalia brownii). The highest TET content was found in Terminalia brownii (125.7 mg/g DM) and Calliandra calothyrsus (122.9 mg/g DM) while the lowest TET content was in Boscia angustifolia (1.5 mg/g DM). Tamarindus indica had the highest TCT (18.05 mg/g DM) while the chemical assay did not detect TCT in Balanites aegyptiaca and Boscia angustifolia. Fractionation of the proanthocyanidins showed that Berchemia discolor had the highest delphinidins compositions while Tamarindus indica had the highest cyanidins. The delphinidins/cyanidins (D/C) ratio varied from 0:100 in G. bicolour, Maerua angolensis, Olea europaea, Persia americana and Tamarindus indica to 87:13 in Zizyphus mucronata.

The phenolics contents of the browse forages were within the range reported earlier for similar forages from Kenya (Osuga et al 2006; Osuga et al 2005; Abdulrazak et al 2000). The tannins content varied from low to moderate except in Calliandra calothyrsus, G. bicolour and Terminalia brownii that had tannin contents of more than 100 mg/g DM. Tannins present in browse forages have been shown to be the major limitation to the use of the forages in livestock diets especially as protein supplements. This is because in ruminants, the polyphenolics tend to alter the composition of rumen micro-organisms, complex and inhibit microbial enzymes, complex with the feed nutrients and their metabolic products may be absorbed from the rumen and produce toxic effects at the tissue level (Mangan 1988). However, low levels (30-40 mg/g DM) of tannins have nutritional benefits for ruminants by protecting dietary proteins from excessive ruminal degradation without affecting forage intake or fibre digestion (Barry et al 1986). Condensed tannin (CT) fractions showed that most of the forages contain a prevalence of procyanidins. However, Berchemia discolor, Terminalia brownii and Zizyphus mucronata tended to show high prevalence of prodelphinidins. The nutritional implications of the differences in delphidins/cyanidin (D:C) ratio are not yet clear (Hedqvist et al 2000). However, Aerts et al (1999) showed that CT with a predominance of prodelphinidins is more effective at reducing rumen microbial protein degradation in vitro than CT with a predominance of procyanidins. Generally, CT with high molecular weight tends to interact more strongly with enzymes and other proteins than CT with low molecular weight (Kawamoto et al 1996; Horigome et al 1988; Makkar et al 1988). The reactivity of CT also increases with increasing prodelphinidin content (Aerts et al 1999).

The in vitro cumulative gas production and the fermentation characteristics of the browse forages are summarized in Table 3. The forages significantly (P<0.05) differed in the gas production and fermentation characteristics. At 24 and 96 h of incubation, Balanites aegyptiaca and Berchemia discolor produced the highest gas production respectively. Terminalia brownii produced the least gas volume after 6 h of incubation. Boscia angustifolia had the highest potential gas production while Terminalia brownii had the lowest potential gas production. The rate of gas production was highest in Balanites aegyptiaca and lowest in Boscia angustifolia.

The results of the gas production and fermentation characteristics of the browse forages are within the range reported earlier for browse species forages from Kenya (Osuga et al 2006; Osuga et al 2005; Abdulrazak et al 2000). The variation in gas production and potential of gas production between the browse species forages can be attributed to compositional differences of the browse forages, especially CP, fibre, nature and concentration of polyphenolics and may be other anti-nutritional components. These factors influence the amount of substrate OM that is fermented and the short chain fatty acids (SCFAs) produced upon fermentation. This is because gas production results from fermentation of the feed OM and CO₂ produced from the buffering of the SCFAs by the bicarbonate buffer. Getachew et al (2000a) noted that presence of tannins in forages depresses in vitro gas and SCFAs production. In the present study, Terminalia brownii produced significantly the least gas volume and had the lowest potential of gas production, which can be attributed to the high content of tannins present in the browse species forage. CT fractionation also found a high predominance of prodelphinidins in Terminalia brownii, which may also have influenced the high reactivity of the tannins present in the browse forage.

Table 4 summarizes the results on the effect of PEG-6000 addition on gas production, in vitro true DM degradability (IVTDMD) and in vitro true OM degradability (IVTOMD) of the browse forages. Addition of PEG-6000 increased gas production in all the species except Boscia angustifolia. However, the increase was more than 100% in Calliandra calothyrsus, Grewia bicolor, Terminalia brownii and T. indica. The IVTDMD and IVTOMD were highest in Maerua angolensis while were lowest in Tamarindus indica and Boscia angustifolia. The effect of PEG-6000 addition on IVTDMD and IVTOMD was only significant in Calliandra calothyrsus, Grewia bicolor, Terminalia brownii and T. indica. The PF ranged from 2.8 to 11.6. Addition of PEG-6000 tended to significantly decrease the PF in all the species except Balanites aegyptiaca, Boscia angustifolia, Maerua angolensis and Olea europea.

Tannins in browse forages have been shown to be the major drawback in the use of the forages in ruminant diets. This is especially important when the tannin concentration in the forages is higher than the beneficial level of less than 40 mg/g DM (Barry et al 1986). However, tannins in browse forages tend to have different biological activities either through direct interaction between tannins and the bacteria cell wall or the effect of tannins on the microbial enzymes (Mangan 1988). PEG-6000 has been shown to be more effective in evaluating the biological anti-nutritive activity of tannins than other methods such as the protein precipitation capacity (Makkar et al 1995; Silanikove et al 1996; Getachew et al 2000a). Silanikove et al (1996) also noted that PEG was more effective in reversing the negative effect of tannins even when the tannins are strongly bound to proteins. Therefore, the increase in gas production on addition of PEG is considered a measure of the biological anti-nutritive activity of tannins. In the present study, Grewia bicolor, Terminalia brownii, Calliandra calothyrsus, Pappea capensis and Tamarindus indica had higher increase in gas production than the other species. This demonstrates that the tannins in the browse species forages would adversely affect their nutritive value. The gas production increase in Grewia bicolor was higher than the increase in Terminalia brownii and Calliandra calothyrsus, which had high TET contents. This could probably be due to higher tannin activity of tannins in the species or presence of some other anti-nutritional factors not measured in this study. However, the other species had low increase, though significant in Berchemia discolor, Persia americana, and Zizyphus mucronata, in gas production which indicates that they would be good supplements to low quality forages.

The forages had moderate to high in vitro DM and OM degradability. This demonstrates the high nutritive value of the browse forages when used in ruminant feeding. Getachew et al (2000b) demonstrated that the browse forages are better used as protein supplements of poor quality roughages such as hay and straws. Addition of PEG tended to significantly (P<0.05) increase both in vitro DM and OM degradability only in Grewia bicolor and Terminalia brownii. However, in the other species, addition of PEG either did not have significant effect or decreased the DM and OM degradability, probably due to the PEG-tannin complexes which are insoluble in neutral detergent solution and thus contributing to the weight of the undegraded residue. Blummel et al (1997) defined PF as the proportion of truly degraded substrate per unit of gas produced, which indicates variation in SCFAs production to microbial biomass production per unit of truly fermented substrate. Addition of PEG decreased the PF in all the species in this study which is in agreement with the results of Singh et al (2005), Baba et al (2002) and Getachew et al (2000b). The higher than the theoretical range of PF (2.75 - 4.41 mg truly degraded substrate/mg gas) suggested by Blümmel et al (1997) may be due to significant loss of detached tannins from fermented substrate, which do not contribute to gas production and non-utilization of the soluble fraction due to tannins inhibition (Baba et al 2002). Since the increase in gas production was not always supported by a proportional increase in substrate degradability, there is need for more studies to establish the relationship between gas production, substrate degradability in the presence and absence of PEG and calculated PF as regards to the inhibitory effects of tannins.

Conclusions

• The browse species evaluated in the current study had high CP content which would make them good protein supplements to poor quality roughages especially during the dry season.

• The moderate to high rumen degradability of the forages would encourage rumen microbial activities when the forages are fed to ruminant animals. However, the presence of high concentration and biological activity of tannins in some of the browse forages, especially Grewia bicolor and Terminalia brownii, would limit their nutritive value.

• Use of tannin binding agents such as PEG would be advantageous in improving the nutritive value of such browse species.

Acknowledgement

Financial aid provided by the Japanese Government through a Mombukagakusho scholarship to the first author during which this work was done is acknowledged.

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Received 16 July 2006; Accepted 3 October 2006; Published 6 December 2006

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