In vitro ruminal fermentation parameters of tanner grass (Brachiaria arrecta) supplemented with leaves from three forage trees

Andell Edwards, V Mlambo, C H O Lallo*, G W Garcia* and M D Diptee**

Department of Food Production [DFP], Faculty of Science and Agriculture [FSA], University of the West Indies [UWI], Hodge Street, St Augustine, Trinidad and Tobago [T & T]
andell_e@hotmail.com
* The Open Tropical Forage-Animal Production Laboratory [OTP-APL], DFP, FSA, UWI, St Augustine, T & T
** School of Veterinary Medicine, Faculty of Medical Sciences, Mt Hope, T & T

Abstract

Grasses in the tropics are generally low in nitrogen (N) and this tends to negatively affect their utilization as ruminant feeds. Protein supplementation using tree forage leaves has the potential to increase the feeding value of grasses and animal productivity. The utility of tree leaves as protein supplements varies with chemical composition, which is in turn influenced by the prevailing biotic and abiotic growth environment of the tree. This study, therefore investigated the effect of incremental levels of Leucaena leucocephala, Gliricidia sepium, and Trichanthera gigantea leaves, grown in a river estate loam soil (pH 5.0 – 6.2) in Trinidad, on in vitro ruminal fermentation parameters of Brachiaria arrecta (tanner grass) utilizing the Reading Pressure Technique (Mauricio et al 1999). The objectives were to determine which supplement is superior and which inclusion level is the most effective. Tanner grass was supplemented in vitro with incremental levels (7.5, 15, 22.5 and 30% w/w) of L. leucocephala (T+Leuc), G. sepium (T+Glir), or T. gigantea (T+Trich) leaves.

Gliricidia sepium supplementation promoted the highest increase (126 %) in gas production from the immediately soluble fraction (a) when included at 15 % level while L. leucocephala promoted the least increase (109 %) at the same level. Gas production from the insoluble fraction (b) declined when leaves of G. sepium, L. leucocephala and T. gigantea were supplemented at 15 %. Gas production rate constant for the insoluble fraction (c) increased by 100 % as the leaves of G. sepium, L. leucocephala and T. gigantea were included at 15 %. Potential gas production (a+b) showed a decline of 87, 86 and 83 % when leaves of G. sepium, L. leucocephala and T. gigantea were included at 15 %. There was a decrease in the lag phase (l) when leaves of all species were used as supplements. At 48-h post inoculation, cumulative gas production and in vitro organic matter degradability (iOMD) were highest (708 ml/g OM) and (545 g/kg DM) respectively, when G. sepium leaves were used as a supplement. Partitioning factors (PF) and predicted ME were highest when leaves of L. leucocephala and T. gigantea were supplemented at the 30 % level. It is concluded that G. sepium is superior to the other tree species when supplemented at the 15 % level.  

Key words: fibrous substrates, in vitro degradability, inclusion rates, protein-rich forages

Introduction

Tanner grass (Brachiaria arrecta) serves as the basal diet for ruminants reared at the University Field Station (UFS), Valsayn, Trinidad and Tobago. Tanner grass is a stoloniferous perennial rooting at lower nodes. It belongs to the Poaceae family and is native to tropical Africa. It grows best in well drained soils and responds well to nitrogen fertilization (Judd 1979). During the dry periods of the year (January to May) in Trinidad and Tobago visual assessment and animal performance seem to suggest that the feeding value (intake, digestibility) of tanner grass is poor. It is important to improve the utilization of tanner grass during the dry periods in order to maintain the condition of animals. One method of increasing the feeding value of tanner grass in the dry season is to supplement it with tree forage leaves. Tree leaves have the potential to provide additional nitrogen needed to enhance rumen microbial activity. When used as supplement in grass-based diets, leguminous forages increase feed conversion, protein and energy intake, rumen function and overall animal performance (Kanani et al 2006). Legume forage supplements have increased intake and digestibility of grass basal diets by ruminants (Getachew et al 1994). Mcleod and Minson (1969) and Preston (1982) reported that the supplementary effect of legumes on the in vivo digestibility of grasses is due to the legume overcoming an N deficiency. Foster et al (2009) reported that feed intake, digestibility, and nitrogen (N) retention were increased by supplementation with legume hay or soybean meal. Akinlade et al (2002) concluded that feeding forage legumes as a supplement can maintain live weight in sheep even in dry periods when feed quality is poor. There are three high protein forages (L. leucocephala, G. sepium and T. gigantea) established at UFS that can serve as suitable N supplements to ruminants on a basal diet of tanner grass.

Garcia (1988) reported on using the in vivo nylon bag digestibility of L. leucocephala and Manihot esculenta to estimate the level of digestibility and by-pass CP value and to estimate the DE value of these forages in sugarcane forage based diets for calves. In a study where G. sepium and L. leucocephala were used to supplement a napier grass (Pennisetum purpureum) diet, increases in total dry matter intake and live weight were recorded (Abdulrazak et al. 1996). Kanani et al (2006) reported that goats fed L. leucocephala had higher average daily gains (93.9 g/day) compared to those fed Medicago sativa, Dolichos lablab, and Desmanthus bicornutus. As a result the authors suggested that L. leucocephala had more potential for feeding growing goats in the tropical regions in comparison to the other forage legumes. However, in vitro studies by Edwards et al (2012) have shown that the in vitro organic matter digestibility of G. sepium leaves is superior to L. leucocephala and T. gigantea. 

In vivo digestibility studies are labour intensive, costly and require sophisticated equipment and animal holding facilities (Khan et al 2003). In vitro ruminal gas production techniques, on the other hand have been used extensively to rapidly evaluate and rank the nutritive value of feedstuffs for ruminant animals (Chenost et al 2001; Degen et al 2010). Information on in vitro ruminal fermentation parameters of tanner grass when supplemented with forage legumes is limited. Incremental levels can be used to determine the minimum quantity of supplement required to maximize rumen microbial activity and hence basal diet utilization. Researchers have utilized varying inclusion levels to evaluate the effect of supplements. Inclusion levels of 0, 10, 20, and 30 % were used to assess the effect of greenleaf desmodium (Desmodium intortum) and sweet potato vine (Ipomoea batatas) supplementation of napier grass (Pennisetum purpureum) on degradation and rumen fermentation in Friesian steers (Kariuki et al 2001). Similarly, Mupangwa et al (2002) employed 0, 10, 20, and 30 % supplementation levels of velvet bean hay (Mucuna pruriens) to estimate the nutrient parameters of natural grass hay offered to sheep. The objectives of this study, therefore, were to evaluate the effects of incremental levels (7.5, 15, 22.5 and 30 %) of L. leucocephala, G. sepium, and T. gigantea on in vitro ruminal fermentation parameters for tanner grass utilizing the Reading Pressure Technique. In addition, it was aimed to determine which species and supplemental level is the most effective. 

Materials and Methods

Study site

Leaf samples were obtained from established trees of G. sepium, L. leucocephala and T. gigantea at The University Field Station (UFS). The UFS (Lat 10° 38’ N Lon 61° 23’ W) has a relatively flat topography with an altitude of 15.2 meters above mean sea level. Average annual rainfall is 1782.9 mm with an average monthly temperature of 27 °C. The soil type is river estate loam. The soil is free draining with a pH range of 5.0 – 6.2. 

Sample preparations 

Fresh leaf materials (leaves with petioles) were harvested from the three forage tree species (L. leucocephala, G. sepium, and T. gigantea) that had been trimmed to a height of 1 meter at the UFS. Harvesting was manually done, 8 weeks after trimming, by cutting branches at a distance of 1 m from the growing tip for G. sepium and 0.5 m for T. gigantea and L. leucocephala.  Leaves from six individual trees for each species were harvested, weighed and stored separately into brown paper bags. Leaf samples were immediately transported to the laboratory and oven dried at 65 °C to a constant weight. The dried samples were then milled to pass through a 1mm sieve using a Wiley Mill (Glen Creston Ltd, Middlesex, UK) and kept at room temperature (25°C, 68 % relative humidity) in separate brown paper bags pending chemical analysis and in vitro ruminal fermentation.

Chemical analyses 

Chemical analyses of forage tree leaves were carried out as part of an earlier study (Edwards et al 2012) and the results are included here (Table 1) for descriptive purposes. Dry matter, organic matter, crude protein, neutral detergent fibre, acid detergent fibre, and acid detergent lignin were determined for tanner grass and tree leaves. Soluble and insoluble condensed tannin content were also determined for tree leaves.

In vitro ruminal gas production 

Rumen microbial fermentation was assessed in vitro using the Reading Pressure Technique (RPT) of Mauricio et al (1999). Ruminal fluid was collected at 7:00 am. The donor was a crossbred Holstein heifer offered tanner grass, G. sepium, L. leucocephala, T. gigantea leaves and a dairy concentrate (Master Mix Feeds Ltd, Trinidad). Rumen digesta from multiple sites within the rumen was sampled by hand and the rumen fluid squeezed into prewarmed thermos flasks, transported to the laboratory, then blended and strained through two layers of warm cheese cloth. The resulting rumen fluid was held under carbon dioxide at 39 °C. About 0.5 g of tanner grass sample was weighed into 125 ml serum bottles in triplicate. Tanner was then supplemented with G. sepium, L. leucocephala, or T. gigantea leaves at 7.5, 15, 22.5 and 30% (w/w) inclusion rates. Using a measuring cylinder, 90 ml reduced RPT buffer (Mauricio et al 1999) was added to each serum bottle (a total of 110 serum bottles). Serum bottles without samples (blanks) were included for each of the two withdrawal periods (24 and 48 post-inoculation) to allow correction for residual DM from rumen fluid that may affect fermentation gas release. Leaf substrates were incubated for 48 h. After adding the buffer, the bottles were sealed and stored at 20 °C before being transferred into the incubators, set at 39 °C, 8 h before inoculation with rumen fluid. The serum bottles were inoculated with 10 ml rumen fluid and incubated at 39 °C for 48 h. Inoculation was completed within 1 h of fluid being prepared and, during this time, anaerobic conditions were maintained by constant flushing of rumen fluid containers and serum bottles with carbon dioxide gas. Serum bottles were withdrawn after 24 and 48 h incubation to estimate extent of degradability. 

Gas measurements 

Headspace gas pressure was measured at 2, 4, 6, 8, 10, 12, 15, 19, 24, 30, 36, 48, 72 and 96 h post-inoculation using a pressure transducer. Gas pressure readings in pounds per square inch (psi) were converted to gas volume (ml) using the following relationship between gas pressure and gas volume that was predetermined for the site:

GP= 0.0022psi² + 2.1244psi + 1.7675

Where  GP = is the predicted gas volume (ml) and psi is the pressure transducer reading (pressure per square inch) at time t

Cumulative gas data were fitted to a non-linear regression model (Orskov and McDonald 1979) using the Curve Fitter (Datafit 9 1999). The model is as follows:

Y= a+b (1-e^-c(t-tl))

Where:

y = gas produced at time “t”; a = gas production from the immediately soluble fraction (ml); b = gas production from the insoluble fraction (ml); c = gas production rate constant for the insoluble fraction (ml/h); e = exponential function; t = incubation time (h); l = lag time 

Estimation of degraded substrate 

In vitro organic matter degradability (iOMD) at 24 and 48 h was determined by recovering the fermentation residues after filtration through sintered glass crucibles (100-160 µm porosity, Pyrex, Stone, UK) under vacuum. Fermentation residues were dried at 105 °C overnight and incinerated in a muffle furnace at 550 °C for 12 h. Loss in weight after incineration was used as a measure of undegradable OM. The iOMD was calculated as the difference between OM content of the substrate and its undegradable OM. Partitioning factors (PF), a measure of fermentation efficiency, were calculated as a ratio of 48 h iOMD (mg) and cumulative gas production (ml/g OM) fermentation parameters. Metabolizable energy (ME) (MJ/kg DM) content of leaf substrates was predicted from 48h organic matter degradability using the following equation (Mc Donald et al 2002):

ME (MJ/kg DM) = 0.016iOMD

where:

iOMD =g digestible organic matter per kg dry matter (DM)  

Statistical analysis 

General linear models (GLM) procedures of Minitab (Minitab 2000, Minitab Inc, State College, PA, USA) were used for the analysis of variance for 24h and 48h cumulative gas production, rate of gas production, iOMD and fitted parameters (a, b and c) data. A 3*4 factorial treatment (3 species combination and 4 inclusion levels) arrangement in a completely randomised design (CRD) was employed using the model of Y_ij = µ + D_{ij +}  F_ij + D_{ij *}  F_ij + e_ij.

where: Y_{ij =}  dependent variable , µ = overall mean, D_ij = species effect (G. sepium, L. leucocephala and T. gigantea), F_{ij =} supplement effect (0, 7.5, 15, 22.5 and 30 %),  D_{ij *}  F_{ij =} species*supplementation effect and e_{ij =} residual error. 

Results

In vitro ruminal fermentation parameters 

Figure 1 shows the effect of species and supplementation level on cumulative gas production. At 48h post inoculation, there was a 146 % increase in cumulative gas production when G. sepium leaves were supplemented at the 15 % level when compared to 0 % supplementation (Figure 1). There was a 128 % increase in 48h cumulative gas production when L. leucocephala leaves were supplemented at 15 % in comparison to 0 % supplementation.

When T. gigantea leaves were used as a supplement at 15 % level, there was a 136 % increase in cumulative gas production at 48-h post inoculation compared to 0 % supplementation. At 48-h post inoculation, cumulative gas production was highest when G. sepium leaves were used as a supplement in comparison to the other species (Figure 1).  

Tables 2 and 3 show the effect of species and inclusion levels on gas production from the immediately soluble fraction (a), gas production from the insoluble fraction (b), gas production rate constant for the insoluble fraction (c), potential gas production (a+b), lag phase (l), in vitro organic matter degradability (iOMD), partitioning factor (PF) and predicted metabolizable energy (ME). There was an increase in gas production from the immediately soluble fraction (a) when leaves of G. sepium (126 %), L. leucocephala (109 %) and T. gigantea (117 %) were included at the 15 % level. However, there was a decrease in (a) when leaves were supplemented at levels beyond 7.5 % for all species. Gas production from the immediately soluble fraction (a) was highest when G. sepium was used as a supplement in comparison with the other species (Table 2).  

There was a decrease of 85 % in gas production from the insoluble fraction (b) when leaves of G. sepium were included at 15 % compared to 0 % supplementation. Supplementing L. leucocephala leaves at 15 % caused gas production from the insoluble fraction (b) to decrease by 86 %. There was a decline of 85 % as T. gigantea leaves were supplemented at 15 %. The gas production rate constant for the insoluble fraction (c) increased by 100 % as the leaves of G. sepium were included at 15 % (Table 2), Supplementing L. leucocephala leaves at 15 % caused gas production rate constant for the insoluble fraction (c) to increase by 100 %. There was an increase of 100 % in the insoluble fraction (c) as T. gigantea leaves were included at 15 % (Table 2). Potential gas production (a+b) showed a decline of 84 % as the leaves of G. sepium were supplemented at 15 %. Leucaena leucocephala leaves caused the potential gas production (a+b) to decrease by 86 % when supplemented at 15 %. There was a decrease in potential gas production (a+b) by 84 % when T. gigantea leaves were included at 15 % (Table 2). Supplementing with tree leaves reduced the lag phase (l). There was a 61 % decline in the lag phase (l) when the leaves of G. sepium were included at the 15 % level, while a 63 % decline was observed with L. leucocephala at 15 %. Trichanthera gigantea leaves caused the lag phase to decrease by 56 % when supplemented at 15 % (Table 2) compared to 0 % supplementation.

At 48-h post inoculation iOMD was highest when G. sepium (545 g/kg DM) and T. gigantea (542 g/kg DM) leaves were used as supplements at the 22.5 % level. There was no difference in partitioning factors (PF) and predicted ME when leaves were used as supplements. 

Discussion

This study was designed to test the hypothesis that in vitro ruminal gas production, fermentation kinetics and organic matter digestibility of tanner grass are influenced by incremental levels of L. leucocephala, G. sepium, and T. gigantea. Data obtained supported this hypothesis as shown in Figure 1 and Tables 2 and 3. In vitro ruminal cumulative gas production of tanner grass at 24-h and 48-h post inoculation increased significantly with supplementation by all forage species (Table 2). This may be attributed to more nutrients having been made available for the microbes to utilize in vitro (Osuga et al 2008). Cumulative gas production, at 48-h post inoculation, was highest for G. sepium supplementation at all inclusion levels, possibly influenced by its high concentration of crude protein (CP), organic matter (OM) and low fibre components (NDF, ADF, ADL) (Table 1). This is consistent with the findings of Kamalak et al (2004) and Karabulut et al (2007) who reported negative correlations between gas production (GP) and cell wall contents (NDF, ADF) and positive relationships between CP, ash and GP of legume hays. In addition, the extent of gas production reflects the efficiency of the degradability of feed OM (Osuga et al 2005). In vitro ruminal cumulative gas production increased dramatically for all species from 24-h to 48-h post inoculation. This is in agreement with Kamalak et al (2004) who reported increases in gas production of tree leaves with increasing incubation time. This demonstrates that the microbial population is larger and more active at 48-h post inoculation. Supplementation with G.sepium leaves recorded the highest percentage increase in gas production from the immediately soluble fraction (a) at the 15 % level (Table 2). This indicates that G. sepium leaves releases more readily available nutrients to the rumen microbes at that level.

All species caused gas production from the insoluble fraction (b) to decline as their leaves were used to supplement tanner grass. Such data may suggest that there was a lack of sustained supply of nutrients from the leaves to allow fermentation of the slowly degradable fractions of the substrates. Supplementation with these forage tree leaves may not have increased the volume of nutrients, particularly N to microbial organisms. Additionally, it is possible that more rumen undegradable crude protein would be made available in the small intestine. The gas production rate constant for the insoluble fraction (c) increased by 100 % as the leaves of G. sepium, L. leucocephala and T. gigantea were included at 15 %, respectively (Table 2). This increase suggests that more soluble carbohydrates and proteins were made available to microorganisms as supplementation increased (Akinfemi et al 2009). This may also explain the increase in cumulative gas production of tanner grass when supplemented at 24 and 48 h as gas production is in direct response to soluble carbohydrates (Chenost et al 2001). The gas production rate constant for the insoluble fraction (c) is critical as it can be associated with dry matter intake, hence providing nutritionist with valuable tools for feeding. There was a decrease in the lag phase (l) as the leaves of all species were used as supplements (Table 2) providing evidence that a greater proportion of nutrients were made available as inclusion levels increased. At 48-h post inoculation iOMD was highest when G. sepium (545 g/kg DM) and T. gigantea (542 g/kg DM) leaves were used as supplements at the 22.5 % level suggesting that more nutrients, particularly N, were being made available to facilitate microbial degradation at this level (Akinfemi et al 2009). Partitioning factors tend to increase with incremental levels of supplement due to increased substrate degradability (Arhab et al 2009). However, this was not the case in this study as increasing supplement levels did not influence PF. 

Forage legumes provide a good source of degradable nitrogen and energy and as a result their inclusion in the diet is expected to increase the population of rumen microbes (Wallace et al 1995). This was not the case in a study by Abdulrazak et al (1996) where neither G. sepium nor L. leucocephala were able to influence apparent digestibility or in sacco DM degradation characteristics of forages. However, Getachew et al (1994) attributed an increase in rumen ammonia to the degradable nitrogen supplied when diets were supplemented with forage legumes. Mcleod and Minson (1969) reported that the supplementary effect of legumes on the in vivo digestibility of grasses is due to the legume overcoming any N deficiency. Preston (1982) has shown that increasing nitrogen in diets of fibrous feeds lead to an increase in total forage and DM intake. 

Though the results were consistent with Akinlade et al (2002) and Foster et al (2009) who reported increases in digestibility of poor quality feed owing to supplementation with legume forages, it is difficult to quantify whether the increase in digestibility from supplementation was due to the supply of nitrogen (N) or a substitution effect due to bulk effect. Minson and Milford (1967) suggested that supplementation with legumes can result in a stimulating effect owing to a supply of N but also a substituting effect due to their bulkiness. Wallace et al (1995) indicated that assessing substitution on a 1:1 basis is misleading since the components of the diet will have different digestibilities and different bulking effects in the rumen. One way to address this is to ensure forage legumes are included at a rate of a third or less of the total dry matter (DM) as was done in this study as this may reduce the effect of bulking. In vitro techniques for evaluating digestibility are arbitrary methods and if their results are to predict in vivo digestibility, standard feeds of known in vivo digestibility should be included with each batch for determination (Mcleod and Minson 1969). The use of such standards was not done for this study but would be explored in future studies. 

Gliricidia sepium seems to have the greatest impact on in vitro ruminal fermentation; however, it shared similar responses to fermentation parameters and organic matter degradability with T. gigantea (Figure 1, Tables 2 and Table 3). This may be attributed to higher levels of degradable nitrogen (Getachew et al 1994; Wallace et al 1995) in the two forages. High degradation in the rumen is not ideal as it indicates smaller proportions of by-pass protein to the duodenum. Supplementation at 22.5% stimulated the highest response from in vitro ruminal fermentation, gas production from the immediately soluble fraction (a) and organic matter digestibility (OM) for G. sepium. However, the highest responses to in vitro ruminal gas production, (a) and (c) for T. gigantea supplementation was attained at the 7.5 % level. Such data suggest that a small quantity of T. gigantea leaves is required for optimum response to fermentation parameters. This may imply that mixtures of the different forage legumes may be a possible way forward (mixing their rumen degradable protein and by-pass protein characteristics to the benefit of the animal’s ruminal and post ruminal nutritional needs). 

Conclusion

• Incremental levels of L. leucocephala, G. sepium, and T. gigantea did have an influence on the in vitro ruminal fermentation parameters of tanner grass utilizing the Reading Pressure Technique. However, when supplemented at the 15 % level, G. sepium proved to be the better supplement. Future research involving in vivo trials must now be conducted to add meaning to these findings.

Acknowledgements

We would like to give our sincerest gratitude to the lab technicians of the Food Production Lab, Department of Food Production, The University of the West Indies for lending their support and expertise. The authors acknowledge funding support for the purchase of chemicals for this study from The School for Graduate Studies, The University of the West Indies (St Augustine Campus) for providing the funding to purchase chemicals.

References

Abdulrazak S A, Muinga R W, Thorpe W and Orskov E R 1996 The effects of supplementation with Gliricidia sepium or Leucaena leucocephala forage on intake, digestion and live-weight gains of Bos Taurus X Bos indicus steers offered napier grass. Animal Science 63, 381-388.

Akinfemi A,  Adesanya A O and  Aya V E 2009 Use of an in vitro gas production technique to evaluate some Nigerian feedstuffs. American-Eurasian Journal of Scientific Research 4 (4), 240-245 

Akinlade J, Smith J W, Larbi A, Archibong I O and Adekunle I O 2002 Forage from cropping systems as dry season supplements for sheep. Tropical Grasslands 36,102-106. 

Arhab R, Macheboeuf D, Aggoun M, Bousseboua H, Vialva D and Besle J M 2009 Effect of polyethylene glycol on in vitro gas production and digestibility of tannin containing feedstuffs from North African Arid Zone. Tropical and Subtropical Agroecosystems 10, 475-486 

Batson H F, Ferguson T U and Archibald K A E 1987 Cultivation of Leucaena with special reference to the Caribbean. Faculty of Agriculture, University of the West Indies, St Augustine, Trinidad. CAEX-TB/3/87, UWI. 

Chenost M,  Aufrere J and  Macheboeuf D 2001 The gas-test technique as a tool for predicting the energetic value of forage plants. Animal Research 50, 349-364 

Datafit 2008 Data curve fitting (nonlinear regression) and data plotting software. Version 9.0. Oakdale engineering, Tomey Road Oakdale, PA 15071, USA.

Degen A A, Kam M, Pandey S B, Upreti C R, El-Meccawi S and Osti N P 2010 In vitro gas production of leaves from fodder trees and shrubs from mid-hills of Nepal using cow, sheep and goat rumen liquor. Journal of Agricultural Science 148, 445-451.

Edwards A, Mlambo V, Lallo C H O and Garcia GW 2012 Yield, chemical composition and in vitro ruminal fermentation of the leaves of Leucaena leucocephala, Gliricidia sepium and Trichanthera gigantea as influenced by harvesting frequency. Journal of Animal Science Advances 2, 321-331.

Foster J L, Adesogan A T, Carter J N, Blount A R, Myer R O and Phatak A C 2009 Intake, digestibility, and nitrogen retention by sheep supplemented with warm-season legume hays or soybean meal. Journal of Animal Science 87: 2891-2898.

Garcia G W 1988 Production of Leucaena (Leucaena leucocephala) and Cassava (Manihot esculenta) forages and their nitrogen utilization by growing dairy cattle fed sugarcane based diets. Ph.D. Thesis. Dept. of Food Production, University of the West Indies, St. Augustine, Trinidad and Tobago. 

Garcia G W, Ferguson T U, Neckles F A and Archibald K A E 1996 The Nutritive value and Forage Productivity of Leucaena leucocephala. Journal of Animal Feed Science and Technology 60, 29-41.

Getachew G, Said A N and Sundstol F 1994 The effect of forage legume supplementation on digestibility and body weight gain by sheep fed a basal diet of maize stover. Animal Feed Science and Technology 46: 97-108. 

Judd I, Benjamin 1979 Handbook of Tropical Forage Grasses. Garland Publishing Inc, New York 

Kamalak A, Canbolat O, Gurbuz Y, Ozay O, Ozkan C O and Sakarya M 2004 Chemical composition and in vitro gas production characteristics of several tannin containing tree leaves. Livestock Research for Rural Development. Volume 16, Article # 6. http://ftp.sunet.se/wmirror/www.cipav.org.co/lrrd/lrrd16/6/kama16044.htm [accessed 24 March 2012]. 

Kamalak A and Canbolat O 2010 Determination of nutritive value of wild narrow-leaved clover (Trifolium angustifolium) hay harvested at three maturity stages using chemical composition and in vitro gas production. Tropical Grasslands 44, 128-133 

Kanani J, Lukefahr S D and Stanko R L 2006 Evaluation of tropical forage legumes (Medicago sativa, Dolichos lablab, Leucaena leucocephala and Desmanthus bicornutus) for growing goats. Small Ruminant Research 65, 1-7.

Karabulut A, Canbolat O, Kalkan H, Gurbuzol F, Sucu E and Filya I 2007 Comparison of In vitro gas production, metabolizable energy, organic matter digestibility, and microbial protein production of some legume hays. Asian-Australian Journal of Animal Science 4, 517-522 

Kariuki J N, Tamminga S, Gachuiri C K, Gitau G K and Muia J M K 2001 Intake and rumen degradation in cattle fed napier grass (Pennisetum purpureum) supplemented with various levels of Desmodium intortum and Ipomoea batatas vines. South African Journal of Animal Science 31 (3), 149-155

Khan, Ajmal, Nisa-UN-Mahr M and Sarwar M 2003 Techniques measuring digestibility for the nutritional evaluation of feeds. International Journal of Agriculture and Biology 5 (1):91-94

Mauricio R  M, Mould F L, Dhanoa M S, Owen E, Channa K S and Theodorou M K 1999 A semi-automated in vitro gas production technique for ruminant feedstuff evaluation. Animal Feed Science and Technology 82, 227-241.

McDade L A 1983 Pollination intensity and seed set in Trichanthera gigantea (ACANTHACEAE). Biotropica 15 (2), 122-124. 

Mc Donald P, Edwards R A, Greenhalgh J F D and Morgan C A 2002 Animal Nutrition. 6^th edition. Longman House, England. 

Mcleod M N and Minson D J 1969 The use of the in vitro technique in the determination of the digestibility or grass/legume mixtures. Agroforestry Journal 44, 4. 

Minson D J and Milford R 1967 The voluntary intake and digestibility of diets containing different proportions of legumes and mature pangola grass (Digitaria decumbens). Australian Journal of experimental Agriculture and Animal Husbandry 7, 546-551.

Mupangwa J F, Ngongoni N T, Daka D E and Hamudikuwanda H 2002 The effect of supplementing a basal diet of veld grass hay with increasing levels of velvet bean hay (Mucuna pruriens) on nutrient parameters in sheep. Livestock Research for Rural Development Volume 14, Article # 4. http://www.lrrd.org/lrrd14/4/mupa144.htm [accessed April 4th 2012].

Osuga I M, Abdulrazak S A, Ichinoe T and  Fujihara T  2005  Chemical composition, degradation characteristics and effect of tannin on digestibility of some browse species from Kenya harvested during the wet season. Asian Australian Journal of Animal Science (18) 1, 54-60

Osuga I M, Wambui C C, Abdulrazak S A, Ichinoe T and Fujihara T 2008 Evaluation of nutritive value and palatability by goats and sheep of selected browse foliages from semiarid area of Kenya. Animal Science Journal 79, 582-589

Orskov E R, McDonald I 1979 The estimation of protein degradability in the rumen from incubation measurements weighted according to rate of passage. Journal of Agricultural Science Cambridge 92, 499-503

Preston T R 1982 Nutritional limitations associated with the feeding of tropical forages. Journal of Animal Science 54: 877-884 

Wallace R J and Lahlou-Kassi A 1995 Rumen Ecology Research Planning. Proceedings of a workshop held at ILRI, Addis Ababa, Ethiopia, 13-18 March 1995. ILRI (International Livestock Research Institute), Nairobi, Kenya. 270 pp. 

Received 5 May 2012; Accepted 10 May 2012; Published 1 June 2012

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