Comparative in vitro evaluation of mixtures of Panicum maximum cv Ntchisi with stylo (Stylosanthes guianensis), Lablab (Lablab purpureus), Centro (Centrosema pubescens) and Histrix (Aeschynomene histrix)

F T Ajayi and O J Babayemi* 

Institute of Agricultural Research and Training, Obafemi Awolowo   University,  Moor Plantation, Ibadan, Nigeria

*Department of Animal Science, University of Ibadan, Ibadan, Nigeria

ajayiajay@yahoo.com 

Abstract

Organic matter digestibility (OMD), metabolizable energy (ME), short chain fatty acid (SCFA) production and degradation constants of Panicum maximum and legume mixtures and Panicum maximum intercrop with legumes were estimated from in vitro gas production parameters.  

Crude protein (CP) content ranged from 7.63 to 23.29%, neutral detergent fibre (NDF) ranged from 50.66 to 64.28%, Ether extract ranged from 2.73 to 9.24% and the ash content values ranged from 6.78 to 12.66%. Phenol content varied from 1.47 – 1.76 %, Tannin value ranged from 1.05 – 1.38 %, Saponin content ranged from 1.38 – 1.72 %, Phytate content ranged from 2.17 – 2.31 % and Oxalate content ranged from 1.54 – 1.73 %. Potential gas production, b from the insoluble fraction of the legumes ranged from 22.38 to 28.46 ml/g DM, the value for the mixtures ranged from 14.55 to 22.10 ml/g DM. The b fraction of the grass in legume intercrop ranged from 20.45 to 24.50.ml/g DM. Increased gas production was observed in the mixtures and intercrop than sole grass. Methane production was higher in legumes than the mixtures. ME, OMD and SCFA values were in the order of mixtures > legumes > sole grass. Grass in legume intercrop had higher ME, OMD, SCFA than sole grass.

This study revealed that grass in legume intercrop and grass/legume mixtures have greater benefit in animal nutrition than grass only.

Keywords: gas production, grass, intercrop, mixtures, Panicum maximum

Introduction

The low productivity of ruminant livestock is as a result of the poor nutritional status in terms of quality (Otchere et al 1987). Tchinda et al (1993) reported that native pastures are the most widely available low cost feeds for ruminants in the tropics. The native pastures deteriorate rapidly especially in the dry season. One of these pastures in use is Guinea grass (Panicum maximum).

Panicum maximum (Panicum) is one of the most common grasses in the derived savannah region of Nigeria. Under good conditions, its nutritional value is high, having up to 12.5 % crude protein, total digestible nutrients (TDN) of 10.2 % and calcium, phosphorus and magnesium (Agishi 1985; McDonald et al 1988).

Pasture can be improved with herbaceous legumes. The use of herbaceous forage legumes in livestock production systems for ruminants in the tropics has increased. They are rich in protein which is usually the most limiting nutrients in tropical animal diets (Andrea and Pablo 1999). Forage legumes can be grazed, harvested and fed fresh or stored as hay or silage (Harricharan et al 1988). A sustainable way of improving the feeding value of poor quality pastures is through supplementation with forage legumes (Andrea and Pablo 1999).

The in vitro gas production method is a laboratory estimation of degraded feeds which are important in livestock nutrition. It is a method that is reproducible and parameters obtained correlate well with in vivo method. In vitro gas production method have the advantage of not only being less expensive and less time consuming, but the method allows experimental conditions more precisely than the in vivo method (Makkar 2002). It is convenient and fast and allows a large number of samples to be handled at a time. It is based on the quantification of substrate degraded and of gas or short chain fatty acid produced in rumen fermentation system based on syringes (Menke et al 1979).

The objectives of this study are to compare the metabolizable energy (ME), organic matter digestibility (OMD), short chain fatty acid (SCFA) and gas production constants of Panicum in combination with Stylosanthes guianensis (Stylo), Centrosema pubescens (Centro), Aeschynomene histrix (Histrix) or Lablab purpureus (Lablab) in vitro. As well as to compare the ME, OMD, SCFA and gas production constants of Panicum intercropped with these herbaceous legumes.
 

Materials and methods  

Two hundred milligrams of ground feed samples were weighed into 100ml calibrated syringes with pistons lubricated with Vaseline. A buffered mineral solution was prepared consisting of NaHCO₃ + Na₂HPO₄ + KCl + NaCl + MgSO₄. 7H₂O + CaCl₂. 2H₂O (1:4, v/v). and stirred at 39 ^oC under continuous flushing with carbondioxide (CO₂). Rumen fluid was collected from three female WAD does that were previously fed concentrate consisting of 20 % maize, 20 % corn bran, 25 % wheat offal, 20 % Palm kernel cake, 10% ground nut cake, 4 % oyster shell, 0.5 % common salt, 0.25 % fish meal and 0.25 % grower premix. In addition, foliages of Gliricidia sepium and Panicum were fed to the animals for two days prior to the collection of rumen liquor.  

The liquor was collected into a pre – warmed thermos flasks and was later filtered through three layers of cheesecloth and flushed with carbondioxide. About 30 ml of buffered rumen fluid was taken into syringes containing the feeds. The syringes were placed in an incubator at 39 ^oC. Gas production rates were recorded at 3, 6, 9, 12, 15, 18, 21 and 24 hour of incubation and each syringe was gently swirled after reading. At the end of 24 hours incubation, 4 ml NaOH was added to the substrate in each syringe to determine the methane production. Rates and extent of gas production were determined for each substrate from the linear equation:

Y = a + b (1 – e^–ct) described by Ǿrskov and McDonald (1979),

Where:

Y = volume of gas produced at time‘t’,

a = intercept (gas produced from the soluble fraction),

b = Potential gas production (ml/ g DM) from the insoluble fraction,

c = gas production rate constant (h ^-1) for the insoluble fraction (b),

t = incubation time.

The metabolizable energy (MJ/Kg DM) and organic matter digestibility (OMD %) were estimated from the volume of gas produced after 24 hr of incubation (GP, ml/200 mg DM) and the proportion of crude protein (CP, g/100 g DM) as established by Menke and Steingass (1988):

OMD = 24.91 + 0.72222GP + 0.0815CP
ME = 2.2 + 0.1357GP + 0.0057CP + 0.0002859 CP²

Short chain fatty acids (SCFA) were calculated as described by Getachew et al (1999). 

The grass (Panicum maximum) and legumes were intercropped and harvested at four weeks interval. 

The treatments were as follows

A. Guinea grass/legume mixtures in ratio 60:40

i.                        Panicum maximum and Aeschynomene histrix

ii.                        Panicum maximum and Stylosanthes guianensis

iii.                        Panicum maximum and Lablab purpureus

iv.                        Panicum maximum and Centrosema pubescens

v.                        Panicum maximum in ratio 100:0

B. Herbaceous legumes in ratio 100:0

vi.                        Aeschynomene histrix

vii.                        Lablab purpureus

viii.                        Stylosanthes guianensis

ix.                        Centrosema pubescens

Chemical analysis  

Ground samples of grass, grass in legume intercrop and sole legumes were analyzed for dry matter, ash, crude protein, crude fiber and ether extract by the procedure of AOAC (1990). NDF, ADF and ADL were determined by the method of Van Soest et al (1991). NFE was calculated using 100 – (% OM, + % CP+ % CF + % EE + % ash).

Saponin was analyzed by method of Okwu and Josiah (2006). Phytate was determined by the method of Maga (1983). Tannin and oxalate were analyzed by method of Beutler et al (1980).

Statistical analysis 

The means were analyzed by the analysis of variance (ANOVA) techniques using the General Linear model procedures of SAS (1998). Treatment means were compared using Duncan Multiple range test (Gomez and Gomez 1984).

Results 

Table 1 reveals the chemical composition of Panicum maximum and herbaceous forage legumes. Dry matter of the forages ranged from 34.16 in Stylo to 43.28 g/100 g DM in Lablab. Among the legumes, crude protein was highest in Lablab (23.29 g/100g DM) and lowest in Stylo (18.05 g/100 g DM). The highest NDF, ADF and ADL mean values were recorded for Panicum maximum but lower values were recorded for the legumes. Among the legumes, Histrix had the highest mean values of NDF and ADF. ADL was highest in Lablab (8.81 g/100 g DM). The EE ranged from 9.24 g/100 g DM in Lablab to 2.76 g/100 g DM in Panicum maximum. The value of ash content was lowest in Panicum maximum but higher values were obtained in the legumes.

Table 2 shows the antinutrients in the Panicum maximum and the herbaceous forage legumes. Among the legumes, Phenol content ranged from 1.47 in Stylo to 1.76 g/100 g DM in Histrix. The tannin content in the forages ranged from 1.01 in Panicum maximum to 1.38 g/100 g DM in Histrix. Saponin content was highest in Lablab (1.72 g/100 g DM) and lowest in Stylo (1.38 g/100 g DM). The Phytate content ranged from 2.17 g/100 g DM in Stylo to 2.31 g/100 g DM in Lablab. Oxalate content was highest in Histrix (1.73 g/100 g DM) and was lowest in Stylo (1.54 g/100 g DM).

Table 3 shows the in vitro gas production characteristics of Panicum maximum and legume mixtures. The mean values differed significantly (P < 0.05) among the treatment.

The gas volume, Y in the sole legume ranged from 12.49 in Histrix to 22.88 ml in Stylo. Similar observations were recorded for the b, a+b and c fractions and the mean values vary significantly (P < 0.05). Among the mixtures, Panicum maximum/Lablab mixture had the highest gas volume, Y (19.50 ml). The lowest value was observed in Panicum maximum/Histrix mixture (12.40 ml). The highest mean value of b fraction was recorded for Panicum maximum/Stylo and Lablab mixtures and were 22.10 and 21.00 ml (P > 0.05) respectively. The lowest value of b obtained was in Panicum maximum/Histrix mixture which was 14.55ml. Similar observation was recorded for the potential extent of gas production, a+b as for the b fraction. The rate of fermentation of the substrates ranged from 0.13 h^-1 in Panicum maximum/ Histrix to 0.17 h^-1 in Panicum maximum/Stylo mixture. Sole Panicum maximum had the lowest mean values of all the fermentation characteristics in the forages evaluated.

Table 4 shows the in vitro gas production characteristics of Panicum maximum intercropped with four herbaceous forage legumes. The mean values of gas production characteristics differed significantly (P < 0.05) among the treatment means. The highest volume of gas produced was observed in Panicum maximum intercropped with Centro (10.40 ml) and was followed by Panicum maximum intercropped with Histrix (9.60 ml).The lowest value of Y was observed in sole Panicum maximum.

The gas produced from the insoluble fraction b ranged from 16.90 ml in Panicum maximum to 20.75 ml in Panicum maximum intercropped with Histrix. The potential extent of gas production, a+b ranged from 18.30 in Panicum maximum to 28.60 ml in Panicum maximum/Histrix. The rate of gas production, c ranged from 0.08 h^-1 in Panicum maximum to 0.12 h^-1 in Panicum maximum intercropped with Lablab.

Figure 1 shows that Panicum maximum and herbaceous forage legumes. Panicum maximum/Stylo and Panicum maximum/Centro had the highest gas production volume. This was followed by Panicum maximum/Lablab. Panicum maximum without legume had the least gas production.

Figure 2 shows that the herbaceous forage legume, Histrix had the lowest gas production volume and was followed by Centro. The peak in gas production was recorded in Stylo and Lablab. Gas production is a reflection of the extent of digestibility.

Figure 3 reveals that Panicum maximum in Stylo intercrop and Panicum maximum in Lablab intercrop gave the highest gas production. The least gas production in the grass in legume intercrop was observed in Histrix. Sole Panicum maximum had the least gas production among the treatments.

Figure 4 shows the methane production of the Panicum maximum and Panicum maximum/ legume mixtures. Legumes were generally higher (p < 0.05) in methane than in sole grass. The mixtures of grass and legumes enhanced the methane production than in grass only but lower than sole legumes.

Fig 5 shows the metabolizable energy (ME) of Panicum maximum, mixtures with legume and sole legume. The grass/legume mixtures had higher ME (P < 0.05) than the sole legume and Panicum maximum. Amongthe mixtures, Panicum maximum/Centro had the highest ME, followed by Panicum maximum/ Stylo mixture. Intercropping both legumes and grass had increased the ME. In the legumes, histrix had the least value of ME but this value was higher than that of the sole Panicum maximum.

Figure 6 shows the organic matter digestibility (OMD) of Panicum maximum/ legume mixture and P .maximum only. The mean values obtained differed significantly (P < 0.05) among the treatments. Panicum maximum/ Histrix mixture had the lowest OMD. It was observed that the mixtures had better OMD when compared with other treatment means. Among the legumes, Histrix also had the least value of OMD % but the value was higher that recorded for sole Panicum maximum as depicted in the graph.

The graph of the short chain fatty acid (SCFA) for the grass/ legume mixtures, sole legume and grass was depicted in figure 7. The mean values obtained varied significantly (P < 0.05) among the treatments. The mixtures had higher SCFA values when compared to the sole legume or grass.

Figure 8 reveals the methane (CH4) production of the Panicum maximum in legume intercrop and Panicum maximum only. The mean values obtained differed significantly (P < 0.05) among the treatment means.

The grass in Lablab intercrop had the highest value of methane production, the least among the intercrop is in the grass in Histrix intercrop but the value was higher than the sole grass. Figure 9 shows the metabolizable energy (ME) of the grass intercropped with the legumes and grass only.

The mean values differed significantly (P < 0.05) among the treatments. Panicum maximum in Stylo intercrop had the highest mean value and was followed by Panicum maximum in Lablab intercrop. The least in the intercrop as depicted in the graph was recorded for Panicum maximum in Histrix intercrop.

Figure 10 shows the organic matter digestibility (OMD) of Panicum maximum in legume intercrop and Panicum maximum only. The organic matter digestibility mean values differed significantly (P < 0.05) among the treatments. Panicum maximum in Stylo intercrop had the highest mean values and was followed by Panicum maximum in Lablab intercrop. The mean values of Panicum maximum in Centro and Histrix were comparable.

The least organic matter digestibility was observed in Panicum maximum only. Figure 11 reveals the short chain fatty acid (SCFA) of the Panicum maximum in legume intercrop and Panicum maximum only. Among the intercrop, Panicum maximum in Lablab intercrop had the highest mean value and was followed by Panicum maximum in Stylo intercrop. Panicum maximum in Histrix intercrop had the least mean value of SCFA but higher than value recorded for sole Panicum maximum.  

Discussion

The DM content obtained in Panicum maximum was higher than 27.30 g/100 g DM reported (Bamikole et al 2003); this was because of the planting season (raining season) and increase biomass yield. The DM obtained in lablab was comparable to value reported (Babayemi et al 2006). The crude protein of lablab was higher than 14.06 g/100 g DM cited by Babayemi et al (2006) and 12.20 g/100 g DM (Mupagwa 2000) but comparable to value of 21.08 g/100 g DM reported by Nworgu and Ajayi (2005). The age of cutting could be adduced for the differences. The crude protein (CP) of centro and histrix compared well with values reported (Nworgu and Ajayi 2005). The CP of Panicum maximum was higher than 7.02 g/100 g DM reported by Bamikole et al (2003) and 8.50 g/100 g DM reported (Arigbede et al 2005). The reason was because of the age of harvesting of the Panicum maximum in this study. The Neutral detergent fibre (NDF) values were within the range of 24 – 61 reported for tropical forages (Topps 1992). The recorded value for phytate and oxalate in this study were within the range of 0.82 – 2.92 mg/100 mg and 0.33 – 2.06 mg/100 mg respectively (Onwuka 1996). The implication of this is that the essential minerals such as Ca, Mg, Fe and Zn required by the animals from the forages would be made available and not impaired during metabolism.

The condensed tannin (CT) content obtained in this study was higher than 30 – 40 g/Kg DM for efficient utilization by ruminants (Barry et al 1986). Stylosanthes guianensis and Centrosema pubescens had the lowest gas production compared to other legumes because of the high CT content in them.  

The gas production from insoluble fraction, b was better in the sole legumes than the mixtures. Similarly, higher rate of gas production observed in the sole legume over the mixtures. The probable reason for this was the low fibre fractions in the legumes. This was an indication that most of the nutrients especially crude protein would be lost without actually benefiting the animals. The high fibre fractions especially ADF and ADL obtained in the Panicum maximum could be adducible to the low b and a+b fractions of fermentation. A feed high in NDF is essential for increase lactation in dairy cow (NRC 2001). The forages are high in NDF which promotes increase gas production. The variations observed in the gas production among the treatments were due to the ADF and lignin contents in the forages. Higher values of fermentation characteristics were obtained in this study than reported values (Babayemi and Bamikole 2006) when gas fermentation from mixture of Panicum maximum and Tephrosia candida were studied. Higher values of rate of fermentation were also obtained than reported values (Krishnamoorthy et al 1995) and (Babayemi and Bamikole 2006).  

Digestibility has been reported to be synonymous to in vitro gas production (Fievez et al 2005) so that the higher the gas production the higher the digestibility. Mixtures of Panicum maximum and histrix and Panicum maximum in histrix intercrop had low gas production volumes when compared to other legumes in this study which might be due to the high tannin content in the forages. Feed that is high in tannin content has low digestibility because it reduces the population of fibre degrading bacteria in the rumen and hence low activity (McSweeney et al 1999). The Panicum maximum in legume intercrop increased in gas production over the Panicum maximum without legume intercrop suggesting that there was a complementary improvement of the Panicum maximum intercrop with legume vis – avis nitrogen and mineral release into the soil for the grass uptake.  

The low methane production observed in this study for the grass and legume mixtures is in order with earlier work (Babayemi and Bamikole 2006). The authors observed lower methane production as the proportion of the browse legume (Tephrosia candida) was reduced and Panicum maximum was increased. Methane production is energy loss to ruminants and also contributes to global warming (Babayemi and Bamikole 2006). The Panicum maximum had the lowest methane production when compared to the mixtures and Panicum maximum in legume intercrop. The Panicum maximum in legume intercrop had higher methane production over the sole Panicum maximum suggesting that the legumes had increased methane production in it. It was also observed that the sole legumes which were high in crude protein produced higher methane (which symbolizes energy loss). This implies that browse or herbaceous legumes would need energy supplements like Guinea grass for adequate utilization by ruminants. 

The ME and OMD of the mixtures of Panicum maximum and legumes were better than sole Panicum maximum or sole legumes. This observation was in line with reported work (Babayemi and Bamikole 2006). The low ME and OMD observed in mixture of Panicum maximum and Histrix and sole Histrix was as a result of the high NDF obtained for Panicum maximum and Histrix. The ME and OMD mean values obtained were higher than range of 2.99 – 4.75 MJ/kg DM and 21.46 – 33.80 % respectively reported (Babayemi and Bamikole 2006). Panicum maximum in legume intercrop had higher ME and OMD over those not cultivated with legumes.  

Short chain fatty acid (SCFA) or volatile fatty acid (VFA) is a reflection of energy availability in a feedstuff. This is one of the end products of rumen fermentation. High volume of gas is produced when substrate is fermented to acetate and butyrate. Relatively lower gas production is associated with propionic production. In vitro gas production had low correlation with volatile fatty acid production particularly that of propionate (Nsamsaeng et al 2006). The mixtures of grass and legumes had higher SCFA and moderate gas production in this study compared to the Panicum maximum or sole legumes. This implies that more energy would be made available to animals when grass is supplemented with legumes in the diet of ruminants. Sole legumes in this study produced much higher gas production which according to Nsamsaeng et al (2006) would lead to the production of more of acetate and butyrate which are not energy giving VFAs.

Conclusions

• Efficient rumen fermentation can be achieved when herbaceous legumes are fed in combination with Panicum maximum.

• Intercropping legumes with Panicum maximum improves the grass proximate composition, organic matter digestibility and metabolizable energy over the grass without legume.

• The reduction in the methane produced during gas fermentation for the sole grass is an indication of its usage as energy feed.

• Legumes are high in methane and so require energy supplement in order to sustain livestock production.
   

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Received 23 November 2007; Accepted 20 March 2008; Published 10 June 2008

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