Seasonal in vitro gas production parameters of three multi - purpose tree species in Abeokuta, Nigeria

O M Arigbede, U Y Anele, J A Olanite, I O Adekunle^*, O A Jolaosho and O S Onifade

Department of Pasture and Range Management, College of Animal Science and Livestock Production,
University of Agriculture, PMB 2240, Abeokuta, Nigeria
^*Department of Soil Science and Land Management, University of Agriculture, PMB 2240, Abeokuta
arigbede2002@yahoo.com

Abstract

Leaves from three indigenous multipurpose tree species (MPTS) namely Moringa oleifera, Millettia griffoniana and Pterocarpus santalinoides were randomly collected in July, October and December 2004, to represent mid wet, late wet and early dry season respectively. The leaves were analyzed to estimate the nutrient composition and in vitro gas production over 3, 6, 9, 12, 15, 18, 21, 24, 48, 72 and 96h post incubation. The gas volumes recorded at these incubation times were fitted to the model P = b (1 - e ^-ct). Influences of chemical constituents on gas production were investigated.

Cumulative gas released at 96h was 35.8ml/200mg DM for Millettia griffoniana whereas Moringa oleifera and Pterocarpus santalinoides recorded 32.2 and 28.7ml/200mg DM respectively. These values were not significant (P>0.05). The highest volume originating from the (b) fraction was recorded for Millettia griffoniana (44.9ml/200mg DM) during the late wet season while the lowest value for this fraction was recorded for Pterocarpus santalinoides (22.5ml/200mg DM) (P>0.05) during the same season. The highest fractional rate of gas production (c) was recorded for Pterocarpus santalinoides (0.058ml/h) whereas Pterocarpus santalinoides and Millettia griffoniana both recorded the lowest fractional rate of production of 0.024ml/h during the mid wet and late wet season respectively. The correlation coefficients between in vitro gas production and the chemical constituents of the forages were generally negative. Significant correlations were observed in the ADF and ash contents of Moringa oleifera and Pterocarpus santalinoides.

The in vitro gas production and the fermentation parameters indicated the presence of potentially degradable nutrients in the MPTS evaluated. This underscores their importance as sources of nutrients for ruminant animals especially during the dry season. It is concluded that the in vitro gas production technique is a potential way of evaluating the nutritional quality of forages consumed by ruminant animals and also invaluable in predicting nutritive value of some of our under - utilized indigenous browse plants.

Key words: Chemical composition, fermentation parameters, In vitro gas production, multipurpose tree species

Introduction

The relevance of evaluating the nutritional value of our indigenous shrubs, trees and browse plants is evident (Cerillo and Juarez 2004; Nherera et al 1999; Topps 1992) as their foliage can make important contributions to the protein and energy consumption of ruminant animals. This is particularly important in the humid zones where forage availability and quality may be severely limited during the dry season (Delgado et al 1999).

The nutritive value of a ruminant feed is determined by the concentration of its chemical components, as well as the rate and extent of digestion. Determining the digestibility of feeds in vivo is laborious, expensive, requiring large quantities of feed, and it is largely unsuitable for single feedstuff thereby making it unsuitable for routine feed evaluation (Getachew et al 2004).

There are a number of in vitro techniques available to evaluate the nutritive value of feeds at relatively low cost. The use of in vitro gas method to estimate the digestion of feed is based on measured relationships between the in vivo digestibility of feeds and in vitro gas production, in combination with the feed's chemical composition (Menke and Steingass 1988).

The in vitro gas production technique developed by Menke et al (1979) is a very useful tool for the rapid screening of feeds to assess their potential as energy sources for ruminant animals (Blummel and Becker 1997), assuming that the volume of gas produced reflect the end result of the fermentation of the substrate to short chain fatty acids (SCFA), microbial biomass and the neutralization of the SCFA. This technique has been used by Blummel and Orskov (1993) to determine gas production at several incubation times, and values obtained could describe the pattern of fermentation of feed by using the model of Larbi et al (1996). In addition, the application of models permits the fermentation kinetics of the soluble and readily degradable fraction of the feed and the more slowly degradable fraction to be described (Getachew et al 1998).

The objective of this study was to estimate the in vitro gas production of three indigenous MPTS namely Moringa oleifera, Millettia griffoniana and Pterocarpus santalinoides with a view of ascertaining their nutritive value.

Materials and methods

The study was conducted at the University of Agriculture, Abeokuta (UNAAB), Ogun State, Nigeria. The site lies within the derived savannah zone of southwestern Nigeria on latitude 7^oN and longitude 3.5^oE. Mean monthly temperatures of the area ranges from 22.5^o - 30.7^oC receiving a mean annual rainfall of about 1037mm. Relative humidity is high during the rainy season with values between 63 and 96% compared to the dry season when lower relative humidity of between 55 and 84% is recorded. The temperature of the soil ranges from 24.5^o - 31.0^oC.

Samples of the MPTS leaves were randomly collected in July, October and December to represent the seasonal variations as follows: mid wet, late wet and late dry. Fresh leaves from each MPTS were weighed and put into separate bags that were well labelled. The samples were then oven - dried at 65^oC to constant weight and the residual weight recorded as the dry matter (DM) content of the leaves. The dried leaf samples were ground with hammer mill to pass through sieve size of 1.0mm and preserved for the determination of the proximate composition (AOAC 1995). The neutral detergent fibre (NDF) and the acid detergent fibre (ADF) contents of the samples were determined using the procedure of Goering and Van Soest (1970).

In vitro gas production

The procedure followed was as described by Menke and Steingass (1988). A sensitive scale was used to measure out 200mg of the milled leaf samples; these were replicated three times and placed into 100ml graduated glass syringe. A total of twenty eight syringes (including a blank syringe, the control) were used for the in vitro gas determination. The rumen fluid (inoculum) was strained through gauze and mixed with sodium and ammonium bicarbonate buffer (35g of NaHCO₃ plus 4g of NH₄HCO₃ per litre) at a ratio of 1:2 (v/v) to avoid lowering the pH of the rumen fluid which will result in decrease in the microbial activities. Thirty millilitres of the buffered inoculum was then added to each syringe and the gas released was read off directly on the graduated syringe. The syringes were positioned vertically in a water bath kept at 39^oC. A blank syringe containing 30ml of the buffered inoculum only was also included as control. All the syringes were gently shaken 30 minutes after commencement of incubation and four times daily at regular intervals thereafter. Gas production was recorded at 0, 3, 6, 9, 12, 15, 18, 21, 24, 48, 72 and 96 hours of incubation. The data obtained were fitted to the non-linear equation:

V (ml/200mg DM) = b (1-e^-ct)

where:

V = potential gas production at time t,
b = the volume of gas that will evolve with time, and
c = the fractional rate of gas production.
Initial gas production rate (Abs_g) was calculated as the product of b and c (Larbi et al 1996).

The data collected were subjected to analysis of variance (ANOVA) (SPSS 1993). Duncan's means separation procedure was used to test the level of significance among means (Duncan 1955). The relationships between the various nutrient components were determined using correlation analysis (SPSS 1993).

Results

The proximate composition is indicated in Table 1

There were steady increases in the volume of gas produced by the three MPTS as incubation period extended from 3 - 96 hrs. Figure 1 shows the total gas production from the MPTS after 96 hours of in-vitro incubation. Millettia griffoniana , Moringa oleifera and Pterocarpus santalinoides produced 35.8, 32.2 and 28.7ml/200mg DM of gas, respectively. The differences in the volumes of the gas produced were however, not significant.

Seasonal in-vitro gas production values showed that Pterocarpus santalinoides recorded the highest volume of 36.0ml/200gm DM while Moringa oleifera and Millettia griffoniana recorded 32.3 and 27.7m/200mg DM, respectively in the mid wet season. Moringa oleifera produced the highest gas volume of 39.3ml/200mg DM in the late wet season while Millettia griffoniana and Pterocarpus santalinoides respectively recorded 38.8 and 22.7ml/200g DM. The highest volume of gas in the early dry season was recorded by Millettia griffoniana with a value of 41.0ml/200gm DM, Moringa oleifera and Pterocarpus santalinoides produced 25.0 and 27.3ml/200mg DM, respectively. Figures 2 - 4 showed the seasonal in vitro gas production by the MPTS at 12, 48 and 96 hours, respectively. There were no significant differences (P > 0.05) in the volume of gas produced within the seasons by the multipurpose trees.

Fermentation parameters from the three MPTS derived by fitting the in vitro gas values into the equation P = b (1 - e -^ct) are presented in Table 2.

The gas volume obtained from the fermentation of the insoluble but degradable fraction (b) produced more gas (P>0.05) in Millettia griffoniana than Moringa oleifera and Pterocarpus santalinoides with values of 39.5, 31.6 and 29.8ml/ 200mg DM, respectively (Table 2). The fractional rate of gas production (c) of the three trees species showed that Moringa oleifera had the highest rate of gas production with a value of 0.046; Pterocarpus santalinoides had production rate of 0.038 while Millettia griffoniana had the lowest rate of production with a value of 0.028, although the differences were not significant (P>0.05) (Table 2). The seasonal fractional rate of gas production of the MPTS increased from mid wet to early dry season except for an insignificant decrease of 0.040 observed for M .oleifera in the early dry and 0.024 for Millettia griffoniana in late wet season. The highest value of seasonal fractional rate of gas production of 0.058 was observed in Pterocarpus santalinoides during the late wet season while the lowest value of 0.024 was recorded for both Millettia griffoniana (late dry season) and Pterocarpus santalinoides (mid wet season) as shown in Table 3.

The result of the correlation analysis between cumulative in-vitro gas production and some chemical components of Moringa oleifera leaves (Table 4) shows that there were positive correlations between in-vitro gas production and crude protein, and ash contents of Moringa oleifera.

The DM, NDF, ADF and EE all had negative relationships with in vitro gas production. The negative relationship between ADF and in vitro gas production is significantly different (P< 0.05). The DM, CP and NDF of Millettia griffoniana had negative relationships with the in vitro gas production while the ADF, ash and EE had positive relationships with the in vitro gas production (Table 2). These correlation values were significantly different from each other. Unlike the DM and NDF of both Moringa oleifera and Millettia griffoniana , the DM, ADF and NDF of Pterocarpus santalinoides had positive correlation with in vitro gas production with the ADF having a significant (P< 0.05) relationship. The CP, ASH and EE of Pterocarpus santalinoides leaves had positive relationships with the in vitro gas production but the ash of Pterocarpus santalinoides had a significant (P< 0.05) relationship with gas production (Table 2).

Discussion

Results obtained for the gas production of the MPTS in the study were similar to those reported by Brenda et al, (1997); Ly et al (1997) and Cerrillo and Juarez (2004) in the leaves of some tropical trees and shrubs. Makkar and Becker (1996) reported a value of 49.5ml/200mg DM for Moringa oleifera leaves. Brenda et al (1997) reported a similar value of 30.5ml/200mg DM for G. sepium and a lower value of 18.4ml/200mg DM for L. leucocephala. Higher values of 45.0 - 52.6ml/200mg DM had earlier being reported for tree species in the semi - arid region of North Mexico (Cerillo and Juarez 2004).

One major advantage of the gas measurement technique is that it focuses on the appearance of fermentation products (soluble but not fermentable products do not contribute to gas production). The other in vitro methods are based on gravimetric measurements which follow disappearance of the substrate (components which may or may not necessarily contribute to fermentation) (Getachew et al 1998). Gas production is a reflection of the generation of Short Chain Fatty Acids (SCFA) and microbial mass (Getachew et al 1998). The range of gas volume recorded for the MPTS indicate that they are capable of producing approximately 50.4mg of microbial mass, 93.6mg of SCFA and gases representing about 60% digestibility (Getachew et al 1998).

The values of 0.024 - 0.058 obtained for the fractional rate of gas production of the multipurpose tree species under investigation show that they are highly digestible as the rate at which a feed or its chemical constituents are digested in the rumen is as important as the extent of digestion. These values were similar to values of 0.026 - 0.059 reported for some MPTS (Makkar and Becker 1996). These values were also similar to 0.048 and 0.055 reported for Leucaena leucocephala and Gliricidia sepium respectively (Brenda et al 1997). Getachew et al (2004) however reported higher rates of 0.056 - 0.17 for corn grain and canola meal probably due to higher nutrient concentration in those feeds than the forage samples used in the present study. The rate at which different chemical constituents are fermented is a reflection of microbial growth and accessibility of the feed to microbial enzymes (Getachew et al 2004). Similarly, Khazaal et al (1996) suggested that the intake of a feed is mostly explained by the fractional rate of gas production (c) which affects the rate of passage of the feed through the rumen, whereas the potential gas production (a + b), is associated with the degradability of the feed. Thus, the higher values obtained for the (c) and (a + b) parameters in the MPTS, may indicate a better nutrient availability for rumen microorganisms in animals fed with these MPTS leaves (Getachew et al 2004).

The result of the correlation between extent of gas production at 96hrs and the various chemical constituents of the MPTS indicates that there were both positive and negative relationships between the volume of gas produced and their chemical constituents. This is consistent with the reports of Nsahlai et al (1994); Getachew et al (2003); Cerrillo and Juarez (2004) and Doane et al (1997). The positive correlation between DM and in vitro gas production of Pterocarpus santalinoides is consistent with Apori et al (1998) who reported a positive relationship between gas production and DM. This could be as a result of the feed constituents, such as NDF and ADF, which contribute more gas if they are degradable as shown by their positive effect on gas production. The positive correlation between CP and gas production of Moringa oleifera leaves is consistent with the reports of Nherera et al (1999) and Larbi et al (1998). The positive relationship (0.27) means that the CP content of the tree (Moringa oleifera) had a contributory effect on the gas production and short chain fatty acid (SCFA) production which implies that 7.29% of variation in the gas production can be explained by variation in the CP concentration of Moringa oleifera. This may be due to the significantly higher CP content of Moringa oleifera than the other trees. The EE of Moringa oleifera and Pterocarpus santalinoides were negatively correlated to the gas production of the species as a result of their higher EE content of 13.2 and 10.7% respectively, which was significantly higher than that of Millettia griffoniana (9.61%) which had a positive relationship with in vitro gas production. Getachew et al (2004) reported that EE contribution to gas production is negligible but the higher significant values of Moringa oleifera and Pterocarpus santalinoides EE might have resulted in the depressing effect on gas production. The lower EE value of Millettia griffoniana might have resulted in the positive relationship observed. In agreement with the negative correlation recorded between NDF and in vitro gas production of Moringa oleifera and Millettia griffoniana , Cerillo and Juarez (2004); Getachew et al(2004) and Nsahlai et al (1994) all reported negative relationship between NDF and gas production in various tropical tree species. The extent of the negative effect of the NDF on gas production was higher in Millettia griffoniana than in Moringa oleifera. This indicates that the effect of NDF on gas production becomes more or less important as the level of NDF declines. The negative correlation between the ADF of Moringa oleifera is similar to the report of Cerillo and Juarez (2004) which recorded a negative highly significant correlation between ADF and in vitro gas production.

Conclusion

• The in vitro gas production and the fermentation parameters indicated the presence of potential degradable nutrients in the MPTS which underscores the importance of these indigenous tree species as sources of nutrition for ruminant animals.

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Received 2 July 2006; Accepted 2 September 2006; Published 5 October 2006

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