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Comparative assessment on chemical compositions and feeding values of leaves of Moringa stenopetala and Moringa oleifera using in vitro gas production method
Department of Animal and Range Sciences, Hawassa University, P.O. Box 05, Hawassa, Ethiopia *Corresponding author: E-mail: email@example.com
(Received in revised form: December 18, 2011)
Evaluating the nutritional value of indigenous shrubs, trees and browse plants is important in Ethiopian situation where availability and quality of forages severely limited during long and dry seasons. A comparative assessment was thus conducted to investigate the nutrient compositions and feeding values of Moringa stenopetala and Moringa oleifera leaves. Feed samples were analyzed for proximate nutrients, minerals and amino acid profiles using official methods. In addition, the metabolizable energy (ME), organic matter digestibility (OMD) and short chain fatty acids (SCFA) were predicated using the Hohenheim in vitro gas test method. Moringa stenopetala had 26.6% crude protein (CP), 3.36% fat,
17.9 KJ/kg DM gross energy, 45% nitrogen free extract (NFE), and 38.4% non fiber carbohydrate (NFC). In Moringa oleifera, the contents of CP, fat, NFE, NFC and gross energy were 28.9%, 6.73%, 45%, 38.4% and 17.9 MJ/kg DM, respectively. Moringa stenopetala leaves contained significantly higher crude fiber, acid detergent fiber (ADF) and cellulose than those of Moringa oleifera. However, the acid detergent lignin (ADL) and hemicelluloses contents of Moringa oleifera were significantly higher than those of Moringa stenopetala. The contents of calcium, phosphorous, magnesium, potassium and sodium in Moringa stenopetala were 2.47%, 0.57%, 0.76%, 2.45% and 0.11%, respectively. The values of the corresponding minerals in Moringa oleifera were 2.62%, 0.43%, 0.56%, 2.0% and 0.03%. The concentrations of essential amino acids were significantly higher in Moringa oleifera than those of Moringa stenopetala and were generally comparable with the contents of soybean meal. The highest in vitro gas production of 47.9 ml was recorded for Moringa stenopetala, being significantly higher than for Moringa oleifera (40.6 ml). Similarly, values of ME
(9.83 MJ/kg DM), OMD (76.4%) and SCFA (101 mmol) in Moringa stenopetala were significantly higher than those of Moringa oleifera. Although not significant, organic matter, CP, fat, ADF and cellulose contents were positively correlated with in vitro gas production while DM, NFE, neutral detergent fiber, ADL and hemicelluloses contents were negatively correlated with gas production. The chemical compositions showed the potential of leaves of both Moringa species to be used as a protein supplement in ruminant and non-ruminant feeding during the dry season. Moreover, the enhanced values of ME, OMD and SCFA in leaf suggest its ability to meet the energy requirements of tropical livestock.
Keywords: Moringa leaves, chemical compositions, in vitro gas production
Inadequate nutrition is one of the factors that generally affect livestock productivity in the tropical countries. Total weight gained by ruminants during the rainy season is lost in the dry season due to feed scarcity. The use of tree leaves as fodder for ruminant livestock has been increasingly important in many parts of the tropics particularly during the dry period. Leaves from browse and fodder trees form major parts of livestock feed in the tropical countries (Woods et al., 1994) and play a major role in improving dietary protein (Kaitho et al., 1998). Tree leaves can be rich in crude protein (CP), minerals and digestible nutrients when compared to grasses. Moringa stenopetala
(Moringa. stenopetala) known as African Moringa has a wide range of adaptation from the arid to humid climates and can be grown in a various land use patterns. It grows in the lowlands of West of the Great Rift Valley Lakes from arid to semi-humid areas altitudinal ranging from 390 m to about 2200 m a.s.l. It is a strategic multi-purpose tree plant in being a unique food tree in drought prone areas and has recently been distributed to other regions of Ethiopia, beyond its place of origin. Leaves are used for human consumption and animal feed (Aberra et al., 2011). A study conducted by Aberra et al. (2009) indicated that the leaves of Moringa stenopetala are rich in crude protein (28.2%) and contain reasonable amounts of essential amino acids.
The Moringa oleifera (Moringa. oleifera) is native to the sub-Himalayan tracts of north-west India, Pakistan, Bangladesh and Afghanistan (Makkar and Becker, 1997). This multipurpose tree has been introduced to Ethiopia over the last few years and is grown on nursery sites parallel to Moringa stenopetala in southern parts of the country. Both Moringa stenopetala and Moringa oleifera trees are the most commonly cultivated Moringa species in the tropics and subtropics which have the potential as alternative animal feed resources during dry periods of the tropics. However, the suitability and digestibility of leaves of both Moringa species in feeding ruminants and non-ruminants under Ethiopian conditions is hardly documented. The objectives of this study were thus to investigate nutrient compositions and feeding values of leaves from Moringa stenopetala and
Moringa oleifera using in vitro gas production techniques.
MATERIALS AND METHODS
Leaf samples of M. stenopetala and Moringa oleifera were collected at Chano Mille nursery site of Southern Agricultural Research located at Arbaminch district of Gamogofa administrative zone, Ethiopia. The altitude is 1100 m a.s.l with anual rainfall of 750-900 mm. The samples were collected from both Moringa trees aged 6 years old in December 2009. Each sample was collected randomly from six different Moringa trees. After removing the petioles, leaf samples were dried at 65 oC for 48 h and ground to pass 1 mm sieve size. Ground feed samples were labeled and kept in air-tight plastic containers until analysis.
Analyses of proximate nutrients and fiber fractions were performed as outlined by Verband Deutscher Landwirtschaftlicher Untersuchungsund Forschungsanstalten (VDLUFA, 2006). The samples were analyzed for DM (method 3.1), ash (method 8.1), crude protein (CP, method 4.1.1, N multiplied by 6.25), petroleum ether extract (EE, method 5.1.1), and crude fiber (CF, method 6.1.1, all VDLUFA, 2006). Neutral detergent fiber (NDF) assayed with a heat stable amylase and acid detergent fiber (ADF) were analyzed according to VDLUFA (2006; methods 6.5.1 and 6.5.2) and were expressed inclusive of residual ash. Cellulose and hemicellulose were computed as ADF minus acid detergent lignin (ADL) and NDF minus ADF, respectively. Non-fiber carbohydrate (NFC) content was calculated as 100(NDF + CP + crude fat + ash) according to NRC (2001). Nitrogen free extract (NFE) was computed by difference of organic matter and the sum of CF, EE and CP. Amino acid contents were measured by ion-change chromatography using an amino acid analyzer L8900 (VWR/Hitachi). After a performic acid oxidation step protein was hydrolyzed with 6 M HCl for 24 h at 113 °C. The amino acids were separated on a cation exchange resin and detected after post column derivatization with ninhydrin reagent using VIS-detection at 570 nm (440 nm for proline). For mineral analysis, samples were incinerated at 550 ◦C, and the remaining ash was treated with 6 mol per L HCl. Minerals were determined from filtered ash solutions using an Inductively Coupled Plasma spectrometer (ICP-OES) (Rodehutscord and Dieckmann, 2005). All chemical analyses were conducted in duplicate on each individual sample.
In vitro studies
Gas production was determined according to the procedure of VDLUFA official method (VDLUFA 2006, method No. 25.1), Menke, and Steingass (1988). About 200 mg of feed sample was weighed in two replicates and transferred into 100 ml calibrated glass syringes, fitted with Vaseline lubricated pistons. To prepare the inoculum, rumen fluid was collected before the morning feeding from two rumencannulated, non-pregnant, non-lactating Holstein Friesian cows, fed on medium quality diet and a concentrate. Details about feeding are described by Steingass and Menke (1986). The rumen fluid was placed directly into pre-warmed thermo flasks and taken immediately to the laboratory. It was then filtered through two layers of cheesecloth and diluted with buffered mineral solution, which was maintained in a water bath at 39 °C under continuous flushing with CO2. A total of 30 ml incubation medium consisting of 10 ml rumen fluid, 5 ml of bicarbonate buffer, 5 ml of macro-mineral solution and 10 ml of distilled water was transferred into a pre-warmed glass syringes containing the samples (200 mg) and blank syringes.
After filling the glass syringes with incubation medium, they were immediately placed in a temperature-controlled incubator preset at 39◦C. Incubation was completed in duplicate within each run and runs were replicated yielding four observations per sample. Three blanks containing 30 ml of medium as well as triplicate samples of reference hay and concentrate feed of known gas production were included. The gas volume was recorded at 2, 4, 6, 8, 10, 12, 14, 18, 24, 30, 36 and 48 hours of incubation according to the time described by Blümmel and Becker (1997). The volume of gas produced (means of two runs) was plotted against the incubation time and the fermentation kinetics and estimated parameters were expressed according to the equation of Beuvink and Kogut (1993) described in detail by Boguhn et al. (2008). The gas produced by test substrates was corrected for that by the blank syringes (containing no substrate), and 24 h gas production was corrected by the standards for the estimation of OMD, ME and SCFA. The Metabolizable energy (ME, MJ/kg DM) and organic matter digestibility (OMD, %) were computed as established by Menke et al. (1979) and Menke and Steingass (1988) and short chain fatty acids (SCFA,
mmol) were calculated as reported by Blümmel et al. (1999) with the following equations: ME (MJ/kg DM) = 2.20 + (0.136*Gv) + (0.0057*CP) + (0.00029*EE); OMD (%) = 14.88 + (0.889*Gv) + (0.45*CP) + (0.651*XA); SCFA=0.0239*Gv-0.0601; Where: Gv, CP, EE and XA are corrected 24 h gas volume (ml/200 mg DM), crude protein, ether extract and ash (g/kg DM) of the incubated samples, respectively.
Results on chemical compositions and calculated gas production parameters were subjected to one-way ANOVA analysis by using SAS GLM procedures (SAS, 2004) and differences of means were separated by Duncan multiple range test. Pearson correlation procedures were also performed with SAS software. Time series measurements of gas volumes from 2-48 hrs of in vitro incubation were used for the curve fitting to mathematically express gas production over incubation time by using the software GraphPad Prism 4.02 for Windows (GraphPad Software Inc. 2004, La Jolla, CA, USA).
RESULTS AND DISCUSSION
Chemical and mineral compositions
The proximate nutrients and fiber fractions of Moringa leaves are presented in Table 1. The contents of crude protein (CP) and fat in Moringa oleifera was significantly (p<0.05) higher than those of Moringa stenopetala. The average CP content in Moringa oleifera was comparable with that of Oduro et al. (2008) for the same Moringa specie. Sanchez et al. (2006) reported CP contents of 22.8% and 23.3 % for Moringa oleifera leaves, respectively, which are lower than those of the current study. Soliva et al. (2005) reported 32.1% of CP for Moringa oleifera leaves whereas Aberra et al. (2011) found 30.6% CP for Moringa stenopetala leaves, which are higher than those of both Moringa species in the current study. The variations in CP contents of the reported values may be due to differences in agro-climatic conditions or to different ages of trees, and possibly due to different stages of maturity. Studies conducted by Yang et al. (2006) indicated that mature leaves contained more CP than young shoots. In general, the CP content of Moringa leaves is higher than those of most tropical forage legumes reported by Babayemi (2007).
The fat and ash contents reported by Gupta et al.. (1989) and Makkar and Becker (1996, 1997) for Moringa oleifera leaves are in good agreement with the current results. However, the fat contents in Moringa oleifera leaves reported by Oduro et al. (2008) are lower than those obtained from the current study. The gross energy values (17.8 MJ/kg DM) reported for Moringa oleifera leaves by Aregheore (2002) agrees with those of
Moringa stenopetala but lower than those of Moringa oleifera. The gross energy contents of
18.7 and 19.4 MJ/kg DM reported by Makkar and Becker (1996, 1997) for Moringa oleifera leaves are slightly higher than those of Moringa stenopetala. The Moringa oleifera leaves contained high acid detergent lignin (ADL) and hemicelluloses contents compared with Moringa stenopetala. However, the contents of fiber fractions (CF, NDF and ADF) and cellulose for Moringa stenopetala were significantly (p<0.05) higher than those of Moringa oleifera. The NDF values in Moringa oleifera are consistent with those of Makkar and Becker (1997), Aregheore (2002) and Sanchez et al. (2006). The ADF contents of Moringa oleifera leaves reported by Makkar and Becker (1996) are in agreement with the present findings. However, ADF and NDF contents in Moringa oleifera leaves in the current study are higher than those of Gupta et al. (1989).
Table 1. Comparative chemical compositions of leaves of Moringa stenopetala and Moringa oleifera (in % on DM basis) Nutrients Moringa stenopetala Moringa oleifera Overall Pooled P
a,bMeans between Moringa species having different letters are significantly (p<0.05) different *computed from 0.0239*CP (g) + 0.0398 * EE (g) + 0.0201*CF (g) + 0.0175*NFE (g)
GE= gross energy; ns= not significant
As presented in Table 2, leaves of Moringa stenopetala contained significantly (p<0.05) higher phosphorous (P), magnesium (Mg), potassium (K) and sodium (Na) than those of Moringa oleifera. However, the contents of calcium (Ca) and ratio of Ca to P were significantly (p<0.05) higher in Moringa stenopetala leaves than those found in Moringa oleifera. The contents of P, K, Mg and Na in Moringa stenopetala are generally higher than those reported by Aberra et al. (2009) for the same species cultivated in the mid-altitude of Hawassa district. However, the Ca content reported by the same authors for Moringa stenopetala leaves is comparable with the present findings. In
Moringa oleifera leaves, Oduro et al. (2008) reported 2.01% of Ca, which is slightly lower than obtained from the present study. The contents of P and especially of Ca and Mg in Moringa leaves are higher than those of native pasture reported by Gizachew et al. (2002). Accordingly, the nutritive value of both Moringa leaves in terms of macro minerals can play a considerable role for supplementing ruminant and nonruminant feeds in the tropics.
Table 2. Comparative compositions of common major minerals in leaves of
Moringa stenopetala and Moringa oleifera (in % on DM basis)
Nutrients Moringa M. oleifera Overall Pooled P stenopetala mean S.E.M
a,bMeans between Moringa species having different letters are significantly (p<0.05) different
Concentrations of amino acids
The concentration of essential amino acids in leaves of Moringa stenopetala and Moringa oleifera has been presented in Table 3. Except for cystein, the concentration of all essential amino acids in Moringa oleifera was significantly (p<0.05) higher than those of Moringa stenopetala. Most of the essential amino acids in Moringa oleifera are slightly higher than those reported by Makkar and Becker (1996) for leaves of the same Moringa specie. The concentrations of leucine, lysine and threonine, reported by Booth and Wickens (1988) for Moringa oleifera leaves are comparable with the current findings. However, the contents of arginine, isoleucine, methionine, phenylanaline and valine reported by the same author were relatively lower than those of the present study. These variations might be possibly attributed to age of the tree, agro-climatic conditions (including altitude) and soil type.
Table 3. Concentrations of essential amino acids in leaves of Moringa stenopetala and
Moringa oleifera (g/kg DM)
Amino acids Moringa stenopetala Moringa oleifera Overall Pooled P mean S.E.M
a,bMeans between Moringa species having different letters are significantly (p<0.05) different
Recent studies conducted by Aberra et al. (2011) indicated that Moringa leaf meal was effectively used to replace soybean meal in the growth performance of growing Rhode Island Red chickens. Moreover, Moringa leaf extracts exhibited anti-microbial activity including inhibition of the growth of Staphylococcus aureus strains isolated from food and animal intestines. Moringa added to fodder can be thus used as a potential bioceutical agent to substitute for antibiotics in livestock production (Yang et al., 2006).
In vitro gas production and calculated parameters
The pattern of in vitro gas production in leaves of Moringa stenopetala and Moringa oleifera is presented in Figure 1. Gas production is basically the result of fermentation of carbohydrates to acetate, propionate and butyrate. Gas production parameters suggest differences in nutritional values that were generally closely related to chemical composition (Cerrillo and Juarez, 2004). Leaves of both Moringa species showed rapid gas production in the early stage of in vitro fermentation which indicates a higher content of rapidly fermentable soluble components.
However, the in vitro gas volume from Moringa oleifera significantly decreased thereafter indicating it may have had more slowly fermentable carbohydrate contents than Moringa stenopetala in the later incubation periods. The high in vitro gas production observed in Moringa stenopetala
The Moringa oleifera leaves had the highest CP content and yet they produced lower gas volume than those of Moringa stenopetala
feedstuffs high in CP produce less gas during fermentation, even if their extent of degradation is high. This is because protein fermentation produces ammonia, which influences the carbonate buffer equilibrium by neutralizing H+ ions from volatile fatty acids without release of carbon dioxide (Cone and Van Gelder 1999). According to Makkar and Becker (1996) about 24% of the CP was soluble in phosphate buffer (pH 7, 0.05 M) for Moringa oleifera leaves. Apart from this, the low gas production from Moringa oleifera leaves could be attributed to the low content of NFC compared with those of Moringa stenopetala. Getachew et al. (2004) reported that NFC was positively correlated with gas production at 6 h, 24 h and 48 h of incubation (De Boever et al. 2005). Furthermore, the lower gas volume of
Moringa oleifera leaves might be further explained by the presence of high fat (6.73% vs. 3.36%), which contribute to negligible gas production as reported by Aberra et al. (2009).
As presented in Table 4, Moringa stenopetala leaves contained significantly (p<0.05) higher calculated ME, OMD and SCFA values compared with those of Moringa oleifera. Similarly, values of estimated parameters for Moringa stenopetala leaves were significantly higher than those of Moringa oleifera. The ME and OMD predicted from the extent of fermentation in the in vitro incubation reported by Makkar and Becker (1996) for Moringa oleifera leaves was 9.5 MJ/kg DM and 74.1%, respectively, which agrees with those of the current findings for Moringa oleifera (9.3 MJ/kg DM and 72.0%). In vitro calculated ME and OMD values reported for Moringa stenopetala leaves by Aberra et al. (2009) are consistent with the present findings for Moringa stenopetala. Moreover, consistent with the present findings of both Moringa leaves, Kiran and Krishnamoorthy (2007) reported an average ME value of 10.2 MJ/kg DM for common protein supplements. In agreement with the current findings, Anele et al. (2009) reported ME values of
9.56 - 10.6 MJ/kg DM for leaves of tropical multi-purpose trees. The calculated SCFA for both Moringa predominance of SCFA in leaves could leaves was much higher than reported probably describe an increased for different forage species by Babayemi proportion of acetate and butyrate but a (2007). Higher production of gas and decrease in propionate production.
Table 4. Calculated metabolizable energy, organic matter digestibility, short chain fatty acids and estimated parameters in leaves of Moringa stenopetala and Moringa oleifera
a,bMeans between Moringa species having different letters are significantly (p<0.05) different ME= metabolizable energy; OMD= organic matter digestibility; SCFA= short chain fatty acids; S.E.M= standard error of the mean 1) Parameters were calculated from 24 hours gas production data. 2) Estimated parameters obtained by fitting gas production data to the equation of Beuvink and Kogut (1993) [where b is the maximum value of gas production; µr (in ml/h) is the rapid gas production rate during early stages of fermentation; µs (in ml/h) is the slower gas production rate during later stages of fermentation].
Correlations of chemical compositions with in vitro gas productions
Results of correlation analysis of chemical compositions with in vitro gas production and estimated parameters are presented in Table 5. Gasmi-Boubaker et al. (2005) reported a positive correlation of CP with gas production at 24 h for tropical browse species which agrees with the findings of the current study. It should be noted that although not significant, CP content showed a trend of positive correlation with gas production at 24 h.
Contrary to the present findings, Getachew et al. (2004) observed a negative correlation between the volume of gas produced and the CP content of the feed. In general, although not significant, the ADF and ADL contents were negatively correlated with gas production, which are consistent with those of Gasmi-Boubaker et al. (2005). De Boever et al. (2005) and Parissi et al. (2005) reported a negative association of gas production with NDF, ADF and ADL contents. These feed constituents are known to be less degradable than soluble carbohydrates and therefore reduce gas production.
Table 5. Correlation coefficients (r) of relationship of chemical composition and in vitro gas production and estimated parameters
Chemical compositions Gv at various incubation times1) Estimated parameters2)
*P<0.05; **P<0.01; ***P<0.001; ns= not significant 1) Gv8= gas produced at 8 h; GP24= gas produced at 24h; GP48= gas produced at 48 h. 2) b (ml) is the maximum value of gas production; µr (mL/h) is the rapid gas production rate during early stages of fermentation; µs (mL/h) is the slower gas production rate during later
stages of fermentation.
A highly significant (p<0.01) positive association was observed between CF and parameter b. However, a significant (p<0.05) negative relationship was found between NDF and estimated parameters µr (rapid gas production rate during early stages of fermentation) and µs (slower gas production rate during later stages of fermentation). In agreement with the findings of Garcia et al. (2005), there were no correlations observed between the maximum values of gas production (parameter b) with some chemical compositions.
Moreover, the positive association values of parameter b with NDF and parameter µr (the rapid gas production rate during early stages of fermentation) with NDF and ADL contents reported by Parissi et al. (2005) are consistent with the current findings. However, the presence of a high negative correlation between the slower gas production rates during later stages of fermentation (parameter µs) with NDF content disagrees with the findings of Getachew et al. (2004). In conclusion, leaves of both Moringa species with high crude protein and essential amino acid with low structured carbohydrate contents had a higher nutritive value in terms of
energy content and in vitro organic matter digestibility. Thus, leaves of both Moringa species have the potential to contribute substantially as feed supplements to ruminants and non ruminants in animal production systems of Ethiopia and other sub-Saharan African countries.
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