Nutritional evaluation of leaves of some salt-tolerant tree species by assessing, in vitro, the ruminal microbial nitrogen and fermentation characteristics

M R Al-Masri

Department of Agriculture, A.E.C., P.O. Box 6091, Damascus, Syria
ascientific@aec.org.sy

Abstract

Leaves of some salt-tolerant tree species (Tamarix articulata Vahl., Tamarix aphylla (L) Karst, Acacia ampliceps Maslin, Casuarina equisetifolia L, Parkinsonia aculeate L, Eucaliptus camaldulensis Dahnhard) were evaluated in terms of microbial nitrogen (MN) and biomass (MBM) production after incubation with rumen fluid and ¹⁵N-tracer for 96 h in the absence or presence of polyethylene glycol (PEG, 6000). The characteristics of fermentation (initial gas produced from soluble fraction; a, gas production during incubation produced from insoluble but fermentable fraction; b, potential gas production; a + b, fractional rate of gas production per hour; c, were assessed using an in vitro incubation technique with rumen fluid. Effective degradability (ED) and short chain fatty acids (SCFA) were also estimated for leaves of the experimental tree species.

The a + b values (mL/g DM) were highest (P<0.05) in A. ampliceps (191), lowest in T. articulate and C. equisetifolia (119), and intermediate in T. aphylla, E. camaldulensis and P. aculeate (158). E. camaldulensis, A. ampliceps and P. aculeate had higher (P<0.05) fractional rate of gas production (0.080/h) than other species (0.061/h). There was a positive correlation between SCFA concentrations and c and a + b values. The ratios of MN and MBM to effective degraded substrate and the values of MN and MBM were significantly higher (P<0.05) in P. aculeate and A. ampliceps compared with other species. Microbial nitrogen and MBM production were positively correlated with a + b, ED and SCFA. The addition of PEG to the plant samples incubated with rumen fluid at a ratio of 2:1 PEG: substrate increased the values of gas production, characteristics of fermentation, MN, MBM, SCFA and ED. The response of leaves of the experimental tree species to PEG treatment in terms of increased gas production varied between species and tended to decline as incubation progressed, with the highest increase during the first 30 h of incubation. Leaves of P. aculeate and A. ampliceps gave highest values of microbial nitrogen and biomass production and therefore may be found useful for supplementation, in vivo of low crude protein forage diets. Feeding of PEG with leaves of tree species containing high levels of tannins can be helpful by increasing the effective degradability of dry matter.

Key words: degradability, gas production, microbial mass, polyethylene glycol, waste

Introduction

Ruminants feeding is mainly dependent on pasture grasses and crop production. The availability of these feeds may be more restricted in arid and semiarid zones, so that a high levels of ruminant production is not always possible. Tree and shrub leaves have the potential for alleviating some of the feed shortages and nutritional deficiencies experienced during the dry season on smallholder farms. Forages from shrubs and trees play an important role in the nutrition of grazing animals in areas where few or no alternatives are available (Meuret et al 1990) and are used to improve livestock performance (Abdulrazak et al 1996; Ben Salem et al 2002). Feeding of tropical tree and shrub foliages could be an attractive strategy for reducing of ruminal methanogenesis for animals fed with low-quality forage diets and for improving their productivity (Delgado et al 2012). However, the use of tree and shrub leaves by herbivores is restricted by deterring mechanisms related to high tannin content (Rubanza et al 2003; Bakshi and Wadhwa 2004). The phenolic compounds (particularly tannins) in some trees and shrubs may bind to protein, thus rendering the protein undegradable by rumen microbes. Polyethylene glycols (PEG) is able to form complexes with tannins (Getachew et al 2000) and has been used to reduce tannin-protein complex formation or to release these complexes (Makkar et al 1995). The effect of PEG on the chemical composition and nutritive value of leaves of some tree species (Tamarix articulata Vahl., Tamarix aphylla (L) Karst, Acacia ampliceps Maslin, Casuarina equisetifolia L, Parkinsonia aculeate L, Eucaliptus camaldulensis Dahnhard) has been investigated (Al-Masri 2013). The addition of PEG increased organic matter digestibility and the response to PEG treatment increased with increased concentration of phenolic compounds in the plant samples.

Compared with other laboratory techniques, the gas-production technique has proved accurate in predicting animal performance and voluntary feed intake of roughages (Blümmel et al 2005) and was suggested as being more efficient than other in vitro techniques for determining the nutritive value of feeds containing anti-nutritive factors and for evaluating the microbial fermentation of ruminant feeds and its impact on fermentation products (Getachew et al 2005). In the gas production method, kinetics of fermentation can be studied by simply reading the increase in gas production at a series of chosen time intervals during incubation with rumen liquor and using the exponential equation P = a + b (1 – e^-ct) (Ørskov and McDonald 1979). Gas production is associated with volatile fatty acid production following fermentation of substrate (Blümmel and Ørskov 1993). In addition, the application of models permits the fermentation kinetics of the soluble and readily degradable fraction of the feeds, and more slowly degradable fraction to be described (Getachew et al 1998).

Objectives

The objectives of the present study were:

• To evaluate the leaves of some multipurpose tree species (T. articulata, T. aphylla, A. ampliceps, C. equisetifolia, P. aculeate, E. camaldulensis) in terms of microbial nitrogen and biomass production.
  
  
• To study the fermentation kinetics using an in vitro incubation technique with rumen fluid.
  
  
• To assess the impact of polyethylene glycol on the aforementioned parameters.
  
  
• To estimate the effective degradability and production of short chine fatty acids.
  
  
• To determine the relationship between gas production and synthesis of microbial biomass.

Materials and methods

Plant materials tested

Six tree species (T. articulata, T. aphylla, A. ampliceps, C. equisetifolia, P. aculeate, E. camaldulensis) grown on a salty soil (salt concentration = 12.39 g/L, pH = 7.65 at 25-100 cm depth), located about 20 km east of Deir Ezzor (39° 41.3 'E; 36.3° 34.1 'N) in north-eastern Syria were selected. Tree species were 7 years old and irrigated by saline water (8.32-9.60 g/L). The leaf samples of each species with 4 replicates (3 trees each) were randomly and manually collected at the vegetative stage from different locations of the tree, dried at room temperature (20-25°C) for one week, ground to pass a 1-mm sieve and stored frozen at -20°C in sealed nylon bags for later analysis and determination. The nutritive components (g/kg DM) in leaves of the tree species were: 96.3, 141, 145, 89.4, 176, 73.8 crude protein, 195, 94.8, 91.4, 84.9, 105, 112 lignin and 55.5, 30.2, 4.5, 6.9, 11.1, 106 tannins for T. articulata, T. aphylla, A. ampliceps, C. equisetifolia, P. aculeate, E. camaldulensis, respectively (Al-Masri 2013).

Study of fermentation kinetics

The experimental samples were incubated in 100-mL calibrated glass syringes at 39°C with the ruminal fluid mixed with the medium, based on a modified procedure of Menke et al (1979) to determine the rate of gas production during 96 h incubation. As a modification, the syringes were incubated standing upright in a water-bath instead of being stacked horizontally on a slowly turning rotor housed in an incubator (Blümmel and Ørskov 1993). The method of Menke et al (1979) was used to study the digestion kinetics of plant samples, utilizing the exponential equation P = a + b (1 – e^-ct) of Ørskov and McDonald (1979), and to evaluate the biological activity of tannins according to Makkar et al (1995) with or without adding polyethylene glycol to the rumen fluid mixture over 96 h of in vitro incubation.

The rumen fluid was collected before the morning feeding from 3 rumen-fistulated Awassi rams, which were principally fed on roughage diet and received 162 g crude protein and 12.8 MJ ME per day (Al-Masri 2013). Rumen fluid samples were taken once every 7 days, 16 h after the last meal. The rumen fluid was homogenised and strained through 100-µm nylon cloth into a warm flask (39°C) filled with CO₂. A total of 30 mL medium, consisting of 10 mL of rumen fluid and 20 mL of bicarbonate-mineral-distilled water mixture (1: 1: 2 by vol.), was pumped with an automatic pipette into the warmed syringes containing the samples (200 mg) and into the blank syringes. The syringes were shaken by hand for a couple of seconds, twice in the first hour and once again after 3, 5 and 8 h of incubation. Gas production with or without adding polyethylene glycol (PEG, 6000; Fluka Firm No. 81260) at a ratio of 2:1 PEG:substrate was recorded after 3, 5, 8, 10, 24, 30, 48, 72 and 96 h of incubation. Gas production from the experimental sample was calculated by subtracting the volume of gas produced from the blank with or without the addition of PEG.

Determination of microbial nitrogen and biomass

¹⁵N-labelled ammonium sulphate (>98% ¹⁵N) was added to 30 mL of the rumen fluid mixture and incubated for 96 h with the samples (200 mg), with or without added PEG, to estimate the microbial nitrogen (MN) and microbial biomass (MBM) production (Al-Masri 2010). Total nitrogen, as well as ¹⁵N atom excess in the N pool of the sample and fluid mixture incubated for 96 h or in the fluid mixture alone (blank) were measured with an emission spectrometer (JASCO N-150, Japan Spectroscopic Com. Ltd, Tokyo, Japan). The following equations were used to estimate MN and MBM production:

MN (mg/96 h/200 mg sample) = [1 – (%¹⁵N atom excess in the N-pool of the sample and fluid mixture incubated for 96 h / %¹⁵N atom excess in the fluid mixture)] * mg N in the sample incubated for 96 h.

MBM (mg/96 h/200 mg sample) = MN / 0.0864

Czerkawski (1986) indicated that the rumen microbes contain 8.64% nitrogen.

Calculations

The effective degradability (ED) of dry matter was calculated assuming that ruminal outflow rate (k) is 0.04/h for sheep (Umunna et al 1995) as: ED (%) = a + [(b * c) / (c + k)]. The volume of gas was based on that produced from 200 mg substrate.

Short chain fatty acids (SCFA) concentration was calculated according to Getachew et al (2002) as: SCFA (m mol/200 mg DM) = 0.0222 GP – 0.00425. Where GP is the net gas production (mL/200 mg DM) after 24 h of incubation.

Data and statistical analyses

Data on gas production were fitted to the exponential equation P = a + b (1 – e^-ct) of Ørskov and McDonald (1979), where P (mL) was defined as gas production at time t, a (mL) was the initial gas produced from soluble fraction, b (mL) was the gas production during incubation which is produced from insoluble but fermentable fraction, a + b (mL) was the potential gas production and c was the fractional rate of gas production per hour.

A factorial design was used in this experiment, with tow fixed factors: (1) plant species (six species); (2) polyethylene glycol treatment (PEG or no PEG). Results were subjected to analysis of variance (ANOVA) using a Statview-IV program (Abacus Concepts, Berkeley, CA, USA) to test the effect of plant species and PEG treatment. Means were separated using the Fisher’s least significant difference test at the 95% confidence level. Regression coefficients (R) between the studied parameters were calculated.

Results and Discussion

Gas production and fermentation kinetics

Changes in gas production from the leaves of the experimental tree species after incubation with or without PEG and their fermentation kinetics are presented in Table 1. The in vitro gas production after 30, 48, 72 and 96 h and the potential gas production (a + b) values were highest (P<0.05) in A. ampliceps, lowest in T. articulate and C. equisetifolia, and intermediate in T. aphylla, E. camaldulensis and P. aculeate. Leaves of E. camaldulensis, A. ampliceps and P. aculeate had the highest (P<0.05) rate of gas production during incubation (0.080/h) than other species (0.061/h). In a study with 7 Mediterranean browse species (Arbutus unedo, Calycotum villosa, Erica arborea, Phillyrea angustifolia, Pistacia lentiscus, Myrtus communis, Quercus suber), Gasmi-Boubaker et al (2005) reported that the Pistacia lentiscus was fermented most slowly with a fractional rate of gas production of 0.013/h and the most rapidly fermented browse was by Calycotum villosa (0.025/h) and the potential gas production ranged from 146 to 224 mL/g DM of all browses. The values of potential gas production (119-191 mL/g DM) or fractional rate of gas production (c) (0.056-0.088) of the experimental tree species are lower or higher to those (228 mL/g DM or 0.015/h, respectively), reported by Sallam et al (2008) for alfalfa hay. In a study with different roughages (oat straw, bean straw, maize stubble, agave bagass), Oritiz-Tovar et al (2007) reported that the c values and potential gas production ranged from 0.028 to 0.076/h and from 110 to 142 mL/g DM for all roughages, respectively. Kafilzadeh and Heidary (2013) indicated that in any evaluation of oat varieties, not only yield and digestibility but also kinetics of fermentation should be taken into consideration. The fractional rate of gas production of forages produced from 18 different varieties of oat (Avena sativa L.) ranged from 0.029 to 0.040/h.

The intake of a feed is mostly explained by the rate of gas production (c) which affects the passage rate of feed through the rumen, whereas the potential gas production (a + b), is associated with degradability of feed (Khazaal et al 1995). Therefore the higher values obtained for the potential gas production in A. ampliceps might indicate a better nutritive availability for rumen micro-organisms. These results are in agreement with Al-Masri (2013) who indicated that A. ampliceps gave significantly (P<0.05) higher values of digestible organic matter and metabolizable energy than other studied tree species.

Figure 1. Cumulative gas production (in vitro) over 96 h from the leaves of the experimental tree species

The amount of gas produced per unit fermented material reflects the level of fermentation of the forages. High CH₄ production from ruminants is undesirable from both economic and environmental aspects. The highest increase in gas production was obtained during the first 30 h of incubation for the experimental samples without addition of PEG (Fig. 1). Gas production parameters suggested differences in nutritional value that were generally closely related to chemical composition (Cerrillo and Juárez 2004; Kamalak et al 2005; Salem 2005; Al-Masri 2010). Indeed the higher lignin (195 g/kg DM) and neutral detergent fibre (NDF) (575 g/kg DM) levels in T. articulate are almost certainly responsible for its reduced gas production versus the other tree species (85-112 and 322-491 g/kg DM, respectively) (Al-Masri 2013). Gas production reflects the degradation of dietary organic matter (OM) and more gas production, more degradation of OM (Groot et al 1996). Differences in potential gas production (a + b; potential degradability) among tree species could also be due to the extent of lignification of NDF. Lower NDF concentrations mean greater amounts of soluble cell walls which are available for fermentation. There is a negative relationship between gas production and cell wall content of diet (Getachew et al 2004). In our results, the a + b values were negatively correlated with lignin (R = -0.48; P = 0.019) and tannins contents (R = -0.13; P = 0.545) and positively correlated with SCFA concentrations (R = 0.97; P = 0.0001). However, there was a positive correlation between c and SCFA values (R = 0.61; P = 0.0016). Al-Masri (2013) indicated that the values of digestible organic matter for the same tree experimental species were negatively correlated with lignin (R = -0.77; P = 0.001) and tannins concentrations (R = -0.53; P<0.02) but positively correlated with crude protein (R = 0.68; P<0.002). A negative correlation between c values and lignin (R = -0.32; P<0.01) and a positive correlation between c values and crude protein concentrations (R = 0.74; P = 0.09) are observed by Al-Masri (2010) for Kochia indica harvested at different stages of maturity.

Figure 2. Increase (% ) in gas production from leaves of the experimental trees species over 96 h incubation, as a result of addition of polyethylene glycol in the fermentation process.

Secondary compounds such as tannins affect ruminal fermentation and forage degradability. Addition of PEG increased the values of gas production and characteristics of fermentation in all species (Table 1). Higher in vitro fermentation, as well as potential degradability of A. ampliceps could be due to its lower secondary compound levels (total tannins) (Al-Masri 2013). Some variations among leaves of the aforementioned experimental species could be due to genotypic characteristics relative to the type of secondary compound affecting digestibility (Muetzel and Becker 2006; Salem et al 2006). Our results indicated that the increase (%) in gas production from each experimental species was not stable over the incubation period (Fig. 2). The response of the leaves of the experimental tree species to PEG treatment in terms of increased gas production varied between species and tended to decline as incubation progressed, with the highest increase during the first 30 h of incubation. This was most obvious with T. aphylla where the increase reached over 80% at 5-10 h but decreased to 8% after 96 h incubation. To some extents similar behaviour was observed with C. equisetifolia and T. articulate. These findings indicate the microbes can adapt or overcome some of the anti-nutritive effects.

Microbial nitrogen and biomass

The levels of MN or MBM produced from g substrate depended on tree species and presence or absence of PEG in the fermentation process (Table 2). Leaves of P. aculeate and A. ampliceps gave highest values of MN and MBM compared with other species. MN and MBM values were negatively correlated with lignin (R = -0.37; P = 0.08) and positively correlated with crude protein concentrations (R = 0.85; P<0.001) for the studied species without addition of PEG. Similar correlations between MN and MBM values and crude fibre (R = -0.73; P<0.01) and crude protein contents (R = 0.84; P<0.01) of some range plants were obtained by Al-Masri (2007).

The net MN or biomass production would depend on the balance between decreased degradable dry matter and higher microbial mass production per unit dry matter digested. Al-Masri (2003) indicated that the amount of MN and MBM produced from 100 mg of truly fermented organic matter of some browses and range plants (Atriplex leucoclada, Prosopis stephaniana, Moringa oleifera, Jatropha curcas) fell in the range of 0.7-2.9 mg and 8-34 mg, respectively. Cone and Van Gelder (2000) established microbial efficiency at the time of estimated maximum microbial biomass and determined efficiency of microbial protein using microbial purines. They observed increased microbial efficiency for substrates with higher fermentation rates and obtained negative microbial efficiency values with some slowly degradable substrates. Substrates with high fermentation rates may have high yield of microbial protein by decreasing the proportion of energy used for maintenance purposes. In our results, the ratios of microbial N or biomass production to effective degraded substrate (EDS) were significantly higher (P<0.05) in P. aculeate and A. ampliceps as compared to other species (Table 2). The values of MN or MBM production were positively correlated with potential gas production and SCFA (R = 0.61; P = 0.0015) and effective degradability (R = 0.59; P = 0.0025).

TThe addition of PEG in the fermentation process increased the values of a + b and SCFA (Table 3). This allied with higher production of microbial nitrogen in the presence of PEG, suggests that the PEG might have bound with tannins, releasing proteins for microbial breakdown. Pritchard et al (1988) indicated that the low intake and feed value of mulga (Acacia aneura) leaf was related to its content of condensed tannins, which bound with proteins in the leaves. Our results indicated that there were negative correlation between MN or MBM values and total tannin concentrations (R = -0.64; P = 0.0007). The higher gas production, SCFA, MN and MBM production following inclusion of PEG in our study are in agreement with Al-Masri (2007).

The degradability of protein in tannin-containing feeds is depressed, resulting in a low NH₃-N concentration. Addition of PEG results in increased levels of NH₃--N. A rapid degradation of nitrogen not matched with energy availability could lead to accumulation of NH₃-N in the in vitro system or to a high absorption of NH₃-N from the rumen in vivo. Synchronization of the rate of degradation of nitrogen and carbohydrate components in the rumen is extremely important for efficient utilization of rumen NH₃-N for synthesis of microbial protein. Therefore, utilization of browses with high tannin levels could be improved by inclusion of tannin-binding agents such as PEG and an additional energy source to trap the nitrogen resulting from fermentation. Addition of PEG can be advantageous if the tannin content of the feed is sufficiently high to the extent that it depresses microbial activity and digestibility of feeds drastically. On the other hand, addition of PEG to low-tannin feeds may result in negative effects by reducing the amount of un-degraded protein and also by decreasing the efficiency of microbial protein synthesis (Getachew et al 2000).

Conclusions

Based on the results of this research it is concluded that:

• Leaves of P. aculeate and A. ampliceps had high values of microbial nitrogen and biomass production and are suitable as nitrogen supplements for low crude protein forage diets for livestock in arid and semiarid zones. This is of economical benefit leading to a reduction in the cost of rations and consequently in the cost of livestock production.
  
  
• The addition of polyethylene glycol to the tree forages containing high levels of tannins increased the values of rumen microbial nitrogen, fermentation characteristics and degradability of dry matter.

Acknowledgements

The author thanks the Director General and Head of Agriculture Department, A.E.C. of Syria, for their encouragement and financial support.

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Received 9 December 2014; Accepted 11 December 2014; Published 4 February 2015

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