In vitro rumen fermentation kinetics and nutritional evaluation of olive tree (Olea europaea L.) pruning residues as affected by cutting regimen

M R Al-Masri

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

Abstract

Nutritive values of the branches (stems with leaves) from Olea europaea trees cut at 25, 50, 75 or 100 cm distance from the tip (cutting length) were evaluated by determination of the 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 which 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), partitioning factor (PF) and short chain fatty aids (SCFA) were also estimated in the experimental olive pruning branches.

There was a negative effect of cutting length on all the studied nutritive parameters and fermentation characteristics. The values of MN, MBM, ED, PF, SCFA, a + b and c declined with the increase in cutting length. The olive branches cut at 25 or 50 cm distance from the tip produced higher (P<0.05) amounts of MN (0.53 mg/g DM) than those cut at 75 and 100 cm lengths (0.30 mg/g DM). The values of ED, SCFA and PF, and the ratios of MN or MBM to effective degraded substrate were significantly higher (P<0.05) in the cutting olive branches at 25 cm length compared to other cutting lengths. There was a positive correlation between MN or MBM production and SCFA, PF, ED and a + b values. The greatest proportion of gas production occurred during the first 48 h of incubation. Microbial N and MBM production were positively correlated with a + b, SCFA, PF and ED.

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 the olive pruning branches to PEG treatment in terms of increased gas production varied between cutting lengths and tended to decline as incubation progressed, with the highest increase (17%) during the first 24 h of incubation. On the basis of the studies on nutritive parameters, olive pruning branches cut at 25 cm distance from the tip in diameter <3 mm were nutritionally well-suited as dietary supplements for ruminants.

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

Introduction

Olea europaea L., an evergreen tree that belongs to the family Oleaceae, is widely spread in the Mediterranean regions. Both olive tree culture and olive oil industry generate substantial amounts of olive by-products, which essentially consist from pruning residues, leaves, stones, pulp, olive cake, and watery waste known as "olive molasses" (Molina-Alcaide and Yáňez-Ruiz 2008). The recycling of this wastes is of great attention as their storage causes serious problems from the environmental, social and economical point of view. An important alternative utilization olive tree pruning by-products and olive leaves would be as roughage feeds for ruminants (Fayed et al 2009; Paiva-Martins et al 2009). Such form of nutrition could improve the economy and the efficiency of animal production (Molina Alcaide and Nefzaoui 1996). Al-Masri (2001) studied the possibility of using olive cake together with animal waste to produce biogas. Olive cake represents a poor agricultural residue that cannot be used alone for ruminant nutrition because of the presence of high amounts of lignocellulosic materials which have low digestibility (Al-Masri and Günther 1999). Removal of the wood from olive cake to obtain olive cake pulp increased the organic matter digestibility, metabolizable energy and the volume of gas produced during fermentation (Al-Masri 2003). The inclusion of olive cake into multi-nutrient feed blocks has proved to be a promising way for their utilization (Ben Salem et al 2003; Molina-Alcaide et al 2005). Olive molasses has been used in diets for pregnant and lactating ewes (Aguilera et al 1992).

In Mediterranean countries, high amounts of olive branches are available following pruning in the late autumn or early winter seasons when natural roughages are not available to animals. Therefore, these by-products could be used as supplements. Olive trees are usually subjected to severe pruning every second year and light pruning in the alternate year at different cutting lengths (20-100 cm) according to the growth period of the tree (fruit or vegetative shoots) (Katana 1978; Albies et al 1985). The pruned branches require an adequate preservation. Air-drying for seven days in the shade at room temperature did not have detrimental effects on the nutritive value of olive leaves and could be a simple and low-cost procedure for their preservation (Martin-Garcia and Molina-Alcaide 2008).

The nutritive quality of the forage and its content of anti-nutritional components (tannins) are influenced by harvest management and cutting regimen (Al-Masri and Mardini 2008; Al-Masri 2010). The phenolic compounds (particularly tannins) in some shrubs and roughages may bind to protein, thus making the protein inaccessible to rumen microbes. Polyethylene glycol (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 different levels of PEG (2-200 g/100 g DM) on the chemical composition and nutritive value of olive leaves has been investigated (Martín García et al 2004). Cell wall constituents in olive leaves decreased and total extractable condensed tannins increased with increasing the amounts of PEG. The addition of PEG (2 g/100 g DM) increased organic matter digestibility of olive leaves. In contrast, Yáňez Ruiz et al (2004) found no effect of PEG (2.1 g/100 g DM) on the condensed tannins and rumen degradability of olive leaves. The effect of PEG on the nutritive value of olive trees pruning residues has been investigated (Al-Masri 2012). The addition of PEG to the samples of olive pruning branches incubated with rumen fluid at a ratio of 2:1 PEG: substrate increased the values of organic matter digestibility, matabolizable energy and net energy lactation.

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 samples of branches from olive trees, cut at 25, 50, 75 or 100 cm distance from the tip, in terms of microbial nitrogen and biomass production after incubation with rumen fluid and ¹⁵N-tracer.
  
  
• 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, partitioning factor and production of short chain fatty acids.
  
  
• To determine the relationship between synthesis of microbial biomass and characteristics of fermentation.

Materials and methods

Plant materials tested

Branches (stems with leaves) of 12-year-old olive (Olea europaea) trees, grown at Der Al-Hajar research station about 30 km south east of Damascus in the arid steppe region, which receives a total annual precipitation of 100-120 mm, were hand-cut using a cutting shear in the routine pruning season at 25, 50, 75 or 100 cm distance from the tip (cutting length) (Photo 1). The pruning branches were less than 3, 4, 5, and 7 mm in diameter, respectively. The branches for each length range were collected with 4 replicates (n = 4) (5 trees each), dried at room temperature (20-25 ^oC) for 6 days, ground to pass through a 1-mm sieve and stored frozen at -20 ^oC in sealed nylon bags for later analysis and determination. The nutritive components (g/kg DM) in olive pruning branches were: 71.9, 66.3, 57.8, 44.6 crude protein, 94.0, 96.9, 97.8, 100 lignin, 406, 434, 474, 496 neutral-detergent fiber, 17.0, 17.4, 17.1, 17.0 tannins and 2.45, 2.26, 2.06, 1.41 buffer soluble nitrogen (BS-N) for the cutting lengths 25, 50, 75 and 100 cm, respectively (Al-Masri 2012).

Photo 1.  Pruned branches of olive trees cut at 25, 50, 75 and 100 cm distance from the tip.

Study of fermentation kinetics

The experimental samples were incubated in 100-mL calibrated glass syringes at 39^oC 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. 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. Details of rumen fluid collection and methods of incubation have been described previously (Al-Masri 2015).

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 96h.

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.

The partitioning factor (PF) is calculated as the ratio of substrate effective degraded dry matter in vitro (mg) to the volume of gas (ml) produced by it after 96 h of incubation. PF is an indicator of forage quality (Blümmel et al 1997), and greater PF reveals the better quality of forage.

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) cutting length; (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 cutting length 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

The changes in cumulative gas production from the branches of olive tree, as a result of cutting length, after incubation with or without PEG for 96 h and their fermentation kinetics are presented in Table 1. Cutting length of the olive branches positively affected the in vitro gas production after 24, 48, 72 and 96 h and the potential gas production (a + b) levels. The values of gas production during incubation (b) and fractional rate of gas production (c) declined with the increase in cutting length (P<0.05). The intake of a feed is mostly explained by the fractional rate of gas production which affects the passage rate of feed through the rumen, whereas the potential gas production, is associated with degradability of feed (Khazaal et al 1995). Therefore the higher values obtained for the fractional rate of gas and potential gas production in the cutting branches at 25 cm length might indicate a better nutritive availability for rumen micro-organisms. These results are in agreement with Al-Masri (2012) who indicated that the olive branches cut at 25 cm distance from the tip gave significantly (P < 0.05) higher values of digestible organic matter, metabolizable energy and buffer soluble nitrogen compared with other cutting lengths (50, 75 or 100 cm), indicating a higher solubility of nitrogen at the former length (25 cm) which included a large amount leaves. The average proportions of leaf (dry matter basis) in the pruned branches cut at 25, 50, 75 and 100 cm of length amounted to 0.69, 0.61, 0.52 and 0.44, 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. 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.

Al-Masri (2015) reported that the values of a + b and fractional rate of gas production of 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) ranged from 24 to 38 mL/g DM and from 0.056 to 0.088/h, respectively. Al-Masri (2010) indicated that the branches of Kochia indica cut at 30 cm distance from the tip had lower c value (0.034/h) than those cut at 15 cm length (0.041/h). The values of potential gas production (36-47 mL/g DM) or fractional rate of gas production (0.042-0.057/h) of the experimental olive pruning branches are lower or higher to those (228 mL/g DM or 0.015/h, respectively), reported by Sallam et al (2008) for alfalfa hay.

The highest cumulative gas production was obtained during the first 48 h of incubation for the experimental pruning samples without addition of PEG (Fig. 1). Gas production reflects the degradation of dietary organic matter (OM) and more gas production, more degradation of OM (Groot et al 1996). The amount of gas produced per unit fermented material reflects the level of fermentation of the roughages. High CH4 production from ruminants is undesirable from both economic and environmental aspects. Olive branches cut at 25 cm distance from the tip produced higher (P<0.05) amounts of gas than those cut at 50, 75 and 100 cm lengths. This may be attributed to the high amounts of crude protein and low concentrations of lignocellolosic materials in the cutting branches at 25 cm length. Indeed the higher lignin (99 g/kg DM) and neutral detergent fibre (NDF) (485 g/kg DM) levels in the cutting branches at 75 and 100 cm lengths are almost certainly responsible for its reduced gas production versus the other cutting lengths (95 and 420 g/kg DM, respectively) (Al-Masri 2012).

Figure 1. Cumulativa gas production (in vitro) over 96 h from the olive tree branches cut 25,50,75 and 100 cm distance from the tip

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). Differences in potential gas production (a + b; potential degradability) among pruning samples 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, thea + b values were negatively correlated with lignin (R = -0.81; P<0.0001 and ) and NDF contents (R = -0.95; P<0.0001) and positively correlated with SCFA concentrations (R = 0.98; P<0.0001). The values of fractional rate of gas production (c) were negatively correlated with lignin (R = -0.43; P = 0.0033) and positively correlated with crude protein concentrations (R = 0.92; P<0.0001). However, there was a positive correlation between c and SCFA values (R = 0.88; P<0.0001). Al-Masri (2012) indicated that the values of digestible organic matter for the same experimental olive pruning branches were negatively correlated with lignin (R = -0.79; P<0.001) but positively correlated with crude protein (R = 0.94; P<0.001).

Figure 2. Increase (%) in gas production from the olive tree branches cut 25,50,75 and 100 cm distance from the tip over 96 h incubation, as a result of addition of polyethylene gycol.

Secondary compounds such as tannins affect ruminal fermentation and forage degradability (Al-Masri 2015). There was a positive significant effect of PEG on the values of gas production and fermentation characteristics of the experimental olive pruning branches (Table 1). Karabulut et al (2006) indicated that PEG supplementation increased the values of organic matter digestibility (OMD), metabolizable energy (ME) and gas production characteristics (a and a + b) expect of gas production rate ( c ) of leaves of Olea europaea. The improvement in gas production, OMD and ME with PEG emphasizes the negative effect of tannins on digestibility. Martín Farcía et al (2004) reported that treatment of olive by-products (olive cake and olive leaves) with PEG (2 g of PEG/100 g of by-product) increased total extractable condensed tannins and in vitro organic matter digestibility and improved nutrient availability either at the rumen and intestine level. Our results indicated that the increase (%) in gas production from the experimental olive branches as a result of PEG treatment was not stable over the incubation period (Fig. 2). The response of the olive pruning branches to PEG treatment in terms of increased gas production varied between cutting lengths and tended to decline as incubation progressed, with the highest increase (17%) during the first 24 h of incubation. This was most obvious with cutting length at 100 cm where the increase reached over 20% at 24 h but decreased to 11% after 96 h incubation. To some extents similar behaviour was observed with cutting branches at 75 cm length. These findings indicate the microbes can adapt or overcome some of the anti-nutritive effects.

Microbial nitrogen and biomass

The average amount of microbial nitrogen (MN) or microbial biomass (MBM) produced from g substrate varied with cutting length of the branches of olive trees (Table 2). The olive branches cut at 25 and 50 cm distance from the tip produced higher (P<0.05) amounts of MN (0.53 mg/g DM) than those cut at 75 and 100 cm lengths (0.30 mg/g DM). Corresponding values for MBM were 6.14 mg/g DM or 3.42 mg/g DM for cutting lengths of the branches performed at 25 and 50 cm or at 75 and 100 cm, respectively. The decline in the MN and MBN values with increase cutting length of the branches were to be expected since there were negative correlations between the MN and MBM values and NDF concentrations (R = -0.63; P<0.01) and positive correlations between MN and MBM values and crude protein contents (R = 0.67; P = 0.0045). Similar correlations between MN and MBM values and lignin (R = -0.89; P<0.001) and crude protein concentrations (R = 0.83; P<0.01) of the branches of kochia indica cut at different lengths (15 or 30 cm) (Al-Masri 2010).

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 (2010) reported that the average amount of MN produced from g substrate of the branches of Kochia indica amounted to 12 mg for the branches cut at 30 cm distance from the tip and 16 mg for cutting length of the branches performed at 15 cm. 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 (Cone and Van Gelder 2000). In our results, the values of effective degradability (ED), partitioning factor (PF), short chain fatty acids (SCFA) and the ratios of microbial N or biomass production to effective degraded substrate (EDS) were significantly higher (P<0.05) in the cutting olive branches at 25 cm length compared to other cutting lengths (Table 2). The values of MN or MBM production were positively correlated with potential gas production (R = 0.58; P = 0.0184), SCFA (R = 0.66; P = 0.0053) and PF and effective degradability ( R = 0.68; P = 0.0035) and negatively correlated with NDF contents (R = -0.63; P<0.01).

The addition of PEG in the fermentation process increased the values of a + b, SCFA and MN for the experimental olive branches and amounted in average to 14 mL/g DM, 0.49 m mol/g DM and 0.21 mg/g DM, respectively (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 (Accacia aneura) leaf was related to its content of condensed tannins, which bound with proteins in the leaves. Our results indicated that the MN or MBM values were negatively correlated with total tannin concentrations (R = -0.32; P = -0.2333) and positively correlated with buffer soluble nitrogen contents (BS-N) (R = 0.89; P = 0.1073). Al-Masri and Mardini (2008) indicated that the BS-N concentrations in leaves and stalks of Sesbania aculeate and Kochia indica harvested at different maturity stages were negatively correlated with condensed tannins (R = -0.81; P = -0.56). The higher gas production, SCFA, MN and MBM production following inclusion of PEG in our study are in agreement with Al-Masri (2015). 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 Getachew et al (2000). 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 NH ₃-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.

Conclusions

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

• Olive pruning branches in diameter <3 mm had high values of effective degradability, partitioning factor and microbial nitrogen and biomass production and could be used as sources of feeds for small ruminants in the Mediterranean zones . This is of economic and environmental benefit leading to a reduction in the cost of the ruminant production and to be a protection of the environment.
  
  
• The addition of polyethylene glycol to the olive pruning branches increased the values of rumen microbial nitrogen, fermentation characteristics and degradability of dry matter.
  
  
• Further investigations of the effect of different levels of PEG on the nutritive value of olive pruning branches in animal studies are necessary to determine if feeding PEG-treated branches is economical.

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 19 April 2016; Accepted 16 July 2016; Published 1 August 2016

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