In vitro rumen fermentation kinetics and nutritional parameters of some native range plants and their relationships to gas production

M R Al-Masri

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

Abstract

The study was achieved to estimate the in vitro rumen fermentation characteristics (initial gas produced from soluble fraction; a, gas produced from insoluble but fermentable fraction; b, potential gas production; a + b, fractional rate of gas production per hour; c), effective degradability, nitrogen solubility and chemical composition of five native drought-tolerant range perennial shrubs, and study their relationships with gas production parameters after incubation with rumen fluid in the absence or presence of polyethylene glycol (PEG, 6000).

The in vitro potential gas production (mL/g DM) and effective degradability of dry matter (%) values were highest (P < 0.05) in Capparis spinosa (250 mL and 31%), lowest in Artemisia herba-alba and Noaea mucronata (149 mL and 20%), and intermediate in Lavandula officialis andAstragalus spinosus (186 mL and 24%), respectively. L. officialis, N. mucronata and A. herba-alba had higher (P < 0.05) fractional rate of gas production (0.087 mL/h) than other species (0.075mL/h). Nitrogen solubility values ranged from 37% to 52%, with C. spinosa having the highest value. The greatest proportion of gas production occurred during the first 24 h of incubation. Gas production after 24 h of incubation and estimated fermentation parameters (a, b and a + b) were positively correlated with nitrogen solubility and crude protein but negatively correlated with tannins, total phenols, lignin and neutral-detergent fibre. The addition of PEG to the plant samples incubated with rumen fluid at a ratio of 2:1 PEG: substrate did no increase the values of gas production, characteristics of fermentation and effective degradability of dry matter. On the basis of the studies on nutritive parameters, the evaluated species were nutritionally well suited as supplements for ruminants in arid regions.

Key words: degradability, gas production, nitrogen solubility, polyethylene glycol

Introduction

In arid and semi-arid zones, livestock feeding is mainly dependent on cereal crop residues so that a high level of ruminant production is not always possible. 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). Some native range plants, which are tolerant to the seasonal changes in environmental conditions, can be used as protein supplements to crop residues for ruminants. Artemisia herba-alba Asso is a perennial shrub that belongs to the Asteraceae family and grows on the steppes of the Mediterranean regions. Noaea mucronata Forssk., a perennial invasive species that belongs to the family Chenopodiaceae, is widely spread on arid and semi-arid rangelands of North Africa and Middle East. Lavandula officialis L. is a strongly aromatic perennial shrub that belongs to the Labiaceae family and distributed in the Mediterranean regions and Arabian Peninsula. Astragalus spinosus Vahl. is an invasive shrub that belongs to the Leguminosae family and wide spread on rocky or silty soils and wastelands. Capparis spinosa L., a drought-tolerant perennial plant that belongs to the family Capparaceae, is utilized for reducing soil erosion and preventing formation of sand dunes in the deserts.

The phenolic compounds (particularly tannins) in some shrubs may bind to protein, thus rendering the protein undegradable by 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). 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).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). In addition, the gas production and fermentation parameters of shrubs and trees might demonstrate differences in their nutritive value that could be closely related to their chemical composition (Cerrillo and Juarez 2004; Kamalak et al 2005). Moreover, estimations of nitrogen solubility and feed degradability are important tools for nutritional evaluation of shrubs for small ruminants.

Objectives

The objectives of the present study were:

• To evaluate, by the use of in vitro incubation techniques with ruminal fluid, some drought-tolerant native range plants (A. herba-alba, N. mucronata, L. officialis, A. spinosus, C. spinosa) in terms of their effective degradability and characteristics of fermentation.
  
  
• To study the relationships between chemical composition, nitrogen solubility and gas production and fermentation parameters.
  
  
• To assess the impact of polyethylene glycol on the aforementioned parameters.

Materials and methods

Plant materials

Different species of native range plants (A. herba-alba, N. mucronata, L. officialis, A. spinosus, C. spinosa), grown naturally on the south-eastern semi-desert of Syria (Gabajeb 35 ^o 16' N, 39^o 42' E and Al-bihsri 35^o 22' N, 39^o 46' E ; 203 m above the sea), were collected from 8 different places of a limited field (about 2500 m²). Plants were harvested at early bloom stage and hand-cut at 25 cm from ground level. The collected plants for each species were mixed well to avoid the variations among the places and 4 representative samples (n = 4), 3 kg each, were randomly taken, dried at room temperature (20-25 ^oC) for one week, ground to pass a 1 mm sieve and stored frozen at – 20 ⁰C in sealed nylon bags for subsequent analysis and evaluation. Table 1 shows the chemical composition of the experimental materials (Al-Masri 2013).

Fermentation kinetics evaluation

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) and Menke and Steingass (1988) 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).

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. The aim of PEG addition was to determine the adverse effect of tannin on the gas production and estimated parameters. 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). Cumulative gas production data 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 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 (mL/h).

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.

Statistical analyses

A factorial design was used in this experiment, with tow fixed factors: (1) plant species (five 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. Correlation coefficients between the studied parameters were calculated.

Results and discussion

The changes in gas production from the evaluated plant species after incubation with or without PEG and their degradability and fermentation kinetics are illustrated in Table 2. The in vitro gas production, effective degradability of dry matter and potential gas production values were highest (P < 0.05) in C. spinosa, lowest inA. herba-alba and N. mucronata, and intermediate inL. officialis and A. spinosus. L. officialis,A. herba-alba and N. mucronata had the highest ( P < 0.05) rate of gas production during incubation (0.087/h) than other species (0.075/h). 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). 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. 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. Sallam et al (2008) reported that the values of a + b and fractional rate of gas production of alfalfa hay amounted to 228 mL/g DM and 0.015/h, respectively. The values of potential gas production (149-250 mL/g DM) and fractional rate of gas production (0.073-0.088/h) of the experimental plant species are higher to those (119-191 mL/g DM or 0.056- 0.088/h, respectively), reported by Al-Masri (2015) for 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).

Crude protein content ranged from 97.0 to 229 g/kg DM, with C. Spinosa having the highest content. The minimum crude protein level of 80 g/kg DM is required for rumen microorganisms function (Norton 2003). Therefore, these experimental range species appear to be good protein source for grazing livestock. The highest cumulative gas production was obtained during the first 24 h of incubation for the experimental plant species (Fig. 1). The in vitro gas produced from the incubated materials after 24 h increased slowly until 48 h, and after that the data were similar up to 96 h of incubation. Thus, the highest rate of organic matter fermentation was during the first 24 h of incubation. This may be attributed to the high concentration s (408-642 g/kg DM) of non-fiber carbohydrates (NFC) in the tested species (Al-Masri 2013) mean greater amounts of soluble carbohydrates which are available for fermentation and a high rate of fermentation during the first 24 h of incubation. Al-Masri (2016) indicated that the greatest proportion of gas production occurred during the first 48 h of incubation for olive tree pruning residues. These residues had low contents (350-377 g/kg DM) of NFC (Al-Masri 2012).

Figure 1. Cumulative gas production (in vitro) over from the experimental plant species.

Indeed the higher lignin (165 g/kg DM) levels in A. herba-alba and N. mucronata are almost certainly responsible for its reduced gas production versus the other plant species (113-148 g/kg DM for L.officialis and A. spinosus and 51.8 g/kg DM for C. spinosa) (Al-Masri 2013). Gas production reflects the degradation of dietary organic matter and more gas production, more degradation of OM (Groot et al 1996). Differences in potential gas production (a + b) and effective degradation of dry matter (ED) among plant species could also be due to the extent of lignification of NDF. Lower NDF and lignin 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). The high values of ED for C. spinosa were expected since there were positive correlations between the ED values and gas production (R = 0.98; P< 0.0001) and negative correlations between the ED values and lignin concentrations ( R = -0.91; P < 0.0001). Lignocellulosic materials, particularly lignin, act as a physical barrier to microbial enzymes. It is generally agreed that the lignin concentration of forages is negatively related to the extent of digestion (Jung et al., 1997). The lignin suppressing effect probably results from a reduction in attachment of ruminal microbes to feed particles and inhibition of microbial growth and microbial enzyme activity (McSweeny et al., 2001).

Gas production parameters suggested differences in nutritional value that were generally closely related to chemical composition (Salem 2005; Al-Masri 2010). Our results indicated that the values of nitrogen solubility ranged from 37% to 52%, with C. spinosa having the highest value. Gas production after 24 h of incubation were positively correlated with nitrogen solubility (Fig. 2) but negatively correlated with lignin (Fig. 3) and total phenols and tannins (Figs. 4 and 5). Al-Masri (2013) indicated that the values of digestible organic matter for the same plant experimental species were negatively correlated with lignin ( R = -0.92; P < 0.001) and neutral-detergent fibre concentrations (R = -0.75; P < 0.001) but positively correlated with crude protein and buffer-soluble nitrogen (R = 0.92; P < 0.0001).

Our results indicated that the estimated fermentation parameters ( a, b and a + b) were positively correlated with crude protein and buffer-soluble nitrogen but negatively correlated with tannins, total phenols, lignin and neutral-detergent fibre (Table 3). These results are in agreement with findings of Kamalak et al. (2004), Abdulrazak et al (2000), and Ndlovu and Nherera (1997). Larbi et al (1998) reported a weak relationship between total condensed tannin and gas production parameters of some shrubs. On the other hand, Al-Masri (2016) observed a negative correlation between c values and lignin (R = -0.43; P = 0.0033) and a positive correlation between c values and crude protein concentrations (R = 0.92; P < 0.0001) for olive ( Olea europaea) pruning branches cut at different distance from the tip.

In our study, the addition of PEG in the fermentation process did not increase the values of gas production, characteristics of fermentation and effective degradability. This might be related to the low concentrations of tannins and total phenols in the tested species. Getachew et al (2002) reported that plant samples containing total phenols and tannin levels (g tannic acid equivalent/kg DM) up to 40 and 20, respectively, are not expected to precipitate protein or cause increases in gas production upon addition of PEG to the in vitro gas production method and, therefore, are not likely to adversely affect ruminant productivity. 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:

• Capparis spinosa had high values of gas production, nitrogen solubility and effective degradability.
  
  
• Gas production and fermentation parameters were positively correlated with nitrogen solubility and negatively correlated with tannins, total phenols, lignin and neutral-detergent fibre.
  
  
• The evaluated species had low concentration of total phenols and tannins; therefore, the addition of polyethylene glycol in the fermentation process did not increase the values of fermentation parameters, gas production and degradability of dry matter.
  
  
• The all evaluated plant species could be used as dietary supplements to make up for dietary deficiencies in protein for grazing animals in arid regions and wastelands.

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 25 April 2017; Accepted 3 May 2017; Published 1 December 2017

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