In vitro nutritional and fermentation characteristics of Medicago arborea at different growth stages and their relationships to gas production

M R Al-Masri

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

Abstract

The objective of this study was to estimate the in vitro ruminal digestion parameters, effective degradability (ED), short chain fatty acids (SCFA), nitrogen solubility and chemical composition of the branches of Medicago arborea harvested at five growth stages and cut at different lengths, and study their relationships with gas production (GP) and fermentation parameters.

There were significant (P<0.05) effects of the growth stage and cutting regimen on the studied parameters. The values of ED and SCFA decreased in the dormant and fruiting stages compared with vegetative, early flowering and vegetative re-growth stages and between cutting length at 50 cm and 25 cm. Cutting length treatments negatively affected all the studied parameters. Fractional rate of gas production and potential gas production values ranged from 0.072 to 0.094/h and from 232 to 275 mL/g DM, respectively. Branches of Medicago arborea harvested at vegetative and early flowering stages had highest values of GP (267 mL/g DM) compared with other stages (225 mL/g). Gas produced from one g effective degraded substrate ranged from 744 to 803 mL for the five studied growth stages. Estimated fermentation parameters and GP were positively correlated with crude protein, nitrogen solubility, ED and SCFA but negatively correlated with neutral-detergent fiber, acid-detergent fiber and lignin. On the basis of the studied parameters, the branches of Medicago arborea cut at 25 cm length and harvested at vegetative and early flowering stages were judged to be nutritionally better than those harvested at dormant and fruiting stages and more suitable as dietary supplements for ruminants in Mediterranean regions.

Key words: cutting, degradability, gas, harvesting

Introduction

In arid and semi-arid regions, small ruminants suffer from under feeding and malnutrition due to the shortage of feeds in most of the year, where drought and dry seasons exist. The ruminant feeding system in these areas depend mainly on rangeland hay and cereal crop residues containing a higher ratio of lingo-cellulosic materials and lower amounts of protein and digestible organic matter which impede ruminants productivity. Tree leaves and leguminous shrubs can be utilized as a low-cost protein, energy and mineral supplement to the low-quality roughages during the feed shortage periods for improving livestock performance and sustainable ruminant production in the tropics (Khan et al 2009, Patra 2010, Khan and Habib 2012, Habib et al 2013, Avornyo et al 2018). Medicago arborea L. is one of the most important fodder shrubs in Mediterranean region because it is preferentially consumed by small ruminants (Amato et al 2004). It is awoody leguminous shrub that belongs to the Fabaceae family, and grows on dry calcareous soils. The nutritive value of shrubby species is strongly linked to the seasonal variation of forage composition which results from the physiological changes that happen in the plants during their growing stages. However, species differ in their reaction to climatic and physiological changes (Dann and Low 1988). The nutritive value of the forage is influenced by harvest time or maturity stage of the plant (Yihalem et al 2005; Al-Masri and Mardini 2008; Nordheim-Viken et al 2009; Foster et al 2012) and cutting regimen (Čop et al 2009; Al-Masri 2009, 2010).

Gas production method has been used for estimation the rumen fermentation parameters and voluntary feed intake of shrubs and trees, and for demonstration the differences in their nutritive value that could be closely related to their chemical composition (Cerrillo and Juarez 2004; Kamalak et al 2005; Blümmel et al 2005). Moreover, estimations of short chine fatty acids, nitrogen solubility and feed degradability are important tools for nutritional evaluation of shrubs for ruminants.

The aims of the current work were to evaluate, by the use of in vitro incubation techniques with ruminal fluid, the branches of Medicago arborea harvested at five growth stages and cut at different lengths in terms of their effective degradability, nitrogen solubility, short chain fatty acids and chemical composition, and study the relationships between the aforementioned parameters and gas production and fermentation characteristics.

Materials and methods

Plant materials tested and their chemical components

Photo 1. M. arborea

Branches of M. arborea (Photo 1) were hand-cut at five growth stages (vegetative, early flowering, fruiting, dormant and vegetative re-growth) at 25 or 50 cm distance from the tip with 4 replicates (n = 4) (4 plants each). The sampled branches were dried at room temperature for one week, ground and stored frozen for later analyses (Al-Masri 2013). Table 1 shows the chemical composition of the experimental materials. Standard methods as described in AOAC (1990) were used for determination of dry matter (DM) and crude protein (CP). Cell-wall constituents (neutral-detergent fiber, NDF; acid-detergent fiber, ADF and lignin) were analyzed (Van Soest et al 1991). Buffer soluble nitrogen (BS-N) was determined according to Makkar and Becker (1996) to estimate N-solubility.

Gas production and fermentation kinetics evaluations

The rates of gas production of the experimental samples incubated over 96 h at 39 ^oC with the ruminal fluid mixed with the medium were determined based on a modified procedure of Menke et al. (1979) and Menke and Steingass (1988). Gas production (GP) was recorded after 3, 5, 8, 10, 24, 30, 48, 72 and 96 h of incubation. Particulars of incubation methods have been described formerly (Al-Masri 2015). Cumulative gas production data were fitted to the exponential equation P = a + b (1 – e^-ct) of Ørskov and McDonald (1979) to study the digestion kinetics of the experimental samples, 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. 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.

Statistical analyses

A factorial design was used in this experiment, with tow fixed factors: (1) growth stage (vegetative, early flowering, fruiting, dormant and vegetative re-growing); (2) cutting length (25 and 50 cm). Results were subjected to analysis of variance (ANOVA) using a Statview-IV program (Abacus Concepts, Berkeley, CA, USA) to test the effect of growth stage and cutting length 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 (GP) from the branches of M. arborea , as a result of growth stage and cutting length and their fermentation characteristics are illustrated in Table 2. There were a significant ( P<0.05) effect of the growth stage and cutting regimen on the studied parameters. Branches of M. arborea harvested at vegetative and early flowering stages had highest values of GP (267 mL/g DM) compared with other stages (225 mL/g). The values of a + b (263 mL/g DM) increased in the vegetative, early flowering and vegetative re-growing stages compared with the dormant and fruiting stages (235 mL/g DM). Increasing cutting length from 25 to 50 cm decreased the values of gas production and a + b from 258 to 226 mL/g DM and from 266 to 239 mL/g DM, respectively. This may be attributed to the low amounts of crude protein and BS-N and high concentrations of NDF and lignin in the cutting branches at 50 cm length. Lignin acts as physical barrier to microbial enzymes. The lignin suppressing effect possibly results from a decrease in attachment of ruminal microbes to feed particles and inhibition of microbial enzyme activity (McSweeny et al 2001).

Fractional rate of gas production (c) and a + b values ranged from 0.072 to 0.094/h and from 232 to 275 mL/g DM, respectively. The a + b value is associated with the feed degradability, whereas the feed intake is mainly explained by the c value which affects the feed passage rate through the rumen (Khazaal et al 1995). Therefore the higher c and a + b values observed in the cutting branches at 25 cm length may point to a greater availability of nutrients for rumen microbes. These results are agreement with Al-Masri (2013) who indicated that the branches of M. arborea cut at 25 cm length gave higher values of digestible organic matter, metabilizable energy and net energy lactation compared with cutting length at 50 cm. Al-Masri (2012) showed that the olive branches cut at 25 cm length gave higher BS-N values compared with the cutting lengths at 50, 75 and 100 cm, indicating a higher N-solubility at the former length (25 cm) which included a large amount leaves. Kafilzadeh and Heidary (2013) indicated that in any nutritional evaluation of plant species, not only chemical composition and degradability of organic matter but also rumen fermentation characteristics should be considered. Al-Masri (2010) observed that the branches of Kochia indica cut at 15 cm length gave higher value (0.041/h) of fractional rate of gas production than those cut at 30 cm length (0.034/h). Our results indicated that the values of potential gas production (232-275 mL/g DM) and fractional rate of gas production (0.072-0.092/h) of the experimental branches of M. arborea were higher than those reported by Sallam et al (2008) for alfalfa hay (228 mL/g DM and 0.015/h, respectively).

Figure 1. Cumulative gas production in vitro over 96 h from the branches of M. arborea harvested at five different growth stages

The greatest cumulative gas production was obtained during the first 48 h of incubation (Fig. 1). Gas production after 3 h increased dramatically up to 24 h than increased markedly until 48 h, and after that remained somewhat constant up to 96 h of incubation. High amounts of soluble carbohydrates which are available for fermentation indicate a high rate of organic matter fermentation during the incubation. Roughages have high contents of lignocellulosic materials (NDF) and low concentrations of soluble carbohydrates need a long incubation period for the fermentation. The concentrations of NDF and non-fiber carbohydrates (NFC) (Al-Masri 2013) in the experimental materials amounted 439-530 and 267-314 g/kg DM, respectively. Al-Masri (2016) indicated that the greatest proportion of gas production occurred during the first 48 h of incubation for olive tree branches cut at 25, 50, 75 and 100 cm distance from the tip. These residues had high concentrations (406-595 g/kg DM) of NDF and low contents (350-377 g/kg DM) of NFC (Al-Masri 2012).

Figure 2. Changes in the effective degradability (ED) of branches of M .arborea, as affected by growth stage and cutting length (25 and 50 cm)	Figure 3. Changes in the short chain fatty acids (SCFA) values of branches of M. arborea, as affected by growth stage and cutting length (25 and 50 cm)

Gas production reflects the degradation of dietary organic matter and more gas production, more degradation of organic matter (Groot et al 1996). The fermentation rate of roughages depends on the gas produced from one unit of fermented substrate. Gas produced from one g effective degraded substrate ranged from 744 to 803 mL for the studied growth stages. The changes in the effective degradability (ED) and short chain fatty acids (SCFA) contents of the branches of M. arborea, as affected by growth stage and cutting length are shown in Figures 2 and 3. The values of ED and SCFA decreased in the dormant and fruiting stages compared with other growth stages and between cutting length at 50 cm and 25 cm. Gas production was positively correlated with crude protein and BS-N (R=0.73; P<0.001) and ED (R=0.95; P<0.001).

Figure 4. Relationship between gas production and nitrogen solubility of the branches of M. arborea cut at different growth stages	Figure 5. Relationship between gas production and short chain fatty acids (SCFA) of the branches of M. arborea cut at different growth stages
Figure 6. Relationship between gas production  (NDF) of the branches of M. arborea cut at differen t growth stages	Figure 7. Relationship between gas production and lignin of the branches of M. arborea cut at different growth stages

The results indicated that the gas production after 48 h of incubation was positively correlated with nitrogen solubility and short chain fatty acids but negatively correlated with neutral-detergent fiber and lignin (Figures 4-7). The relationships between the estimated fermentation parameters ( b, a + b and c) and the nutritional components are illustrated in Table 3. These results are in agreement with finding of Al-Masri (2017), Kamalak et al (2004), and Abdulrazak et al (2000). Al-Masri (2016) observed a negative correlation between c values and lignin and a positive correlation between c values and crude protein concentrations for olive pruning branches. Preston et al (2019) found that there were positive linear relationships between (i) the percentage of methane in the gas and the rate of gas production; and (ii) between methane production and the solubility of leaf protein.

Conclusions

• Gas production, effective degradability (ED), short chain fatty acids (SCFA) and fermentation characteristics decreased in the dormant and fruiting stages compared with other growth stages and between cutting length at 50 cm and 25 cm.
  
  
• Estimated fermentation characteristics and gas production were positively correlated with crude protein, nitrogen solubility, ED and SCFA but negatively correlated with neutral-detergent fiber and lignin.
  
  
• Branches of Medicago arborea cut at 25 cm length and harvested at vegetative and early flowering stages were better in terms of nutritional value than those harvested at dormant and fruiting stages and could be used as sources of feeds for ruminants during the dry season in Mediterranean regions.

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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