Available Online: http://www.ejpau.media.pl/volume18/issue2/art-03.html
IMPACT OF SEASON OF HARVEST ON POTENTIAL NUTRITIVE VALUE, METHANE PRODUCTION AND CONDENSED TANNINS CONTENT OF CALOBOTA SAHARAE IN M´SILA, NORTH-CENTRAL ALGERIA
Samir Medjekal1, Mouloud Ghadbane2, Hacène Bousseboua3
1 Department of Applied Microbiology, University Mentouri of Constantine, Algeria
2 Department of Microbiology and Biochemistry, Faculty of Science, University of Mohamed Bouadiaf M’sila, Algeria
3 Ecole Nationale Supérieure de Biotechnologie Ville Universitaire Ali Mendjeli, Algeria
The current trial was conducted to study the effect of season on the potential
nutritive value, methane production and condensed tannin of Calobota saharae shrub
harvested at three different seasons, in winter (mid-January), spring (mid-May)
and summer (end of July). Leaves, thin twigs, some flower and seeds (when existing)
were clipped with scissors from the aerial part of the plants then dried and
analysed for chemical composition. Gas and methane production were determined
at 24 h incubation time. Season of harvest had a significant effect (p < 0.05)
on the chemical composition, gas production and in vitro digestibility
of Tilley and Terry but no effect on metabolisable energy. Neutral detergent
fibre (NDF), Acid detergent fibre (ADF) and acid detergent lignin (ADL) of Calobota
saharae were high during summer (dry season) and low in spring and winter
(wet season). The NDF, ADF and ADL contents ranged from 463.05 to 616.82 and
352.27 to 488.21 and 121.19 to151.73 (g/kg DM) respectively. The CP content
was lower (p < 0.05) in summer (96.84 g/kg DM) versus
spring (138.85 g/kg DM)
and winter (139.42 g/kg DM). The gas and methane production at 24 h
incubation varied between 65.75 to 77.75 and 6.50 to 10.17 (ml/g DM) respectively
and decreased significantly (p <0.05) from spring to summer. In conclusion,
season had a significant effect on the nutritive value of Calobota saharae shrub. Calobota
saharae should be grazed or harvested during winter and spring since these
seasons provide this shrub with high ME and CP content for ruminant.
The current trial was conducted to study the effect of season on the potential nutritive value, methane production and condensed tannin of Calobota saharae shrub harvested at three different seasons, in winter (mid-January), spring (mid-May) and summer (end of July). Leaves, thin twigs, some flower and seeds (when existing) were clipped with scissors from the aerial part of the plants then dried and analysed for chemical composition. Gas and methane production were determined at 24 h incubation time. Season of harvest had a significant effect (p < 0.05) on the chemical composition, gas production and in vitro digestibility of Tilley and Terry but no effect on metabolisable energy. Neutral detergent fibre (NDF), Acid detergent fibre (ADF) and acid detergent lignin (ADL) of Calobota saharae were high during summer (dry season) and low in spring and winter (wet season). The NDF, ADF and ADL contents ranged from 463.05 to 616.82 and 352.27 to 488.21 and 121.19 to151.73 (g/kg DM) respectively. The CP content was lower (p < 0.05) in summer (96.84 g/kg DM) versus spring (138.85 g/kg DM) and winter (139.42 g/kg DM). The gas and methane production at 24 h incubation varied between 65.75 to 77.75 and 6.50 to 10.17 (ml/g DM) respectively and decreased significantly (p <0.05) from spring to summer. In conclusion, season had a significant effect on the nutritive value of Calobota saharae shrub. Calobota saharae should be grazed or harvested during winter and spring since these seasons provide this shrub with high ME and CP content for ruminant.
Key words: Calobota saharae, Condensed tannin, In vitro gas production, Nutritive value, Methane production, Rumen fermentation.
ADF (acid detergent fibre); ADL (acid detergent lignin); AOAC (Association of official analytical chemists); CP (crude protein); DM (dry matter); EE (ether extract); FCT (fibre-bound condensed tannins); Free CT (free condensed tannins); GC (gas chromatography); GP24 (gas production 24hour); HCL (hemicellulose); IVD-TT (in vitro digestibility of Tilley and Terry); ME (metabolisable energy); NDF (neutral detergent fibre); PCT (protein-bound condensed tannins); SDS (sodium dodecyl sulfate); TCT (total condensed tannins).
Large tracts of desert and semi-desert lands of African and Middle Eastern countries cannot be cultivated but can be used to raise livestock, mainly sheep, goats and camels. Even in favourable rainfall years, animal performance can be poor due to a lack of adequate dietary protein and energy intake in the dry season. Furthermore, the herbaceous forage is fibrous and of low quality, and in the event of drought, livestock mortality can be high . Feed supplementation during drought is infeasible for livestock producers in resource-poor countries due to high feeding costs . Thus, and alternative approach for overcoming such feed constraints is the propagation of nutrients local fodder shrubs on degraded land. One ligneous species suitable for browse in areas of severe aridity is Calobota saharae (Coss. & Durieu) Boatwr. & B-E. van Wyk (formerly Genista saharae or Spartidium saharae). Calobota have a great ecological significance in Mediterranean countries.
They colonize degraded forests and deforested areas that characterize the landscape . The genus Calobota consisting of 87 species  among these species 23 grow in Algeria [24, 31]. The nutritive value of a ruminant feed is determined by the concentrations of its chemical components, as well as their rate and extent of digestion. Determining the digestibility of feeds in vivo is laborious, expensive, requires large quantities of feed, and is largely unsuitable for single feedstuffs thereby making it unsuitable for routine feed evaluation . In vitro methods provide less expensive and more rapid alternatives. Both in vitro gas production and the ANKOM Technology (Automated instrumentation for the food analysis) devices can be used as rapid evaluation tools to assess nutritional quality of feeds . In Algeria, there is limited information on the nutritive value of local shrubs and both in vitro and in vivo studies are not available. Moreover there is limited information on the impacts of seasonal variations on nutritional values of local most grazed leguminous shrub. A study was therefore carried out to evaluate the effects of season on chemical composition, in vitro gas production and organic matter degradability of indigenous shrub Calobota Saharae.
MATERIALS AND METHODS
Collection of Calobota saharae
The study was carried out during 2009 in a medium-sized shrub-grassland between El Maader and Bousaada district located in the north central Algeria (N35° 26' 07,9''; E004°20'52,8'') (Fig. 1), at an altitude of about 398 m above sea. The area is an arid high plateau with steppe like plains and extensive barren soils. Its climate is continental, semi-arid, subjected partly to Saharan influences with an annual average temperature of 21°C (average temperature of 34°C in summer and 10°C in winter), and a low and irregular rainfall not exceeding 250 mmper year. M’Sila experiences high temperature between June and September, and rainfalls between September and December (Fig. 2).
Representative samples from the aerial parts of plants were randomly hand clipped with scissors along a transect of about 2 km, in winter (mid-January), spring (mid-May) and summer (end of July) during the year 2009. Between six and ten specimens of each plant species were sampled to obtain a representative aliquot of the edible biomass. Leaves, thin twigs (young stem) and some flower and seeds (when existing) were clipped with scissors from the aerial part of the plants, and taken immediately to the laboratory where the samples from different specimens were pooled, oven dried at 50°C , and subsequently ground to a 1 mm screen.
|Fig. 1. Location of the study areaion of the tarsocrural joint, characteristic of the third peroneal muscle rupture|
|Fig. 2. Precipitation and Temperature 2008–2010 (Tocyo Climate Center, http://ds.data.jma.go.jp/tcc/tcc/ )|
Chemical composition and condensed tannins
Ash (Ash method ID 942.05), ether extract (EE, method ID 7.045) and crude protein by Kjeldhal (CP, method ID 984.13) in samples were determined by the procedures of the Association of Official Analytical Chemists . Neutral detergent fibre (NDF), acid detergent fibre (ADF) and acid detergent lignin (ADL) contents were analysed following the methodology described by Van Soest et al.  using an ANKOM Model 220 Fibre Analyser (Macedon, NY, USA).
Levels of condensed tannins of each sample were determined using the method described by Terrill et al. . Free tannins are estimated by HCL-butanol, after the extraction of this fraction with diethyl ether to remove lipids and interfering pigments. The residue from the acetone extraction is treated with sodium dodecyl sulfate (SDS) and the extract obtained reacts again with HCl-butanol assay protein bound tannins. After SDS extraction the remaining residue is also treated with HCL-butanol to assay fibre-bound condensed tannins . Solution of purified quebracho tannin (1 mg/ml aqueous acetone, 700 ml/l) was the standard. Absorbance was measured against a blank at 550 nm. The CH4 concentration was determined by gas chromatography (GC) using a HP Hewlett 5890, Packard Series II gas chromatograph (Waldbronn, Germany). A sample of 0.5 ml of gas was injected using a 1 ml Sample-Lock® syringe (Hamilton, Nevada, USA).
In vitro gas production (GP24h), In vitro dry matter digestibility
of Tilley and Terry (IVD-TT) and metabolizable energy (ME) MJ/kg
Four adult and mature Merino sheep (body weight 48.3±3.45 kg) fitted with a permanent ruminal cannula were used for the extraction of rumen fluid to carry out the in vitro incubations (gas production and digestibility) of the browse material. Animals were fed twice a day (9 a.m. and 4 p.m.) a diet that consisted of alfalfa hay and grain oats in a proportion 60:40 at approximately and had free access to water and mineral/vitamin licks. A sample of rumen contents was withdrawn prior to morning feeding. Of each of the animals takes a similar amount of rumen contents, which is transferred into thermos flasks and taken immediately to the laboratory. Animals were cared for by trained personnel in accordance with the European Convention for the Protection of Vertebrates used for Experimental and other Scientific Purposes . Rumen fluid from the four sheep was mixed, strained through various layers of cheesecloth and kept at 39°C under a CO2 atmosphere. Batch cultures of mixed rumen micro-organisms were used to study the ruminal fermentation, gas and methane production. The experimental procedure was based on Theodorou et al.  protocol with some modifications. Three identical 48 h incubation runs were carried out in three consecutive weeks. The buffer solution of Goering and Van Soest , (1970) was previously prepared into an Erlenmeyer flask under a CO2 stream and kept one hour with an O2-free headspace after the resazurine color turnover showed an O2-free solution. Particle-free ruminal fluid was mixed with the buffer solution in a proportion 1:4 (vol/vol) at 39°C under continuous flushing with CO2. Buffered ruminal fluid (50 mL) was added into each bottle under CO2 flushing. Bottles were sealed with butyl rubber stoppers and aluminum caps and placed in a water bath at 39°C. Serum bottles of 120 mL were used. In each incubation run triplicate samples (0.5 g dry matter, DM) were placed into the bottle and incubated. Pressure in the bottle headspace and volume of gas produced were measured at 24 inoculation using a Wide Range Pressure Meter (Spec Scientific LTD, Scottsdale, AZ, USA) and a calibrated glass syringe as described by Theodorou et al.  respectively. An aliquot of the gas produced was taken in a 10 mL vacuum tube (Venoject®, Terumo Europe N.V., Leuven, Belgium) for CH4 concentration analysis. Fermentation flasks without samples (i.e., blanks) were included to allow correction for gas produced directly from rumen fluid.
Analysis of in vitro dry matter (DM) digestibility (IVD-TT) followed the method of Tilley and Terry . A culture medium containing macro- and micro-mineral solutions, resazurin and a bicarbonate buffer solution was prepared as described by Van Soest et al. . Rumen fluid was then diluted into the medium in the proportion 1:5 (v/v). Samples (400 mg) were weighed out into artificial fibre bags (size 5 × 5 cm, pore size 20 m) which were sealed with heat and placed in incubation jars. The jars were then placed in a revolving incubator (Ankom Daisy II digestion system, ANKOM Technology Corp., Fairport, NY, USA) at 39°C, with continuous rotation to facilitate the effective immersion of the bags in the rumen fluid. After 48 h of incubation in buffered rumen fluid, samples were subject to a 48 h pepsin-HCl digestion as described by Tilley and Terry .
ME [MJ/kg DM] content of Calobota saharae samples was calculated using equation of Menke et al  as follows:
ME [MJ/kg DM] = 2.20 + 0.136 GP + 0.057 CP,
GP = 24 h net gas production [ml/200 mg];
CP = Crude protein.
All data obtained were subjected to analysis of variance (ANOVA) using the randomized completed block design. Significance between individual means was identified using the Tukey’s multiple range tests. Mean differences were considered significant at P < 0.05. Analysis of variance (PROC ANOVA) was performed using the SAS software package .
The changes in chemical composition of Calobota saharae at different seasons of harvest are presented in Table 1. There were differences between growth seasons in ash, cell wall components, CP, EE and condensed tannins contents (p < 0.05). The CP content was lower in summer (96.84 g/kg DM) versus spring (138.85 g/kg DM) and winter (139.42 g/kg DM). In winter, Calobota saharae had lower (p < 0.05) NDF, ADF, ADL and HCL content than in summer, and intermediate values were observed in spring. In the other hand, high values of ash, EE and CTC were observed during spring versus summer and winter with values ranging between 3.92 and 5.44, 20.64 and 23.79 and 25.33 and 34.72 g/kg DM respectively.
The 24h volume of gas produced (GP24), methane (CH4 24h) in vitro digestibility of Telly and Terry in each season are in Table 2. There are marked decreases in gas production, methane and IVD-TT from spring to summer.
Table 1. Chemical composition and condensed tannins [g/kg DM] contents of Calobota saharae harvested at three different seasons (n = 6) for all chemical parameters.
a, b, c Row means with common superscripts do not differ (P<0.05);
S.E.M.: standard error mean; Ash: Ash %; NDF: Neutral detergent fiber, ADF: Acid detergent fiber, ADL: Acid detergent lignin, HCL: Hemicellulose, CP: Crude protein, EE: Ether extract, Free CT: Free condensed tannins, PCT: Protein-bound condensed tannins, FCT: Fibre-bound condensed tannins, TCT: Total condensed tannins;
*** – P < 0.05
Table 2. Gas production (GP24 [ml/g DM] methane 24h [ml/g] in vitro digestibility [%] and metabolic energy [MJ/KG DM]; n = 3 for all parameters)
a, b, c Row means with common superscripts do not differ (P<0.05);
S.E.M.: standard error mean; GP24: gas production 24hour [ml/g], CH4: methane production 24 hour [ml/g], IVD-TT: in vitro digestibility of Tilley and Terry [%], ME: Metabolisable energy [MJ/KG DM], NS: Non-significant
In the current study, large differences in the nutritive value of Calobota saharae (as assessed by gas production and DM degradability) were observed, which were primarily the results of changes in maturity of the collected leaves. Reduction (p < 0.05) in CP content of Calobota saharae in summer versus other seasons, is consistent with other studies, as was the observation that the minimum CP content of fodder tree leaves in the dry season was more than twice that of grasses in the wet season [9, 33]. However, the CP content of Calobota saharae remained relatively high (96.84 g/kg DM) during summer which is higher than the minimum level of 7–8 % DM required for optimum rumen function and feed intake in ruminants livestock . In addition, the higher CP content (193.42 g/kg DM) during winter compared with the other two seasons as a result of higher moisture content and nitrogen uptake being more rapid than dry matter accumulation agrees with [3, 19] who reported that seasonal variation occur between plant species and between seasons with higher values reported for seasons with higher moisture levels. The lower CP content during summer may be largely due to moisture stress experienced by Calobota saharae during this period and build-up of lignocellulosic fibre structures of the plants, diluting the nitrogen . The wide variations in NDF, ADF, ADL and HCL contents of Calobota saharae in winter and summer are consistent with the report of Onwuka et al. , on browse species in the humid lowlands of West Africa and in other parts of the tropics [8, 37]. The seasonal differences in cell wall constituents may relate to the differences in elements of the weather between seasons, and their effects on cell wall lignifications as well as translocation of nutrient to the different part of the plant . Moreover, cell wall concentration in shrub fodder is negatively correlated with palatability [14, 18]. Therefore, the observed differences in NDF, ADF, ADL and HCL among the seasons of harvest could have implications for the use of Calobota saharae as a fodder. Condense tannin had an important role in forages depending on the amount. Low level tannin (2–3% of DM) may have beneficial effect since the level of tannin in diets prevents the CP from extensive degradation through formation of protein-tannin complexes . In addition, condensed tannins in fodder trees and shrubs could also, help in the control of gastro intestinal parasites because they have biological properties that decrease fecal egg count in sheep and goats, and hatch rate and Laval development in feces . On the other hand, high tannin level (5% of DM) in diets may result in the increased indigested CP due to excessive formation of tannin-protein complexes . As can be seen from Table 1, the observed condensed tannin levels of Calobota saharae harvested at three different seasons were low magnitude. Therefore, low condense tannin of Calobota saharae seems to have a potential for beneficial effect when included into ruminant diet as it can increase rumen undegradable CP without decreasing digestibility . The gas production is closely associated with the amount of fermented substrate in diets . The low gas production for Calobota saharae in the dry season (summer) may be attributed to the relatively higher NDF, ADF, ADL and the low CP content. Our observation are similar to Evitayani et al. , who found that in vitro gas production and in vitro dry matter digestibility of some legume species increased in wet season (winter) compared with the dry season (summer). Similar observations with maturity were also observed by kamalak et al.  in Gundelia tournifortii hay and Mahmut et al.  in Sanguisorba minor hay. There were no differences in ME of Calobota saharae obtained in the current study this could be due to the little variations of gas production between the three seasons, since, ME energy values were estimated using the gas production and CP content. According to Fagg and Stewart , indigenous legumes of arid regions such as Calobota saharae, because of their nitrogen fixing symbiosis with legume nodulating bacteria, collectively called rhizobia, contribute to soil fertility by enhancing soil nitrogen content and organic matter. They provide high-quality animal fodder, prevent erosion and contribute to soil stabilization and ecosystem restoration.
Methane is a product of microbial fermentation, particularly in the rumen. At the end of a complex interplay of microbiological and chemical processes, methanogens use hydrogen to reduce carbon CO2 to CH4 . Lopez et al.  suggested that the methane reduction potential of any feedstuffs can be estimated from the percentage of methane of in vitro gas production and the feedstuffs can be arbitrarily divided in three groups, low potential (% methane in gas between >11% and ≤14%), moderate potential (% methane in gas between >6% and <11%), high potential (% methane in gas between >0% and <6%). Therefore, Calobota saharae, shrub had no methane reduction potential since the percentage of methane for all the seasons is between 10 to 13%. The recent interest in methanogenesis reflects effects to identify mitigation strategies to reduce output of this greenhouse gas from livestock and manure. It also reflects an interest in reducing a source of energy loss that arises when feed is digested by ruminants.
The results presented in this study indicate that the major differences in nutritive value of Calobota saharae shrub, in terms of chemical composition and rumen microbial fermentation, are caused by the effect of the growing season. Calobota saharae should be grazed or harvested during winter and spring since these season provides this shrub with high ME and CP content for ruminant. This study has practical implications for the development of agro-forestry technologies. Calobota saharae shows potential as a fodder shrub for revegetation projects in degraded ecosystems in arid and semiarid lands.
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Department of Applied Microbiology, University Mentouri of Constantine, Algeria
route de Ain El-Bey
Department of Microbiology and Biochemistry, Faculty of Science, University of Mohamed Bouadiaf M’sila, Algeria
28 000 M’sila
Ecole Nationale Supérieure de Biotechnologie Ville Universitaire Ali Mendjeli, Algeria
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