Essay: Vitro screening of Algerian steppe browses species

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  • Vitro screening of Algerian steppe browses species
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The aim of theses studies was to screen the nutritive value and the effects of anti-nutritionnal secondary compounds (condensed tannins) on in vitro rumen fermentation and methane reduction of eight substrates. Polyethylene glycol (PEG), a tannin binding agent is used to measure the biological activity of tannins from algerian forage species (Albizia julibrissin pods, Acacia nilotica pods, Punica granatum leaves and pericarp, Vicia faba leaves, Artemisia herba alba whole plant without roots, Attriplex halimus leaves and Calligonum azel barks.
The percent increase in cumulative gas production at 48 h of incubation with and without PEG was greatest for Calligonum Azel (894.6) (p<0.05) at 6hours. Acacia nilotica pods gives the greatest % increase (171.3 ; 129.76 ; 122.10) (p<0.05) at 12h, 24h and 48, respectively. The addition of PEG had a significant (p<0.05) effect on asymptotic gas production in Pinuca granatum pericarp and Acacia nilotica. The best methane reduction (p<0.05) were observed with Attriplex halimus, Acacia nilotica and Calligonum Azel in the absence and presence of PEG (0.93-2.00 ; 1.72-3.81 and 0.009-0.135 mmol/g DM), repectively. A significant (p<0.05) positive correlation were observed between Total Condensed Tannin (TCT) and percent increment at 12h, 24h, and 48h (p<0.01) and A (Asymptotic gas production) (p<0.05).

Key words : Nutritive value, Plant secondary compounds metabolites, Condensed Tannins, Polyethylen Glycol

Algerian steppe constitutes a buffer zone between the Sahara desert and the green belt in the North Covering more than 30 millions ha of land this areas are used mainly for sheep production with local breeds well adapted to the extreme environmental conditions and showing a particular productive performance. Droughts occur frequently, associated with extensive breeding, and had a critical influence on vegetation and thus on rangelands . Actually, steppe rangelands are in a process of degradation due to the fragility of the physical environment, changes in the pastoral methods are accelerating this process (Aidoud, 1994).
Perrenial Acacia and shruby plants from saharian areas can be used to mitigate desertification, allowing soil fixation, enhancing restoration of the vegetation and rehabilitation of rangelands. Trees, especially those of the Acacia genus, are adapted to a low moisture environment. Acacia leaves and pods are a potential source of protein.especially when herbaceous vegetation becomes withered during droughts (Osuji and Odenyo, 1997).
Like other leguminous, Acacia genus is rich in protein. Adversly, their high contain in anti-nutrional factors such as secondary compounds, especially condensed tannin limits their use as forages (Waghorn et al. 2002). Condensed tannins have various effects on rumen fermentations. Several studies focused on the utilisation of tannin rich plant to reduce methane production by ruminants (Woodward et al. 2004 ; Graiger et al. 2009 ; Naumann et al. 2018).
Methane emission from ruminants has received global attention because of its contribution to the greenhouse effect and global warming greater than CO2 (UNFCC, 2005).
The digestion of feed by rumen microbes (archaea, bacteria, protozoa and fungi) under anaerobic conditions results in the production of volatile fatty acids (VFA), ammonia, carbon dioxide and methane (Martin et al. 2010). Rumen methane is a by-product produced during the process of microbial digestion and I s equivalent to a loss of 2-12% of the feed energy ( Boali et al. 2004). Reduction ruminal CH4 improves the efficiency of nutrient utilization and also helps to protct the environment.
To mitigate enteric CH4 emissions from ruminants several dietary strategies have been suggested (Boadi et al. 2004; Beauchemin et al. 2008). Therefore, decreasing CH4 production from ruminants is desirable for reducing greenhouse gas emissions and increasing utilization of the digested energy. Plant secondary metabolites have been suggested as effective alternatives to antibiotics to suppress rumen methanogenesis through their antimicrobial activity (Jayanegara et al. 2009). The addition of polyethylene glycol has been used in order to decrease the tannins activity in plants, then, an increase of in vitro GP indicates the effect on the activity of tannins in substrates (Getachew et al. 2000a).
The goals of these experiments were to screen the nutritive value and to observe the effects of their tannin content (essentially from pods, pericarp and barks) on in vitro fermentation and methanogenesis of these browse species collected from Algerian steppic areas.
Materials and methods
Source of shrubby samples
Pods without seeds from Acacia nilotica and Alibizia julibrissin, Pinuca Granatum fruit and leaves, Artemisia herba alba (whole plant without roots), Atriplex halimus, leaves of Vicia faba from the steppe area of Djelfa district and Barks of Calligonum azel are taken from saharian area of the region of Nakhla (Oued Souf district). Samples from different specimens were pooled, oven-dried at 55ºC (Makkar 2003b).
Animals and extraction of rumen fluid
Three mature Merino sheep (body weight 49.4 ± 4.23 kg) fitted with a permanent ruminal cannula (60 mm diameter) were used for the extraction of rumen fluid. Animals were fed with lucerne hay ad libitum (167 g CP, 502 g NDF, 355 g ADF and 71 g ADL kg−1 DM).
Chemical analysis
Dry matter (DM), ash and crude protein (CP) contents were determined following the methods of AOAC (2002).
Phenolic compounds were extracted following the procedures described by Makkar (2003b).
Tannins bioassay experiment
The bioassay for the assessment of the activity of tanninss has been described in detail by Ammar et al. (2004). The increase in gas on addition of PEG is a measure of tannins activity, where the protocol used was similar to that described in Makkar et al. (1995).
In vitro digestibility
In vitro DM digestibility was determined using the ANKOM-DAISY procedure (Ammar et al. 1999). The procedure followed general conditions described in the standard in vitro fermentation method (Goering and Van Soest 1970).
VFA and methane production analysis
Gas production, for methane analysis and VFA profiles, obtained using an adaptation of the technique described by Theodorou et al. (1994).
Methane content in fermentation gas was determined by gas chromatography (GC) using a Shimadzu GC-14 B GC (Shimadzu, Japan) equipped with CarboxenTM 1000, 45/60, 2m×1/8 in. column (Supelco, USA) and flame ionization detector (FID) according to Van Nevel et al. (1970) procedures.
Volatile fatty acid analysis was performed by gas chromatography using crotonic acid as internal standard (Ottenstein and Bartley 1971).

Gas production technique datas were fitted using the exponential model proposed by France et al. (200):
G = A [1 – e−k(t-L) ] for t ≥ L
G (mL/g) denotes the cumulative gas production at time t; A (mL.g-1) is the asymptotic gas production; c (h) is the fractional rate of substrate fermentation and L (h) is the lag time.
The energy value and digestibility of feedstuffs were calculated from the amount of gas produced at 24 h of incubation with supplementary analyses of crude protein and ash, as follows (Menke and Steingass, 1988).
ME (MJ/kg DM) = 2.2 + 0.136 x G24 + 0.057 x CP + 0.029 x CP2
OMD (%) = 14.88 + 0.889 x G24 + 0.45 x CP + 0.0651 x ASH
Were ME is the metabolisable energy
G24 is 24h net gas production (mL/200 mg DM) CP is crude protein (% of DM)
OMD is organic matter digestibility
ASH is ash (% of DM)

Statistical analysis
One way analysis of variance was performed on in vitro digestibility, methane production and VFA data, with browse species as the only source of variance (fixed effect) with source of inoculum as a blocking factor (random effect). Tukey’s multiple comparison test was used to determine which means differed from the rest (P<0.05). The chemical fractions of the forages were related to fermentation variables through Pearson correlation.Analysis of variance was performed using the GLM procedure of the SAS software package (SAS Institute 2000).

Results and discussion
Chemical composition of the plant material collected from the different species is shown in table 1. The CP content of the plant species sample varied widely, being particularly high for deseeded pods of A.julibrissin and V.faba (142 and 194 g kg-1 DM, respectively) and low for the pericarp of P.granatum and barks of C.azel (33.9 and 57 g kg-1), repectively). The cell wall content ranged from 277 to 513 g NDF/kg DM, and from 169 to 408g ADF/kg DM. The highest value of ADL was observed for barks of C.azel (222.8 g ADL/kg DM). CP and NDF concentrations influence the amount of substrate organic matter (OM) fermented with the VFA that are produced (Njidda and Nasiru, 2010)
The highest content of Total Phenol (TP) and Total Tannins (TT) were observed in P.granatum leaves and pericarpe with (312; 306 and 286; 281 g/kg M, standard equivalent, respectively). Whereas A.halimus showed lower concentrations (26 and 22 g/kg DM, standard equivalent, respectively) (table 2). In contrary, A.nilotica and C.azel showed the highest results for TCT 851.3 and 719 g/kg standard equivalent, respectively) and A.halimus the lowest value with (49g/kg standard equivalent). The highest proportion of TCT was recovered as Free Condensed Tannins(FCT) in A.nilotica (703 g/kg standard equivalent). Barks of C.azel are used for tanning leather (Pottier-Alapetite, 1979).
For screening plants for antimethanogenic activity, the best way is to perform tannins bioassay followed by TP and TT analysis (Jayanegara, et al. 2009).Table 3 shows the effect of including PEG during the in vitro incubation of species. Polyethylene glycol inclusion increased significantly (p<0.05) cumulative gas production of all substrates. The greatest and significant (p<0.05) response to PEG was recorded after 6 h incubation with C.azel barks and A. nilotica pods (895 and 89 % increase, respectively). The response to PEG treatment, represented by the % increase, showed significant (p<0.05) fluctuation with incubation time 12h, 24h and 48 for all samples. The response to PEG treatment declined with incubation time, except for A.nilotica. Screening plants for antimethanogenic activity associated to the biological activity of tannins bioassay (gas and methane production with and without PEG) seemed to be a better alternative than TP and TT analysis (Mlambo et al. 2009). Condensed Tannins limit in vitro fermentation, thus the increase in gas production following inclusion of PEG provides a measure of potential effects of tannins on nutrient degradability (Makkar 1988). The highest gas production value (p<0.05) was observed in V.faba (table3). This can be attributed to its lower condensed tannins and lignin but highest CP content (Table 1 and 2). The magnitude of gas decrease was higher for condensed tannins than those of hydrolysable tannins (Jayanegara et al. 2015). Comparable results were obtained with pods and leaves of aciacias species (Alam et ai. 2007; Mlambo et al. 2008; Bouazza et al. 2012). Biological activity of tannins has been reported to vary between forage species due to the chemical structure and nature of tannins (Dalzell and Kerven, 1998; Rubanza et al. 2005), degree of polymerization (Schofield et al. 2001). The PEG-based in vitro tannin bioassay complements the chemical method because it soecifically highlights the presence of potential active tannins because tannins concentration is not a good predictor of biological activity of tannins (Mlambo et al. 2009; Vitti et al. 2005). The TCT , builds different complexes with different cell plant constituents including enzymes and decreasing digestibility (Waghorn 2008).Polyethylen Glycil suppress the effect of CT, the decrease of gas production is possible through interactions between tannins and feed constituents such as structural and non-structural carbohydrate (cellulose, hemicellulose, pectin) and all proteins (McSweeney et al. 2001; Jayanegara et al. 2012). The main interaction between tannins and those macromolecules is via hydrogen bon (Mueller-Hrvey 2006). Such bind prevents degradation and fermentation of the molecules partially to form gas and thus mitigates its production (Jayanegara et al. 2015).
Table 4 shows that the addition of PEG affect significantly (p<0.05) the Asymptotic gas production (A) for A.nilotica and P.granatum pericarp. V.faba present the highest (p<0.05) gas production from the slowly fermentable organic matter (A value) in presence and absence of CT (table 4). The lowest (p<0.05) A value was obtained for C.azel in absence and presence of tannins too. The rate of GP (c) was the highest (14.06% h-1) in Artemisia herba alba and the lowest (2.6% h-1 ) in Vicia faba. The study conducted by Guimarãs-Beelen et al. (2006) observed that if the rate of gas production is reduced, the bacteria proliferation is restricted. Elahi et al. (2014) explained that complexes generating between PEG and CT conduct to steric obstruction which does not permit and/or limit the fixation of bacteria to the feeds components.
Despite the large increase in gas production upon addition of PEG-6000, AIVD and TIVD in the presence of PEG-6000 was not significantly (P < 0.05) different from that observed in the absence of PEG-6000 (Table 5). Makkar et al. (1995), Getachev et al. (2000), Osuga et al. (2008) in a study using six Acacias forages from Kenya and Bouazza et al. (2014) with five Acacias leaves from Algeria also made similar observations. This could be due mainly to the tannin-PEG complexes, wich become insoluble in neutral detergent solution, thus distorting the weight of degraded sample (Osuga et al. 2008). This is not in agreement with other studies (Barry and McNabb, 1999; Alam et al. 2007; Barman and Rai, 2008). However, the chemical structure, concentration and biological effects of tannins in forages, and their nutritional value, show large variability (McSweeney et al. 2001).
Effects of PEG treatement on in vitro organic matter digestibility (g/kg DM) and feed metabolisable energy (MJ/Kg DM) at 24h incubation are reported in table 5. Variable responses of in vitro digestibility among our selected browses species could due to phenolics and tannins, or to variations in tannin anti nutritive activity. The highest response on in vitro gas production, OM digestibility and metabolisable energy estimates due to addition of PEG in A.nilotica pods could be related to reversed tannin anti-nutritive activity.
A decrease of methane emission was observed with C. azel and A.nilotica only (P<0.05) (Table 5). In vitro studies show that some tannin is more active than others (Aerts et al. 1999; Osborne and McNeill 2001; Bueno et al. 2008). The inhibitory effects of Condensed tannins on methanogenesis have been attributed to their direct effects on rumen methanogenic archaea and protozoa, indirectly leading to a depression of fibre degradation (Tiemann et al. 2008; Patra and Saxena 2011). Any reduction in fibre degradation is likely to reduce methane formation because fibrolysis delivers H2 as a substrate for methanogenesis in forming acetate from pyruvate (Moss et al. 2000; Jayanegara et al. 2011). Tannins are known to decrease protozoa populations (Bhatta et al. 2009) in which part of the methanogens are living together (Morgavi et al. 2010). The magnitude of decrease due to CT in the present study was relatively higher than other studies using tannins extracted and purified from plant leaves (Jayanegara et al. 2015). Different forms of tannins (whole plants or extracted tannins) may influence the CH4 emissions and rumen fermentations parameters differently, most probably with respect to the magnitude of the effects (Jayanegara et al. 2011). In the whole plants, the presence of other components might negatively or positively influence CH4 emissions such as fiber (Beauchemin et al. 2008), lipids (Machmüller et al. 2000), saponins (Hess et al. 2003) and essential oils (Benchaar et al. 2008).
Ruminal VFA concentrations mainly indicate the degradation patterns of carbohydrates by microbes. The addition of PEG had improved signifantly (p<0.05) the production of total VFAs (Table 6). Vicia faba leaves had produced the largest amount, of total VFA, acetate and propionate. The highest acetate to propionate ratio was recorded from Calligonum azel barks. High level of acetate to propionate ration is an indication of a more acetogenic fermentation, due to the activity of fibrolytic bacteria degrading substrates rich in structural carbohydrates (Gatachev et al. 20044) The absence of tannins effect resulting from addition of PEG had resulted in a substantial increase in total and individual VFA production for Acacia nilotica pods. This finding was consistent with the finding of other researchers using tannins-rich species in their studies (Getachev et al. 2008; Singh et al. 2012=. Goel and Makkar (2012) concluded that tannins-rich tropical plants reduce total VFA production. In contrast, studies with sheep (Priolo et al. 2000) and goats (Silinakov et al. 2006) reported that VFA concentrations are not affected when animals received the same diets but supplemented with PEG concentrations. The decrease of VFA concentration could also due to the presence of other secondary metabolites that interact with CT and can negatively affect VFA concentrations (Rira et al. 2015)

In conclusion, browses species used in our study showed that according to their CP, NDF, and CT content, Acacia nilotica and Albizia julibissin pods can be used as alternative for ruminant nutrition and showed the best methane reduction potential during the in vitro screening. Condensed tannins from all substrates tested had significant effects on total gas production.
These results suggest the potential for CT in gorages studied such as pods from A.nilotica, A.julibrissin and Calligonum azel barks to minimize methane emission by ruminants, probably in addition to other benefits when included at studied proportion of the diet.

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Table 1 : Chemical composition of browse plants (mean) used in the study (n=3)
Albizia julibrissin 904 927 365 278 85 242
Pinuca granatum P 919 956 277 169 50 34
Pinuca granatum L 915 911 222 155 95 109
Vicia faba 888 857 179 121 40 194
Atriplex halimus 871 815 253 113 47 157
Acacia nilotica 909 929 323 227 94 167
Artemisia herba alba 901 920 359 273 115 123
Calligonum azel 916 891 513 407 223 57
DM, Dry Matter; OM, Organic Matter; CP, Crude Protein; ADF, Acid Detergent Fiber; NDF, Neutral Detergent Fiber; ADL, Acid Detergent Lignin

Table 2 : Secondary compositions of studied browses
sample Total Phenol Total non Tannin
(PVPP) Total Tannin Bound Condenced
Tannin Free Condenced
Tannin Total Condenced Tannin
Albizia julibrissin 77 15 62 110 545 655
Pinuca granatum P 286 5 281 37 188 224
Pinuca granatum L 312 6 306 68 11 79
Vicia faba 65 17 48 157 21 178
Atriplex halimus 26 3 22 46 3 49
Acacia nilotica 114 3 111 148 703 851
Artemisia herba alba 41 4 37 47 25 72
Calligonum azel 146 9 137 148 570 719
PVPP, polyvinyl polypyrrolidone; SEM, standard error of the mean.
Total phenols and tannins were expressed as tannic acid equivalent and condensed tannins as leucocyanidin equivalent.

Table 3 : Gas production (mL/400 mg DM) of studied browse plants
Time of incubation (h)
6h 12h 24h 48h % Increase3
ID -PEG1 +PEG2 -PEG +PEG -PEG +PEG -PEG +PEG 6h 12h 24h 48h
Albizia julibrissin 17.17d 37.61c 56.6d 96.35c 109.41c 144.64c 131.44c 172.83bc 54,1 41,2c 24,4c 23,92bc
Pinuca granatum P* 43.61b 55.93b 96.59b 109.94b 136.24b 162.75b 149.25bc 196.72ba 22,0 12,2d 16,3c 24,22bc
Pinuca granatum L* 30.22c 34.75c 75.72c 84.8c 131.73b 137.91c 160.93b 171.39bc 13,1 10,7d 4,51d 6,15bc
Vicia faba 80.30a 78.81a 149.86a 148.74a 192.23a 193.397a 208.13a 225.28a -1,9 -0,8d 0,61d 7,510bc
Atriplex halimus 9.37de 8.11e 50.09d 46.83d 109.25c 108.010d 149.18bc 150.45cd -16,4 -7,2d -1,21d 0,56c
Acacia nilotica 4.02ef 36.09c 23.72e 89.7c 63.94d 144.813c 84.29d 179.57bc 89.3 171,3b 129,76b 122,10ab
Artemisia herba alba 17.94d 20.83d 54.84d 59.48d 107.64c 113.797d 133.56bc 134.88d 35,9 18,1d 11,178d -7,96c
Calligonum azel -11,77g -1.56f -5.61f 13.03e 1.54e 27.977e 8.92e 44.55e 894.6a 150,6a 94,55a 80,69a
SEM 5.2 4.9 8.9 8.12 10.9 8.6 11.7 10.7
Specie <.0001 <.0001 <.0001 <.0001
PEG <.0001 <.0001 <.0001 <.0001
Specie*PEG <.0001 <.0001 <.0001 <.0001

(-PEG)1Incubated without polyethylene glycol. (+PEG)2 Incubated with polyethylene glycol. P*: Pericarp; L*: Leaves; a, b, c, d, means in a the same row with different superscripts are significantly different (P<0.05);(3) % 3increase = (+ PEG gas volume (ml) – – PEG gas volume (ml)) X 100/ – PEG gas volume (ml).

Table 4 : In vitro gas production characteristics of the studied browse plants
A c L
Albizia julibrissin 145.40bx 184.68bcx 0.1449a 0.01482bc 1.965b 3.9357ab
Pinuca granatum P* 151.81bx 212.42aby 0.1077a 0.03308bc 3.019ab 2.6086b
Pinuca granatum L* 164.78bx 182.10bcdx 0.1079a 0.05207bc 2.143b 3.2752ab
Vicia faba 211.65ax 239.28ax 0.0262a 0.00000c 3.179ab 2.9374b
Atriplex halimus 157.71bx 160.00cdx 0.0916a 0.10191ab 3.215ab 2.7107b
Acacia nilotica 90.50cx 190.99bcy 0.1345a 0.04537bc 5.366ab 3.5042ab
Artemisia herba alba 136.19bx 139.86dx 0.1406a 0.14949a 2.262ab 2.5279b
Calligonum azel 30.90dx 56.62ex 0.1081a 0.05105bc 6.988a 6.0960a
SEM 11.060 0.0139 0.0110 0.4510 0.2931
Specie <.0001 0.0267 0.0003
PEG <.0001 0.0023 0.8710
Specie*PEG <.0001 0.3360 0.4220

(-PEG)1Incubated without polyethylene glycol. (+PEG)2 Incubated with polyethylene glycol.; A: Asymptotic gas production, c: Fractional rate of fermentation; L : Lag time.P*: Pericarp; L*: Leave; a, b, c, d, means in a the same row with different superscripts are significantly different (P<0.05). x, y means in the same column with different superscripts are significantly different (P<0.05). SEM: Standard error of the mean

Table 5 : Effects of polyethylene glycol l (PEG) treatment on the Digestibilities, predicted metabolizable energy (ME; MJ kgDM) and methane (mmol/g DM)
Albizia julibrissin 68.765ab 70.665a 73.855c 75.650b 75.915b 80.152ab 2.814a 2.879a 2.5535b 3.2185c
P. granatum P* 59.635bc 59.240bc 74.430c 75.985b 64.035c 73.078b 0.997e 1.1350e 2.6255b 2.449d
P. granatum L* 53.330cd 59.045b 79.985b 82.225a 55.593de 50.269c 1.165d 1.0835e 1.125d 0.880f
Vicia faba 71.980a 72.530a 87.370a 86.795a 90.457a 89.54a 2.428b 2.4135b 4.9190a 4.6665a
A. halimus 66.310ab 69.080a 83.625ab 85.570a 62.234 60.807c 1.618c 1.5965d 0.927d 2.002e
Acacia nilotica 37.535e 39.845c 51.130e 54.290d 52.287e 73.637b 1.571 1.8975c 1.7195c 3.8065b
Artemisia herba alba 45.615de 47.960c 60.445d 60.440c 54.073de 51.914c 1.233d 1.1995e 1.6315c 1.044f
Calligonum azel 27.140f 23.955d 44.315f 43.800e 32.421f 36.694d 0.5645f 0.6295f 0.009e 0.135g
SEM 3.89 4.134 3.80 3.839 4.171 4.286 0.1803 0.182 0.352 0.38405
Specie <.0001 <.0001 <.0001 <.0001 <.0001
PEG 0.1065 0.0145 0.0022 0.0005 0.0022
Specie*PEG 0.4100 0.3297 <.0001 <.0001 <.0001

(-PEG)1Incubated without polyethylene glycol. (+PEG)2 Incubated with polyethylene glycol.; AIVD: Apparent In Vitro Digestibility; TIVD: True In Vitro Digestibility; OMD: Organic Matter Digestibility; ME: Metabolisable Energy; CH4: Methane; P*: Pericarp; L*: Leave; a, b, c, d means in a the same row with different superscripts are significantly different (P<0.05); x, y means in the same column with different superscripts are significantly different (P<0.05). SEM: Standard error of the mean.

Table 6: Volatile fatty acid (mM/L) production, in supernatant after 24 h incubation of 400 mg DM of browses with or without PEG

Acetate Propionate Isobutyrate Butyrate Isovalerate Valerate TVFA Ac/Pr
A.julibrissin 31.51b 32.29b 11.78a 12.21a 0.75b 0.875a 2.64d 3.34d 0.94c 1.23b 0.54b 0.71b 48.1bx 85.31by 2.68f 2.58ef
P. granatum P* 23.01de 27.1d 6.93c 8.37c 0.38d 0.57c 3.66b 4.33b 0.52e 0.68d 0.33d 0.43c 34.83dx 66.81cdy 3.32cde 2.75e
P. granatum L* 21.38ef 19.55f 6.12d 5.170e 0.46cd 0.495c 2.83d 2.8e 0.685d 0.73d 0.360cd 0.395c 31.83ex 55.71dy 3.5bcd 4.14b
V. faba 38.45a 38.57a 12.18a 12.12a 0.94a 0.89a 4.69a 4.67a 1.305a 1.345a 1.36a 1.395a 58.92ax 105.765ay 3.16def 3.17d
A. halimus 26.86c 27.6cd 7.35bc 7.56d 0.57bc 0.625bc 3.23c 3.38d 0.935c 0.885c 0.53b 0.495c 39.47cx 72.33bcy 3.66abc 3.56c
Acacia nilotica 21.23f 28.52c 7.49b 9.45b 0.54cd 0.80a 1.69e 3.675c 0.76d 1.22b 0.47bc 0.78b 32.18ex 64.16cdy 2.84ef 2.25f
A. h.alba 24.43d 22.19e 6.24d 5.39e 0.75b 0.76ab 2.69d 2.51f 1.18b 1.145b 0.5b 0.5c 35.78dx 63.09cdy 3.92ab 4.54a
C.azel 13.635g 16.68g 3.35e 3.97f 0.49cd 0.555c 1.675e 2.2g 0.68d 0.995c 0.33d 0.38c 20.16fx 32.42ey 4.08a 3.44cd
SEM 1.798 1.70 0.714 0.754 0.046 0.038 0.241 0.207 0.065 0.059 0.082 0.082 2.81 5.21 0.122 0.189
Specie <.0001 <.0001 <.0001 <.0001 <.0001 <.0001 <.0001 <.0001
PEG <.0001 <.0001 <.0001 <.0001 <.0001 <.0001 <.0001 0.0228
Specie*PEG <.0001 <.0001 0.0016 <.0001 <.0001 0.0005 <.0001 0.1786
(-PEG)1Incubated without polyethylene glycol. (+PEG)2 Incubated with polyethylene glycol. P*: Pericarp; L*: Leave; Ac/Pr : Acetate/Propionate; a, b, c, d means in a the same row with different superscripts are significantly different (P<0.05); x, y means in the same column with different superscripts are significantly different (P<0.05). SEM: Standard error of the mean.

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