The present study was undertaken to investigate the effect of crude seed powder (CSP) and gross saponins extract (GSE) of seeds of
Methane production during anaerobic fermentation of nutrients in the rumen is an essential metabolic but nutritionally wasteful process which represents 2 to 15% of gross energy loss [
Seeds of
Seed powder was defatted in petroleum ether (boiling range 40–60°C) refluxing for 6 h in a soxhlet’s apparatus at 45°C. The contents were filtered and 25 g fat-free sample was diluted with absolute methanol taken in 1 : 10 ratio. The contents were shaken at 25°C and 120 rpm for 24 h followed by centrifuge for 20 min at 3500 rpm for 20 min. Methanol extract was filtered through Whatman filter paper Number 1 and dried under rotary evaporator. Dried methanolic plant extract was dissolved in distilled water (10 mL), transferred in to a separating funnel, and extracted with equal volume of
Gross saponins extract (GSE) was extracted by previously described method [
Total saponins contents of
Two Gram-positive and two Gram-negative bacteria and two fungi species were employed to determine the antimicrobial action of
Antibacterial and antifungal activities of the crude seed powder (CSP) and gross saponins extract (GSE) were tested using agar well diffusion method as described previously [
Suspensions of fungal spores were prepared from 5–7 days old cultures that grew at 28°C on a SDA plates which were prepared by pour plating using fungal spores of
The substrates used in incubation were prepared by taking different roughage and concentrate ratio, that is, high fiber diet (
All the treatments, that is, 6% (DM basis) of gross saponins extract (GSE) and saponin fractions A and B were arranged in factorial randomized block design (RBD) with three replicates. Sets were also incubated devoid of substrate with and without supplementation which served as blanks for particular treatment and values were corrected for different parameters with blanks. The experiment was conducted in 100 mL calibrated glass syringes containing 200 ± 5 mg of substrates with 6% GSE and 6% of saponins fraction A and B, respectively, and then 30 mL reduced buffer medium [
After 48 h incubation, total gas (TG) was estimated by the extent of displacement of piston of glass syringes. TG produced due to fermentation of substrate was corrected by subtracting TG produced in blank syringe (containing inoculum and buffer but not the substrate) from total gas produced in the syringe containing substrate, inoculum, and buffer. Methane concentration in representative gas samples was estimated by using gas chromatograph (Nucon-5765, India) equipped with flame ionization detector (FID) and stainless steel column packed with Porapak-Q (length 6′; o.d.1/8” i. d. 2 mm; mesh range 80–100). The gas flow rates for nitrogen, hydrogen, and air were 30, 30, and 300 mL/min, respectively. Temperature of injector oven, column oven, and detector were 40, 50, and 50°C, respectively.
The true DM degradability of feed sample of each syringe containing residues after incubation was estimated as per method [
For the estimation of individual volatile fatty acids, 4 mL of 25% metaphosphoric acid was added to 1 mL of incubation sample; the mixture was mixed uniformly and left as such for 3-4 h at ambient temperature [
The PF is calculated as the ratio of substrate truly degraded
Content of the glass syringes containing
The total gas production kinetics and cumulative methane gas production were carried out in
Experimental data of different parameters were analyzed in randomized block design with three replicates for analysis of variance [
The crude seed powder (CSP) and gross saponins extract (GSE) of
Tested microorganisms | Zone of inhibition (CSP) | Zone of inhibition (GSE) | Positive control | ||||||||||
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Types | Names | Specimen number | Concentration (µg) | ||||||||||
500 | 250 | 100 | 50 | 25 | 500 | 250 | 100 | 50 | 25 | ||||
Gram-negative bacteria |
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NCDC 135 | 7.2 | 4.8 | 3.3 | 1.3 | nz | 12.8 | 8.9 | 7.3 | 4.0 | 1.9 | 17.7a |
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NCDC 113 | 3.9 | 2.6 | 1.1 | nz | nz | 9.2 | 5.9 | 5.7 | 2.8 | nz | 14.7a | |
Gram-positive bacteria |
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MTCC 1144 | 3.4 | 2.1 | 0.9 | nz | nz | 7.4 | 6.2 | 5.0 | 2.3 | nz | 9.3b |
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NCDC 240 | 7.8 | 4.4 | 2.3 | 1.8 | nz | 13.3 | 10.1 | 6.3 | 3.3 | 2.0 | 16.7b | |
Fungi |
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NCDC 280 | 0.8 | nz | nz | nz | nz | 3.2 | 1.2 | nz | nz | nz | 6.0c |
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NCDC 315 | 6.7 | 4.3 | 1.0 | nz | nz | 11.8 | 8.5 | 3.0 | 1.4 | nz | 13.2c |
Sterile broth medium as negative control.
Nz: No zone.
The results of present experiments indicated that the zones of inhibition for Gram-negative and Gram-positive bacteria were increased with increasing of concentration of treatments, that is, CSP and GSE. Gram-negative bacteria
The ingredient and chemical compositions of diets containing different roughage and concentration ratio were presented in Table
Chemical composition of diets.
Diets | Chemical constituents of diets (g/kg on DM basis) | ||
---|---|---|---|
(D1) |
(D2) |
(D3) | |
OM | 867.6 | 878.4 | 875.6 |
CP | 108.6 | 125.3 | 142.7 |
EE | 23.4 | 30.4 | 34.8 |
NDF | 623.1 | 604.5 | 538.7 |
ADF | 372.0 | 329.5 | 298.7 |
HC | 251.1 | 275.0 | 240.0 |
TA | 132.4 | 121.6 | 124.4 |
D1: high fiber diet, D2: medium fiber diet, D3: low fiber diet, OM: organic matter, CP: crude protein, EE: ether extract, NDF: neutral detergent fiber, ADF: acid detergent fiber, HC: hemicelluloses, TA: total Ash.
Effects of
Parameters | Diets | ||||||||||||||
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D1 |
D2 |
D3 |
SEM | ||||||||||||
Control | GSE | Sap A | Sap B | Control | GSE | Sap A | Sap B | Control | GSE | Sap A | Sap B | Diet | Treatment | D * T | |
IVDMD% |
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0.645 | 0.744 | 1.289 |
PF |
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0.054 | 0.062 | 0.107 |
MBM |
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1.763 | 2.036 | 3.526 |
CH4 |
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2.875 | 3.319 | 5.750 |
CH4 |
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0.115 | 0.133 | 0.230 |
M/TDS |
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0.021 | 0.024 | 0.041 |
D1: high fiber diet, D2: medium fiber diet, D3: low fiber diet, GSE: gross saponins extract, IVDMD:
Effects of
Parameters | Diets | ||||||||||||||
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D1 (60 |
D2(50 |
D3(40 |
SEM | ||||||||||||
Control | GSE | Sap A | Sap B | Control | GSE | Sap A | Sap B | Control | GSE | Sap A | Sap B | Diet | Treatment | D * T | |
Acetate |
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0.165 | 0.191 | 0.330 |
Propionate (mM/100 mL) |
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0.064 | 0.077 | 0.133 |
Butyrate |
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0.046 | 0.053 | 0.092 |
C2 : C3 |
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0.119 | 0.137 | 0.238 |
NH3-N |
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0.092 | 0.106 | 0.183 |
D1: high fiber diet, D2: medium fiber diet, D3: low fiber diet, GSE: gross saponins extract, C2 : C3: acetate to propionate ratio, NH3-N: ammonia nitrogen, SEM: standard error of means.
Effects of
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| |
Control |
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0.993 |
GSE |
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0.989 |
Sap A |
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0.986 |
Sap B |
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0.988 |
GSE: gross saponins extract,
In the current experiment, results indicated that IVDMD values were increased as compared to control and the differences among treatments values were significant (
In present study the partition factor (PF) values and microbial biomass (mg) production decreased with all saponins supplementation in
Results of present study indicated that methane production was decreased in saponins extracted from
Results of the present experiment showed that the reduction of methane production per 200 mg of truly digested substrate (TDS) was highest in saponin fraction A with all diets, when compared with control (Table
In the rumen, the methane production also depends upon the association between methanogens, protozoa, and rate of methane production per methanogenic cell [
The individual volatile fatty acids (IVFAs) concentration varied among the treatments (Table
Results of current experiment indicated that the concentration of propionate slightly was affected by
The ammonia nitrogen (mg/100 mL) was decreased due to the
Results related to gas kinetics in
Effects of
In current study, the results of cumulative methane gas production (mL/gDM) were presented in Table
Effects of
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Control |
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0.992 |
GSE |
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0.982 |
Sap A |
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0.979 |
Sap B |
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0.981 |
GSE: gross saponins extract,
Effects of
In present study, results of quantification of methanogens and protozoal population are presented in Table
Real-time PCR quantification of changes in rumen microbial population on supplementation of
Microbial groups | Control diet (D2) | D2 + GSE | D2 + Sap A | D2 + Sap B | SEM | CD |
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MMB | 1.00 | 0.318 | 0.484 | 1.581 | 0.091 | 0.321 |
Protozoa | 1.00 | 0.940 | 0.649 | 0.985 | 0.070 | 0.247 |
mcrA | 1.00 | 0.584 | 3.198 | 3.392 | 0.881 | N.S. |
MMB: methanomicrobiales; mcr A: total methanogens quantified using mcr A gene; in control diet population of all microbes considered as 1.00 then relative abundance is calculated with treatment.
In present study it is concluded that saponins fraction A of
The authors declare that there is no conflict of interests regarding the publication of this paper.
Authors are thankful to National Fund for Basic and Strategic Research in Agriculture (NFBSRA), Indian Council of Agricultural Research (ICAR), New Delhi, for provide the financial support under Grant F. no. NFBSRA/PCN/AP-10/2006-07 to carry out this research work.