Collection of forages and preparation of complete feed block
Conventional cowpea hay (Vigna sinensis) and polyphenolrich plants Acacia nilotica and Ziziphus nummularia leaves were harvested from the Agricultural Farm area of Central Sheep and Wool Research Institute, Avikanagar and dried in shade. Three different complete feed blocks (CFB) were prepared by incorporating 30 parts of these forages with concentrate mixture (65 parts) and molasses (5 parts). The composition (kg/100 kg) of concentrate mixture was maize 40, barley 36, groundnut cake 14, mustard cake 3, til cake 4, mineral mixture 2 and common salt 1. The molasses moiety was first mixed with the concentrate mixture, which followed subsequent mixing with the forages in a mechanical mixer. The composite mixture was then subjected to preparation of CFB by compressing at 5000 psi (351.5 kg/cm2) using a horizontal CFB making machine developed by NARP, Department of Agricultural Research and Education (DARE), New Delhi.
Sample preparation and analysis
Representative samples of A. nilotica and Z. nummularia leaves, cowpea (V. sinensis) hay and CFB were collected and dried in hot air oven at 50–55 °C (~ 48 h) till constant weight. The dried samples were ground to pass 1 mm screen and stored in screw capped polycarbonate vials for further analysis. Chemical composition i.e. DM, EE, ash were analyzed by the methods of AOAC [7]. Nitrogen (N) content of the sample was estimated by distilling the digested sample in distillation unit (Gerhardt Vapodest 45 s, Germany) attached to auto titrator (TitroLine easy). Fiber fractions (i.e. neutral detergent fiber, NDF; acid detergent fiber, ADF) were determined by following the method of Van Soest et al [8]. Acid detergent residue was treated with 72% H2SO4 (w/w) and ashed for acid detergent lignin (ADL) estimation. Polyphenol fractions were analyzed by the methods described by Hagerman et al [9]. Folin-Ciocalteu method was used for the determination of total phenols [10]. The condensed tannins (CT) content was analyzed with the help of butanol- HCl reagent in the presence of ferric ammonium sulphate, and CT (g/Kg DM) is expressed as leucocyanidin equivalent.
$$ \mathrm{CT}\ \left(\mathrm{g}/\mathrm{Kg}\right)=\left({\mathrm{A}}_{550}\mathrm{nm}\times 78.26\times \mathrm{Dilution}\ \mathrm{factor}\right)/\left(\%\mathrm{Dry}\ \mathrm{matter}\right)\times 10\mathrm{a} $$
Where, A550 nm is absorbance at 550 nm.
For non-tannin phenolics (NTP) estimation, accurately weighed 100 mg PVPP, 1 mL each of distilled water and tannin-containing extract was transferred to a 15 mL test tube. Thereafter, the tubes were vortexed and kept at 4 °C for 15 min. and then centrifuged at 3000 rpm for 10 min to collect the supernatant, which was estimated for NTP by Folin-Ciocalteu method [10] and expressed as g/Kg DM. Total tannin phenols (TTP) were calculated as the difference between TP and NTP. Hydrolysable tannin (HT) was calculated as the difference between TTP and CT.
In vitro gas production (IVGP) test
In vitro gas production (IVGP) technique of Menke et al [11] was followed for ruminal fermentation in glass syringes. Rumen liquor was collected from adult male rams being fed near maintenance in the morning (before feeding) with the help of stomach tube attached to a suction pump. It was transferred to a pre-warmed CO2 filled thermos, and immediately carried to the laboratory (max - 30 min). Rumen fluid from different rams collected was mixed in equal proportion, and filtered through four layered muslin cloth under continuous flushing of CO2 to maintain anaerobiosis. Oven-dried samples (200 mg) in triplicate were weighed into 100 mL calibrated glass syringes fitted with plungers. Syringes were filled with 30 mL of medium consisting of 10 mL rumen fluid and 20 mL buffer solution. Three blank syringes were also incubated with only 30 mL of the medium. The syringes were placed in hot water bath cum shaker maintained at 39 °C. Gas production (GP) was recorded after 2, 4, 6, 8, 10 12, 18, 24, 30, 36, 48 72 and 96 h of incubation. Net GP by each sample during the above mentioned period was calculated by subtracting the gas produced of the blank. The data so generated was processed as per Sigmastat Software (version 3.5) for calculating time to reach half asymptote (t1/2; h), potential GP (mg/200 mg substrate), rate constant (c) and lag phase (L; h). The GP kinetic parameters were calculated from the time dependent (0 to 96 h) in vitro cumulative GP data by applying single pool logistic model as depicted below. The assumptions were made that the rate of GP is proportional to both the accumulated microbial mass and to the amount of digestible substrate remaining [12].
$$ \mathrm{Y}\ \left(\mathrm{t}\right)=\mathrm{b}/\left[\right(1+\exp\ \left\{\right(2+4\mathrm{c}\ \left(\mathrm{L}\hbox{-} \mathrm{t}\right)\left\}\right] $$
Where, Y (t) = GP (mL) after time t, b = asymptotic value of the component (total potential GP, mL), c = specific rate of fermentation and L = lag time (the time axis intercept).
Gas production and methane assay
After calculation of fermentation constants, two sets of samples each in triplicate were run simultaneously. In set one 200 mg and in set two 400 mg of oven dried samples weighed in to 100-mL glass syringes fitted with waxed plungers and were incubated with rumen buffer medium. In set one 30 mL and in set two 40 mL of the medium (with double strength buffer) was added as per the modified method of Menke and Steingass [13]. Samples were incubated in hot water bath cum shaker maintained at 39 °C up to 24 h and total GP was recorded. The gas sample from the first set was analyzed for methane concentration and fermentation was terminated by keeping the syringes in ice water. Methane (CH4) was analyzed by Gas Chromatograph (Model 1000, Series 011124002 of DANI make, Italy) using FID with PTV column. The temperature of injection port was 120 °C; column 50 °C; detector 120 °C. The flow rate of carrier gas (nitrogen) was 30 mL/min; hydrogen 30 mL/min; air 300 mL/min. The standard gas used for methane estimation composed of 99.998% methane (Sigma-Aldrich; Missouri, United States). Methane concentration was calculated by comparing the peak area of standard with samples. Methane production was calculated by applying the following equation.
$$ {\displaystyle \begin{array}{l}{\mathrm{CH}}_4\left(\mathrm{mL}\right)/100\ \mathrm{mg}\ \mathrm{digested}\ \mathrm{OM}=100\times \Big\{\left(\mathrm{GP}\ {\mathrm{t}}^{1/2}\times \left[{\mathrm{CH}}_4{\mathrm{t}}^{1/2}\right]\right)\ \\ {}-\left(\mathrm{GP}\ \mathrm{in}\ \mathrm{blank}\ {\mathrm{t}}^{1/2}\times \left[{\mathrm{CH}}_4\mathrm{of}\ \mathrm{blank}\ {\mathrm{t}}^{1/2}\right]\right)\Big\}/\mathrm{mg}\ \mathrm{digested}\ \mathrm{OM}\end{array}} $$
Substrate degradability
The sample in set two (400 mg sample) syringes after incubation for 24 h was transferred to 600 mL spoutless beaker and 100 mL of neutral detergent solution was added after washing the in vitro syringes with the same solution (for ensuring quantitative transfer) and refluxed for 1 h as practiced during NDF assay. The samples were then filtered and washed through pre-weighed sintered glass crucibles (G-1) and the residue was dried in hot air oven at 100 °C for 24 h and weighed. The crucible with residue was incinerated in muffle furnace at 600 °C for 4 h and weighed next day after cooling. OM digestibility was calculated after necessary corrections for the blank samples.
Statistical analysis
Analysis of variance was employed for data analysis following General Linear Model (GLM) procedure using statistical software SPSS (version 16). Tukey’s test was utilized to compare significant differences (P < 0.05) among the means for the two set of substrates (roughages and CFBs).