In vitro rumen fermentation kinetics, metabolite production, methane and substrate degradability of polyphenol rich plant leaves and their component complete feed blocks

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.

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.

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) 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 (t^1/2; 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].

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).

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.

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.

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).

The nutrient composition of V. sinensis hay, A. nilotica leaves, Z. nummularia leaves revealed similar CP contents (140–145 g/Kg) with a varied fiber fractions, viz. comparatively high NDF, ADF and lignin in polyphenol-rich forages than V. sinensis hay (Table 1). The CFB’s containing aforementioned roughages followed a similar pattern for these nutrients. The polyphenolic fractions of these forages revealed low total phenol (TP) in V. sinensis (3.9 g/Kg) compared to A. nilotica (163 g/Kg) and Z. nummularia (52.5 g/Kg). A. nilotica had high TTP and HT whereas NTP and CT contents were higher in Z. nummularia (21.4 and 28.3 g/Kg). The polyphenolic composition of CFB followed a similar trend as it was for the component roughage moiety.

In vitro fermentation constants, gas, methane production and substrate degradability of roughages revealed (Table 2) highest gas production (GP) in A. nilotica leaves (151 mL/g DM) followed by V. sinensis hay (137 mL/g DM) and Z. nummularia leaves (126 mL/g DM). Potential gas production (mL/200 mg DM) was significantly higher (P < 0.05) for V. sinensis hay (42.1) and A. nilotica (40.9) as compared to Z. nummularia leaves (37.9). The t½ was significantly lower (P < 0.05) in A. nilotica leaves as compared to V. sinensis hay and Z. nummularia leaves. Total methane produced per g of substrate was 28.4, 12.7 and 23.0 mL in these feed resources. DMD and OMD were higher in A. nilotica leaves (65.0, 68.8%) followed by V. sinensis hay (57.7 and 64.6%) and lowest value was observed in Z. nummularia leaves (54.9 and 60.2%). Significant (P < 0.05) difference were observed in methane production per g digestible DM/OM in different feed resources, viz. V. sinensis hay produced highest methane per unit of digestible DM and OM (49.1, 49.8 mL) followed by Z. nummularia leaves (41.9, 43.8 mL) and lowest in A. nilotica leaves (19.6, 22.3 mL). Amongst the CFBs, highest (P < 0.05) GP was recorded in CFB2 (233 mL/g DM) followed by CFB1 (221 mL/g DM) and CFB3 (184 mL/g DM) (Table 3). Potential gas production (mL/200 mg DM) was significantly lower (P < 0.05) in CFB3 as compared to CFB1 and CFB2. There was no significant difference observed for t½, rate constant and lag phase among CFBs. Methane produced per g of substrate was 50.8, 29.8 and 34.0 mL in CFBs.

Degradability of DM and OM was higher in CFB2 (82.0 and 86.6%) followed by CFB1 (79.5 and 81.8%) and lowest in CFB3 (75.6 and 80.3%) (Table 3). Similarly, significant (P < 0.05) difference were observed in methane production per unit digestible substrates, wherein CFB1 produced highest methane per unit of degradable DM and OM (63.9 and 68.3 mL) followed by CFB3 (47.5 and 47.0 mL) and lowest value (34.3 and 38.3 mL) was recorded in CFB2. The trend was in line with the roughage components (Table 2). The partitioning factor (PF) was low in A. nilotica leaves compared to other two roughages. However, the feed blocks containing these roughages showed a different pattern, being higher in CFB3 compared to CFB1 and CFB2.

A. nilotica leaves showed higher (P < 0.05) total N and NH₃-N values than the other two substrates (Table 4). TCA precipitable N content was higher (P < 0.001) in Z. nummularia leaves than A. nilotica leaves, which was higher than V. sinensis hay. The VFA profile showed higher (P < 0.05) TVFA including acetate, propionate and other VFA fractions in A. nilotica leaves as compared to Z. nummularia and V. sinensis hay. The ratio of nonglucogenic to glucogenic VFA and acetate and propionate ratio were higher (P < 0.05) in A. nilotica leaves than the Z. nummularia leaves and V. sinensis hay.

The pH was similar and total N (mg/dL) values ranged from 91.3 (CFB2) to 115.1 (CFB3) showing significant difference between the CFBs (Table 5). Conversely, the ammonia N concentration was higher (P < 0.05) in CFB2 than CFB1 and CFB3. The TCA precipitable N ranged from 44.6 in CFB2 to 78.8 in CFB1. Total VFA (mM/L) production was highest from CFB2 (48.7) followed by CFB1 (42.7) and CFB3 (25.4) and the concentration of acetate, propionate, BcFA and other FA followed a similar trend. The acetate: propionate ratio was higher (P < 0.05) in CFB3, but the ratio of nonglucogenic to glucogenic VFA was non-significantly different between the CFB types. The proportion of propionate was higher (P < 0.05) in CFB1 and CFB2 than CFB3.