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Background: This experiment aimed at assessing polyphenol-rich plant biomass to use in complete feed making for the feeding of ruminants. Methods: An in vitro ruminal evaluation of complete blocks (CFB) with (Acacia nilotica, Ziziphus nummularia leaves) and without (Vigna sinensis hay) polyphenol rich plant leaves was conducted by applying Menke’s in vitro gas production (IVGP) technique. A total of six substrates, viz. three forages and three CFBs were subjected to in vitro ruminal fermentation in glass syringes to assess gas and methane production, substrate degradability, and rumen fermentation metabolites. Results: Total polyphenol content (g/Kg) was 163 in A. nilotica compared to 52.5 in Z. nummularia with a contrasting difference in tannin fractions, higher hydrolysable tannins (HT) in the former (140.1 vs 2.8) and higher condensed (CT) tannins in the later (28.3 vs 7.9). The potential gas production was lower with a higher lag phase (L) in CT containing Z. nummularia and the component feed block. A. nilotica alone and as a constituent of CFB produced higher total gas but with lower methane while the partitioning factor (PF) was higher in Z. nummularia and its CFB. Substrate digestibility (both DM and OM) was lower (P < 0.001) in Z. nummularia compared to other forages and CFBs. The fermentation metabolites showed a different pattern for forages and their CFBs. The forages showed higher TCA precipitable N and lower acetate: propionate ratio in Z. nummularia while the related trend was found in CFB with V. sinensis. Total volatile fatty acid concentration was higher (P < 0.001) in A. nilotica leaves than V. sinensis hay and Z. nummularia leaves. It has implication on widening the forage resources and providing opportunity to use forage biomass rich in polyphenolic constituents in judicious proportion for reducing methane and enhancing green livestock production. Conclusion: Above all, higher substrate degradability, propionate production, lower methanogenesis in CFB with A. nilotica leaves may be considered useful. Nevertheless, CFB with Z. nummularia also proved its usefulness with higher TCA precipitable N and PF. It has implication on widening the forage resources and providing opportunity to use polyphenol-rich forage biomass for reducing methane and enhancing green livestock production. Keywords: Polyphenol, Methane production, Fermentation metabolites, degradability * Correspondence: firstname.lastname@example.org Animal Nutrition Division, ICAR- Indian Veterinary Research Institute, 243122, Izatnagar, UP, India Full list of author information is available at the end of the article © The Author(s). 2018 Open Access This article is distributed under the terms of the Creative Commons Attribution 4.0 International License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated. Aderao et al. Journal of Animal Science and Technology (2018) 60:26 Page 2 of 9 Background subsequent mixing with the forages in a mechanical Browses and trees invariably find their place in ruminant mixer. The composite mixture was then subjected to ration in most of the tropical countries. Polyphenols are preparation of CFB by compressing at 5000 psi one of the principal components of these feed resources, (351.5 kg/cm ) using a horizontal CFB making machine which have multiple di- and/or tri-hydroxyphenyl units developed by NARP, Department of Agricultural Re- to classify them as 1) condensed tannins, 2) hydrolysable search and Education (DARE), New Delhi. tannins and 3) phlorotannins . These constituents have huge structural diversity and their interactions with Sample preparation and analysis intrinsic (plant) and extrinsic (microbes) proteins have a Representative samples of A. nilotica and Z. nummu- profound effect on the outcome of expected positive ef- laria leaves, cowpea (V. sinensis) hay and CFB were col- fects on ruminal fermentation attributes and implica- lected and dried in hot air oven at 50–55 °C (~ 48 h) till tions of their use in preparation of complete feed for constant weight. The dried samples were ground to pass ruminants. Exploitation of plant biomass rich in one or 1 mm screen and stored in screw capped polycarbonate the other component polyphenols to manipulate rumen vials for further analysis. Chemical composition i.e. DM, microbial population including methanogens and EE, ash were analyzed by the methods of AOAC . Ni- thereby harnessing positive rumen fermentation metabo- trogen (N) content of the sample was estimated by distil- lites is considered a useful approach. Methane emission ling the digested sample in distillation unit (Gerhardt from enteric fermentation and manure management are Vapodest 45 s, Germany) attached to auto titrator some of the major sources of livestock GHG emission (TitroLine easy). Fiber fractions (i.e. neutral detergent and several studies have been conducted to evaluate ef- fiber, NDF; acid detergent fiber, ADF) were determined fect of various plant secondary metabolites (PSM) for by following the method of Van Soest et al . Acid de- reducing methane production [2–4]. Various phyto- tergent residue was treated with 72% H SO (w/w) and 2 4 chemicals like saponins and tannins have been shown to ashed for acid detergent lignin (ADL) estimation. Poly- modulate rumen fermentation favourably, and to inhibit phenol fractions were analyzed by the methods de- methane production in the rumen [3, 4]. scribed by Hagerman et al . Folin-Ciocalteu method Leaves with high nutrient content, digestibility and was used for the determination of total phenols . low methane production can be used by marginal and The condensed tannins (CT) content was analyzed with landless farmers as supplementary feed resource for the the help of butanol- HCl reagent in the presence of fer- feeding of small ruminants. Use of browse species con- ric ammonium sulphate, and CT (g/Kg DM) is expressed taining secondary compounds as feed supplement for ru- as leucocyanidin equivalent. minants in many parts of the tropics is increasing in CTðÞ g=Kg ¼ðÞ A nm 78:26 Dilution factor = order to improve animal performance by diverting en- 550 ergy loss through methane towards production [5, 6]. ðÞ %Dry matter 10a The present study was thus aimed at evaluating the ef- fect of polyphenol rich plant leaves alone or as compo- Where, A nm is absorbance at 550 nm. nent of complete feed block (CFB) on in vitro rumen For non-tannin phenolics (NTP) estimation, accurately fermentation attributes, methane production and sub- weighed 100 mg PVPP, 1 mL each of distilled water and strate degradability. tannin-containing extract was transferred to a 15 mL test tube. Thereafter, the tubes were vortexed and kept Methods at 4 °C for 15 min. and then centrifuged at 3000 rpm for Collection of forages and preparation of complete feed 10 min to collect the supernatant, which was estimated block for NTP by Folin-Ciocalteu method  and expressed Conventional cowpea hay (Vigna sinensis) and polyphe- as g/Kg DM. Total tannin phenols (TTP) were calcu- nolrich plants Acacia nilotica and Ziziphus nummularia lated as the difference between TP and NTP. Hydrolys- leaves were harvested from the Agricultural Farm area able tannin (HT) was calculated as the difference of Central Sheep and Wool Research Institute, Avikana- between TTP and CT. gar and dried in shade. Three different complete feed blocks (CFB) were prepared by incorporating 30 parts of In vitro gas production (IVGP) test these forages with concentrate mixture (65 parts) and In vitro gas production (IVGP) technique of Menke et al molasses (5 parts). The composition (kg/100 kg) of con-  was followed for ruminal fermentation in glass sy- centrate mixture was maize 40, barley 36, groundnut ringes. Rumen liquor was collected from adult male cake 14, mustard cake 3, til cake 4, mineral mixture 2 rams being fed near maintenance in the morning (before and common salt 1. The molasses moiety was first feeding) with the help of stomach tube attached to a mixed with the concentrate mixture, which followed suction pump. It was transferred to a pre-warmed CO 2 Aderao et al. Journal of Animal Science and Technology (2018) 60:26 Page 3 of 9 filled thermos, and immediately carried to the laboratory estimation composed of 99.998% methane (Sigma-Al- (max - 30 min). Rumen fluid from different rams col- drich; Missouri, United States). Methane concentration lected was mixed in equal proportion, and filtered was calculated by comparing the peak area of standard through four layered muslin cloth under continuous with samples. Methane production was calculated by ap- flushing of CO to maintain anaerobiosis. Oven-dried plying the following equation. samples (200 mg) in triplicate were weighed into hi 1=2 1=2 CHðÞ mL =100 mg digested OM ¼ 100 f GP t CH t 4 4 100 mL calibrated glass syringes fitted with plungers. Sy- hi 1=2 1=2 ringes were filled with 30 mL of medium consisting of GP in blank t CH of blank t g=mg digested OM 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 Substrate degradability cum shaker maintained at 39 °C. Gas production (GP) The sample in set two (400 mg sample) syringes after in- was recorded after 2, 4, 6, 8, 10 12, 18, 24, 30, 36, 48 72 cubation for 24 h was transferred to 600 mL spoutless and 96 h of incubation. Net GP by each sample during beaker and 100 mL of neutral detergent solution was the above mentioned period was calculated by subtract- added after washing the in vitro syringes with the same ing the gas produced of the blank. The data so generated solution (for ensuring quantitative transfer) and refluxed was processed as per Sigmastat Software (version 3.5) for 1 h as practiced during NDF assay. The samples were 1/2 for calculating time to reach half asymptote (t ; h), po- then filtered and washed through pre-weighed sintered tential GP (mg/200 mg substrate), rate constant (c) and glass crucibles (G-1) and the residue was dried in hot air lag phase (L; h). The GP kinetic parameters were calcu- oven at 100 °C for 24 h and weighed. The crucible with lated from the time dependent (0 to 96 h) in vitro cumu- residue was incinerated in muffle furnace at 600 °C for lative GP data by applying single pool logistic model as 4 h and weighed next day after cooling. OM digestibility depicted below. The assumptions were made that the was calculated after necessary corrections for the blank rate of GP is proportional to both the accumulated mi- samples. crobial mass and to the amount of digestible substrate remaining . Statistical analysis Analysis of variance was employed for data analysis fol- YtðÞ ¼ b=½ð1 þ exp fð2 þ 4c ðL‐tÞg lowing General Linear Model (GLM) procedure using statistical software SPSS (version 16). Tukey’s test was Where, Y (t) = GP (mL) after time t, b = asymptotic utilized to compare significant differences (P < 0.05) value of the component (total potential GP, mL), c = spe- among the means for the two set of substrates (rough- cific rate of fermentation and L = lag time (the time axis ages and CFBs). intercept). Results Gas production and methane assay Nutrient and polyphenolic composition After calculation of fermentation constants, two sets of The nutrient composition of V. sinensis hay, A. nilotica samples each in triplicate were run simultaneously. In leaves, Z. nummularia leaves revealed similar CP contents set one 200 mg and in set two 400 mg of oven dried (140–145 g/Kg) with a varied fiber fractions, viz. compara- samples weighed in to 100-mL glass syringes fitted with tively high NDF, ADF and lignin in polyphenol-rich for- waxed plungers and were incubated with rumen buffer ages than V. sinensis hay (Table 1). The CFB’scontaining medium. In set one 30 mL and in set two 40 mL of the aforementioned roughages followed a similar pattern for medium (with double strength buffer) was added as per these nutrients. The polyphenolic fractions of these for- the modified method of Menke and Steingass . Sam- ages revealed low total phenol (TP) in V. sinensis (3.9 g/ ples were incubated in hot water bath cum shaker main- Kg) compared to A. nilotica (163 g/Kg) and Z. nummu- tained at 39 °C up to 24 h and total GP was recorded. laria (52.5 g/Kg). A. nilotica had high TTP and HT The gas sample from the first set was analyzed for me- whereas NTP and CT contents were higher in Z. nummu- thane concentration and fermentation was terminated laria (21.4 and 28.3 g/Kg). The polyphenolic composition by keeping the syringes in ice water. Methane (CH )was of CFB followed a similar trend as it was for the compo- analyzed by Gas Chromatograph (Model 1000, Series nent roughage moiety. 011124002 of DANI make, Italy) using FID with PTV column. The temperature of injection port was 120 °C; In vitro gas and methane production column 50 °C; detector 120 °C. The flow rate of carrier In vitro fermentation constants, gas, methane produc- gas (nitrogen) was 30 mL/min; hydrogen 30 mL/min; air tion and substrate degradability of roughages revealed 300 mL/min. The standard gas used for methane (Table 2) highest gas production (GP) in A. nilotica Aderao et al. Journal of Animal Science and Technology (2018) 60:26 Page 4 of 9 Table 1 Chemical and polyphenol constituents (g/kg dry matter) of roughage components and complete feed block (CFB) Constituents Vigna sinensis hay Acacia nilotica leaves Ziziphus nummularia leaves CFB 1 CFB 2 CFB 3 DM 892 902 865 888 902 899 OM 882 876 874 909 896 902 Ash 118 124 126 91 104 97.8 CP 144 145 140 153 155 150 EE 25.4 25.7 32.1 52.1 53 54.8 NDF 608 695 657 374 400 389 ADF 451 468 473 252 257 259 ADL 108 124 196 60.4 65 86.6 Hemicellulose 157 227 184 122 143 130 Cellulose 343 344 277 192 192 172 Total phenols 3.9 163 52.5 2.7 45.2 16.3 Total tannin phenols 2.9 148 31.1 2.1 40.4 9.4 Non-tannin phenols 1 15 21.4 0.6 4.8 6.9 Condensed tannins 1.2 7.9 28.3 1.8 2.3 8.2 Hydrolysable tannins 1.7 140.1 2.8 0.3 38.1 1.2 CFB1 Concentrate mixture + Vigna sinensis hay (70:30), CFB2 Concentrate mixture + Acacia nilotica leaves (70:30), CFB3 concentrate mixture + Ziziphus nummularia leaves (70:30) leaves (151 mL/g DM) followed by V. sinensis hay leaves (65.0, 68.8%) followed by V. sinensis hay (57.7 and (137 mL/g DM) and Z. nummularia leaves (126 mL/g 64.6%) and lowest value was observed in Z. nummularia DM). Potential gas production (mL/200 mg DM) was leaves (54.9 and 60.2%). Significant (P < 0.05) difference significantly higher (P < 0.05) for V. sinensis hay (42.1) were observed in methane production per g digestible and A. nilotica (40.9) as compared to Z. nummularia DM/OM in different feed resources, viz. V. sinensis hay leaves (37.9). The t½ was significantly lower (P < 0.05) in produced highest methane per unit of digestible DM A. nilotica leaves as compared to V. sinensis hay and Z. and OM (49.1, 49.8 mL) followed by Z. nummularia nummularia leaves. Total methane produced per g of leaves (41.9, 43.8 mL) and lowest in A. nilotica leaves substrate was 28.4, 12.7 and 23.0 mL in these feed re- (19.6, 22.3 mL). Amongst the CFBs, highest (P < 0.05) sources. DMD and OMD were higher in A. nilotica GP was recorded in CFB2 (233 mL/g DM) followed by Table 2 In vitro fermentation constants, degradability and methane production of different forages Attributes Vigna sinensis Hay Acacia nilotica leaves Ziziphus nummularia leaves SEM Significance (P value) Fermentation kinetics Potential gas production (ml/200 mg DM) 42.1 40.9 37.9 0.926 0.021 Rate constant (c) 0.044 0.061 0.049 0.005 0.001 1/2 Half time (t , h) 15.6 11.4 14.0 0.71 0.018 Lag phase(L, h) 1.90 3.33 5.37 1.16 0.012 Degradability and methane production b a c DMD % 57.7 65.0 54.9 0.31 < 0.001 b a c OMD % 64.6 68.8 60.2 0.59 < 0.001 b a c Gas production (mL/g) 137 151 126 3.1 < 0.001 a ab b Gas production (mL/g DDM) 237 232 229 2.5 0.024 Gas production (mL/g DOM) 240 250 239 5.2 0.541 a c b Methane production (mL/g DM) 28.4 12.7 23.0 0.95 < 0.001 a c b Methane production (mL/g DDM) 49.1 19.6 41.9 1.36 < 0.001 a c b Methane production (mL/g OMD) 49.8 22.3 43.8 0.70 < 0.001 b a b Partitioning factor (g/mL) 4.16 3.99 4.18 0.038 0.042 a, b, c Means bearing different superscript ( ) differ significantly μm = maximum rate of GP (b × c) At the inflection point (Y = b/2), i.e. inflection occurs halfway to the maximum gas volume and thus, t =L + (μm × b/2) 1/2 Aderao et al. Journal of Animal Science and Technology (2018) 60:26 Page 5 of 9 CFB1 (221 mL/g DM) and CFB3 (184 mL/g DM) and other VFA fractions in A. nilotica leaves as compared (Table 3). Potential gas production (mL/200 mg DM) to Z. nummularia and V. sinensis hay. The ratio of non- was significantly lower (P < 0.05) in CFB3 as compared glucogenic to glucogenic VFA and acetate and propionate to CFB1 and CFB2. There was no significant difference ratio were higher (P <0.05) in A. nilotica leaves than the observed for t½, rate constant and lag phase among Z. nummularia leaves and V. sinensis hay. CFBs. Methane produced per g of substrate was 50.8, The pH was similar and total N (mg/dL) values ranged 29.8 and 34.0 mL in CFBs. from 91.3 (CFB2) to 115.1 (CFB3) showing significant difference between the CFBs (Table 5). Conversely, the Substrate degradability and partitioning factor ammonia N concentration was higher (P < 0.05) in CFB2 Degradability of DM and OM was higher in CFB2 (82.0 than CFB1 and CFB3. The TCA precipitable N ranged and 86.6%) followed by CFB1 (79.5 and 81.8%) and low- from 44.6 in CFB2 to 78.8 in CFB1. Total VFA (mM/L) est in CFB3 (75.6 and 80.3%) (Table 3). Similarly, signifi- production was highest from CFB2 (48.7) followed by cant (P < 0.05) difference were observed in methane CFB1 (42.7) and CFB3 (25.4) and the concentration of production per unit digestible substrates, wherein CFB1 acetate, propionate, BcFA and other FA followed a simi- produced highest methane per unit of degradable DM lar trend. The acetate: propionate ratio was higher and OM (63.9 and 68.3 mL) followed by CFB3 (47.5 and (P < 0.05) in CFB3, but the ratio of nonglucogenic to glu- 47.0 mL) and lowest value (34.3 and 38.3 mL) was re- cogenic VFA was non-significantly different between the corded in CFB2. The trend was in line with the roughage CFB types. The proportion of propionate was higher components (Table 2). The partitioning factor (PF) was (P < 0.05) in CFB1 and CFB2 than CFB3. low in A. nilotica leaves compared to other two rough- ages. However, the feed blocks containing these rough- Discussion ages showed a different pattern, being higher in CFB3 The nutrient composition of the three roughage moieties compared to CFB1 and CFB2. in the CFB, V. sinensis hay, A. nilotica leaves and Z. nummularia leaves are in line with the documented in- Rumen fermentation metabolites formation [14, 15]. Total polyphenolic constituents in A. A. nilotica leaves showed higher (P < 0.05) total N and nilotica was higher than Z. nummularia leaves, but the NH -N values than the other two substrates (Table 4). tannin polyphenolic fractions (g/Kg DM) revealed a dif- TCA precipitable N content was higher (P < 0.001) in Z. ferent pattern, the former was rich in HT (140) while nummularia leaves than A. nilotica leaves, which was the later was rich in CT (28.3). Similar polyphenolic higher than V. sinensis hay. The VFA profile showed composition in these tree leaves has also been reported higher (P < 0.05) TVFA including acetate, propionate earlier [16, 17] and in CFB, the level of polyphenolic Table 3 In vitro fermentation constants, degradability and methane production of complete feed block (CFB) Attributes CFB1 CFB2 CFB3 SEM Significance (P value) Fermentation kinetics Potential Gas Production (ml/200 mg DM) 61.9 61.4 55.4 1.096 0.031 Rate Constant (c) 0.059 0.064 0.060 0.005 0.082 1/2 Half time (t h) 11.7 10.8 11.6 1.12 1.121 Lag phase (h) 1.69 3.23 3.75 1.607 0.094 Degradability and methane production a a c DMD % 79.5 82.0 74.0 0.609 < 0.001 b a c OMD % 81.8 86.6 80.3 0.379 < 0.001 a a b Gas production (mL/g) 221 233 184 5.34 < 0.001 Gas production (mL/g DDM) 278 284 246 6.43 0.152 a a b Gas production (mL/g DOM) 297 300 254 6.45 0.012 a c b Methane production (mL/g DM) 50.8 29.8 34.0 1.64 < 0.001 a c b Methane production (mL/g DDM) 63.9 36.3 45.9 2.11 < 0.001 a c b Methane production (mL/g DOM) 68.3 38.3 47.0 0.809 < 0.001 a a b Partitioning factor (g/mL) 3.36 3.33 3.94 0.016 < 0.001 a, b, c Means bearing different superscript ( ) differ significantly CFB1 Concentrate mixture + Vigna sinensis hay (70:30), CFB2 Concentrate mixture + Acacia nilotica leaves (70:30), CFB3 Concentrate mixture + Ziziphus nummularia leaves (70:30) Aderao et al. Journal of Animal Science and Technology (2018) 60:26 Page 6 of 9 Table 4 In vitro ruminal fermentation metabolites of different forages Attributes Roughage SEM Significance (P value) Vigna sinensis hay Acacia nilotica leaves Ziziphus nummularia leaves c b a Total N (mg/dL) 47.3 57.1 60.5 0.459 < 0.001 b a b Ammonia N (mg/dL) 16.4 17.3 16.4 0.209 0.046 b c a TCA precipitable N (mg/dL) 15.7 10.8 21.8 0.971 < 0.001 b a c Acetic acid (mM/L) 26.3 31.3 11.1 0.759 < 0.001 a a b Propionic acid (mM/L) 5.31 5.06 2.60 0.159 < 0.001 a a b Isobutyric acid (mM/L) 0.88 0.89 0.26 0.021 < 0.001 a a b Butyric acid (mM/L) 2.89 2.68 2.28 0.069 0.003 a a b Isovaleric acid (mM/L) 0.75 0.89 0.24 0.039 < 0.001 Valeric acid (mM/L) 0.37 0.34 0.64 0.103 0.137 b a c Total VFA (mM/L) 36.5 41.2 17.1 0.911 < 0.001 a a b Branch-chain fatty acids (mM/L) 2.00 2.13 1.15 0.119 0.002 b a c Acetate: propionate ratio 4.96 6.18 4.25 0.11 < 0.001 b a b Non-glucogenic: glucogenic VFA ratio 4.66 5.40 4.47 0.11 0.002 a, b, c Means bearing different superscript ( ) differ significantly TCA Trichloroaceticic acid, VFA Volatile fatty acids constituents followed the trend as that in respective leaves and V. sinensis hay. A similar trend in OM degrad- roughage source. ability between the substrates could also be attributed to The three roughage substrates were degraded differently this variation in chemical constituents. Alteration in sub- in the in vitro ruminal fermentation system. The substrate strate degradability due to differences in chemical com- from A. nilotica leaves was degraded at the highest rate position is quite obvious [3, 16]. Consequently, t was and it was not affected by the increased level of HT. On lowest in A. nilotica leaves followed by Z. nummularia the contrary, Z. nummularia leaves showed a lower de- leaves and highest in V. sinensis hay. This was in direct re- gradability value compared to conventional V. sinensis lation with the degradability, as stated earlier by Sahoo et hay, due probably to the presence of higher CT and lignin al  that halfway time to maximum gas volume is posi- content . These values were comparable in A. nilotica tively correlated with speed of microbial attachment and Table 5 In vitro ruminal fermentation metabolites of complete feed block (CFB) Attributes Complete feed block CFB1 CFB2 CFB3 SEM Significance (P value) b c a Total N (mg/dL) 103 91.3 115 1.52 < 0.001 b a b Ammonia N (mg/dL) 18.1 20.8 17.8 0.209 < 0.001 a c b TCA precipitable N (mg/dL) 78.8 44.6 70.3 1.67 < 0.001 a a b Acetic acid (mM/L) 29.8 33.6 17.9 1.73 < 0.001 a a b Propionic acid (mM/L) 6.53 7.44 3.47 0.351 < 0.001 a a b Isobutyric acid (mM/L) 0.95 1.00 0.37 0.029 < 0.001 b a c Butyric acid (mM/L) 4.19 5.03 2.50 0.179 < 0.001 b a c Isovaleric acid (mM/L) 0.89 1.09 0.29 0.029 < 0.001 b b a Valeric acid (mM/L) 0.41 0.45 0.95 0.041 < 0.001 a a b Total VFA (mM/L) 42.8 48.6 25.5 2.2 < 0.001 b a c Branch-chain fatty acids (mM/L) 2.24 2.54 1.61 0.063 < 0.001 b b a Acetate: propionate ratio 4.54 4.54 5.13 0.148 0.047 Nonglucogenic: glucogenic VFA ratio 4.59 4.64 4.82 0.108 0.38 a, b, c Means bearing different superscript ( ) differ significantly TCA Trichloroaceticic acid, VFA Volatile fatty acids CFB1 Concentrate mixture + Vigna sinensis hay (70:30), CFB2 Concentrate mixture + Acacia nilotica leaves (70:30), CFB3 Concentrate mixture + Ziziphus nummularia leaves (70:30) Aderao et al. Journal of Animal Science and Technology (2018) 60:26 Page 7 of 9 rate of degradation, which ultimately decides substrate de- dissimilar phytochemicals. The concentration of ammo- gradability of forages. The rate constant was higher for A. nia N and TCA-precipitable N did not show any definite nilotica and Z. nummularia leaves and lowest in V. sinen- correlation. A higher ammonia N in A. nilotica leaves sis hay. Different feedstuffs always demonstrate difference with intermediate total N concentration led to lower in rate constants during kinetic assay . A high rate TCA-precipitable N compared to V. sinensis hay and Z. constant for A. nilotica leaves is justifiable from their nummularia leaves. This reduction in TCA-precipitable higher GP. However, higher rate constant for V. sinensis N may be due to the harmful effects of higher HT and hay may be ascribed to lowest NDF and high lignin. total polyphenol content on ruminal microbes responsible A direct correlation does exist between substrate de- for metabolism of N fractions. Moreover, negative correl- gradability, short-chain fatty acid production and IVGP ation between polyphenol content and TCA-precipitable . Accordingly, A. nilotica leaves and CFB2 revealed N has also been reported . Total VFA concentration higher GP. Expression of GP per unit digestible DM and was in line with substrate fermentation that degraded at OM registered higher values but the differences between higher rate to produce more gas. Similar correlation was the substrates narrowed down where all the three sub- observed earlier [3, 18, 20] and it is emphasized that the strates had similar values because of variability in digest- amount of gas produced from feeds depends largely upon ibility. This could be attributed to intrinsic higher CT chemical composition and rate and extent of degradability  and lignin  content in Z. nummularia leaves of feeds and production of VFA and their proportion. The compared to V. sinensis hay and A. nilotica leaves. These proportion of acetate and other branched chain fatty acids CT is having negative effect on GP because of their abil- (BcFA) that revealed better acetate: propionate ratio and ity to interact with protein and fiber fractions, thereby nonglucogenic: glucogenic VFA ratio could be attributed reducing microbial enzymatic degradation (precipitate to interaction of CT and other non-polyphenolic polymer microbial enzymes) and microbial growth [3, 22, 23]. lignin. Evidently, it was associated with higher proportion Highest DM and OM degradability in CFB2 followed by of propionate, butyrate and other BcFA in Z. nummularia CFB1 and lowest in CFB3 was in line with CFBs with re- leaves. A different trend in CFB with Z. nummularia spective constituent roughages. Higher degradability was leaves commensurate with higher CT and lignin content. evidenced by higher GP in CFBs. Similar results were Preparation of total mixed ration based compact feed also observed by Bhatta et al. . On similar line, low block involving crop residues, tree leaves and browses pre- methane production by A. nilotica leaves may be attrib- sents altogether a different substrate for ruminal microbial uted to its higher TTP compared to other forages. In degradation that have different proportional distribution tropical legumes, content of tannins and other secondary of soluble and insoluble carbohydrates, proteins and fats. metabolites is higher that affects NDF digestibility and Moreover, ruminal fermentation is a dynamic process in- reduce methane production . Methane inhibition ac- volving production of fermentation metabolites, microbial tivity observed in Z. nummularia leaves could thus be synthesis and multiplication associated with gradual de- attributed to presence of CT. This alternatively produced cline in substrate availability consequent upon its degrad- positive fermentation pattern with better acetate: propi- ability and ensuing alteration in the total fermentation onate and nonglucogenic: glucogenic VFA ratio . process. Consequently, the PF that demonstrates parti- Plants rich in tannins  and saponins  have poten- tioning of nutrients in to microbial protein synthesis  tial for enhancing flow of microbial protein from rumen, may become a critical determinant to establish correlation increasing efficiency of feed utilization and decreasing between true substrate degradability and production of methanogenesis. The CFB containing these plant bio- shortchain fatty acids, fermentable gas and methane mass recorded a similar pattern and it was observed production. earlier too . A lower methane emission for some shrubs (e.g. Z. nummularia) than other forages could Conclusion thus be attributed to presence of various phytochemicals The complete feed with HTrich A. nilotica leaves exhib- [3, 23, 27]. It has been suggested that the action of tan- ited higher substrate degradability, propionate produc- nins on methanogenesis is attributed to direct inhibitory tion and lower methanogenesis. On the other hand, CFB effects on methanogens  depending upon the chem- with CTrich Z. nummularia leaves produced lower fer- ical structure of tannins, and also indirectly by decreas- mentable gas, VFA with higher PF to emphasize its use- ing fiber degradation [3, 26, 29]. fulness. This conflict could only be addressed by in vivo The data on fermentation metabolites was inconsistent experiments. Nevertheless, this in vitro ruminal assess- in both roughage components and the CFBs. Variation ment of polyphenol-rich plant biomass and the CFBs in total N concentration in the digesta as against rela- could certainly elaborate the diverse effects on ruminal tively similar N content in the three roughages was indi- fermentation behaviour between polyphenolic constitu- cative of different fermentation behaviour effected by ents HT and CT. Aderao et al. Journal of Animal Science and Technology (2018) 60:26 Page 8 of 9 Therefore, judicious incorporation of A. nilotica and Z. 2. Kamra DN, Agarwal N, Chaudhary LC. Inhibition of ruminal methanogenesis by tropical plants containing secondary compounds. Int Cong Ser. 2006; nummularia leaves in complete feeds would promise bet- 1293:156–63. ter ruminant production. Besides, inclusion of these leaves 3. Pal K, Patra AK, Sahoo A. Evaluation of feeds from tropical origin for in vitro as part of total mixed ration would eventually help in de- methane production potential and rumen fermentation in vitro. Span J Agric Res. 2015;13:e0608. livering positive fermentation attributes by minimizing 4. Jadhav RV, Kannan A, Bhar R, Sharma OP, Gulati A, Rajkumar K, Verma MR. their antinutritional effects. Finally, polyphenol rich plant Effect of tea (Camellia sinensis) seed saponins on in vitro rumen leaves can be used for strategic reduction of methane fermentation, methane production and true digestibility at different forage to concentrate ratios. J Appl Anim Res. 2018;46:118–24. emission from the animals thereby diverting the energy 5. Abdulrazak SA, Fujihara T, Ondiek JK, Ørskov ER. Nutritive evaluation of for higher production with better feed efficiency. some Acacia tree leaves from Kenya. Anim Feed Sci Technol. 2000;85: 89–98. Abbreviations 6. Waghorn GC, McNabb WC. Consequences of plant phenolic compounds for ADF: Acid detergent fiber; ADL: Acid detergent lignin; BcFA: Branched-chain productivity and health of ruminants. Proc Nutr Soc. 2003;62:383–92. fatty acids; BW: Body weight; CFB: Complete feed block; CP: Crude protein; 7. AOAC. Official Methods of Analysis, 17th edn. Gaithersburg: Association of CRD: Complete randomized experimental design; CT: Condensed tannins; Official Analytical Chemists; 2000. DM: Dry matter; DM: Dry matter; DWG: Daily weight gain; EE: Ether extract; 8. Van Soest PV, Robertson JB, Lewis BA. Methods for dietary fiber, neutral FA: Fatty acids; FID: Flame-ionization detector; GP: Gas production; detergent fiber, and nonstarch polysaccharides in relation to animal HCl: Hydrochloric acid; HT: Hydrolysable tannins; IVGP: In vitro gas nutrition. J Dairy Sci. 1991;74:3583–97. production; ME: Metabolizable energy; N: Nitrogen; NDF: Neutral detergent 9. Hagerman A, Harvey-Mueller I, Makkar HPS. Quantification of tannins in tree fiber; NTP: Non-tannin phenols; OM: Organic matter; PTV: Programmable foliage–a laboratory manual. Vienna: FAO/IAEA; 2000. p. 4–7. temperature vaporizer; TCA: Trichloro acetic acid; TP: Total phenols; TTP: Total 10. Makkar HPS. Quantification of tannins in tree and shrub foliage: a laboratory tannin phenols; VFA: volatile fatty acids manual. Dordrecht: Kluer Academic Publishers; 2003. 11. Menke KH, Raab L, Salewski A, Steingass H, Fritz D, Schneider W. The Acknowledgements estimation of the digestibility and metabolizable energy content of Authors are thankful to ICAR-Central Sheep and Wool Research Institute, Avi- ruminant feeding stuffs from the gas production when they are incubated kanagar and ICAR- Indian Veterinary Research Institute, Izatnagar for provid- with rumen liquor in vitro. J Agri Sci. 1979;93:217–22. ing necessary facilities to carry out this work. 12. Schofield P, Pitt RE, Pell AN. Kinetics of fibre digestion from in vitro gas production. J Anim Sci. 1994;72:2980–91. Funding 13. Menke KH, Steingass H. Estimation of the energetic feed value obtained No external source of fund is received for this experimentation. from chemical analysis and in vitro gas production using rumen fluid. Anim Re Dev. 1988;28:7–55. Availability of data and materials 14. ICAR. Nutrient Requirement of Sheep, Goat and Rabbit, second ed. New Authors approved availability of data and materials. Delhi: Indian Council of Agricultural Research; 2013. 15. Sharma SC, Sahoo A. Promising Feed & Fodder Resources for Dry Areas. Authors’ contributions Avikanagar: Central Sheep and Wool Research Institute; 2017. GNA implemented the experimental protocol and drafted the manuscript; 16. Singh B, Sahoo A, Sharma R, Bhat TK. Effect of polethylene glycol on gas AS designed the experiment, monitored implementation and interpreted the production parameters and nitrogen disappearance of some tree forages. result; RSB helped in implementation and data analysis; PKK helped in Anim Feed Sci Technol. 2005;123:351–64. chemical analysis and LS helped in chemical analysis. All authors read and 17. Rana KK, Wadhwa M, Bakshi MPS. Seasonal variations in tannin profile of approved the final manuscript. tree leaves. Asian-Australas J Anim Sci. 2006;19:1134–8. 18. Sahoo A, Ogra RK, Sood A, Ahuja PS. Nutritional evaluation of bamboo Ethics approval cultivars in sub_Himalayan region of India by chemical composition and in All experimental procedures involving animals were duly approved by vitro ruminal fermentation. Grassl Sci. 2010;56:116–25. Institute (CSWRI, Avikanagar) Animal Ethics Committee (IAEC) (approval 19. Blummel M, Bullerdick P. The need to complement in vitro gas number IAEC-CSWRI/2017/IXX13455) by following the guidelines of measurements with residue determination from in sacco degradabilities to Committee for the Purpose of Control and Supervision of Experiments on improve the prediction of voluntary intake of hays. Anim Sci. 1997;64:71–5. Animals (CPCSEA). 20. Blümmel M, Makkar HPS, Becker K. In vitro gas production: a technique revisited. J Anim Physiol Anim Nutr. 1997;77:24–34. Consent for publication 21. Nsahlai IV, Siaw D, Osuji PO. The relationships between gas production and Not applicable. chemical composition of 23 browses of the genus Sesbania. J Sci Food Agric. 1994;65:13–20. Competing interests 22. Tavendale MH, Meagher LP, Pacheco D, Walker N, Attwood GT, The authors declare that they have no competing interests. Sivakumaran S. Methane production from in vitro rumen incubations with Lotus pedunculatus and Medicago sativa, and effects of extractable condensed tannin fractions on methanogenesis. Anim Feed Sci Technol. Publisher’sNote 2005;123:403–19. Springer Nature remains neutral with regard to jurisdictional claims in 23. Bhatta R, Saravanan M, Baruah L, Sampath KT. Nutrient content, in published maps and institutional affiliations. vitro ruminal fermentation characteristics and methane reduction potential of tropical tannin-containing leaves. J Sci Food Agric. 2012; Author details 92:2929–35. Animal Nutrition Division, ICAR- Indian Veterinary Research Institute, 243122, 24. Archimede H, Eugene M, Marie Magdeleine C, Boval M, Martin C, Izatnagar, UP, India. Division of Animal Nutrition Division, ICAR- Central Morgavi DP, Lecomte P, Doreau M. Comparison of methane production Sheep and Wool Research Institute, Avikanagar, Rajasthan 304501, India. between C3 and C4 grasses and legumes. Anim Feed Sci Technol. 2011;166:59–64. Received: 4 July 2018 Accepted: 29 October 2018 25. Parmar P, Bhatt S, Dhyani S, Jain A. 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Journal of Animal Science and Technology – Springer Journals
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