Nutritional potentialities of some tree leaves based on polyphenols and rumen in vitro gas production

Aim: The study was conducted to evaluate eight tree leaves based on polyphenolic content and rumen in vitro incubation and gas production technique (RIVIGPT) for their nutritive potentiality. Materials and Methods: Eight selected tree leaves, namely Sesbania grandiflora, Melia dubia, Dillenia spp., Artocarpus heterophyllus, Commiphora caudata, Moringa oleifera, Leucaena leucocephala, and Acacia auriculiformis, were selected for proximate composition, forage fiber fractions, total phenolics (TPs), non-tannin phenols (NTPs), total tannins (TTs), condensed tannins (CTs), and hydrolysable tannins (HTs); RIVIGP with and without polyethylene glycol (PEG); and in vitro dry matter digestibility (IVDMD) (modified in vitro two stage) analysis was conducted. On the basis of RIVIGPT, the in vitro digestible organic matter (IVDOM) and dry matter intake (DMI) was calculated. Results: Crude protein (CP) content of tree leaves ranged from 9.59 to 25.81%, neutral detergent fiber (NDF) 28.16 to 53.33%, acid detergent fiber (ADF) 21.26 to 41.7%, acid detergent lignin (ADL) 3.62 to 21.98%, TP 1.83 to 17.35%, TT 0.40 to 15.47%, and CTs 0.02 to 15.26%. IVDMD (%) was ranged from 64.95 to 88.12. The mean metabolizable energy (ME) (MJ/Kg) of tree leaves estimated with and without PEG was 7.75±0.56 and 8.75±0.39, in vitro gas production at 24 h (IVGP24) (ml) 31.06±4.14 and 37.09±2.64, initial gas production (a) (ml) 0.49±0.63 and 1.33±0.72, potential gas production (D) (ml) 38.74±4.27 and 43.79±2.44, rate of gas production (k) (h−1) 0.11±0.02 and 0.11±0.013, t1/2(ml) 9.81±2.41 and 7.42±0.80, in vitro gas production at 96 h IVGP96 (ml) 39.50±4.430 and 45.14±2.65, the predicted IVDOM (%) 55.44±4.15 and 61.98±3.03, and DMI (g/Kg W0.75) 103.1±14.76 and 104.3±10.16, respectively. The addition of PEG showed an improvement in IVGP24, IVGP96, ME, predicted IVDOM, and predicted DMI. CP was positively correlated with ME, IVGP24, IVGP96, a+b, k (r=0.749, p<0.05), IVDMD, IVDOM, and DMI (r=0.838, p<0.05) and negatively correlated with a and t1/2. NDF, ADF, and ADL contents were negatively correlated with ME (r=0.899, p<0.05), IVGP24 (r=−0.867, p<0.05), IVGP96 (r=−0.858, p<0.05), a+b (p<0.05), k (r=−0.828, p<0.05), IVDMD, IVDOM (r=−0.853, p<0.05), and DMI and positively correlated with a and t1/2. TP, TT, and CT were negatively correlated with ME, IVGP, IVGP96, a+b, k, IVDMD, IVDOM, and DMI and positively correlated with a (r=0.808, p<0.05) and t1/2. ME (MJ/Kg) was positively correlated with IVGP24 (r=0.938, p<0.05), IVGP96 (r=0.875, p<0.05), a+b (r=0.813, p<0.05), k (r=0.731, p<0.05), IVDMD, IVDOM (r=0.985, p<0.05), and DMI (r=0.727, p<0.05) and negatively correlated with a and t1/2. Conclusion: In the present study, the potentiality of tree leaves was assessed based on CP, ADF, ADL, TP, CT, IVGP, ME, IVDMD, predicted IVDOM, and predicted DMI. Based on this, it can be concluded that S. grandiflora, M. dubia, M. Oleifera, and L. leucocephala were graded as best; A. heterophyllus and C. caudata as moderate; and Dillenia spp. and A. auriculiformis as lowest potential ruminant feed.


Introduction
The inadequacy of nutrients is a major limitation for livestock as a deficit of 728 mT (64%) of green fodder and 157 mT (25%) of dry fodder with 27% of crude protein (CP) and 24% of total digestible nutrients in India which is expected by 2020 [1]. This can partly be overcome by feeding tree leaves, as huge quantity of biomass is available from fodder trees, which provide nitrogen, energy, minerals and vitamins and additionally have laxative effect and reduce cost of feeding [2]. Suitability of inclusion in the feeding can be assessed either by in situ or rumen in vitro incubation and gas production techniques (RIVIGPT), which would be complementary to traditional chemical measurements [3]. In comparison to in vivo feeding trial, the RIVIGPT is simple, less expensive, less time consuming and allow more control over experiment. As being more efficient than other in vitro techniques, it is suggested for determining the nutritive value of feeds containing anti-nutritive factors and for evaluating the microbial fermentation of ruminant feeds and its impact on fermentation products [4,5].
Therefore, the present study was conducted to evaluate nutritive potentialities of eight tree leaves such as Sesbania grandiflora, Melia dubia, Dillenia spp., Artocarpus heterophyllus, Commiphora caudata, Moringa oleifera, Leucaena leucocephala, and Acacia auriculiformis of different parts of Karnataka state based on polyphenolic content and RIVIGPT. The selected tree leaves are grown in this region and used in animal feeding. Except S. grandiflora, A. heterophyllus, M. Oleifera, and L. leucocephala, other tree leaves are new to investigation and there is no literature with regard to nutritive value, and hence, this study will be useful in comparing among the tree leaves.

Ethical approval
The Institutional Animal Ethics Committee approved to collect rumen fluid from the cannulated cow. were chosen from different parts of Karnataka state. About 2-3 Kg of tree leaves from the single source were hand plucked, oven dried at 55°C for 48 h, and grounded to pass through size of 1 mm sieve for further analysis (Kan. regional language, Kannada).

RIVIGP
The selected tree leaves were subjected to RIVIGP (with and without PEG) for estimating metabolizable energy (ME) and rate of gas production. A lactating dairy cow producing 3 Kg of milk per day, fitted with a flexible rumen cannula of large diameter (Bar Diamond Inc., USA), receiving a basal diet consisting of finger millet straw and CFM (maize 60%, WB 35%, mineral mixture 2%, urea 2%, and salt 1%) was used as donor cow for rumen fluid. For RIVIGPT, rumen fluid was collected before offering CFM.
Feed samples (200±10 mg) were incubated with and without PEG in 100 ml calibrated glass syringes in triplicate with 30 ml mixed rumen suspension with three blank incubations and standards [6]. For PEG treatment, PEG is added twice the amount of feed sample, and blank samples with PEG were also incubated for estimating corrected gas production. Cumulative gas production was recorded after 2, 4, 6,8,12,16,24,36,48,60,72, and 96 h of incubation.
Data on gas production were fitted to the exponential equation Y=a+b (1−e −ct ), where Y (mL) was defined as gas production at time t, a (mL) was the initial gas production, b (mL) was the gas production during incubation, a+b or D (mL) was the potential gas production, and c (mL/h) was the fractional gas production.
The equations used to estimate the IVDOM and ME [5] [8] was estimated using the following equation: DMI=18.9-0.23 (a+b)+687(c)+0.11CP(g/Kg DM) Where a (mL) was the initial gas production, b (mL) was the gas production during incubation, and c (mL/h) was the fractional gas production.

IVDMD
The IVDMD was carried out using Ankom 200 Fiber Analyzer where F57 Ankom Filter Bag (porosity: 25 µ) was used for extraction. Bags were made up of N-free Monofilament Polyester Screen Printing Fabrics.
About 400mg of dry forage samples (2 mm) were weighed into F57 Ankom Filter Bags and subjected for 48 h incubation in Mold's buffer/rumen fluid mixture in sealed Erlenmeyer flasks followed by treatment with NDS. The dry residues were weighed, and digestibility was calculated using the following equation [7,9].

Polyphenolic fractions
TPs were estimated by Folin-Ciocalteu reaction [4] using gallic acid as a standard. For the CT fraction, the extract was treated with butanol-HCl in the presence of ferric ammonium sulfate, and CT expressed as leukocyanidin equivalent as A550 nm×782.6 weight of sample DM, where A550 nm is absorbance at 550 nm assuming that the effective E1%, 1 cm, 550 nm of leukocyanidin is 460. The phenolic content of the supernatant after precipitating with polyvinylpolypyrrolidone (100 mg) was measured by the Folin-Ciocalteu reaction and this was regarded as the NTP. TTPs were calculated as the difference between TP and NTP. HTs were calculated as the difference between TTP and CT.

Statistical analysis
The data on RIVIGP were subjected to nonlinear regression using GraphPad Prism software to assess gas kinetics (exponential decay equation model). Pearson correlation analysis was used to assess the relationship between chemical composition and ME, IVGP 24 (in vitro gas production at 24 h), IVGP 96 (in vitro gas production at 96 h), IVDMD, IVDOM, and DMI of the tree leaves.

Gas production kinetics
RIVIGP values of tree leaves with or without PEG are shown in Table-3. The IVGP 24 (ml) was more in M. oleifera (42.63) and less in A. auriculiformis (8.68). The IVGP 96 (ml) was higher for S. grandiflora (50.54) and less for A. auriculiformis (10.27). The D (ml) value was more in S. grandiflora (47.48) and lower for A. auriculiformis (9.64). k was higher for M. dubia and lower for Dillenia spp., and t 1/2 (h) was highest for Dillenia spp. and least for M. dubia. Among the evaluated tree leaves, energy content (ME, MJ/Kg) was higher in S. grandiflora (9.87) and lower in A. auriculiformis (5.03).

IVDMD
IVDMD of tree leaves was analyzed using Ankom 200 Fiber analyzer based on modified in vitro two-stage method which is shown in Table-4. IVDMD of tree leaves ranged from 64.95% (A. auriculiformis) to 88.12% (M. dubia) and an average of 76.02%.

Prediction of IVDOM and DMI
The IVDOM and DMI were predicted based on gas production and chemical composition of tree leaves which are presented in Table-4. IVDOM was ranged from 32.52 (A. auriculiformis) to 69.05 (S. grandiflora) and the predicted DMI (g/Kg W 0.75 ) was ranged from 41.9 g Dillenia spp. to 136.8 g (M. dubia).

Effect of PEG on gas production kinetics, IVDOM, and DMI
The PEG inclusion during incubation resulted in increased ME, a, D, IVGP 24 , k, IVGP 96 , IVDOM, and DMI of tree leaves and decreasedt 1/2 . In Dillenia spp., A. heterophyllus, and A. auriculiformis tree leaves, it was more pronounced.

Chemical composition
In the present study, CP varied from 9.59 to 25.81%, and it was in similar range for tree leaves as in published reports [12][13][14][15][16][17]. Similarly, the NDF, ADF, and ADL contents of analyzed tree leaves were in a instead of the similar range [13,18,19].
In the present study, there were considerable variations in chemical compositions between the tree leaves. This was due to differences in genotype, environment, stage of maturity, and harvesting [20][21][22]. The multipurpose tree leaves contained moderate levels of CP, minerals, and vitamins that are deficient in many low-quality roughages and it was found that CP level above the threshold level (11-12%) is required for the moderate level of ruminant production [23].

Polyphenolic fractions
The literature on the polyphenolic content of tree leaves depicts that the concentration remarkably varies from source to source. The polyphenolic content estimated was lower [17,24,25] than the present values, whereas a similar range of values was also observed in the past [15,16,[26][27][28].
Tannin composition in plants depends on type of plant, photosynthetic capacity, soil fertility, environmental conditions, maturity of the leaves, and processing and analytical method employed in analysis [4]. In general, the intake of CT below 5% improves the utilization of feed by ruminants, mainly because of a reduction in ruminal protein degradation and, as a consequence, greater availability of mainly essential amino acids for absorption in the small intestine. Values of CTs exceeding 5% on dry matter basis could inhibit microbial activity, depress dry matter digestibility, and reduce voluntary intake [29].

IVDMD and prediction of IVDOM and DMI
IVDMD of tree leaves ranged from 64.95% (A. auriculiformis) to 88.12% (M. dubia) and an average value of 76.02%, whereas in previous study, it was found the range of IVDMD in tree leaves between 22.70 and 58.72 % that was lower than the present study which may arise due to lower quality of tree leaves [19]. The predicted IVDOM ranged from 32.52 (A. auriculiformis) to 69.05 (S. grandiflora) with an average value of 55.43%, and the predicted DMI (g/Kg W 0.75 ) was lowest in Dillenia spp. (42.2 g) with an average value of 103.13 g.

Effect of PEG on kinetics of gas production
The improvement in gas production in Dillenia spp., A. heterophyllus, and A. auriculiformis tree leaves was due to affinity of PEG to tannins [32][33][34][35]. It was also found that there was only minor improvement in IVGP 24 and IVGP 96 in L. leucocephala and C. caudata due to the low level of CT in them. Addition of PEG also increased the ME, IVDOM % content, and DMI (g/KgW 0.75 ) in these tree leaves.
In general, the improvement in fermentation in each species by adding PEG almost certainly reflects its deactivation of secondary compounds [11,35,36]. High CT (proanthocyanidins) and fiber (NDF and ADF) contents reduce digestibility [31,32] while low CP affects the acceptability of browse [25]. Similarly, in the present study, lower level of IVGP was observed in those tree leaves which are containing more CT, NDF, ADF, and ADL.

Conclusion
In the present study, the nutritive potentiality of tree leaves was assessed based on CP, ADF, ADL, TP, CT, IVGP, ME, IVDMD, IVDOM, and DMI. It can be concluded that S. grandiflora, M. dubia, M. oleifera, and L. leucocephala were graded as the best due to higher CP, ME, IVGP, IVDMD, IVDOM and DMI and lower polyphenols and fiber fractions; A. heterophyllus and C. caudata were graded as the moderate potential and Dillenia sp. and A. auriculiformis were graded as the lowest potential as a ruminant feed. While the addition of PEGin Dillenia spp. and A. auriculiformis improved the RIVIGP but it did not impact their overall nutritive ranking. However, more studies are required to characterize these feeds better All values were mean of triplicates. +=Indicates with PEG,-=Indicates without PEG. ME=Metabolizable energy through in vivo feeding trials with respect to palatability and intake.   24 =Gas production volume (ml/0.2 g DM) after 24 h of incubation, IVGP 96 =Gas production volume (ml/0.2 g DM) after 96 h of incubation, a=Rapidly produced gas (ml), a+b = Potential cumulative gas production (ml), k=Rate of gas production/h, t1/2=Half-life, IVDMD=In vitro DM digestibility (g/Kg DM), IVDOM=In vitro digestibility of organic matter (%), DMI=Dry matter intake (g/Kg W 0.75 ), PEG=Polyethylene glycol