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Exploring feeding value of oak (Quercus incana) leaves:Nutrient intake and utilization in calves

R.K. Sharma a,b, B. Singh a,c, A. Sahoo a,⁎

a Indian Veterinary Research Institute, Regional Station, Palampur—176 061 (HP), Indiab Faculty of Veterinary Science & Animal Husbandry, SKUAST, Jammu, India

c Dairy Cattle Nutrition Division, National Dairy Research Institute, Karnal, India

Received 18 August 2006; received in revised form 24 January 2008; accepted 25 January 2008

Abstract

For evaluating the feeding value of oak leaves as a supplement to wheat straw an in vivo study was conducted involving fifteenmale calves (aged 6–9 months, average body weight 84 kg,) distributed randomly into three equal groups. The calves in group 1(G1) received a maintenance diet of concentrate (C) and wheat straw (WS) as the sole roughage; in group 2 (G2), C, WS and oakleaves (OL) at a ratio of 31:39:30 on dry matter (DM) basis and in group 3 (G3), C, WS and OL at a ratio of 33:45:22. The feedinglasted for 4 weeks and included a metabolism trial of 6 days duration. The DMI (kg/day) increased (41 and 38% in G2 and G3,respectively) with supplementation of oak leaves, being higher in G2 (2.90) followed by G3 (2.82) and G1 (2.05). Digestibility (%)of DM, organic matter and neutral detergent fiber was higher in G2 (51.5, 55.8, 49.0) compared to G1 (43.9, 49.3, 43.7) and G3(45.4, 50.1, 44.0) contributing to higher ME intake (17%). Increased N utilization efficiency (20.6, 67.7 and 55.1% in G1, G2 andG3, respectively) and microbial protein production (180, 209, 194 g/day) coupled with a decrease in plasma urea level (about 25%)was observed in oak leaf supplemented groups. Supplementation of WS/OL ratio of 44:56 enhanced DMI, digestibility, efficiencyof N utilization and body weight gain compared with a WS/OL ratio of 100/0.© 2008 Elsevier B.V. All rights reserved.

Keywords: Oak leaves; Wheat straw; Tannins; Calves

1. Introduction

The strategy for increased animal productivity incountries with scarce feed resources is to find forages(trees/shrubs/pods) or fruits/seeds that are high inprotein and minerals and that can be used as supple-

ments to improve the nutritional value of conventionalpoor quality roughages. Awide variety of tree leaves canbe used as nitrogen (N) sources to supplementconventional fibrous crop residues (Devendra, 1990).Tree forages also provide energy, minerals, vitamins,besides many advantages i.e. on-farm availability andeasy accessibility to farmers, laxative influence on thealimentary system, and scope of variety in the diet. Buttannins in tree forages affect nutrient utilization indiverse ways (Singh and Bhat, 2001), i.e. in largequantities, tannins reduce forage quality and adversely

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⁎ Corresponding author. IVRI, Mukteswar Campus—263138,Nainital (Uttarakhand), India. Tel.: +91 5942 286562; fax: +91 5942286347.

E-mail address: sahooarta@yahoo.com (A. Sahoo).

1871-1413/$ - see front matter © 2008 Elsevier B.V. All rights reserved.doi:10.1016/j.livsci.2008.01.022

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affect herbivore nutrition. However, the condensedtannins (CT) are useful as they prevent bloat, protectproteins, improve N utilization and inhibit gastrointest-inal parasitism (Barry and McNaab, 1999). Therefore,there seems to be a scope for exploiting the use of treeforages in ruminant production.

Oak (Quercus incana) leaves (OL) form the bulk oflivestock feed during winter in the Western Himalayas(Makkar and Singh, 1991b).However, poisoning from theconsumption of OL (Garg et al., 1992) limits its use as asole feed in cattle. Therefore, incorporation of OL in poorquality roughage-based diet could be a useful strategy, andthe present study is aimed at exploring the feeding value ofOL at optimized ratio in male zebu calves.

2. Materials and methods

2.1. Treatments and feeding

In a completely randomized design fifteen male Zebu calves(Bos indicus) of 6–9 months of age were distributed into threegroups of five animals each with an average body weight (BW)of 84 kg. The control group 1 (G1) was fed a maintenance dietbased on wheat straw (WS, chaffed to b3 cm) and concentratemixture (groundmaize 360, deoiled groundnut cake 200, deoiledrice bran 125, rice husk 125, molasses 70 and mineral mixture20 g/kg) at 60:40 ratio by assuming dry matter intake (DMI) at2.5% of calf's BW. Accordingly, the concentrate (C) level wasfixed at 1% with WS ad libitum (110%). The two experimentalgroups G2 and G3 were supplemented with OL at 50:50 and37.5:62.5 ratios with WS, respectively. The animals were fedindividually with a weighed quantity of the ration once in themorning (9.30–10.00 A.M.). Feed offered and residues left wererecorded daily. Care was taken to allow sufficient time for theconsumption of concentrate before WS/OL were offered. Fresh,clean water was offered ad libitum twice a day. The feeding trialwas continued for a period of 28 days followed by a metabolismtrial involving an adaptation period of 2 days with collectionbags and 6 days actual collection of feed and biological samples.Quantitative record of the feed offered, and refusals wasmaintained during the trial. The residual feed, faeces and urinewere collected at 24 h intervals. Aliquots (as a fraction of totalweight) of faeces (10–20 g) and urine (5–10 ml) were preservedin 10% sulphuric acid for N estimation. Feed, residue and faecessamples were dried to constantweight at 55–60 °C in a forced airoven and pooled over the collection period. The dried sampleswere ground (≤1 mm) and stored in screw-capped polycarbo-nate container until analysis.

The animals were weighed weekly on two consecutive daysbefore feeding and watering in the morning. The BW of theanimals was also recorded before and after the metabolic trial.

2.2. Urine and blood parameters

An aliquot of urine sample was collected daily over theperiod ofmetabolism trial and diluted 5 timeswith distilledwater

to prevent precipitation of uric acid during the storage (−20 °C).Blood samples were collected from jugular veins in the morningprior to feeding (0 h) and 3 h post-feeding (after N80% of OLconsumption) for two consecutive days following the metabolictrial. About 4 ml blood was placed into tubes containinganticoagulant (sodiumEDTA and sodium fluoride mixture) withthe remainder (about 6ml) being placed into a non-anticoagulanttube. Blood in the non-anticoagulant tube was left to clot atroom temperature for 1 h. Tubes with anticoagulated and non-anticoagulated blood were centrifuged at 500 ×g for 10min. Theplasma/serum was removed and stored frozen (−70 °C) untilanalysis.

2.3. Chemical analysis

Standard methods as described in AOAC (1984) werefollowed for the determination of proximate composition, viz.dry mater (DM, method no. 7.003), ash (method no. 7.009),crude protein (CP, method no. 7.021), ether extract (EE, methodno. 7.062). Organic matter (OM) was calculated by difference.

Fiber composition was determined as per the methods of VanSoest et al. (1991), where neutral detergent fiber (NDF) wasestimated without sodium sulphite. Nitrogen in NDF and aciddetergent fiber (ADF) was determined by the Kjeldahl methodand represented as NDIN and ADIN, respectively. Aciddetergent residue was treated with 72% H2SO4 (w/w) for lignin(sa) estimation. Nonstructural carbohydrate (NSC)was estimatedanalogously, viz. NSC=OM–CP–EE–(NDF–CP in NDF).

Tomeasure tannins, 200mgof feedwasweighed into a 50mlconical flask. The pigments and fats were removed with 20 mlof diethyl ether containing 1% acetic acid. The residue free ofdiethyl ether was extracted with 10 ml of 70% aqueous acetonein an orbital shaker (30 °C, 130 rpm) for 2 h, followed by cen-trifugation for 20 min at 5000 ×g and the supernatant wascollected for tannin analysis. Total phenols were estimated byFolin–Ciocalteu reaction (Makkar, 1994). For CT fraction, theextract was treated with butanol-HCl in presence of ferricammonium sulphate and expressed as leucocyanidin equivalent.The non-tannin phenols were estimated after binding the tanninswith polyvinyl–polypyrrolidone. Total tannin phenols (TTP)and hydrolysable tannins (HT) were calculated by difference.

2.4. Urinary purine derivatives

The urine samples that were previously stored at −70 °Cwere thawed and diluted 25 times with distilled water tomeasure allantoin and uric acid. Purine derivatives (PD) inurine were determined by the methods described in IAEA-TECDOC-945 (1997).

2.4.1. AllantoinThe method of Young and Conway (1942) was employed.

Briefly, to 1.0 ml samples, standard (allantoin, 10–60 μg/ml)or blank in a test tube, 5.0 ml distilled water and 1.0 ml 0.5 MNaOH was added. The content was vortex mixed and kept inboiling water bath for 7 min. Then 1.0 ml 0.5 M HCl and1.0 ml 0.023 M phenylhydrazine were added after cooling.

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The solution was then mixed and transferred again to boilingwater bath for 7 min., immediately followed by cooling inan icy alcohol bath. Then concentrated HCl (3.0 ml) and potas-sium ferricyanide (0.05 M, 1.0 ml) were added, mixed and theabsorbance read at 522 nm after exactly 20 min.

2.4.2. Uric acidThe method of Fujihara et al. (1987) was employed, in

which the concentration of uric acid was measured aftertreatment with uricase. Briefly, 1.0 ml samples, standard (5–40 μg/ml) or blank was mixed with 2.5 ml phosphate buffer(KH2PO4 0.67 M, pH 9.4) in two sets of tubes, 150 μl bufferwere added to one set and 150 μl uricase solution (fromporcine liver, Sigma Chem. Ltd.) (0.12 U/ml buffer) to theother set were incubated in a water bath (37 °C) for 90 minfollowed by reading the OD at a wavelength of 293 nm. Acurvilinear relationship resulted between uric acid concentra-tion and absorbance that was transformed into a linear relation-ship after concentrations were logarithmized.

2.4.3. Microbial NPrediction of microbial N supply in calves was assessed

from the urinary purine derivatives (PD) based on the relation-ship derived by Chen and Orskov (2003), as

MN ¼ X � 70ð Þ= 0:116� 0:83� 1000ð Þ ¼ 0:727XX ¼ Y � 0:147W0:75

� �� �=0:85

where, MN, intestinal flow of microbial nitrogen (g per day)X, microbial purines absorbed (mmol per day)Y, excretion of PD (mmol per day).

N.B.The equation is based on the assumptions that i) Digest-

ibility of microbial purines is 83%; ii) N content of purines is70 mg N/mmol; iii) ratio of purine-N:total-N in mixed rumenmicrobes is 11.6:100 in cattle fed on maintenance and/or nearmaintenance ration; and iv) endogenous excretion of PD islower in Zebu (B. indicus) than in European cattle (Bos taurus)(Pimpa et al., 2001).

2.5. Blood biochemical constituents

Glucose in the plasma was determined by the O-toluidinemethod (Hultman, 1959). Creatininewasmeasured by themethodofBones andTaussky (1945).Ureawas determined by themethodof Rahmtullah and Boyde (1980). Serum protein was determinedby the biuret reaction according to Gornall et al. (1949).

2.6. Statistical analysis

The data were analysed using the statistical software SPSS(version 11.0). Differences among treatments were analysed byfixed effects (Model I) ANOVA (Snedecor and Cochran, 1989).Polynomial contrasts with linear and quadratic functions wereapplied to explain any trend at different levels of OL feeding.

Yijk ¼ Aþ Tiþ e ijð Þ

where, Yij=dependent variables; μ=overall mean; Ti=effectof treatment i, e(ij)= residual.

3. Results

3.1. Chemical composition

The DM per kg OL was 600 g. The CP content (g/kgDM) in C, WS and OL was 165, 35 and 96, respectively(Table 1). OL had a high lignin content (226 g/kg DM),and also had considerable amounts of NSC compared toWS (227 vs 13 g/kg DM). The tannin constituents (g/kgDM) in oak leaves were TTP, 58.2; HT, 24.8; and CT,33.4.

3.2. Intake, digestibility and plane of nutrition

Animals in OL supplemented groups (G2 and G3)consumed more DM (38–41%) than the animals incontrol (Table 2) and the digestibility (g/kg) was alsosignificantly higher, the difference between G2 and G3being non-significant. The digestibilities of EE andADF were non-significantly affected. Digestibility ofCP did not differ (PN0.05) between the groups, butwas higher (P=0.027) in G2 than in G3 when correctedfor faecal NDIN values. The CP and total digestiblenutrients (TDN) concentration (g/kg DM) of diets were89.0 and 463.3 in G1, 93.1 and 541.6 in G2 and 90.3and 481.7 in G3, respectively. An increase in NDFdigestibility was observed in G2. Intake of DM, protein

Table 1Chemical composition (g/kg DM) of fed feeds

Attributes Concentrate Wheat straw Oak leaves

Replicates 3 3 6OM 897.4 (0.31) 901.8 (0.38) 958.4 (0.94)CP 164.7 (0.91) 34.8 (2.87) 96.0 (3.73)EE 32.2 (3.06) 8.9 (1.35) 52.1 (3.69)NDF 446.1 (0.85) 867.2 (1.04) 660.7 (1.05)ADF 226.1 (1.59) 605.2 (1.04) 466.7 (1.17)Lignin (sa) 67.0 (3.28) 84.7 (3.07) 226.5 (1.53NDIN 6.3 (1.90) 3.5 (2.57) 12.4 (3.22)ADIN 1.9 (2.68) 2.4 (3.33) 5.6 (2.37)NSC 293.8 (1.85) 12.8 (3.83) 227.1 (2.02)Total phenolics – – 68.0 (2.46)Non-tannin phenols – – 9.8 (0.97)Total tannin phenols – – 58.2 (2.85)Hydrolysable tannins – – 24.8 (3.16)Condensed tannins – – 33.4 (3.73)

Values in parentheses represents coefficient of variance (c.v.).ADF, acid detergent fiber; ADIN, acid detergent insoluble N; CP,crude protein; NDF, neutral detergent fiber; NDIN, neutral detergentinsoluble N; NSC, non-structural carbohydrate; OM, organic matter.

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and energy (metabolizable energy, ME) in OL supple-mented groups was higher than the control. HigherDM intake in G2 and G3 was mainly due to additionalconsumption of DM from supplemental OL (0.87 and0.62 kg, respectively), which accounted for 30 and 22%of the total diet, respectively. This resulted in a sig-

nificantly higher intake of OM, digestible OM and NSCin the supplemented groups. DMI per 100 kg BWand theintake of nutrients per kgW0.75 also revealed significantdifferences between control and supplemented groups.Between the two supplemented groups, G2 had relativelyhigher intakes of DM (5%), CP (7%), TDN (18%) and

Table 2Intake and digestibility of nutrients in calves during metabolism trial

Attributes G1 G2 G3 SEM ‘P’value

Significance of effects

Linear Quadratic

Feed intake (DMI, kg/day)Concentrate 0.88 0.90 0.93 0.074 0.927 NS NSWheat straw 1.17 1.13 1.28 0.145 0.903 NS NSOak leaves 0.00a 0.87b 0.62b 0.054 b0.001 ⁎⁎⁎ ⁎⁎⁎

Total 2.05a 2.90b 2.83b 0.251 0.044 ⁎ ⁎

Roughage (%) 57.1a 67.7b 67.1b 3.161 0.040 ⁎ ⁎

Oak leaves: straw 0:100 44:56 33:67 – NA NA NAConcentrate: oak leaves: straw 43:0:57 31:30:39 33:22:45 – NA NA NA

Nutrient digestibility (%)DM 43.86a 51.46b 45.36a 1.29 0.003 ⁎⁎ ⁎⁎⁎

OM 49.32a 55.84b 50.09a 1.18 0.004 ⁎⁎ ⁎⁎⁎

CP 39.11 43.40 39.53 1.38 0.092 NS ⁎

(70.70ab) (74.90b) (67.24a) (2.45) (0.027) (⁎) (⁎⁎)NDF 43.72a 48.97b 43.95a 0.97 0.004 ⁎⁎ ⁎⁎⁎

ADF 40.75 43.99 40.22 1.19 0.092 NS ⁎

Nutritive value of dietCP (g/kg DM) 89.0 93.1 90.3 0.043 0.943 NS NSTDN (g/kg DM) 463.3a 541.6b 481.7a 1.215 0.023 ⁎ ⁎⁎

ME(MJ/kg DM)# 6.98a 8.16b 7.26a 0.183 0.023 ⁎ ⁎⁎

Plane of nutritionNutrient intake (kg/day)

OM 1.85a 2.66b 2.58b 0.229 0.041 ⁎ NSDOMI 0.91a 1.48b 1.29b 0.076 0.001 ⁎⁎⁎ ⁎⁎⁎

NDF 1.44a 1.97b 1.95b 0.162 0.047 ⁎ NSADF 0.99 1.30 1.29 0.126 0.107 NS NSNSC 0.27a 0.47b 0.43b 0.048 0.039 ⁎ NSTDN 0.95a 1.57b 1.36b 0.119 0.039 ⁎ ⁎⁎

ME (MJ) 14.31a 23.65b 20.53b 1.792 0.039 ⁎ ⁎⁎

CP (g) 182a 270b 256b 20.21 0.045 ⁎ NSDMI/100 kg BW 2.37 3.17 3.10 0.085 0.004 ⁎⁎ ⁎

Nutrient intake/kgW0.75

DM (g) 71.7a 97.7b 93.4b 4.54 0.003 ⁎⁎ ⁎⁎

TDN (g) 33.2a 53.03b 44.9b 2.75 0.001 ⁎⁎⁎ ⁎⁎⁎

ME (MJ) 0.50a 0.80b 0.68b 0.041 0.001 ⁎⁎⁎ ⁎⁎⁎

CP (g) 6.42a 9.23b 8.62b 0.327 0.001 ⁎⁎⁎ ⁎⁎⁎

Liveweight (BW) gainInitial BW (kg)Final BW (kg) 88.2 94.4 92.6 10.80 0.663 NS NSADG (g) 136a 386b 300ab 80.07 0.047 ⁎ ⁎⁎

G1, no oak leaves (control); G2, oak leaves (50%)+wheat straw (50%); G3, oak leaves (32.5%)+wheat straw (67.5%).ADF, acid detergent fiber; CP, crude protein; NDF, neutral detergent fiber; NSC, non-structural carbohydrates; OM, organic matter; DMI, dry matterintake; DOMI, digestible OM intake; TDN, total digestible nutrients (digestible CP+digestible carbohydrates+2.25×digestible ether extract); ME,metabolizable energy (TDN×15.062 MJ/kg).Values in parentheses: CP digestibility corrected for faecal NDIN (faecal NDIN is substracted from total faecal N for microbial correction).Means bearing different superscripts in a row differ significantly (Pb0.05); NS, non-significant, ⁎Pb0.05, ⁎⁎Pb0.01, ⁎⁎⁎Pb0.001, NA—not applicable.

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ME (18%). The average daily gain in G2 (386 g/day) wassignificantly higher than in G1 (136 g/day). There wasa linear increase in intake of nutrients (OM, TDN/ME)

with the increase in OL feeding, but some parametersshowing quadratic effect indicating an optimized responseof diet with C, OL and WS at 31:30:39 ratio.

Table 3Nitrogen balance of calves in different groups

Attributes G1 G2 G3 SEM ‘P’value

Significance of effects

Linear Quadratic

N intakeg/day 29.12a 44.21b 41.25b 3.31 0.046 ⁎ NSg/kgBW0.75 1.035a 1.528b 1.391b 0.060 0.001 ⁎⁎⁎ ⁎⁎⁎

Faecal N excretiong/day 17.66a 25.18b 24.84b 2.02 0.044 ⁎ NSg/kgBW0.75 0.631a 0.858b 0.840b 0.025 0.001 ⁎⁎⁎ ⁎⁎⁎

%N Intake 60.89 56.60 60.47 1.38 0.092 NS ⁎

Urinary N excretiong/day 9.23 6.48 6.85 0.985 0.059 NS ⁎

g/kgBW0.75 0.317 0.211 0.249 0.055 0.420 NS NS%N Intake 30.63b 13.87a 18.00ab 4.19 0.038 ⁎ ⁎⁎

N balanceg/day 2.21a 12.56b 9.55b 2.01 0.010 ⁎⁎ ⁎⁎

g/kgBW0.75 0.087a 0.458b 0.302b 0.063 0.005 ⁎⁎ ⁎⁎⁎

%N intake 8.49a 29.54b 21.53ab 4.47 0.019 ⁎ ⁎

%N absorbed 20.63a 67.67b 55.09b 10.66 0.023 ⁎ ⁎

(11.70a) (39.36b) (31.96b) (6.14) (0.021) (⁎) (⁎)

G1, no oak leaves (control); G2, oak leaves (50%)+wheat straw (50%); G3, oak leaves (32.5%)+wheat straw (67.5%).Values in parentheses for %N absorbed are corrected for faecal NDIN (the faecal NDIN values is substracted from total faecal excretion for microbialcorrection).Means bearing different superscripts in a row differ significantly (Pb0.05); NS, non-significant, ⁎Pb0.05, ⁎⁎Pb0.01, ⁎⁎⁎Pb0.001.

Table 4Urinary purine derivatives and microbial protein production of calves in different groups

Attributes G1 G2 G3 SEM ‘P’value

Significance of effects

Linear Quadratic

Urinary purine derivativesmmol/day

Allantoin 33.50 36.17 35.68 4.921 0.952 NS NSUric acid 4.35 7.31 4.99 1.570 0.465 NS NSTotal purines 37.85 43.48 40.67 3.504 0.921 NS NS

mmol/kgBW0.75

Allantoin 1.181 1.216 1.201 0.029 0.700 NS NSUric acid 0.149 0.242 0.164 0.032 0.069 NS ⁎⁎

Total purines 1.330 1.458 1.365 0.044 0.148 NS NS

Purine absorbedmmol/day 39.63 46.03 42.73 3.844 0.519 NS NSmmol/kgBW0.75 1.393 1.543 1.433 0.052 0.148 NS NS

Microbial protein productiong/day 180.1 209.2 194.2 17.47 0.519 NS NSg/kgBW0.75 6.33 7.01 6.51 0.235 0.148 NS NS

G1, no oak leaves (control); G2, oak leaves (50%)+wheat straw (50%); G3, oak leaves (32.5%)+wheat straw (67.5%).NS, non-significant, ⁎Pb0.05, ⁎⁎Pb0.01.

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3.3. N balance

There was an increased intake of N in animals in G2and G3 compared to those in G1 (Table 3). Excretion ofN in faeces was significantly higher in G2 and G3, buttended to be lower in urine compared to G1 (P=0.059).Balance of N (expressed as g/day, g/kgW0.75, %Nintake, %N absorbed, or %N absorbed corrected forfaecal NDIN) was higher in G2 and G3 than in G1. Thedifference in N balance parameters between G2 and G3was non-significant. However, the efficiency of Nutilization expressed as per cent of N intake or absorbedwas about 22% higher in G2 than G3.

3.4. Urinary purine derivatives and microbial proteinproduction

Allantoin excretion (mmol/day) in G1, G2 and G3was33.5, 36.2 and 35.7, respectively and the differencesamong the groups were non-significant (Table 4). Excre-tion of uric acid (mmol/kgW0.75) revealed a quadraticresponse (Pb0.01) with about 62% higher excretion thuscontributing to only 10% higher excretion of total purines

in G2 than G1. Thus the MCP production (g/day) calcu-lated from the PD excretion was also similar for the threegroups (180.1, 209.2 and 194.2).

3.5. Blood metabolites

None of blood biochemical parameters except ureashowed significant difference between the groups(Table 5). The animals in G2 and G3 exhibited a signifi-cant reduction in urea (mg/dl) both pre (19.7, 20.3) andpost-feeding (24.0, 25.0) compared to control (27.1 and31.6, respectively).

4. Discussion

The fiber and tannin constituents of OL indicated thatleaves were mature (Makkar and Singh, 1991a) andsuitable for feeding calves without the risk of toxicity(Garg et al., 1992). A higher amount of NSC in OLsuggested that these must be supportive in providingdegradable source of energy for ruminal microbial proteinsynthesis. On the contrary, NDF, lignin, NDIN and ADINcontents of both OL andWS increased the total bulk. TheN in oak leaves was mostly fiber-bound and availableonly to a limited extent as evidenced from the data onNDIN and ADIN content, high faecal N output, and lowapparent N digestibility and this could also be attributed tothe effects of tannins. The ADIN, considered relativelyindigestible (Waters et al., 1992; AFRC, 1993) constituted36.5% of total-N in OL. In the Metabolizable ProteinSystem, true digestibility of undegraded dietary N is esti-mated from the concentration of ADIN on the assumptionthat ADIN is completely undegradable and indigestible.However, the fate of ADIN in the digestive tract ofruminants fed top feeds is largely unknown.

For the straw-based basal feeds that are low in N,digestible nutrients and minerals, an ideal feed supple-ment should compensate for the nutrient deficiency andan increased intake of basal diet should be achieved.Improved performances have been frequently observed inanimals fed with tree leaves (Devendra, 1990; Leng,1997). Despite of the bulky nature of OL (Table 1), theDMI in G2 and G3 increased by 41 and 38% compared toG1. Tannins are known to affect feed utilization when thelevel is over 4% (Min et al., 1999), but at lower levels (1.0and 0.73% of DM in G2 and G3, respectively as in thisstudy) has a positive effect. It was probably the contri-bution of NSC and palatability compared to dry poorquality roughages which improved total voluntary feedintake (VFI) in OL supplemented groups (G2 and G3).The increase in total DMI was reported to be associatedwith improved N and energy supply to cellulolytic

Table 5Changes in blood parameters of calves in different groups

Parameter G1 G2 G3 SEM ‘P’value

Significance ofeffects

LinearQuadratic

Glucose mg/dl)Pre-feeding(0 h)

65.30 67.32 70.09 1.53 0.166 NS NS

Post-feeding(3 h)

76.82 73.91 73.51 3.78 0.802 NS NS

Urea (mg/dl)Pre-feeding(0 h)

27.12b 19.69a 20.33a 1.74 0.042 ⁎ NS

Post-feeding(3 h)

31.58b 23.97a 24.95a 1.80 0.048 ⁎ NS

Protein (g/dl)Pre-feeding(0 h)

7.48 7.71 7.70 0.227 0.738 NS NS

Post-feeding(3 h)

7.48 7.85 7.60 0.250 0.590 NS NS

Creatinine (mg/dl)Pre-feeding(0 h)

2.73 3.53 3.07 0.431 0.470 NS NS

Post-feeding(3 h)

3.35 3.95 3.48 0.449 0.636 NS NS

G1, no oak leaves (control); G2, oak leaves (50)+wheat straw (50);G3, oak leaves (32.5)+wheat straw (67.5).Means bearing different superscripts in a row differ significantly(Pb0.05); NS, non-significant, ⁎Pb0.05, ⁎⁎Pb0.01.

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bacteria (Chakeredza et al., 2002), leading to increaseddegradation rate of poor quality roughage, and to a higherdigesta passage rate (Goodchild and McMeniman, 1994).A high DM intake associated with increased intake ofother nutrients particularly those of CP and NSC, lead tobetter digestibility. Significantly higher digestibility in G2may be indicative of a possible better level of supple-mental OL (30%) than in G3 (22%). It is interesting tonote that animals hardly consumedOL in amounts greaterthan 50% of roughage during the adaptation period (1wk)and therefore, the observed level of 30% in the total dietinducing a positive effect on intake and digestibility ofnutrients may be considered as the optimal level. This wascontrary to a study of Nahed et al. (2003) in sheep, inwhich different amounts of Buddleia skutchii tree leaveswith Kikuyu (Pennisetum clandestinum) grass hay werefed and in which positive effects were found if only 25%of the total ration were fed. The reduced performance athigher levels of inclusion is attributed to higher lignincontent in the tree leaves. However, in the present exper-iment, OL with higher lignin content than WS increasedintake and digestibility of DM and other nutrients. Thiswas probably due to the enhanced intake of N and energyconstituents from OL at the specified level (30% of totalfeed DM) of inclusion.

Increased intake of N in G2 and G3 compared to G1was due to supplemental OL. The faecal N excretionincreased with higher CP intake. In G2, faecal N wasabout 4% lower as per cent of N intake and a compara-tively higher CP digestibility after correction for faecalNDIN indicated better utilization compared to the othertwo groups. In the present study, calves in G2 and G3consumed 30 and 22% of DM from OL and thusconsumed CT at 1.00 and 0.73% of DMI, respectively.Higher faecal N excretion, a tendency for lower urinary Nexcretion and a significantly lower plasma urea Nconcentration are suggestive of the role of OL tannins inprotecting proteins from ruminal degradation (Singh andBhat, 2001). Higher N efficiency observed in G2 and G3was perhaps an indication of the availability of tanninprotected proteins after passage of the rumen. A lack ofdifference in PD excretion among the groups in spite ofsignificantly higher DMI, suggests that the OL supple-mentation did not increase microbial protein flow tothe duodenum. An increase in energy intake in G2, ifregarded as originating from rumen fermentation, reflectsthe lower efficiency of MCP synthesis in the rumen. Butthe tannins at this level are reported to increase microbialefficiency. It is possible that tannins protected both proteinand carbohydrates from ruminal degradation. In thepresence of CT, dietary N is partitioned towards faecesdue probably to a lesser proteolysis in the rumen and

consequent reduction in ammonia production (Perez-Maldonado and Norton, 1996; Waghorn and Mcnabb,2003). NDIN minus ADIN represents slowly degradableprotein (fraction B3), the fraction which normallybypasses the rumen and is also digestible (Licitra et al.,1996). Further, CT from OL might also have protectedother dietary protein fractions leading to higher non-ammonia N at the intestinal level and thus a possibleincrease in N utilization efficiency.

The TDN intake of 33.2, 53.0 and 44.9 g/kg W0.75 inG1, G2 and G3, respectively corresponds to requirementfor maintenance, maintenance plus 300 g average dailygain (ADG) and maintenance plus 200 g ADG as recom-mended by NRC (2001). The observed daily gain for thethree groups was 136, 386 and 300, respectively. Theincreased gain in the OL fed group may be partly at-tributable to the tannins. The optimum levels of tannin/CTrequired to have a positive response on nutrient utilizationare still a matter of speculation. In the present study, thesupplemented tannins were very low (1.75% in G2 and1.28% in G3) and CT contributed to ≤1.0% of DM. Theassociative effects of feeds, which were due to one orseveral intrinsic and extrinsic factors, may have con-tributed to the contribution of structural and non-structuralcarbohydrates, rumen degradable and undegradableproteins, tannins and other plant factors. Sahoo et al.(2000) observed an associative increase in nutrientutilization in male crossbred calves fed on cereal greenfodder mixed with wheat straw. According to Lykos et al.(1997) efficiency of conversion of ruminally digestedOMto microbial protein increases with the increased ruminalNSC availability. In the present study theNSC availabilityin G2 was about 10% higher than in G3. It may thus beconcluded that improvement in digestibility of nutrients,coupled with increased intake of energy and possibly,optimized the supply of degradable and undegradableintake protein and resulted in improved performance ofcalves supplementedwithOL at 44:56 ratio withWS (G2)compared to the other two groups.

Allantoin, the principal purine metabolite in urine isconsidered an indicator of microbial purine catabolismand hence reflectsMCP synthesis in ruminants (Lindberg,1989). Excretion of allantoin, uric acid and total PDis highly correlated with rumen degradability of protein(Matras et al., 1991; De Boever et al., 1998). Relativelyincreased excretion of PD in G2 (15% more comparedto G1) may thus be indicative of some protection of feedprotein against ruminal degradation in the presence of OLtannins. Thus the MCP values based on PD revealed 16%increased availability in G2 compared to G1, while it wasonly 8% in G3. Chen et al. (1992) found a more positivecorrelation between DMI, PD excretion and MCP.

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However, a proportionate increase in PD with respect toincrease in DMI was not observed in the present expe-riment. But, microbial N supply per kg of digestible OMintake (y), was closely correlatedwith DMI per kg BW (x)(y=57.76−1.105x; R2, 0.95). Thus, efficiency of micro-bial N synthesis (EMNS) seemed to be a function of theDMI:BW ratio, which increased with the feed intakerelative to BWof the animal. Similarly, Mupangawa et al.(2000) also did not observe any significant difference intotal urinary PD excretion and microbial N supply in dif-ferent treatments groups with higher DMI and digestibility.Furthermore, observations on microbial protein synthesisin relation to ration composition are still confounding andfurther studies are needed to elucidate the microbial popu-lation and substrates involved in the rumen fermentationprocess. Osuji et al. (1993) and Boero et al. (2001) havediscussed the importance of dietary differences influencingthe proportion of salvaged purines and their contribution tothe microbial nucleic acid pool in the rumen.

The increase in post-feeding (3 h) plasma urea con-centrationwas as expected and was in response to ruminaldegradation of dietary intake protein. However, a lesslinear progression in high protein fed animals (G2 andG3) may have been due to high NDIN content in OL. Ithas been reported that CT diminish rumen proteolysis,and also limit microbial activity in the rumen (Bae et al.,1993; McAllister et al., 1994). The resulting lower rumenammonia concentration may thus have contributed to thelow plasma urea level. The alterations in other metaboliteswere similar in all the three groups and the values werewithin the normal range (Kaneko, 1989).

5. Conclusion

The OL supplementation on a WS and concentrate-based diet influenced feed intake, nutrient digestibility andretention, and MCP production resulting in increased per-formance atWS:OL ratio of 56:44. A reduced blood ureaNconcentration revealed a significant effect of tannins fromOL on protein degradation. The tree leaves contain usuallyless CP than conventional cakes, but they may be effect-ively used to substantially replace common protein ingre-dients of commercial concentrates. Considering the goodpalatability, easy on-farm accessibility and increased use oftree forages inHill regions duringwinters, OL appears to bea promising forage supplement to straw-based diet.

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