APPENDIX I. Detailed procedure for estimation of purine derivatives : Determination of allantoin by a colorimetric method

Transcription

1 APPENDIX I Detailed procedure for estimation of purine derivatives : I Determination of allantoin by a colorimetric method In this procedure, allantoin is first hydrolysed under a weak alkaline condition at 100 C to allantoic acid which is hydrolysed to urea and glyoxylic acid in a weak acid solution. The glyoxylic acid reacts with phenylhydrazine hydrochloride to produce a phenylhydrazone derivative of the acid. The product forms an unstable chromophore with potassium ferricyanide. The colour is read at 522 nm. Apparatus: Spectrophotometer, Boiling water bath, Ultrasonic bath (optional), 1 ml and 5 ml pipettes Chemicals 1 Sodium hydroxide (NaOH) 0.5 M. 2 NaOH 0.01 M 3 Hydrochloric acid (HCl) 0.5 M 4 Concentrated hydrochloric acid (11.4 N) cooled at -20 C at least 20 min before use 5 Potassium ferricyanide 0.05 M freshly prepared before use 6 Phenylhydrazine hydrochloride M, freshly prepared before use 7 Alcohol bath, 40% (v/v) alcohol, kept at -20 C. You may use 40% NaCl solution instead of alcohol solution 8 Allantoin (From Sigma or BDH) Preparation of standards Prepare a stock allantoin solution 100 mg/l. Dilute it to give working concentrations of 10, 20, 30, 40, 50 and 60 mg/l.

2 1 Weigh 50 mg of allantoin and transfer it to a 500 ml volumetric flask. Dissolve in about 100 ml 0.01 M NaOH, and top up to volume with distilled water. The addition of NaOH is only to help to dissolve allantoin. 2 To prepare 50 ml of the working standards 10, 20, 30, 40, 50 and 60 mg/l respectively, accurately weigh 5, 10, 15, 20, 25 and 30 g of stock solution into 50 ml volumetric flasks and make up to volume with distilled water. 3 Store each working standards as small aliquots in the freezer. Only the necessary quantities are thawed and any left over discarded. This ensures that fresh standards are used for each analysis run. Preparation before analysis 1.Put the alcohol both into freezer over night. 2.Put the concentrated HCl into the freezer just before the beginning of the analysis. 3.Switch on the water bath. 4.Prepare the fresh solutions of Phenylhydrochloride and Potassium ferricyanide. Phenylhydrazine hydrochloride and Potassium ferricyanide solutions: Prepare solutions of phenylhydrazine hydrochloride and potassium ferricyanide on the day of analysis (keep these solutions in fridge before use). Fifty ml is enough for 10 samples in duplicate. Procedure: Weigh g of phenylhydrazine hydrochloride, dissolve in a small beaker and transfer to a 50 ml volumetric flask. Top up to volume with distilled water. Weigh 0.835g potassium ferricyanide and transfer to a 50 ml volumetric flask, dissolve and make up to volume with distilled water.

3 Procedures This procedure requires critical timing of the reactions. The reading of standards and samples absorbance must be done within a shortest possible time-span, since absorbance decreases with time (see FIG I). Therefore, no more than 10 samples in duplicate should be processed in each run. A set of standards and a blank (using distilled water) in duplicate, are processed. 1 Pipette 1 ml of sample, QC sample, standard or distilled water (blank) into 15 ml tubes. (Courtesy : IAEA) 2 Add 5 ml of distilled water and 1 ml of 0.5 M NaOH. Mix well using a vortex mixer. 3 Place the tubes in the boiling water bath for 7 min. Remove from the boiling water and cool the tubes in cold water 4 Add to each tube 1 ml of HCl (0.5 M). The ph after adding the HCl must be in the range 2-3. Note:If this is the first time you do this assay, you need to check the ph. Add more HCl if necessary. The same amount can then be used in later runs.

4 5** Add 1 ml of the phenylhydrazine solution. Mix and transfer the tubes again to the boiling water for exactly 7 min. 6 Remove tubes from the boiling water and place immediately in the icy alcohol bath for 10 min. 7 Pipette 3 ml of precooled concentrated HCl and 1 ml of potassium ferricyanide. Perform this for all samples within the shortest possible time span. 8 Mix thoroughly and transfer to 4.5 ml cuvettes at room temperature. 9 Zero the absorbance reading with the blank. Read the absorbance at 522 nm after exactly 20 min of addition of potassium ferricyanide reagent. Once started, do it as quickly as possible (because the colour will fade gradually). It is important that absorbance for samples and standards be read within the shortest possible time span. ** Please note: Steps 5-9 should not be interrupted. Calculation Standard curve : The standard curve is linear (See FIG I). Therefore, we can fit a linear regression between the known allantoin concentrations (standards) (X) and the corresponding absorbance (Y). The linear equation is in the following form: Y = a + b X, where a is the intercept and b is the slope of regression.

5 FIG I: Allantoin standard curve

6 Worksheet example for Allantoin estimation in lambs : (OD at 522 nm) Sample Test tube No. OD at 522nm Mean of OD value (X) Ae (mg/liter) a Ae(mmol/day) b Blank B B Standards 10 mg/l 20 mg/l 30 mg/l 40 mg/l 50 mg/l Reference (QC) Sample Test Sample 1 Test sample a a a a a a a a a Equation used to calculate Ae (mg/liter) (Y) = X b Ae (mmol/day) = D1 x D2 x C 158 Where, D1 is dilution made at farm to the total voided urine (2 liters in case of lambs) D2 is dilution made for the second time at laboratory (1:15 in case of lambs) C is concentration of allantoin in mg/liter 158 is molecular weight of allantoin

7 II Determination of uric acid by uricase method Uric acid absorbs UV at 293 nm, although other compounds may also absorb at this wavelength. When samples are treated with uricase, uric acid is converted to allantoin and other compounds that do not absorb UV at 293 nm. Therefore, the reduction in absorbance reading after treatment with uricase is correlated with the concentration of uric acid in the sample. After treatment, the absorbance of the standards should be zero if the conversion is complete. Apparatus: UV Spectrophotometer, Temperature-controlled water bath, Ultrasonic bath (optional), 200 µl, 1 ml and 5 ml pipettes Chemicals 1. KH 2 PO 4 buffer, 0.67 M, ph 9.4. Adjust the ph with 5M KOH solution. 2. Uricase from porcine liver (e.g. SIGMA Cat. No U-9375, 33 unit/g solid). Stored frozen. 3. Uric acid Preparation of standards Prepare a stock uric acid solution of 100 mg/l. Dilute it to give working concentrations of 5, 10, 20, 30, 40, and 50 mg/l. 1 Prepare a 100 mg/l stock solution of uric acid Weigh 50mg uric acid, transfer to a 500ml volumetric flask, add about 400 ml distilled water, and then add about 100 µl of 0.01M NaOH to help dissolve the uric acid. Make up to volume with distilled water when uric acid is fully dissolved. 2 Dilute the stock solution in distilled water to give working concentrations of 5, 10, 20, 30, 40, 50 mg/l.to prepare 50 ml of the working standards 5, 10, 20, 30, 40, 50 mg/l respectively, weigh 2.5, 5, 10, 15, 20 and 25 g of stock solution into 50 ml volumetric lasks and make up to the volume with distilled water.

8 3 Store each working standards as small aliquots in the freezer. Only the necessary quantities are thawed and any left over discarded. This ensures that fresh standards are used for each analysis run Preparation before analysis Before the analysis : Start heating up the water bath at 37 C Enzyme preparation :Prepare a uricase enzyme solution of 0.12 IU/ml in the phosphate buffer. If it is not used immediately, keep the enzyme solution in fridge (4 C). Samples: Dilute the urine so that the final uric acid concentration is within the range of the standards Procedures 1 Pipette 2.5 ml of urine, the QC sample, standard or blank (distilled water) into 10 ml tubes. Prepare two sets of tubes. To all tubes, add 1 ml phosphate buffer and mix.

9 (Courtesy : IAEA) 2 To one set, add 150 µl buffer and to the other add 150 µl of uricase solution. Mix well. 3 Incubate in the water bath at 37 C for 90 min. 4 Remove from water bath, mix (shake) and transfer the solutions to cuvettes and read the absorbance at 293 nm. If the enzymatic conversion is complete, the absorbance of the standards with uricase added should be zero. If not, incubate in water bath for an additional 30 min and read again. Standard curve and calculation 1.Use the absorbance reading of the set without addition of uricase for constructing the standard curve. Standard curve is curvilinear over the standard range of 5-40

10 mg/l. When both X and Y are transformed to natural log (Ln) functions, Ln (Y) is linearly correlated to Ln (X). This relationship is then used for the calculation of the concentrations of the samples from their absorbance readings. Ln(Y) = a + b Ln(X) 2 Calculate the net reduction in absorbance ( ) for the samples due to uricase treatment. 3 Calculate the uric acid concentration from based on the established standard equation. C = EXP((Ln( ) - a) b) F where C is the concentration of unknown, is the net reduction in optical density after uricase treatment of the unknown/qc sample, F is the dilution factor.

11 III Determination of xanthine plus hypoxanthine by enzymatic method In this method, xanthine and hypoxanthine are converted to uric acid by treatment with xanthine oxidase and thus determined as uric acid which is monitored by its absorbance at 293 nm. The absorbance at 293 nm increases after the enzyme treatment. The net increase in absorbance is linearly correlated with the amount of uric acid formed. Apparatus: UV Spectrophotometer, Temperature-controlled water bath, Ultrasonic bath (optional), 200 µl, 1 ml and 5 ml pipettes Chemicals 1 KH 2 PO 4 buffer, 0.2M ph 7.35; adjust ph with a solution of either H 3 PO 4 or KOH. 2 L-histidine 4.3 mm. 3 Xanthine oxidase (XO). Stored in fridge. 4 Uric acid Preparation of standards Prepare a stock uric acid solution of 100 mg/l. Dilute it to give working concentrations of 10, 20, 30, 40, 50 and 60 mg/l. Procedure: Refer the section 8.2 for standards preparation. Preparation Before the analysis Before the analysis : Start heating up the water bath at 37 C Enzyme preparation : Dilute the xanthine oxidase in the phosphate buffer to obtain a concentration of 0.16 IU/ml. keep the enzyme solution in fridge if it is not used immediately.

12 Samples: Dilute the urine so that the final uric acid + xanthine +hypoxanthine concentration is within the range of the uric acid standards Procedures 1. Pipette 1 ml of urine, the QC sample, standard or blank (distilled water) into 10 ml tubes. Prepare two sets of tubes. To all tubes, add 2.5 ml phosphate buffer; add 0.35 ml L-histidine solution. Mix well. 2. To one set, add 150 µl buffer and to the other add 150 µl of xanthine oxidase solution. Mix well. 3. Incubate in the water bath at 37 C for 60 min. 4. Remove from water bath, mix (shake) and transfer the solutions to cuvettes and read the absorbance at 293 nm.

13 (Courtesy : IAEA) Note: Use the standards of xanthine to check whether the conversion of xanthine to uric acid is complete by xanthine oxidase treatment. The conversion of xanthine and hypoxanthine to uric acid is complete when the absorbance of the samples remains constant.

14 The activity of xanthine oxidase can be inhibited by excess amount of substrate in the sample. The addition of L-histidine reduces this inhibition. Standard curve and calculation 1. Use the absorbance reading of the set without addition of xanthine oxidase for constructing the standard curve. Standard curve is curvilinear over the standard range of 5-40 mg/l. When both X and Y are transformed to Ln functions, Ln (Y) is linearly correlated to Ln (X). Ln(Y) = a + b Ln(X) This relationship is then used for the calculation of the concentrations of the samples from their absorbance readings. 2 Calculate the net increase in absorbance ( ) for the samples due to XO treatment, i.e. the difference between two sets with and without XO addition: = absorbance with XO - absorbance without XO. 3 Calculate the corresponding concentration of uric acid from based on the

15 above standard curve. This increment in uric acid concentration corresponds, on a molar basis, to the sum of xanthine and hypoxanthine present in the samples. C = EXP((Ln( ) - a) b) where C is the concentration of unknown, after XO treatment of the unknown/qc sample. is the net increase in absorbance 4 Xanthine can also absorb UV at 293 nm although the absorbance is 10 times lower than uric acid at the same concentration. Hypoxanthine does not absorb UV at this wavelength. The calculated in Step 2 has thus been underestimated. Therefore it is necessary to correct for the contribution of absorbance from the xanthine (OD x ). We have to assume hypoxanthine has the same absorbance as xanthine since in this assay xanthine and hypoxanthine can not be separated. To correct for the absorbance due to xanthine, we need to establish the absorbance response factor for xanthine. This can be done in one of the following ways: (1) Use xanthine solutions of concentrations ranging from 10 to 50 mg/l, and go through the above procedure (without XO added). Fit the absorbance of xanthine

16 (ODx) into a linear function of xanthine concentration. A xanthine standard may not be needed for every run. Response factor: d= the slope of the xanthine standard curve (2) Single point measurement. Measure the absorbance of xanthine standard 50 mg/l. Response factor: d= absorbance xanthine standard concentration The contribution of absorbance from xanthine in the unknown samples can then be estimated: OD x = d C where C is the xanthine+hypoxanthine concentration calculated in Step 3 5 Re-adjust the net increased in absorbance of the unknown samples/qc samples. Re-calculated the concentration: C = EXP((Ln( ) - a) b) F where C is the corrected concentration of unknown, F is the dilution factor.

17 Worksheet example for Uric acid estimation in lambs : (OD at 293 nm) Sample Blank Stds 5 mg/l 10 mg/l 20 mg/l 30 mg/l 40 mg/l Ref. (QC) sample Test sample 1 Test sample 2 Test tube No. OD before treatment OD after treatment B B Mean of OD before treatment Ln (x) (concentration) Ln (y) (OD of before treatment 1a a a a a ΔOD a Ln ΔOD X Exp (X) Ue (mmol/ day) a a a a ΔOD = OD before incubation (3.65/3.5) x OD after incubation; X = (Ln ΔOD a) / b, Where a is intercept ( ) and b is slope (0.9461); Ue (mmol/day) = {Exp (x) x 10} / 168, Where 10 is dilution factor and 168 is molecular weight of uric acid

18 Worksheet example for Xanthine and Hypoxanthine estimation in lambs : (OD at 293 nm) Sample Blank Stds 10 mg/l 20 mg/l 40 mg/l 60 mg/l 80 mg/l Ref. (QC) sample Test sample 1 Test sample 2 Test tube No. OD before treatment (A) B B OD after treatment (B) Mean of OD before treatment Ln (x) (concentration) Ln (y) (OD of before treatment 1a a a a a OD1 (B-A) C1 ODx OD2 X&HXe (mmol/ day) a a a C1 = Exp [{Ln(OD1) a} / b], Where a is intercept ( ) and b is slope ( ); ODx = d x C1, Where d is the slope of the xanthine standard curve (0.0027) or absorbance xanthine standard concentration; OD2 = OD1 + ODx ; X & HXe (mmol/day) = ([Exp ({Ln (OD2) a} / b)] x 10)/ 168, Where 10 is dilution factor and 168 is molecular weight of uric acid.

19 Standard curve for Xanthine standard : (OD at 293 nm) Sample Test OD before OD after tube No. incubation incubation Blank B B Stds. 10 mg/l Mean of OD after incubation (X) d = X/C 1a mg/l a mg/l a mg/l a mg/l a d = X/C, Where X is absorbance of standards after incubation and C is concentrations of standards. Equation for xanthine standard : a = , b = , r = {Either d or slope (b) of the equation may be used for the calculation}

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21 APPENDIX - II Table 8.1 Average DMI (g/d) of individual lambs for the 4-groups during feeding and metabolism trial for the 4-periods. Group Period-1 Period-2 Period-3 Period-4 Anim. No. RH CS Total RH CS Total RH CS Total RH CS Total D D D D Mean ±SE ±27.85 ±27.74 ±55.58 ±29.49 ±28.39 ±57.88 ±29.47 ±28.61 ±58.09 ±31.64 ±29.92 ±61.56 D D D D Mean ±SE ±23.88 ±24.04 ±47.92 ±27.73 ±26.91 ±54.64 ±32.78 ±32.09 ±64.86 ±35.71 ±33.55 ±69.26 D D D D Mean ±SE ±9.44 ±9.52 ±18.96 ±6.87 ±6.72 ±13.59 ±7.08 ±6.84 ±13.92 ±5.52 ±5.24 ±10.75 D D D D Mean ±SE ±7.54 ±7.67 ±15.21 ±9.07 ±8.61 ±17.68 ±7.66 ±7.42 ±15.08 ±5.61 ±5.26 ±10.86

22 Table 8.2 AverageDMI (% of B.wt.) of individual lambs for the 4-groups during feeding and metabolism trial for the 4-periods. Period-1 Period-2 Period-3 Period-4 Group Anim.No. RH CS Total RH CS Total RH CS Total RH CS Total D D D D Mean ±SE ±0.0 ±0.0 ±0.0 ±0.0 ±0.0 ±0.0 ±0.0 ±0.0 ±0.0 ±0.0 ±0.0 ±0.0 D D D D Mean ±SE ±0.0 ±0.0 ±0.0 ±0.0 ±0.0 ±0.1 ±0.0 ±0.0 ±0.0 ±0.0 ±0.0 ±0.0 D D D D Mean ±SE ±0.0 ±0.0 ±0.1 ±0.0 ±0.0 ±0.0 ±0.0 ±0.0 ±0.0 ±0.0 ±0.0 ±0.0 D D D D Mean ±SE ±0.0 ±0.0 ±0.0 ±0.0 ±0.0 ±0.0 ±0.0 ±0.0 ±0.0 ±0.0 ±0.0 ±0.0 Table 8.3 Average OMI (g/d) of individual lambs for the 4-groups during feeding and metabolism trial for the 4-periods.

23 Group Period-1 Period-2 Period-3 Period-4 Anim. No. RH CS Total RH CS Total RH CS Total RH CS Total D D D D Mean ±SE ±25.83 ±26.31 ±52.14 ±27.14 ±25.97 ±53.11 ±27.27 ±26.77 ±54.05 ±29.14 ±26.74 ±55.88 D D D D Mean ±SE ±22.15 ±22.05 ±44.20 ±25.52 ±25.25 ±50.77 ±30.33 ±28.81 ±59.14 ±32.89 ±31.50 ±64.39 D D D D Mean ±SE ±8.75 ±8.95 ±17.70 ±6.32 ±6.04 ±12.36 ±6.56 ±6.51 ±13.06 ±5.08 ±4.78 ±9.86 D D D D Mean ±SE ±7.00 ±6.89 ±13.89 ±8.34 ±8.16 ±16.50 ±7.09 ±6.85 ±13.94 ±5.16 ±4.90 ±10.06

24 Table 8.4 Average DOMI (g/kg) of individual lambs for the 4-groups during metabolism trial for the 4-periods. Period-1 Period-2 Period-3 Period-4 Group Anim.No. DOMI DOMI DOMI DOMI D D D D Mean ±SE ±29.44 ±29.61 ±32.72 ±32.04 D D D D Mean ±SE ±30.26 ±35.39 ±41.98 ±51.02 D D D D Mean ±SE ±10.79 ±9.05 ±13.01 ±18.32 D D D D Mean ±SE ±8.49 ±20.61 ±15.52 ±7.71

25 Table 8.5 Average CPI (g/d) of individual lambs for the 4-groups during feeding and metabolism trial for the 4-periods. Period-1 Period-2 Period-3 Period-4 Group Anim.No. RH CS Total RH CS Total RH CS Total RH CS Total D D D D Mean ±SE ±1.63 ±5.38 ±7.01 ±1.80 ±5.82 ±7.62 ±1.86 ±6.14 ±8.00 ±1.88 ±6.05 ±7.93 D D D D Mean ±SE ±1.39 ±5.32 ±6.71 ±1.69 ±5.64 ±7.34 ±2.07 ±6.43 ±8.50 ±2.12 ±6.69 ±8.81 D D D D Mean ±SE ±0.55 ±1.92 ±2.48 ±0.42 ±1.34 ±1.76 ±0.45 ±1.37 ±1.82 ±0.33 ±1.05 ±1.38 D D D D Mean ±SE ±0.44 ±1.60 ±2.04 ±0.55 ±1.71 ±2.27 ±0.48 ±1.52 ±2.00 ±0.33 ±1.09 ±1.43

26 Table 8.6 Average NDFI (g/d) of individual lambs for the 4-groups during feeding and metabolism trial for the 4-periods. Group Period-1 Period-2 Period-3 Period-4 Anim. No. RH CS Total RH CS Total RH CS Total RH CS Total D D D D Mean ±SE ±23.38 ±10.12 ±33.50 ±26.94 ±11.86 ±38.80 ±26.42 ±11.68 ±38.10 ±28.80 ±12.59 ±41.39 D D D D Mean ±SE ±20.06 ±9.15 ±29.21 ±25.33 ±9.81 ±35.14 ±29.38 ±13.57 ±42.95 ±32.50 ±9.19 ±41.69 D D D D Mean ±SE ±7.93 ±3.81 ±11.74 ±6.28 ±2.91 ±9.19 ±6.35 ±2.03 ±8.38 ±5.02 ±2.04 ±7.06 D D D D Mean ±SE ±6.33 ±3.10 ±9.43 ±8.28 ±2.69 ±10.98 ±6.87 ±3.03 ±9.90 ±5.10 ±1.91 ±7.01

27 Table 8.7 Average ADFI (g/d) of individual lambs for the 4-groups during feeding and metabolism trial for the 4-periods. Group Period-1 Period-2 Period-3 Period-4 Anim. No. RH CS Total RH CS Total RH CS Total RH CS Total D D D D Mean ±SE ±14.39 ±2.68 ±17.07 ±16.50 ±5.00 ±21.50 ±16.43 ±4.68 ±21.11 ±16.47 ±16.47 ±32.94 D D D D Mean ±SE ±12.34 ±4.12 ±16.47 ±15.52 ±4.31 ±19.82 ±18.27 ±7.54 ±25.82 ±18.59 ±18.59 ±37.18 D D D D Mean ±SE ±4.88 ±1.53 ±6.41 ±3.84 ±1.54 ±5.38 ±3.95 ±0.63 ±4.57 ±2.87 ±2.87 ±5.74 D D D D Mean ±SE ±3.90 ±1.70 ±5.60 ±5.07 ±0.82 ±5.89 ±4.27 ±1.27 ±5.54 ±2.92 ±2.92 ±5.84 Table 8.8 (a) Quantity of dung voided (g/d) and its chemical composition (% DM) for individual lambs during metabolism trial

28 for the 4-groups. Period - 1 Period - 2 Quantity (g/d) Quantity (g/d) Group Anim. DM TA OM CP NDF ADF DM TA OM CP NDF ADF No. Wet Dry Wet Dry D D Mean ±SE ±4.83 ±86.97 ±25.49 ±0.23 ±0.23 ±1.00 ±1.50 ±0.93 ±4.55 ±86.77 ±27.06 ±0.23 ±0.23 ±0.63 ±0.36 ±0.67 D D Mean ±SE ±0.99 ±36.29 ±17.37 ±0.50 ±0.50 ±1.17 ±0.47 ±0.28 ±1.04 ±39.52 ±17.79 ±0.32 ±0.32 ±0.27 ±1.08 ±1.24 D D Mean ±SE ±2.16 ±24.89 ±9.27 ±0.19 ±0.19 ±0.47 ±0.69 ±0.69 ±0.74 ±19.68 ±7.61 ±0.23 ±0.23 ±0.26 ±0.62 ±1.37 D D Mean ±SE ±1.34 ±5.69 ±8.03 ±0.29 ±0.29 ±0.68 ±0.98 ±0.33 ±1.00 ±35.33 ±12.98 ±0.43 ±0.43 ±0.94 ±0.78 ±0.32 Table 8.8 (b) Quantity of dung voided (g/d) and its chemical composition (% DM) for individual lambs during metabolism trial

29 for the 4-groups. Group Anim. No. DM Period - 3 Period - 4 Quantity (g/d) Quantity (g/d) TA OM CP NDF ADF DM TA OM CP NDF ADF Wet Dry Wet Dry D D Mean ±SE ±3.22 ±76.53 ±24.36 ±0.20 ±0.20 ±0.51 ±0.38 ±1.12 ±2.41 ±75.42 ±27.92 ±0.26 ±0.26 ±0.32 ±0.59 ±0.85 D D Mean ±SE ±0.85 ±48.26 ±22.78 ±0.27 ±0.27 ±0.23 ±0.77 ±0.21 ±2.03 ±47.08 ±15.99 ±0.30 ±0.30 ±1.02 ±1.25 ±1.18 D D Mean ±SE ±1.73 ±42.22 ±12.77 ±0.03 ±0.03 ±0.31 ±1.12 ±0.49 ±2.55 ±54.72 ±15.10 ±0.20 ±0.20 ±0.85 ±0.99 ±0.74 D D Mean ±SE ±1.00 ±25.12 ±8.41 ±0.41 ±0.41 ±1.01 ±0.84 ±0.54 ±0.61 ±20.00 ±7.50 ±0.43 ±0.43 ±0.99 ±1.20 ±0.64

30 Table 8.9 (a) Digestibility of nutrients (%) in individual lambs during metabolism trial for the 4-groups. Period-1 Period-2 Group Anim.No. DM OM CP NDF ADF DM OM CP NDF ADF D D Mean ±SE ±1.09 ±1.10 ±3.97 ±0.87 ±0.43 ±0.80 ±0.84 ±2.60 ±0.89 ±0.55 D D Mean ±SE ±0.48 ±0.67 ±3.64 ±0.74 ±0.78 ±0.79 ±0.76 ±0.45 ±1.27 ±2.28 D D Mean ±SE ±0.80 ±0.83 ±1.56 ±1.33 ±1.65 ±1.08 ±1.00 ±0.94 ±1.39 ±0.53 D D Mean ±SE ±0.63 ±0.58 ±2.17 ±1.14 ±0.64 ±2.85 ±2.86 ±1.30 ±3.45 ±3.63

31 Table 8.9 (b) Digestiblity of nutrients (%) in individual lambs during metabolism trial for the 4-groups. Period-3 Period-4 Group Anim.No. DM OM CP NDF ADF DM OM CP NDF ADF D D Mean ±SE ±0.49 ±0.52 ±1.47 ±0.31 ±0.62 ±0.09 ±0.09 ±1.04 ±0.31 ±0.80 D D Mean ±SE ±1.68 ±1.68 ±2.23 ±1.93 ±2.00 ±1.78 ±1.79 ±4.97 ±2.34 ±1.58 D D Mean ±SE ±2.00 ±1.88 ±1.77 ±2.48 ±2.73 ±2.69 ±2.60 ±1.85 ±3.35 ±2.48 D D Mean SE ±1.73 ±1.77 ±1.92 ±2.29 ±1.88 ±0.89 ±0.97 ±3.09 ±1.52 ±1.04

32 Table 8.10 Average daily body weight gain (g/day) in lambs during the feeding trial for the 4-groups Period-1 Period-2 Period-3 Period-4 Group Anim.No. Gain, g/d Gain, g/d Gain, g/d Gain, g/d D D D D Mean ±SE ±2.06 ±1.19 ±6.30 ±12.88 D D D D Mean ±SE ±6.30 ±14.63 ±10.17 ±15.61 D D D D Mean ±SE ±15.48 ±1.19 ±4.29 ±11.36 D D D D Mean ±SE ±7.24 ±10.38 ±6.63 ±3.57

33 Table 8.11 Average daily urine voided (ml/day) by individual lambs during the feeding trial for the 4-groups Group Anim.No. Period-1 Period-2 Period-3 Period-4 D D D D Mean ±SE ±28.40 ±42.18 ±25.53 ±32.39 D D D D Mean ±SE ±30.74 ±43.98 ±75.67 ±41.36 D D D D Mean ± ±SE ± ±30.75 ±34.43 D D D D Mean ±SE ±90.51 ±38.25 ±43.62 ±13.86

34 Table 8.12 (a) N-balance ( g/d) for individual lambs during metabolism trial. Period-1 Group Anim. No. N-intake (g/d) N-outgo (g/d) RH CS Total Dung Urine Total Balance N-retained % of intake D Mean ±SE ±0.3 ±0.9 ±1.1 ±0.7 ±0.2 ±0.9 ±0.3 ±2.6 D Mean ±SE ±0.2 ±0.9 ±1.1 ±0.4 ±0.7 ±0.8 ±0.3 ±1.0 D Mean ±SE ±0.1 ±0.3 ±0.4 ±0.3 ±0.1 ±0.4 ±0.0 ±1.4 D Mean ±SE ±0.1 ±0.3 ±0.3 ±0.3 ±0.1 ±0.4 ±0.1 ±1.5

35 Table 8.12 (b) N-balance ( g/d) for individual lambs during metabolism trial. Period-2 Group Anim. No. N-intake (g/d) N-outgo (g/d) RH CS Total Dung Urine Total Balance N-retained % of intake D Mean ±SE ±0.3 ±1.0 ±1.3 ±0.7 ±0.3 ±0.9 ±0.4 ±2.0 D Mean ±SE ±0.3 ±0.9 ±1.1 ±0.3 ±0.6 ±0.9 ±0.3 ±2.0 D Mean ±SE ±0.1 ±0.2 ±0.3 ±0.2 ±0.3 ±0.5 ±0.3 ±2.9 D Mean ±SE ±0.1 ±0.3 ±.4 ±0.2 ±0.5 ±0.7 ±0.6 ±6.2

36 Table 8.12 (c) N-balance ( g/d) for individual lambs during metabolism trial. Period-3 Group Anim. No. N-intake (g/d) N-outgo (g/d) N-retained RH CS Total Dung Urine Total Balance % of intake D Mean ±SE ±0.3 ±0.9 ±1.2 ±0.7 ±0.4 ±0.9 ±0.7 ±6.0 D Mean ±SE ±0.3 ±1.1 ±1.4 ±0.5 ±0.9 ±1.4 ±0.6 ±4.5 D Mean ±SE ±0.1 ±0.2 ±0.3 ±0.3 ±0.2 ±0.4 ±0.2 ±1.9 D Mean ±SE ±0.1 ±0.3 ±0.3 ±0.4 ±0.2 ±0.4 ±0.3 ±2.8

37 Table 8.12 (d) N-balance ( g/d) for individual lambs during metabolism trial. Period-4 Group Anim. No. N-intake (g/d) N-outgo (g/d) N-retained RH CS Total Dung Urine Total Balance % of intake D Mean ±SE ±0.3 ±1.0 ±1.3 ±0.6 ±0.4 ±1.0 ±0.3 ±0.7 D Mean ±SE ±0.3 ±1.1 ±1.4 ±0.3 ±0.5 ±0.7 ±0.8 ±4.6 D Mean ±SE ±0.0 ±0.2 ±0.2 ±0.3 ±0.3 ±0.5 ±0.5 ±3.4 D Mean ±SE ±0.1 ±0.2 ±0.3 ±0.5 ±0.2 ±0.3 ±0.1 ±1.3

38 Table 8.13 (a) Purine derivatives (mmol/day) and microbial N supply (g/day) in lambs Period-1 Group Anim.No. Bodywt. Ae, Ue, XHe, PDe Pabs MNS MN,g/kg MN, g/kg mmol/d mmol/d mmol/d mmol/d mmol/d g/day DOMR ADOM D Mean ±SE ±1.93 ±0.82 ±0.06 ±0.04 ±0.85 ±1.11 ±0.81 ±3.03 ±1.97 D Mean ±SE ±1.80 ±0.48 ±0.07 ±0.04 ±0.58 ±0.76 ±0.56 ±1.82 ±1.18 D Mean ±SE ±0.45 ±0.18 ±0.02 ±0.09 ±0.25 ±0.37 ±0.27 ±1.99 ±1.29 D Mean ±SE ±0.59 ±0.13 ±0.03 ±0.03 ±0.14 ±0.21 ±0.15 ±1.36 ±0.90

39 Table 8.13 (b) Purine derivatives (mmol/day) and microbial N supply (g/day) in lambs Period-2 Group Anim.No. Bodywt. Ae, Ue, XHe, PDe Pabs MNS MN,g/kg MN, g/kg mmol/d mmol/d mmol/d mmol/d mmol/d g/day DOMR ADOM D Mean ±SE ±1.90 ±0.47 ±0.08 ±0.19 ±0.72 ±0.95 ±0.69 ±2.50 ±1.63 D Mean ±SE ±2.13 ±0.51 ±0.08 ±0.04 ±0.57 ±0.75 ±0.54 ±1.89 ±1.22 D Mean ±SE ±0.43 ±0.10 ±0.01 ±0.06 ±0.14 ±0.20 ±0.14 ±0.92 ±0.49 D Mean ±SE ±0.49 ±0.47 ±0.11 ±0.14 ±0.69 ±0.96 ±0.70 ±3.46 ±2.25

40 Table 8.13 (c) Purine derivatives (mmol/day) and microbial N supply (g/day) in lambs Period-3 Group Anim.No. Bodywt. Ae, Ue, XHe, PDe Pabs MNS MN,g/kg MN, g/kg mmol/d mmol/d mmol/d mmol/d mmol/d g/day DOMR ADOM D Mean ±SE ±2.05 ±0.64 ±0.13 ±0.17 ±0.91 ±1.16 ±0.84 ±2.22 ±1.39 D Mean ±SE ±2.35 ±0.28 ±0.05 ±0.06 ±0.37 ±0.45 ±0.32 ±2.11 ±0.62 D Mean ±SE ±0.36 ±0.20 ±0.03 ±0.06 ±0.23 ±0.32 ±0.23 ±1.27 ±0.81 D Mean ±SE ±0.37 ±.35 ±0.09 ±0.05 ±0.42 ±0.57 ±0.42 ±2.53 ±1.60

41 Table 8.13 (d) Purine derivatives (mmol/day) and microbial N supply (g/day) in lambs Period-4 Group Anim.No. Bodywt. Ae, Ue, XHe, PDe Pabs MNS MN,g/kg MN, g/kg mmol/d mmol/d mmol/d mmol/d mmol/d g/day DOMR ADOM D Mean ±SE ±2.35 ±0.56 ±0.15 ±0.10 ±0.73 ±0.92 ±0.67 ±0.81 ±0.53 D Mean ±SE ±2.75 ±0.74 ±0.13 ±0.28 ±0.63 ±0.78 ±0.57 ±0.88 ±0.58 D Mean ±SE ±0.24 ±0.49 ±0.06 ±0.22 ±0.77 ±1.04 ±0.75 ±4.07 ±2.68 D Mean ±SE ±0.35 ±0.28 ±0.04 ±0.03 ±0.30 ±0.39 ±0.29 ±0.93 ±0.61

42 Table 8.14 (a) Average DMI (kg/day) of individual cows for the 3-groups during feeding trial for the 3-periods Period-1 Group Anim. No. Week-1 Week-2 Week-3 Week-4 Week-5 Average FMS CS Total FMS CS Total FMS CS Total FMS CS Total FMS CS Total FMS CS Total D Mean ±SE ±0.3 ±1.6 ±1.3 ±0.0 ±1.6 ±1.7 ±0.0 ±1.6 ±1.7 ±0.1 ±1.6 ±1.8 ±0.8 ±1.6 ±2.5 ±0.2 ±1.6 ±1.8 D Mean ±SE ±0.3 ±1.4 ±1.1 ±0.2 ±1.4 ±1.1 ±0.4 ±1.4 ±1.0 ±0.3 ±1.4 ±0.9 ±0.5 ±1.4 ±0.8 ±0.3 ±1.4 ±1.0 D Mean ±SE ±0.4 ±0.2 ±0.2 ±0.1 ±0.2 ±0.0 ±0.1 ±0.2 ±0.1 ±0.1 ±0.2 ±0.1 ±0.3 ±0.2 ±0.1 ±0.2 ±0.2 ±0.0

43 Table 8.14 (b) Average DMI (kg/day) of individual cows for the 3-groups during feeding trial for the 3-periods Period-2 Group Anim. No. Week-1 Week-2 Week-3 Week-4 Week-5 Average FMS CS Total FMS CS Total FMS CS Total FMS CS Total FMS CS Total FMS CS Total D Mean ±SE ±0.8 ±0.9 ±1.8 ±0.6 ±0.9 ±1.5 ±1.3 ±0.9 ±2.2 ±1.2 ±0.9 ±2.1 ±1.2 ±0.9 ±2.2 ±1.0 ±0.9 ±2.0 D Mean ±SE ±0.3 ±1.3 ±1.1 ±0.2 ±1.3 ±1.1 ±0.4 ±1.3 ±1.0 ±0.4 ±1.3 ±1.0 ±0.5 ±1.3 ±0.8 ±0.4 ±1.3 ±1.0 D Mean ±SE ±0.3 ±0.2 ±0.1 ±0.2 ±0.2 ±0.0 ±0.3 ±0.2 ±0.1 ±0.1 ±0.2 ±0.1 ±0.0 ±0.2 ±0.3 ±0.2 ±0.2 ±0.1