18
D663 = spectrophotometer on 633 nm wave length D645 = spectrophotometer on 645 nm wave length
3.4.4 The analyses of forage quality
Since the feed was not only focused on the quantities rather its quality. Moreover the main process of growth from forage was obtained form
photosynthesis process. However, the product of photosynthesis would be transferred into the organ for the growth and maintenance. The product of
photosynthesis was the dry matter production and others nutrients compound. Hence, the nutrients content analyses were measured on moisture analysis, dry
matter production, ash analysis, fat content fiber content and protein content respectively.
3.4.4.1 Dry Matter Analysis
Dry matter analysis was measured based on AOAC method Association of Official Analytic Chemist. The Total Dry Matter by Oven Drying at 105
o
C for 16 hr. The principle mechanism was emphasized on the heat treatment 105
C. Water would evaporated by heat treatment, remaining the rest material dry
matter that would be weighted formerly. Total dry matter is determined gravimetrically as residue remaining after drying. Weighing made on hot sample
or after cooling in desiccator. The calculation of moisture content could be seen on equation:
Where W
1
= dry weight of sample and container with cover in grams W
2
= tare weight of container with cover in grams W
3
= dry weight of sample in grams Percent Total Moisture was calculated as:
Total Moisture = 100 - Total DM
19
3.4.4.2 Ash Analysis
Ash of Animal Feed. 942.05 Official methods of Analysis. 1990. Association of Official Analytical Chemists, 15th Edition. Ashing is the process
of mineralization for preconcentration of trace substances prior to chemical analysis. The ash content is an approximate measure of the mineral content and
other inorganic. Remove crucibles with cover which have been dried for at least 2 hr at 100
C from oven, to
desiccator. Cool, and record weight of crucibles with cover to the nearest 0.1 mg W1. Weigh 1.5 to 2.0 g of sample into the crucible,
recording weight of crucible with cover and
sample to the nearest 0.1 mg W2. Ash in furnace at 600
o
C for 2 hr after the furnace reaches temperature. Allow crucibles to cool in furnace to less than 200
o
C and place crucibles with cover in
desiccator with vented top. Cool and weigh crucible with cover and ash to the
nearest 0.1 mg W3.
3.4.4.3 Fat Analysis
Fat analysis was conducted based on AOAC method. It emphasized on the fat extraction measurement. Sample was weight the ground dry sample into the
extraction thimble. The extraction thimble was closed with fat free cotton wad, and inserted into the Soxhlet extractor. Fill the solvent into the solvent vessel, and
extracted at a temperature of 50
O
C for 16 hours. The solvent drained into a suitable container by opening the spigot on the Soxhlet extractor. The solvent
vessel was continued for heating until all the solvent has been evaporated and condensed in the Soxhlet extractor. Vessel contained fat residue was placed in a
drying oven 105
O
C and heat to constant weight indicating evaporation of all solvent. The vessel containing the fat was allowed to cool to room temperature
about 30 minutes. Compute the fat content according to the following formula
F = x 100
20
Where m
1
= weight the dry empty vessel gram m
2
= weight of the vessel containing fat residue after evaporation of the solvent gram
E = the sample weight gram
3.4.4.4 Protein Analysis
Protein Crude Determination in Animal Feed: Copper Catalyst Kjeldahl Method. 984.13. Official Methods of Analysis. 1990. Association of Official
Analytical Chemists. The Kjeldahl method is the standard method of nitrogen determination dating back to its development in the late 1800s. The method
consists of three basic steps: 1 digestion of the sample in sulfuric acid with a catalyst, which results in conversion of nitrogen to ammonia; 2 distillation of the
ammonia into a trapping solution; and 3 quantification of the ammonia by titration with a standard solution.
Digestion
Weigh approximately 1g ground sample into digestion flask, recording weight Wto nearest 0.1 mg. Include reagent blank and high purity lysine HCl as check
of correctness of digestion parameters. Weigh a second subsample for laboratory dry matter determination. Add 15 g potassium sulfate, 0.04 g anhydrous copper
sulfate, 0.5 to1.0 galundum granules. Place flask on pre heated burner adjusted to bring 250mL water at25oC to rolling boilin 5 min. Heat until white fumes clear
bulb of flask, swirl gently, and continue heating for 90 minfor copper catalyst or 40 min for CuSO4TiO2 mixed catalyst. Cool, cautiously add 250 mL distilled
water and cool to room temperature 25oC. Note: If bumping occurs during distillation, volume of water may be increased to ca. 275 mL.
Distillation:
Prepare titration flask by adding appropriate volume VHCl accurately measured acid standard solution to amount of water so that condenser tip is immersed try
15 mL acid and 70 mL water if undecided. For reagent blank, pipet 1 mL of acid and add approximately 85 mL water. Add 3 to 4 drops methyl red indicator
21 solution. Add 2 to 3 drops of tributyl citrate or other anti foam agent to digestion
flask to reduce foaming. Add another 0.5 to1.0 galundum granules. Slowly down side of flask, add sufficient 45 sodium hydroxide solution approximately80
mL to make mixture strongly alkali. Do not mix until after flask is connected to distillation
apparatus or ammonia will be lost. Immediately connect flask to
distillation apparatus and distill at about 7.5 boil rate
temperature set to bring 250 ml at 25
o
C to boil in 7.5 min, until at least 150 ml, distillate was collected in titrating flask. Remove digestion flask and titrating flask from unit, rinsing the
condenser tube with distilled
water as the flask is being removed.
Titration
Titrate excess acid with standard sodium hydroxide solution to orange endpoint color change from red to orange to yellow and record volume to nearest 0.01 mL
VNaOH. Titrate the reagent blank B similarly. The calculation of N :
[ ]
V
NaOH
= mL standard NaOH needed to titrate sample V
HCl
= mL standard HCl pipetted into titrating flask for sample V
NaOH
= Normality of NaOH
V
HCl
= Normality of HCl V
BK
= mL standard NaOH needed to titrate 1 mL standard HCl minus B B
= mL standard NaOH needed to titrate reagent blank carried through method and distilled into 1 mL standard HCl
1.4007 = milliequivalent weight of nitrogen x 100
W = sample weight in grams
Percent Crude Protein CP CP DM basis= N DM basis X F
Where F = 6.25 for all forages and feeds except wheat grains
