Basic Laboratory Manual: Analysis of Animal Feed and Physical Evaluation

2.1. Determination of Moisture in Feedstuffs

The amount of free water that is present in any feedstock is referred to as moisture. Any feedstock sample can be kept free of moisture by placing it in an oven. "Dry Matter" refers to the amount that is left over after this process.

2.1.1. Apparatus and Equipment

• Metal Tong
• Heat resistant gloves
• Spatula
• Permanent markers
• Hot air oven
• Petri-dish
• Desiccators
• Balance machine

2.1.2. Procedure

Step 1. Petri-dish preparation

Place the clean glass petri-dish (120 mm in diameter) in oven and dried in 105⁰c for 20 minutes. Keep the lid opened and separated. Take out the petri-dish from the oven and put into the desiccator to cool.

Step 2. Sample preparation

The petri-dish is ready to use in the analysis of moisture. Calibration status of the balance should check before weighting. Use petri-dish of 120mm in diameter to take 10g of sample.

Step 3. Drying on hot air oven

Place the petri-dish with sample inside the hot air oven carefully. Close the door tightly. Set the temperature at 130⁰c for 2hr. after 2hr open and put the petri-dish from oven into desiccator.

Step 4. Final weight

Now take the final weight of the dish containing dried sample. Clean the balance after measuring.

Step 5. Calculation

$$Moisture={\displaystyle \frac{Ws-(W1-W2)}{Ws}}$$

W_s= weight of sample

W₁= weight of dish

W₂= weight of dish after drying

2.2. Determination of Dry Matter (DM)

Dry matter is the portion of forages that have been dehydrated. Dry matter content is the foundation for all nutritional analyses. The Animal Oxygen Analysis and Chemistry (AOAC) method for determining the moisture content of animal feed has been modified for the Wollo University Animal Nutrition Laboratory to work differently.

2.2.1. Equipment

• Silica crucibles
• Desiccators
• Hot air oven
• Balance machine

2.2.2. Procedure

• ✓ Dried and grinding samples
• ✓ Dry empty crucibles or container overnight at 105 °C
• ✓ Cool samples in desiccators to room temperature
• ✓ Measure oven-dry crucible (W_t)
• ✓ Add approximately 2g of ground sample; record weight (Ws)
• ✓ Dry overnight at 105 °C for 24hr
• ✓ Allow the desiccators to cool down to room temperature
• ✓ Weight oven dry crucible and sample = (W0)

$$\mathrm{\%}\mathrm{D}\mathrm{M}={\displaystyle \frac{\mathrm{W}0-\mathrm{W}\mathrm{t}}{\mathrm{W}\mathrm{s}}}x100$$

2.3. Determination of Ash

2.3.1. Equipment

• Sensitive balance (Figure 2)
• Muffle furnace (550 °C) (Figure 6)
• Desiccator (Figure 2)
• Porcelain or silica crucibles (Figure 5)

Figure 1. Grinding Machine.

Figure 1. Grinding Machine.

Figure 2. Balance machine and Dissector.

Figure 2. Balance machine and Dissector.

Figure 3. Heat sensitive glove, spatula, and metal tong.

Figure 3. Heat sensitive glove, spatula, and metal tong.

Figure 4. Hot air oven and Petri-dish.

Figure 4. Hot air oven and Petri-dish.

Figure 5. Silica crucibles.

Figure 5. Silica crucibles.

Figure 6. Muffle furnace.

Figure 6. Muffle furnace.

2.3.2. Procedure

• Ignite dry matter samples overnight at 550 °C for 2:30hr in muffle furnace
• Allow the desiccators to cool down to room temperature
• Weigh ignited crucible and sample (Wa)
• Weight oven dry crucible and sample = (W₀)
• Weight oven-dry crucible (W_t)

$$\mathrm{\%}\mathrm{A}\mathrm{s}\mathrm{h}={\displaystyle \frac{\mathrm{W}\mathrm{a}-\mathrm{W}\mathrm{t}}{\mathrm{W}\mathrm{o}-\mathrm{W}\mathrm{t}}}x100$$

2.3.3. Precaution

• The ash is highly hygroscopic and thus weighing should be done quickly

2.4. Determination of Organic Dry Matter (ODM)

Organic dry matter of feedstuff can be calculated by using the following formula adopted from AOAC.

$$\mathrm{\%}\mathrm{O}\mathrm{D}\mathrm{M}=100-\%Ash$$

2.5. Determination of Crude Protein

It is every nitrogenous substance found in the feedstock sample. True protein and non-true protein (non-protein nitrogen), like urea, are included in it. When it comes to farm animals' nutrition, crude protein is regarded as a significant component. The Kjeldahl method is used to calculate total nitrogen, or crude protein.

2.5.1. Equipment

• Digestion rack
• Balance machine (Figure 2)
• Spatula (Figure 3)
• Acid proof glove (Figure 3)
• Funnel
• Kjeldahl flask
• Mixer machine
• Dropper
• Pipette
• Sample shaker
• Conical flask
• Volumetric flask
• Measuring cylinder
• Hot plate with magnetic stirrer

2.5.2. Chemicals/Reagent Preparation

1. Catalyst (potassium sulphate + copper sulphate + selenium sulphate

KSo₄ + CuSo₄ + SeO₂

Ratio 5 3 1

Procedure

• Clean everything what you need to prepare the catalyst
• Use clean and separated spatula for weighting different reagent/chemicals
• Transfer into same mixer chamber to mix catalysts
• Close the mixer chamber tightly with lid
• Mix all the tree chemicals using the mixer machine

2. Sulfuric Acid (concentrated 95-98%)

3. 40% Sodium Hydroxide solution

Procedure

• Take weight of 40g of NaOH pellet
• Transfer the weighted NaOH into the flask and shake slightly to mix
• Take 80ml of distilled water into the flask
• Label the flask with 40% NaOH, the wait too dissolve all the pellets and cool at room temperature
• After cooling, add water to make the final 100ml volume

4. 4% Boric Acid solution preparation

Procedure

• Weight 4g of boric acid powder
• Transfer the boric acid powder into 40ml of some hot distilled water
• Stir with a clean glass rod to dissolve boric acid well. Tur off the hot plate and cool the boric acid solution
• Label a 100ml volumetric flask with 4% boric acid solution
• Take the cooled boric acid solution into the volumetric flask
• Add distilled water to volume up to 100ml, and then rotate the flask to mix 4% of boric acid solution.

5. 0.1N hydrochloric acid (standardized)

Procedure

Step 1. Phenolphthalein indicator preparation

• Dissolve 2g phenolphthalein indicator powder into 100ml of ethanol and mix well by shaking

Step 2. Dilute 0.83ml of HCl (concentrated) with distilled water to make the total volume of 100ml.

• Label a 100ml volumetric flask with 0.1N HCl
• Drop 80ml of distilled water into 100ml volumetric flask
• Pipette 0.83ml of concentrated HCl (37%) into the flask
• Add distilled water enough to make 100ml of the final volume
• Shake the flask to mix the HCl with distilled water

Step 3. Standardized newly prepared 0.1N HCl with standard 0.1N NaOH solution and find the actual normality.

• Take 0.1N standard NaOH solution into burette
• Take the initial burette reading
• Measure 20ml of newly prepared HCl solution and take into conical flask
• Add 3-4 drops of phenolphthalein indicator into conical flask
• Titrate it with standard 0.1 NaOH solution
• Take final burette reading after the color changed

Step 4. Calculation for standardization of 0.1N HCl

• Burette reading of NaOH (V2)
• Normality of NaOH (N2)
• Volume of prepared HCl (V1)
• Normality of HCl (N1)

$$N_1={\displaystyle \frac{V_{2}X{N}_2}{N_1}}$$

Rewrite the actual normality of HCl from the calculation of standardization

6. Methyl red indicator

• Dissolve 100mg Methyl red indicator powder into 100ml of methanol and mix well by shaking

Determination of total nitrogen (crude protein) using the Kjeldahl method

2.5.3. Procedures

• 1. Digestion

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Label the kjeldahls flask with the sample number

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Take the weighted sample into the flask

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Again, weight 3g of catalyst

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Take the catalyst into the flask to mix sample

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Take 20ml of concentrated H₂SO₄ and pour the acid into the sample flask

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Shake the flask gently to mix the acid with sample and catalyst

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Place the flask on digestion unit carefully

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Turn on the digester power and set the temperature at 230⁰ c and water circulation open

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After 2hr clean green color solution indicates the end of digestion

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Turn off the digester and wait to cool the flask

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Now, the digested sample diluted with distilled water

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Add 20ml of distilled water into the flask, mix and pour the digested sample into 100ml volumetric flask

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Add enough water to make the final volume of 100ml

• 2. Distillation

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Measure 30ml of 4% boric acid and pour into a conical flask

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Place the flask on the distillate collection unit

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Take 10ml of digested sample to transfer into distillation flask

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Now, add 50ml of 40% NaOH

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Add another 50ml of distilled water

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Run the distillation at 200⁰c for 1hr

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Turn off the distillation after collect approximately 100ml of distillated

• 3. Titration

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Take 0.1N HCl into burette

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Note the initial burette reading

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Add few drops of methyl red indicator into the conical flask and mix well

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Start titration-adding 0.1N HCl

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Place a white background at bottom of the flask to transparence colors

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Start titration adding 0.1N HCl

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Stop the titration if the color is changed into orange

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Note final reading of burette

• 4. Calculation

Finally calculate nitrogen and crude protein

$$N\mathrm{\%}={\displaystyle \frac{V_1+n_{1}x{F}_{1}x{M}_{wn}}{W_{s}x10}}$$

$$\mathrm{C}\mathrm{r}\mathrm{u}\mathrm{d}\mathrm{e}\mathrm{protein}\mathrm{\%}=N\mathrm{\%}xFactorx{F}_2$$

Where

V₁= Volume of 0.1N HCl (final burette reading –initial burette reading)

n₂= Normality of HCl

F₁= Acid factor

F₂= Dilution Factor

Mwn=Molecular weight of nitrogen =14.007

2.6. Determination of Crude Fat or Ether Extract

Crude fat or ether is estimated by extracting the feed sample using continues evaporation and condensation of fat solvent like petroleum ether, diethyl ether, benzene, hexane etc. In special made extraction apparatus, that is soxhlate apparatus. Lipids are a group of materials that are insoluble in water but soluble in ether, chloroform, and benzene. The ether extraction procedure itself is quite simple and usually involves a reflux apparatus in which the ether is boiled, condensed, and allowed to pass through the feed sample.

2.6.1. Apparatus and Equipment

• Soxhlet apparatus
• Soxhlet extractor
• Filter paper
• Measuring cylinder (Figure 8)
• Thimbles

Figure 7. Funnel and Kjeldahl digester unit.

Figure 7. Funnel and Kjeldahl digester unit.

Figure 8. Hot plate with magnetic stirrer and measuring cylinder.

Figure 8. Hot plate with magnetic stirrer and measuring cylinder.

Figure 9. Soxhlet appatus and extractor.

Figure 9. Soxhlet appatus and extractor.

2.6.2. Chemical/Reagent

• n-hexane 95%

2.6.3. Procedure

Step 1. Thimble Preparation

• Gather everything you need to make a thimble with filter paper
• Make a thimble with filter paper
• Place the thimble on balance machine
• Take weight of a thimbles

Step 2. Sample Preparation

• Grind the sample if it is solid
• Put about 4.5g of sample into the thimble.
• Take note weight of the sample
• Place small amount of cotton into thimble in a way that covers the sample
• Fold the thimble to enclose the sample
• Take a cellulose thimble (sample holder)
• Label the thimble contain sample with sample number and put inside the cellulose thimble
• Take a cleaned and dried flat bottom flask
• Take a weight of the flask placing in a balance machine

Step 3. Fat extraction

• Set up soxhlet extraction unit placing the sample in it
• Add sufficient amount of n-hexane
• Run the water through the condenser of soxhlet extractor
• Turn on the power and active for 6hr
• Take out the sample from thimble
• Rotate the flask to evaporate the excess n-hexane

Step 4. Taking final weight

• Place the flask inside the oven to remove moisture and hexane
• Set the temperature at 1100c for 30 minutes
• Take out the dried flask and place in desiccator to cool for 20 minutes
• Measure the final weight of flask after cooling

Step 5. Calculation

$$\mathrm{\%}\mathrm{C}\mathrm{r}\mathrm{u}\mathrm{d}\mathrm{e}\mathrm{F}\mathrm{a}\mathrm{t}={\displaystyle \frac{W_2-W_1}{W_s}}x100$$

Where

W₁= weight of flask

W₂= weight of flask and fat

W_s= weight of sample

2.7. Determination of Crude Fiber

2.7.1. Reagents

• Sulfuric acid solution, 0.255N, 1.25 g of H2SO4/100 mL
• Sodium hydroxide solution, 0.313N, 1.25 g of NaOH/100 mL, free of Na2CO3 (concentrations of these solutions must be checked by titration)
• Alcohol - Methanol, isopropyl alcohol, 95% ethanol, reagent ethanol
• Bumping chips or granules - antifoam agent (decaling)

2.7.2. Apparatus

• Digestion apparatus
• Ashing dishes
• Desiccator
• Filtering device
• Suction filter: to accommodate filtering devices. Attach suction flask to trap in line with aspirator or other source of vacuum with valve to break vacuum.

2.8. Determination of NFE (Nitrogen Free Extract)

Nitrogen free extract (NFE) represents the soluble carbohydrate fraction of the feed. In the Weende’s system of analysis, NFE is not estimated but calculated.

NFE on as feed basis = 100 – (Moisture+ Crude protein +Ether extract + Crude fiber + Total ash)

NFE on dry matter basis = 100 – (Crude protein + Ether extract + Crude fiber + Total ash)

3.1. Preparation of the Ingredients

The weight of the block to be made determines the amount of each ingredient to be mixed. Using the following proportion, UMB can be produced by thoroughly mixing the exact quantities of the components;

• Molasses (34%)
• Urea (10%)
• Cement (15%)
• Wheat bran (25%)
• Noug seed cake (13%) and
• Common salt (3%).

3.2. Apparatus

• Molding instrument
• Ingredients
• Mixing equipment
• Weighing scales.

3.3. Procedure

1. Collect the following ingredients and prepare based on the required nutrient block. First, all of the ingredients are weighed out and placed in sacks, plastic bags, or buckets.

• Molasses
• Urea
• Cement
• wheat bran
• Noug seed cake
• Common salt

2. The cement and water are mixed in the tank by hand or by using a wooden paddle

3. The salt, molasses and urea are then added and similarly mixed

4. Finally the bran is added quite slowly as all the ingredients are mixed together

5. The mixed ingredient will shoveled into the moulds where it is tamped to displace the air

6. After moulding, the blocks are usually left for 24 hours before being placed in storage.

Precautions While Supplementing Urea Molasses Block

It is essential to note the following while supplementing Urea Molasses Block

• Ø Feed to ruminants only (sheep, goats and cattle).
• Ø Do not feed to monogastrics, (i.e., horses, donkeys, or pigs).
• Ø Do not feed to young ruminants less than six months of age (kids, lambs)
• Ø Blocks should be used as a supplement and not as the basic ration
• Ø A minimum of coarse forage in the rumen is essential
• Ø Never give blocks to an emaciated animal with an empty stomach. There is the risk of poisoning due to excessive consumption
• Ø The amount of blocks fed to sheep and goats should be limited to 100 grams/day while for cattle it should be limited to 700 grams/day.
• Ø The blocks should never be supplied in ground form or dissolved in water as this can result in over consumption
• Ø Supply sufficient amount of water ad lib