Shah Nawaz Dahri Hi

For Which parameter? for example protein determination or calcium determination?

Article ASSESSMENT OF THE QUALITY OF THE POULTRY FEED AND ITS EFFECT...

Feed evaluation is the testing of feed quality, providing information on the composition of feed or feed ingredients as well as their suitability for poultry. Poultry feed is made up of many ingredients, which are broadly grouped into providers of energy (fats, oils and carbohydrates), protein (amino acids), vitamins, minerals and product quality enhancement. Typically, cereals such as wheat, barley, sorghum and maize will provide energy while soybeans, lupins, canola and peanuts provide protein. These ingredients are then combined in such a way as to provide the energy, protein, vitamin and mineral requirements for poultry through the process of feed formulation. In order to know what amounts of these ingredients should be included in the diet, the ingredients are first evaluated, to see what nutrients they contain in what quantities. After the diet has been prepared, it may also be necessary to evaluate the complete product, to determine its suitability for the class of poultry that will be fed (such as egg layers, meat chickens or breeders).

Feed evaluation is a key process in the poultry industry. Feed ingredients need to be tested in order to formulate the complete diet, and diets have to be evaluated to determine their suitability for poultry. Evaluation provides different types of information, as required by nutritionists and farmers. In general, the range of tests that can now be performed is wide and it is now possible to obtain results rapidly.

Measures of feed quality

Feeds and feed ingredients can be evaluated physically as well as chemically. The physical evaluation of feed mostly provides preliminary information on the quality of the material. It involves assessing physical qualities such as weight, colour, smell and whether the material has suffered from any contamination by other materials. Chemically, feed is made up of water and dry matter. The dry matter contains organic and inorganic compounds. The organic part of feed is made of mainly carbohydrates, proteins, vitamins and fats and oils. The inorganic part is made of mineral elements, also known as ash. Feed or feed ingredients can be analysed to provide values of each of these components. Apart from obtaining values of chemical composition, the extent of utilisation of these components by the bird, termed nutritive value, is also measured.

How feed quality is measured

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Feed samples (Source: Aust. Chicken Meat Fed.)

Feed quality is measured by chemically breaking up the food into the components mentioned above. In the industry, it is sometimes necessary to break down these large components into smaller analytical fractions. Thus, values of starch and the non-starch component (called fibre) of carbohydrates may be provided. Proteins are made of amino acids, 10 of which must be present in poultry diets, so their amounts should be indicated during feed evaluation.

In the past, feed evaluation was a cumbersome process, requiring days to complete. However, newer equipment and procedures have been developed, which enable the rapid evaluation of most materials. For example, starch is determined using a ready-to-use kit and protein is rapidly determined on Leco® machines, which eliminate time-consuming digestion of feed with strong acids and reaction of material with acids and bases. Near-infrared reflectance spectroscopy (NIRS) is one of the latest techniques by which feed ingredients can be evaluated with the most minimal preparation of the sample. NIRS provide the capability to rapidly measuring crude protein, fibre,  fat, total and digestible amino acids, calcium, total and available phosphorus and also the energy value (ME) of individual ingredients.

Of greater importance in feed evaluation is the response of poultry to particular feeds. This is regarded as the real nutritive value of the feed, and must be measured as part of feed evaluation. Nutritive value does not necessarily entail animal growth or egg production. It gives information on how much of each of the fractions in feed, i.e. starch or energy, protein (amino acids), fats, minerals or vitamins, was used by the bird. When feed is given to poultry, they are able to break down only a fraction of the feed and absorb it into the body for growth and egg production. The rest is voided in faeces and urine, which are excreted together by poultry. The amount of nutrients retained by the bird is an indication of the nutritive value of the feed.

Importance of feed evaluation

Feed evaluation is important because ingredients that belong to the same class contain different nutrients; for example, maize provides more energy than wheat while soybeans contain more proteins than lupins and canola. The same ingredient varies from one supplier to the other, and between years. In drought years, cereals fill poorly and are therefore lower in quality. Most importantly, if feeds are not evaluated, it is not possible to tell if the material will be suitable for feeding poultry. Feeding standards have already been set for different types of poultry, so the requirements for different nutrients must be met precisely. It is possible, with the current state of knowledge, to predict poultry growth or egg production by modelling feed quality, type of housing, class of poultry and duration of feeding. The central key issue in these models is feed quality, which can only be obtained through feed evaluation.

Journal of Poultry Science and Technology | April-June, 2016 | Volume 04 | Issue 02 | Pages 28-38 © 2016 Jakraya JOURNAL OF POULTRY SCIENCE AND TECHNOLOGY Journal homepage: www.jakraya.com/journal/jpst ORIGINAL ARTICLE Laboratory Protocols of Wet Analysis for Poultry Feed and Raw Materials Md. Saiful Islam*1 , Md. Moinul Haque2 and Md. Shakhawat Hossain3 *1Department of Applied Chemistry and Chemical Engineering, University of Rajshahi, Rajshahi-6205, Bangladesh. 2Department of Chemistry, Dhaka City College, Dhaka-1205, Bangladesh. 3Bangladesh Sericulture Research and Training Institute, Rajshahi-6207, Bangladesh. *Corresponding Author: Md. Saiful Islam E-Mail: [email protected] Received: 04/06/2016 Accepted: 26/06/2016 Abstract Quality control of both feed and raw materials is essential for every poultry feed industry. But quality control department is fully dependent on the analysis protocols. Analysis results in expressing the raw material as well as product quality whether it is accepted or rejected. In the feed mill laboratory, the chemist analyzes the different nutrients such as protein, calcium, fat, salt, ash as well as moisture content. Depending on the analyzed nutrient values, the nutritionist makes the formula for feed production. After that the nutrient values are rechecked in the final product in the laboratory and also compared with the standard value. It is noticed that, the analysis protocols help to evaluate the formulation as well as the quality of both feed and raw materials. Keywords: Poultry, Feed, Raw materials, Wet analysis, Laboratory protocol. 1. Introduction The goal of feed manufacturing is to produce feed that meets intended specifications in nutritional composition. Today the feed manufacturing is a very competitive activity and consistent feed quality is a key growth driver. Quality control and assurance have emerged as a critical feature in the business of feed manufacturing. A quality control system involves personnel being properly trained to ensure a high level of organization, documentation and policing of various procedures and processes necessary to guarantee the basic quality of feed ingredients and feed. Laboratory analysis is a definitive aspect of a quality control. The analysis of raw materials can help the feed manufacturer in several ways, such as: a) Prediction of nutritive values of feed ingredients and feed b) Avoiding contaminants c) Detecting adulterants Knowledge of feed ingredient composition is vital for the nutritionist in order to meet precisely the nutrient requirements of livestock. The nutrient values in any feed ingredient vary from season to season, source to source; batch to batch as also within a batch therefore feed ingredients need to be analyzed carefully for their nutritive value before they are incorporated into the diet. Even a slight variation in quality of feed ingredients or feed, affects animal performances. It is difficult to have a general assumption of the published values because a wide variability is observed in the nutrient content of raw materials (Islam et al., 2015a; Islam et al., 2015c). Whilst under formulating a ration will reduce animal performance and is an obvious quality deficiency, over formulation also deviates from quality since it results in losses of revenue. The establishment of a feed mill laboratory can ensure feed quality by controlling the quality of feed ingredients and by assuring the quality of final feed (Benton et al., 2004). Every quality control program should include periodic laboratory analysis of ingredients and feed (Briggs et al., 1999; Cooper et al., 2002; Defoor et al., 2000). To achieve optimal animal performance well balanced diets that satisfy nutrient requirements of the animal is mandatory and for producing these diets accurate formulation is essential. The most accurate formulations result when laboratory analysis of ingredients is available. Success of a quality control program hinges on collection of representative samples for laboratory analysis. Great care should be taken to ensure samples are representative of material so that lab results reflect the nutrient content of the ingredient or feed being sampled. Compound feeds are composed as a blend of various raw materials and/or by-products. Whilst formulating feed, the feed manufacturer needs to find Islam et al… Laboratory Protocols of Wet Analysis for Poultry Feed and raw materials Journal of Poultry Science and Technology | April-June, 2016 | Volume 04 | Issue 02 | Pages 28-38 © 2016 Jakraya 29 an optimum balance amongst different materials that will satisfy the requirements of specific animals in a specific physiological state. In order to achieve this, the manufacturer needs to keep an eye on the chemical composition and nutritional value of feedstuffs (Fairchild et al., 1999; Owens et al., 1997; Shamsudeen et al., 2013). Hence a laboratory at feed mill and right use of it can optimize both the quality and economics of feed manufacturing. A lab has a very key role to play right from purchasing of raw materials to dispatch of finished feed. It requires a small investment relative to the enormous benefit of "customer satisfaction" and "enhanced productivity" through "Quality Feed". 2. Materials and Method Laboratory analysis is an essential part for poultry feed industry. The performance of bird is totally dependent on the quality of finished feed. The following tests are important to decide the quality of feed. Every batch of feed must be analyzed for its proximate principals. Normally some routine analysis is performed one by one in the laboratory as mentioned in below: a) Moisture content b) Crude protein c) Crude fat d) Crude fibre e) Calcium f) Salt g) Ash content 2.1 Moisture Content Moisture is the important factor for both feed and raw materials (Islam et al., 2015b; Willits CO, 1951; Stock et al., 1991). This method is used for testing the moisture content of feed and raw materials except Dicalcium phosphate, Rock phosphate, Fat and Oil. Some conditions for Hot Air Oven baked according to the type of raw materials and feed as follows: a) In case of raw materials and animal feeds at 105±2ºC temperature for 16 hours, b) For corn, wheat, millet, oats, rye, rice, broken rice at 131±2ºC for 4 hours, c) For corn and baked beans at 131±2ºC for 38 hours. d) For wheat, wheat by product at 65±2ºC for 16 hours and will have a sample in vacuum Oven atmospheric pressure less than 20 mm. Hg. e) For the molasses at 60±2ºC for 16 hours and will have a sample in Vacuum Oven atmospheric pressure less than 20 mm. Hg. 2.1.1 Equipments and Accessories a) An oven with fan (Forced Convection Hot Air Oven) that can set the temperature at 103±2ºC, respectively. b) An oven with vacuum (Vacuum Oven) that can set the temperature at 65±2ºC and 60±2ºC, respectively. c) Petri dishes with diameter 9.5 cm. d) Scales are 4 positions. e) Container with the lid for closed and the surface area for spreading a sample of about 0.3 g/cm include aluminum dish with a diameter of approximately 5 cm. f) Liner (Wypall plus), or other paper, which applied to a filter paper liner. g) Eye glasses h) Aluminum tray i) Desiccator j) Gloves for holding the sample container. 2.1.2 Sample Preparation a) Raw materials and feed samples are ground to a size less than 1.0 mm was used as homogenous. b) For corn mill reduced the sample size by grinding with a coarse grind. Then crushed by a mesh size less than 1.0 mm and taken to achieve a homogeneous mixture. 2.1.3 Procedure a) At first the Petri dish is cleaned by distilled water and kept it into dryer for 10 minutes after that the petri dish is transferred into desiccators and waited until cool. b) The weight of dry petri dish is taken (recorded as M1). c) Grounded sample about 2-5 g is taken in the dry petri dish (recorded as M2). d) The oven temperature is set at 105±2ºC temperature for 16 hours. e) Then the petri dish is kept inside the oven for 16 hours. f) After that the petri dish is transferred to a desiccator and waited until cool. g) The final weight is taken quickly and recorded as M3. 2.1.4 Calculation % moisture of both feed and raw materials can be calculated using the following equation Where, M1= Weight of empty petri dish M2 = Weight (empty petri dish+Sample) before dry. M3 = Weight (empty petri dish+Sample) after dry. Islam et al… Laboratory Protocols of Wet Analysis for Poultry Feed and raw materials Journal of Poultry Science and Technology | April-June, 2016 | Volume 04 | Issue 02 | Pages 28-38 © 2016 Jakraya 30 2.2 Crude Protein Generally crude protein is measured by the micro Kjeldahl method. The Danish Chemist Johan Kjeldahl developed this method for analyzing nitrogen in organic substances (Shukla et al., 2001; Marc et al., 2002). The original method has been continuously improved environmental and personal aspects, increased the speed and versatility of the method and simplified the entire analytical procedure. The Kjeldahl method has three different steps, digestion, distillation and titration. 2.2.1 Equipment’s a) Grinder Retsch ZM 200 b) Electronic Balance ABS-220-4 Digit c) Spoon d) Tracing Paper e) Graduated Measuring Cylinder, of capacity 25ml. f) Pro Laboratory Fume Hood g) Digestion System Foss TecatorTM 8 digestor h) ScrubberTM 2501 Tecator i) Digestion tube, of capacity 250 ml, suitable for use in digestion block. j) Conical Flask k) Distillation System KjeltecTM 8100 l) Eyela-CA-1112E Water Cooler System m) Puris-Expe RO Water System n) Burrette, of capacity 50ml o) Pipette (5 ml, 10 ml, 25 ml) p) Reagent Bottle (250 ml, 500 ml) q) Volumetric Flask (100 ml, 250 ml, 500 ml, 1000 ml) 2.2.2 Reagents 1. For digestion: a. Catalyst Mixer (CuSO4+K2SO4) b. Concentrated H2SO4 (98%) 2. For distillation: a. Mixed indicator (Methyl red + Methylene blue) b. NaOH 50% solution c. HBO3 4% solution 3. For Titration: a. 0.2 N H2SO4 solution or HCl acid solution. 2.2.3 Preparation of Solution 1. Catalyst mixer 8 g CuSO4 + 70 g K2SO4 + Mixed well 2. Phenolphthalein Indicator 0.5 g Phenolphthalein + 50 ml 95% Ethanol 3. 0.1 N NaOH 4 gNaOH + distilled water = Total volume will be 1000 ml. 4. Mixed Indicator 0.2 g Methyl Red + 0.1 g Methylene Blue + 150 ml 95% Ethanol + Mixed well 5. 4% Boric acid solution 40 g Boric acid + distilled Water = Total volume will be 1 lit. + 10 ml Mixed Indicator. 6. 50% NaOH 500 gNaOH + distilled water = Total volume will be 1000 ml. 7. 0.2 N H2SO4 11 ml conc. 98% H2SO4 + 1989 ml distilled water = Total volume will be 2000 ml. 2.2.4 Standardization of 0.2 N H2SO4 Solution 0.1 g pure and dry Na2CO3 + 50 ml distilled water + 1 drop methyl red indicator + titration with H2SO4 solution in a 250 ml conical flask. 2.2.5 Standardization of 0.1 N NaOH Solution 10 ml NaOH + 1 drop Phenlophthalein Indicator + Titration with 0.2 N H2SO4 2.2.6 Procedure for Digestion a) At first the FOSS Digestion unit is turned on and the temperature is set at approximately 420ºC using temperature set on controller. Then allowed time for unit to reach temperature. b) The digestion tubes are inserted into the holes provided in the flask rack. A digestion tube must be set in all positions (even in empty) for the exhaust manifold to work properly. c) Approximately 0.5 to 1 g of ground homogenous sample is taken in a piece of weighing paper. The paper is folded carefully so that the sample is well contained. The folded paper is placed into digestion tube. d) When the digestion unit has been on for an adequate warm-up period, 15-20 ml of concentrated H2SO4 and also 4 g of catalyst mixer are added to each tube. e) The scrubber unit is turned on to eliminate evaporation of acid to environment. In this system 8% acid is lost during the initial 15 minutes. f) The sample is digested at 420ºC for approximately 60-70 minute or until the sample have a clear blue-green appearance. g) After complete the digestion, the flask rack containing tubes is removed from the digester. Islam et al… Laboratory Protocols of Wet Analysis for Poultry Feed and raw materials Journal of Poultry Science and Technology | April-June, 2016 | Volume 04 | Issue 02 | Pages 28-38 © 2016 Jakraya 31 h) The tubes are allowed cooling for 15 to 20 minute. When the tubes have cooled, the flask rack containing tubes is removed from the fume hood. The fume removal system is placed on the rack provided under the fume hood and the water supply is turned off to the fume removal system. i) Then the digested sample is diluted with 50 ml of distilled water when the tubes are cool enough to handle. The digestion tube is shacked until fully dissolved. 2.2.7 Procedure for Distillation a) The cooling water is turned on to the system of distillation unit. b) The distillation vessel is placed with digested sample on the apparatus, twisting the tube to ensure a complete seal. c) A receiver 250 ml. Erlenmeyer flask (Receiving Flask) containing 25 ml of 4% boric acid indicator is inserted, making sure that delivery tube is fully submerged. d) Approximately 50 ml of NaOH solution is added by slowly pushing down the alkali dispenser handle, and the dispenser handle is allowed to return to its closed position. e) Open the system valve to start steam generation. If desired, set the time for approximately 4 minutes. f) Produced all NH3 gas is collected to approximately 150 ml distillate in the receiver flask. g) Then the receiving flask is removed with the content and the waste is drained out from the distillation unit. h) The content is then titrated with standardized 0.2 N sulfuric acid solutions. 2.2.8 Crude Protein (% CP) Calculation Crude protein is an estimate for total protein. A crude protein contains nitrogen from not only protein but non-protein sources as well. Crude Protein (CP) is based on a laboratory nitrogen analysis, from which the total protein content in a feedstuff can be calculated by multiplying the nitrogen figure by 100/16 or 6.25 (Daniel et al., 1996). This is from the assumption that nitrogen is derived from protein containing 16 % nitrogen (AOAC, 1984). However, some portion of the N in most feeds is found as non-protein nitrogen (NPN) and, therefore, the value calculated by multiplying N x 6.25 is referred to as crude rather than true protein. Protein is made up of amino acids. Formula: Where, A-Volume of Sulfuric acid (ml) for sample titration. B-Volume of Sulfuric acid (ml) for blank titration. 2.3 Crude Fat Fat is extracted of measured for both feed and raw materials as crude fat. Sample is weighted into a sample bag and defatted with petroleum ether solvent at a temperature of 90ºC for 1 h and then evaporated petroleum ether. Then the sample bag is dried and weighed after extraction of fat. Weight lost is fat (Crude Fat) (State of California-business, 1999; Sun et al., 2012; Thiex et al., 2003). In case of high fat, the fat can be measured with or without previous hydrolysis with hydrochloric acid. 2.3.1 Equipment a) Grinder Retsch ZM 200-(Net size-1.0 mm). b) Analytical Balance-capable of weighing 0.1 mg. c) Oven-capable of maintaining a temperature of 105±2.0ºC. d) Extraction instrument-capable of extracting at 90±2.0ºC (XT10, ANKOM Technology). e) Fat Filter Bags XT4-constructed from chemically inert and heat resistant filter media, capable of being heat sealed closed and able to retain 1 micron particles while permitting solution penetration (XT4, ANKOM Technology). f) Heat sealer-sufficient for sealing the filter bags closed to ensure complete closer. g) Desiccator pouch-collapsible sealable pouch with desiccant inside that enables that removal of air from around the filter bags (Moisture Stop weigh pouch, ANKOM Technology). h) Marking pen-solvent resistant (F08, ANKOM Technology) i) ANKOM Fat analyzer XT10 (low fat analyzer) j) ANKOM HCl Hydrolysis System (high fat analyzer) k) Scales capable of reading 4 positions. 2.3.2 Reagents a) Petroleum Ether (B.P. 35-65ºC). Depending on the method used, other solvents such as hexanes and ethyl ether may be used. Petroleum Ether purified for fat extraction (i.e. evaporation residue shall be less than 20 mg/lit) b) 2 M Hydrochloric acid Notes: Most fat solvents are extremely flammable. Avoid static electricity and use in fume hood. Islam et al… Laboratory Protocols of Wet Analysis for Poultry Feed and raw materials Journal of Poultry Science and Technology | April-June, 2016 | Volume 04 | Issue 02 | Pages 28-38 © 2016 Jakraya 32 2.3.3 Procedure a) At first the sample is ground and the particle size should be less than 1.0 mm, the amount of at least 100 grams of a mixture to be homogeneous. b) The sample is placed into a labeled filter bag. c) The weight of samples is taken approximately 1.5 g (W1) into a sample bag. d) The filter bag is closed with a heat sealer within 4 mm of the top to encapsulate the sample. e) The sample is dried in the oven at about 103ºC for at least 2 hours, then left to cool in desiccators. Desiccant Pouch for about 15 minutes and then weighed (W2). f) Then the sample is put into the sample bag. They were then put into the machine. g) Operation time is set about 60-minute to extract. h) Extraction temperature is set at 90ºC. i) After extraction, the sample is allowed to cool about 20 minutes. j) The defatted samples are dried at 105±2ºC in the oven for at least 1 hour, and then cool at room temperature in Desiccant Pouch for about 15-30 minutes. k) The weight is taken (W3) for final calculation. Calculation: % crude fat can be calculated using the following equation Where, W1 = Original weight of sample. W2 = Weight of sample with the filter bag after drying. W3 = Weight of dried sample and filter bag after extraction. 2.4 Crude Fibre This method determines crude fiber which is the organic residue remaining after digesting with 0.255N H2SO4 and 0.313 N NaOH. The compounds removed are predominantly protein, sugar, starch, lipid and portion of both the structural carbohydrates and lignin (Crude fibre analysis: Official Method AOCS Ba 6- 84/AOAC 962.09). This method is applicable for all feed materials such as grains, meals, pet food, mixed feeds, forages and the following oilseeds: corn and soybeans. 2.4.1 Equipment a) Scales-capable of reading 4 positions. b) Oven-(Hot Air Oven) that can set the temperature at 105±2ºC, respectively. c) Furnace-(Muffle furnace) is controlled by the electric current. Maintain the temperature at 650±05ºC. d) Digestion instrument-Device fiber, which is digested at a temperature of 100±0.5ºC and maintain a pressure of 10-25 psi and a similar flow around each sample to ensure the uniformity of extraction (ANKOM 200I). e) Filter bags-can be sealed with heat, F57 ANKOM Technology. The sample bags (F57) may be produce up to 0.5% unit’s lower values on finely ground samples (Finely ground samples are samples with fiber particles less than 25 microns). f) Heat sealer-sufficient for sealing the filter bags closed to ensure complete closer (LOVRO R - SK-210). g) Desiccators’ pouch-collapsible sealable pouch with desiccant inside that enables that removal of air from around the filter bags (Moisture Stop weigh pouch, ANKOM Technology). h) Marking pen-solvent resistant (F08, ANKOM Technology) i) Crucible 5.9 cups j) Grinder Retsch ZM 200 5:10 (Sieve size-1.0 mm). 2.4.2 Reagents a) Acetone b) Petroleum Ether (B.P. 35-65ºC). c) H2SO4 Solution - 0.255±0.005N. 1.25 g H2SO4/100 ml. Concentration must be checked by titration. d) NaOH Solution - 0.313±0.005N. 1.25 g NaOH/100 ml. Concentration must be checked by titration. 2.4.3 Procedure 1. At first the sample is ground and the particle size should be less than 1.0 mm, the amount of at least 100 grams of a mixture to be homogeneous. 2. Solvent and acid resistant marker is used to label the filter bags. The filter bag weight is taken as (W1). 3. Weight is taken is about 0.95-1.00g of prepared sample (W2) directly in filter bag. 4. The filter bag closed by heat sealer within 4 mm of the top to encapsulate the sample. 5. Weight is taken one blank bag and included in run to determine blank bag correction (C1). 6. Fat is extracted from samples by placing all bags into a 250 ml container. Enough petroleum ether is added to cover bags and soak for 10 minutes. Pour off solvent and the bagsis allowed Islam et al… Laboratory Protocols of Wet Analysis for Poultry Feed and raw materials Journal of Poultry Science and Technology | April-June, 2016 | Volume 04 | Issue 02 | Pages 28-38 © 2016 Jakraya 33 to air dry. The sample is spread uniformly inside the filter bag by shaking and flicking the bag to eliminate clumping. 7. Maximum 24 bags are placed into the bag suspender. All 9 trays are used regardless of the number of bags being processed. Place 3 bags per tray and then stack trays on center post with each level rotated 120 degrees. The bag suspender is inserted with bags into the fiber analyzer vessel and places the bag suspender weight on top of the empty 9th tray to keep it submerged. 8. The instructions are followed displayed on the screen of the ANKOM 200I. a) Select crude fiber. b) Close lid c) Confirm hot water on (>70ºC) d) Press start. 9. When the crude fiber extraction and rinsing process is completed, the lid is opened and removed the samples. Excess water is pressed out from the bags and it is placed in a 250ml beaker and added enough acetone to cover bags and soaked for 3-5 minutes. 10. The bags are removed from acetone and place on a wire screen to air dry. Completely dried in an oven at 105±2ºC (most ovens will complete drying within 2-4 hours). 11. The bags are removed from oven, placed directly into a collapsible desiccant pouch and flatten to remove air. Cool to ambient temperature and weight is taken of bags. 12. The bag is putted in a cup crucible then baked in the oven for at least 30 minutes at 105±2ºC and the temperature drop in room temperature afforded. Weighted sample bag with a cup crucible recorded in the current test for fiber. 13. The sample in pre-weighted crucible is placed in furnace for 2 hours at 650±15ºC and cool in desiccators and weight is taken to calculate loss of weight of organic matter (W3). Calculation % crude fat can be calculated using the following equation Where, W1 = weight of the bag sample (g) W2 = weight of sample (g) W3= Weight of organic matter (Loss of weight on ignition of blank bag and fiber) (g) C1 = Ash corrected blank bag factor. 2.5 Calcium Normally for calcium analysis at first the sample is digested by nitric acid in the case of organic sample (Trace amount) (State of California-business, 1999; Sun et al., 2012; Thiex et al., 2003; Harris, 2003). Most common acid for oxidation of organic matrices with this typical reaction: (CH2)X + 2HNO3 -> CO2 (g) +2NO + 2H2O; Nitric acid is used for removing organic gases and to avoid further problem with adding perchloric acid. After removing gas, perchloric acid is added then heat is applied. 2.5.1 Equipment a) Scales-capable of reading 4 positions. b) Grinder Retsch ZM 200 5:10 (Sieve size-1.0 mm). c) Burette size 25 or 50 ml. d) Electric stove (Hotplate). e) Filter paper Whatman No. 1 or equivalent. f) Glassware g) Volumetric flask (100 ml, 250 ml, 500 ml) h) Pipette (5 ml, 10 ml, 25 ml) i) Water bath j) Conical flask 2.5.2 Chemicals and Preparation of Solution a) Ammonium hydroxide; NH4OH (1:1) 500 ml NH4OH + 500 ml Distilled water = Total volume will be 1 liter. b) Ammonium Oxalate, saturate; (NH4)2C2O4 42 g (NH4)2C2O4 + Distilled water = Total volume will be 1 liter. c) Sodium Oxalate; Na2C2O4 d) Hydrochloric Acid; HCl (1:3) 100 ml concentrated HCl + 300 ml distilled water = Total volume will be 1 liter. e) Sulfuric Acid; H2SO4 (1:5) 100 ml concentrated H2SO4 + 500 ml distilled water, f) Deionized distilled water g) Nitric acid (HNO3) - 1:1 h) Perchloric acid (HClO4) i) Methyl red indicator 0.5g methyl red indicator + 95% ethanol = Total volume will be 1 liter. j) 0.1 N. Potassium Permanganate; KMnO4 k) 3.2 g potassium permanganate (KMnO4) is dissolved in 1000 ml distilled water and after filter the solution is stored in dark bottle. l) 0.2 g dried sodium oxalate (Na2C2O4) is taken into 500 ml conical flask. Islam et al… Laboratory Protocols of Wet Analysis for Poultry Feed and raw materials Journal of Poultry Science and Technology | April-June, 2016 | Volume 04 | Issue 02 | Pages 28-38 © 2016 Jakraya 34 m) 100 ml sulfuric acid; H2SO4 (1:5) is added to the conical flask and stirred until the solid dissolved. n) Then the solution is titrated with potassium permanganate (KMnO4) until the solution turned pink. Calculation: 2.5.3 Procedure A. Roasting Method: for example, fodder or feedstuff a) Around 2-2.5 g sample is taken into a crucible. b) Then it is placed into the muffle furnace at 600ºC for nearly 2 hours. c) The crucible is allowed to cool, then filled with HNO3 ratio of 1:1 volume 10 ml and 40 ml HCl (1:3) and boiled gently on the hot plate. d) The solution is then transferred into 100 ml volumetric flask and diluted to full volume. B. Dissolution Method: For examples of inorganic compounds (Limestone, DCP, TCP, Oyster shell, CuSO4, MnSO4, FeSO4.H2O, ZnO, MCP, etc.) a) Around 0.5-1.0 g of sample is taken into dry 100 ml volumetric flask. b) 10 ml of concentrated HCl is added to dissolve the sample and boiled in water bath with occasional swirling until no more reaction occurs. c) Finally, the content is diluted to 100 ml with distilled water. C. Digestion method: For example; feed, premix type of organic compounds. a) 1.0 g of sample is taken into a Kjeldahl flask. b) 20 ml of concentrated HNO3 is added to the flask and heated until nearly dry. c) The content is then allowed to cool and 10 ml of concentrated HClO4 is added. d) The flask is reheated until the white smoke and clear solution. e) Small amount of cold water is added heated to boiling solution. f) The allowed to cool, then the content is transferred into a 100 ml volumetric flask. g) Finally, the content is diluted to 100 ml with distilled water. 2.5.4 Procedure for the Preparation of Mineral (Ca) a) After preparing the roasting, dissolution or digestion, 5 ml of the prepared sample is taken in the conical flask and the volume is adjusted to 100 ml with distilled water. b) 2 drops of methyl red indicator are added and stirred. c) NH4OH (1:1) is added dropwise until the solution color changed from pink to yellow. d) HCl (1:3) is added dropwise until the solution color changed from yellow to pink. e) Distilled water is added until a volume of 150 ml sample solution. f) Then the solution is boiled gently in the hot plate then slowly 10 ml of saturated (NH4)2C2O4 is added with shaking, if the solution is not pink then HCl (1:3) is added again drop wise until pink. g) The conical flask is left in the water bath for 2 hours at 70-80ºC for precipitation and then filtered with the paper Whatman No 1. h) The precipitate is washed with hot water at least 10 minutes and transfer filter paper including precipitate (CaC2O4) into original flask. i) 100 ml H2SO4 (1:5) is added to the conical flask and boiled at 70ºC. j) Then the solution is titrated with 0.1 N potassium permanganate (KMnO4) solution while heating until the solution turned pink. (KMnO4 used ml = S) k) A Blank titration is performed along with the sample. (KMnO4 used ml = B). Calculation % calcium can be calculated using the following equation Where, S = volume of std. KMnO4 for sample (ml) B = volume of std. KMnO4 for blank (ml) N = Normality of std. KMnO4 X = ml. dilution of sample solution Y = ml. aliquot of sample solution. 2.6 Salt The NaCl content is determined by the wellknown Volhard method. The sample is treated with AgNO3, then wet-ashed and the excess AgNO3 is back titrated with NH4SCN. The AgNO3 solution must be added first, followed by the concentrated HNO3. This order of addition is critical to ensure complete precipitation of the chlorides (Harris, 2003). If HNO3 is added first, loss of chloride by volatilization as HCl could occur because HCl has a higher vapor pressure than HNO3. The volume of AgNO3 solution added must be in excess of that required to react with the chloride Islam et al… Laboratory Protocols of Wet Analysis for Poultry Feed and raw materials Journal of Poultry Science and Technology | April-June, 2016 | Volume 04 | Issue 02 | Pages 28-38 © 2016 Jakraya 35 in the sample. Following boiling, cooling and dilution, then the excess AgNO3 is back titrated with NH4SCN solution, employing ferric ammonium sulfate as an indicator. The FeNH4 (SO4)2 react with an excess of thiocyanate, forming the salmon colored complex, ferric thiocyanate FeSCN++, indicating the endpoint. 2.6.1 Equipment a) Scales-capable of reading 4 positions. b) Oven-(Hot Air Oven) that can set the temperature at 105±2ºC, respectively. c) Burette-Class A, Kimax. d) 250 ml Erlenmeyer flask (Conical flask) - Pyrex e) Pipettes-(10 ml, 5 ml) - Class A, Pyrex f) Boiling Chips - Ceramics g) Grinder Retsch ZM 200 5:10 (Sieve size - 1.0 mm). 2.6.2 Reagents a) FeNH4 (SO4)2 . 12H2O (Ammonium ferric sulfate or Ferric alum indicator)-99% b) Nitric Acid (HNO3)-65% c) Ammonium Thiocyanate (NH4SCN)-98% d) Di-Ethyl ether (C2H5)2O-99.5% e) Silver Nitrate (AgNO3)-93.5-100.5% f) Potassium Chromate (K2CrO4)-95% g) Water (Distilled or Deionized) 2.6.3 Solution Preparation a) 0.1N AgNO3 17.04 g AgNO3 is dissolved in 1lit distilled water. b) Ferric indicator 80 g of ammonium ferric sulfate FeNH4 (SO4)2 . 12H2O in 1 lit distilled water. c) N NH4SCN 7.613 g NH4SCN is dissolved in 1 lit distilled water. d) Two percent (2%) Potassium Chromate 2 g potassium chromate is dissolved in 100 ml distilled water. e) N NaCl 2.923 gdried NaCl is dissolved in 100 ml distilled water. 2.6.4 Standardization of AgNO3 Solution Silver nitrate solutions of known concentration can be prepared from known mass of dried AgNO3. However, if we don't have access to the high purity reagent, or if we have a solution of unknown concentration, we can easily standardize it against sodium chloride. Reaction taking place during titration is: AgNO3 + NaCl → AgCl↓ + NaNO3 2.6.5 Procedure to Follow (If Prepared from 0.1N NaCl Solution) a) 25 ml 0.1N NaCl (sodium chloride) is taken into 250 ml conical flask. b) Drops of 2% potassium chromate solution are added to the conical flask. c) Then the solution is titrated with 0.1N AgNO3 solution till the first color change (Permanent light brown/salmon colored). Calculation 2.6.6 Procedure to Follow (If Prepared from Dried NaCl) a) 0.1 g of dried NaCl is taken into 250 ml conical flask. b) 50 ml of distilled water is added to the conical flask. c) 2 drops of 2 % potassium chromate solution is added. d) Then the solution is titrated with 0.1N AgNO3 solution till the first color change (Permanent light brown/salmon colored). Calculation: 2.6.7 Standardization of NH4SCN Solution a) 5 ml 0.1N AgNO3 solution is taken into 250 ml conical flask. b) 7.5 ml HNO3is added to the conical flask. c) The solution is boiled at least 10-15 minutes and kept it for cool at least 5-10 minutes. d) 50 ml of distilled water is added to the solution. e) Then 2.5 ml ferric indicator is added. f) Then the solution is titrated with 0.1N NH4SCN solution till the first color change (Permanent light brown/salmon colored). Calculation: 2.6.8 Procedure a) The sample is ground and the particle size should be 1.0 mm, the amount of at least 100 grams of a mixture to be homogeneous. Accurately 0.5 g sample is taken into a 250 ml conical flask. b) 5 ml 0.1N AgNO3 solution is added to the conical flask and the flask is swirled until sam- Islam et al… Laboratory Protocols of Wet Analysis for Poultry Feed and raw materials Journal of Poultry Science and Technology | April-June, 2016 | Volume 04 | Issue 02 | Pages 28-38 © 2016 Jakraya 36 Table 1: Nutrient values of raw materials Raw material name Nutrient Values (%) Moisture Crude Protein Crude Fat Crude Fibre Calcium Salt Ash Corn 10.32 7.55 4.21 3.55 - - 1.00 Soybean Meal 10.66 46.63 2.20 3.50 - - 6.44 Meat and Bone Meal 5.78 50.12 7.50 4.20 10.18 - 36.25 Fish Meal 4.40 62.18 10.25 4.23 4.77 3.44 12.22 Rice Bran Fine 7.58 12.65 16.11 7.24 - - 11.32 Rice Bran Coarse 6.72 7.88 9.88 37.20 - - 15.15 Full Fat Soya 10.98 36.78 18.56 7.25 - - 5.11 Wheat Grain 7.86 11.28 1.55 2.70 - - 3.68 Wheat Bran Coarse 9.82 12.66 4.50 10.12 - - 10.05 Mustard Meal 8.88 37.25 1.25 12.10 - - 9.34 Table 2: Common nutrient values of different feed Raw material name Nutrient Values (%) Moisture Crude Protein Crude Fat Crude Fibre Calcium Salt Ash Broiler Starter 11.55 21.52 3.50 5.00 0.80 0.42 7.00 Broiler Grower 11.45 20.25 3.00 5.00 0.70 0.36 7.00 Broiler Finisher 10.98 19.15 3.00 5.50 0.70 0.37 8.00 Layer Starter 11.25 20.32 5.00 5.00 0.80 0.44 8.00 Layer Grower 11.10 18.19 2.50 5.40 0.80 0.36 10.00 Layer Finisher 10.86 15.55 2.50 8.00 0.80 0.31 10.00 ple and solution are in intimate contact. c) 7.5 ml concentrated HNO3 is added to it. d) Then the solution is boiled 10-15 minutes and then it is cooled. e) Finally, 50 ml H2O and 2.5 ml ferric indicator is added to the conical flask. f) Then the solution is titrated with 0.1N NH4SCN solution till the first color change (Permanent light brown/salmon colored). g) Required volume of 0.1N NH4SCN = A. (If first drop of titration receives endpoint, then restart from second step and more volume of 0.1N AgNO3 is added. h) A blank titration is performed along with sample. Required volume of 0.1N NH4SCN = B. Calculation: %NaCl can be calculated using the following equation 2.7 Ash Content Ash is the weight of the remaining after burning the sample in a furnace (Muffle Furnace) at a temperature of 650ºC for 2 hours. 2.7.1 Equipment and Accessories a) Muffle furnace that can set the temperature at 650±20ºC. b) Oven (Hot Air Oven) that can set the temperature at 105±2ºC, respectively. c) Digital scales with 4 positions. d) Ceramic cup with a surface area of approximately 20 cm2 and height of about 2.5 cm. e) Desiccator f) Container vise g) Gloves for holding the sample container. 2.7.2 Test Procedure a) At first the sample is ground to a size less than 1.0 mm used as homogenous. b) A ceramic cup is dried in the oven at 105±2ºC for about 30 minutes. c) At the end of 30 minutes, the cups are then placed it in a desiccator and the weight of empty cup is taken as (M0). d) Weight of sample is taken in crucible as (M1) and is recorded this for determination of ash. Islam et al… Laboratory Protocols of Wet Analysis for Poultry Feed and raw materials Journal of Poultry Science and Technology | April-June, 2016 | Volume 04 | Issue 02 | Pages 28-38 © 2016 Jakraya 37 e) The ceramic cups with sample are burned in a furnace at a temperature of 650ºC for 2 hours. f) After the time prescribed the ceramic cup burned out and placed in desiccators or kept at room temperature. g) The weight of ceramic cups is taken with the burnt sample as (M2). Calculation: % Ash can be calculated using the following equation Where, M0 = weight of ceramic cup roasted (g) M1 = weight of sample (g) M2 = weight of ceramic cup containing the sample after burning (g) 3. Results and Discussion Laboratory analysis is the key point for the quality control of a poultry feed industry. Quality of the outcome feed depends on its nutrient values. The feed cannot be a balanced diet for chicken without proper combination of nutrient values in feed. Proper combination of nutrients also a cost-effective mater. Therefore, determination of nutrient values in both feed and raw materials is very important by which balanced as well as quality feed can be produced. These analysis protocols have been used effectively for the determination of nutrient values of some raw materials and feed. Results are shown in the Table-1 and Table2. 4. Conclusion Raw materials and feed analysis is the essential part for every feed mill. Nutrient values can be measured by these protocols for both raw materials and feed. Feed formulation can be done by the nutritionist depending on the analysis record of raw materials. Quality control department can compare the analysis results with the standard for both feed and raw materials. Acknowledgement The authors are very much grateful to the Polymer and Pharmaceutical Research Laboratory of the Department of Applied Chemistry and Chemical Engineering, University of Rajshahi, Central Science Laboratory of Rajshahi University and Research Laboratory of Poultry Feed Factory, Rajshahi, Bangladeshfor assisting and providing facilities to carry out the research. We are also grateful to the teachers and some research students of the Department of Applied Chemistry and Chemical Engineering, University of Rajshahi. References AOAC (1984). Official methods of analysis of associaion of official analytical chemists international. (14th Edn), Washington DC. Benton JR, Klopfenstein TJ and Erickson GE (2004). In situ estimation of dry matter digestibility and degradable intake protein to evaluate the effects of corn processing method and length of ensiling. Journal of Animal Science, 82(Suppl 1): 463. Briggs JL, Maier DE, Watkins BA and Behnke KC (1999). Effect of ingredients and processing parameters on pellet quality. Poultry Science, 78: 1464-1471. Cooper RJ, Milton CT, Klopfenstein TJ, Scott TL, Wilson CB and Mass RA (2002). Effect of corn processing on starch digestion and bacterial crude protein flow in finishing cattle. Journal of Animal Science, 80: 797- 804. Crude fibre analysis: Official method AOCS Ba 6-84/AOAC 962.09. Daniel MB, Michael DR and Stuart JE (1996). Protein methods (2ndEdn.), Wiley Publishers. ISBN 0-471- 11837-0. Defoor PJ, Galyean ML, Cole NA and Jones OR (2000). Effects of planting density and processing method on laboratory characteristics of grain sorghum for ruminants. Journal of Animal Science, 78: 2032-2038. Fairchild F and Greer D (1999). Pelleting with precise mixer moisture control. Feed International, 20(8): 32-36. Harris DC (2003). Quantitative chemical analysis (6th Edn.). San Francisco: W.H. Freeman. pp. 142-143. ISBN 0- 7167-4464-3. Islam MS, Haque MM and Hossain MS (2015a). Estimation of moisture and crude protein content of locally available raw materials for poultry feed. International Journal of Pharmaceutical Sciences and Research, 6(5): 1000-07.doi:10.13040/IJPSR.0975-8232.6 (5).1000-07. Islam MS, Haque MM and Hossain MS (2015b). Effect of corn moisture on the quality of poultry feed. Journal of Poultry Science and Technology, 3(2): 24- 31. Islam MS, Noor MA, Hossain MS, Saha KC and Seal H (2015c). Chemical investigation of bioactive compounds of black pepper. International Journal of Pharmaceutical Sciences and Research, 6(4): 1000- 06.doi: 10.13040/IJPSR.0975-8232.6 (4).1000-06. Marc A, Rubio R, Compa R and Casals I (2002). Comparison of the Kjeldahl method and a combustion method for total nitrogen determination in animal feed. Talanta, 57: 1019-1026. Owens FN, Secrist DS, Hill WJ and Gill DR (1997). The effect of grain source and grain processing on Islam et al… Laboratory Protocols of Wet Analysis for Poultry Feed and raw materials Journal of Poultry Science and Technology | April-June, 2016 | Volume 04 | Issue 02 | Pages 28-38 © 2016 Jakraya 38 performance of feedlot cattle: A review. Journal of Animal Science, 75: 868-879. Shamsudeen P, Shrivastava HP, Singh R and Deo C (2013). Effect of chelated and inorganic trace minerals on aflatoxin synthesis in maize. Journal of Poultry Science and Technology, 1(1): 13-16. Shukla R and Cheryan MZ (2001). The industrial protein from corn. Industrial Crops and Production, 13: 171- 192. State of California-business (1999). Transportation and housing agency, California Test, 226. Stock RA, Sindt MH, Cleale-IV RM and Britton RA (1991). High-moisture corn utilization in finishing cattle. Journal of Animal Science, 69: 1645-1656. Sun T, Liu Z, Qin L and Long R (2012). Aspects of lipid oxidation of meat from free-range broilers consuming a diet containing grasshoppers on alpine steppe of the Tibetan Plateau. Poultry Science, 91: 224-231. Thiex NJ, Anderson S and Gildemeister B (2003). Crude fat, diethyl ether extraction, in feed, cereal grain, and forage (Randall/Soxtec/submersion method): collaborative study. Journal of AOAC International, 86(5): 888-98. Willits CO (1951). Methods for determination of moistureoven drying. Analytical Chemistry, 23(8): 1058-1062; DOI: 10.1021/ac60056a003.

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