ADDITIVES

FIELD OF THE INVENTION

The invention relates to a process for preparing feed additives. In particular, the invention relates to a process for preparing calcium supplement for feed additives.

BACKGROUND TO THE INVENTION

The following discussion of the background to the invention is intended to facilitate an understanding of the present invention only. It should be appreciated that the discussion is not an acknowledgement or admission that any of the material referred to was published, known or part of the common general knowledge of the person skilled in the art in any jurisdiction as at the priority date of the invention.

Food shortage has become a serious problem, which affects lives of human beings. Food shortage is caused by an increase in the world's population, pollution and global warming. These result in a decline in agricultural productivity, thereby leading to a shortage in supply of raw materials in food production for both humans and their livestock. Therefore, there have been many technology developments which emphasize on increasing food and agricultural productivity in order to meet the demands of growing populations worldwide.

The animal feed industry is a very important supply chain component in the livestock industry sector such as poultry, pig farming and fishery. Calcium carbonate (CaC0 ₃ ) is a dietary source which has numerous crucial benefits for animal growth in addition to soybean and corn, which are typical nutrient sources of protein and carbohydrate, respectively. Calcium carbonate is an essential mineral for the metabolic process. It serves as a structural element in bones and also aids in the strengthening of avian egg shells.

Sources of calcium carbonate commonly used in animal feed industry are powdered seashells, corals and animal bones due to their calcium content, which can be as high as about 38% and also because they are available as fine grains. Limestone is another source of calcium carbonate and its geological supply is abundant in nature. Moreover, calcium carbonate that contains no heavy metal such as Arsenic (As) Cadmium (Cd), Lead (Pb), and Mercury (Hg) , is beneficial to animal growth performance and enhances the use of calcium in animal feed effectively.

Chinese patent number CN 203400768U discloses the development of a ball mill grinding system which can efficiently grind calcium carbonate into small particle sizes. However, the prior art system still faces many challenges which make it unsuitable for use in preparing a calcium supplement as an animal feed raw material. The present invention relates to a process for preparing a calcium supplement for animal feed additive. The present invention seeks to address and/ or ameliorate the problems in the prior art.

SUMMARY OF THE INVENTION

An object of the present invention is to develop a process for preparing the calcium supplement for animal feed additive; calcium supplement for animal feed additive comprising the steps of

(a) extracting the limestone containing calcium carbonate of more than 95% by dry weight;

(b) drying the said limestone by convective hot air until the moisture content of between 0.01 and 0.05% by dry weight has been obtained;

(c) comminuting the said limestone by vertical shaft impactor to obtain ground calcium carbonate; and

(d) classifying the particle size of the said ground calcium carbonate by using sieving apparatus, centrifugal and/or cyclonic suction type machines. characterized in that the obtained ground calcium carbonate to be used as animal feed additive are round-shaped particles with low angularity or sharpness. According to one alternative aspect, the present invention relates to the animal feed additive preparation process for calcium supplementation in which classifying the particle size of the ground calcium carbonate by using sieving apparatus, centrifugal and/ or cyclonic suction type apparatus into a variety of grades consists of the 1 ^st grade having particle sizes between 1.4 and 3 millimeters, the 2 ^nd grade having particle sizes between 0.7 and 1.4 millimeters, the 3 ^rd grade having particle sizes between 0.5 and 0.7 millimeters, the 4 ^th grade having particle sizes between 150 micron and 0.5 millimeters, the 5 ^th grade having particle sizes between 75 and 150 microns, and the 6 ^th grade having particle sizes between 0.01 and 75 microns.

Furthermore, the present invention also relates to animal feed formulation comprising of:

(a) base materials;

(b) additive materials; and

(c) 1- 10% by dry weight of ground calcium carbonate particles to be used as animal feed additive characterized in that the said ground calcium carbonate particles to be used as animal feed additive contain calcium carbonate of more than 95% by dry weight, moisture content of between 0.01 and 0.05% by dry weight, heavy metal contents of 0- 10 parts per million (ppm) and are of rounded shapes with low angularity or sharpness. These results from the comminution by vertical shaft impactor and subsequent particle size classifying of the said ground calcium carbonate particles by sieving apparatus, centrifugal and/or cyclonic suction type apparatus

According to one alternative aspect, the present invention relates to the animal feed formulation, base materials can be selected from the group consisting of corn, rice bran, soybean, steamed soybean, peanuts, corn dreg, rice bran dreg, soybean dreg, peanut dreg, sunflower seed dreg, and a combination thereof.

According to one alternative aspect, the present invention relates to the animal feed formulation, additive materials can be selected from the group consisting of vitamin substances, mineral substances, growth stimulants, feed preservatives, feed additives, enzymes, probiotic substances, hormonelike (parahormone) substances, semi- hormone substances, insect larvae suppressants, and a combination thereof.

According to another alternative aspect, the present invention relates to the animal feed formulation, which can be used as broiler chicken feed, egg-laying chicken feed, broiler duck feed, egg- laying duck feed, Muscovy duck feed, quail feed, beef cattle- fattening cattle feed, dairy cattle feed, goat feed, sheep feed, fish feed, shrimp feed, mantis shrimp feed or domestic pet food.

The resultant animal feed additive has round- shaped calcium carbonate particles with low angularity or sharpness ( i. e. sharp edges), low moisture content and high purity, while maintaining the level of heavy metals ( such as arsenic, cadmium, lead and mercury) in compliance with established standards. Therefore, the resulting feed additive becomes well-suited as a raw material in animal feed.

BRIEF DESCRIPTION OF THE DRAWINGS The present invention will become more fully understood from the detailed description given herein and the accompanying drawings which are given for the purpose of illustration only, wherein like numerals indicate like elements throughout, wherein:

Fig. 1 shows an overall schematic diagram of a calcium carbonate grinding system according to an embodiment of the present invention; Fig. 2 shows scanning electron microscopy images of calcium carbonate particles obtained from a process according to an embodiment of the present invention. The calcium carbonate particles are considered to have a relatively rounded shape with low angularity or sharpness.

EMBODIMENTS OF THE INVENTION Particular embodiments of the present invention will now be described with reference to the accompanying drawings. The terminology used herein is for the purpose of describing particular embodiments only and is not intended to limit the scope of the present invention. Additionally, unless defined otherwise, all technical and scientific terms used herein have the same meanings as commonly understood by one or ordinary skill in the art to which this invention belongs. Where possible, the same reference numerals are used throughout the figures for clarity and consistency. Throughout the specification, unless the context requires otherwise, the word "comprise" or variations such as "comprises" or "comprising", will be understood to imply the inclusion of a stated integer or group of integers but not the exclusion of any other integer or group of integers.

Furthermore, throughout the specification, unless the context requires otherwise, the word "include" or variations such as "includes" or "including", will be understood to imply the inclusion of a stated integer or group of integers but not the exclusion of any other integer or group of integers.

An object of the present invention is to develop a process for preparing a calcium supplement as an animal feed additive, and/or a calcium supplement for animal feed additive. The process comprises the steps of:

(a) extracting the limestone containing calcium carbonate of more than 95% by dry weight;

(b) drying the said limestone by convective hot air until the moisture content of between 0.01 and 0.05% by dry weight has been obtained; (c) comminuting the said limestone by vertical shaft impactor to produce the ground calcium carbonate; and

(d) Classifying the particle size of the said ground calcium carbonate by using sieving apparatus, centrifugal and/or cyclonic suction type apparatus, characterized in that the said ground calcium carbonate particles to be used as animal feed additive is round-shaped, with low angularity or sharpness.

According to an aspect of the present invention, there is a process for preparing a calcium supplement as an animal feed additive, the process comprising the steps of: (a) selecting a limestone comprising more than about 95% by dry weight of calcium carbonate;

(b) drying the limestone;

(c) comminuting the limestone to obtain calcium carbonate particles which are substantially round-shaped with low angularity or sharpness; and

(d) separating and classifying the calcium carbonate particles based on particle sizes,

characterized in that step (b) comprises drying the limestone to obtain a moisture content of about 0.01% to about 0.05% by dry weight. Problems encountered in calcium carbonate grinding processes disclosed in the prior art include high energy consumption in the grinding process, the obtained calcium carbonate particles having sizes which are only within a specific small size range, lack of a variety of particle sizes, the particles having a variety of shapes with angularity and sharpness ( i. e. sharp edges), and having impurities in the additive, for example having a level of heavy metal (e. g. arsenic, cadmium, lead and/ or mercury) contamination beyond the established standard limits. Therefore, use of the ordinary calcium carbonate as a feed additive for calcium supplementation has so far been rendered unsuitable and detrimental to for example, animal growth and quality of avian eggs.

In an aspect of the present invention and with reference to Fig. 1, the step of limestone preparation comprises the drying of the limestone by hot air convection (20). Limestone is stored in a limestone storage tank (10), where it is thereafter dried by hot air convection drying (20) at drying temperature of about 100-160 degree Celsius (°C). Examples of the limestone drying apparatus include but are not limited to a conical dryer, a horizontal shaft dryer, a rotary dryer, a vacuum dryer, a belt dryer, or a combination thereof. The drying apparatus in this step is designed to obtain and maintain the moisture content of limestone of about 0.01% to about 0.05% by dry weight, so that the subsequent grinding process can be carried out with maximized efficiency. This is because a reduction of the moisture of the limestone will facilitate the grinding (comminuting) process, which will decrease electricity consumption of the same. Hot air drying also helps to reduce and remove low boiling point impurities.

In an aspect of the present invention, the step of limestone comminution comprises the use of a vertical shaft impactor (30) to produce ground calcium carbonate (i.e. calcium carbonate particles) . The vertical shaft impactor is a type of grinding equipment or material size reducing system capable of reducing materials down to a diverse range of particle sizes. Such grinding method takes place in a closed system thereby alleviating dust or particulate air pollution. The principle of grinding rocks by centrifugal throw in a vertical shaft impactor can minimize machinery wear more favorably compared to roller mill or ball mill grinding. Calcium carbonate particles produced by this process have a relatively, preferably substantially rounded shape, low angularity and sharpness, with a large variety of different particle sizes or grades to meet requirements of various different applications. Therefore, it is necessary that the calcium carbonate particles proceed to the next step of particle size separation for the purposes of use in different applications.

Calcium carbonate that has undergone limestone comminution by the vertical shaft impactor results in particles having a large variety of different particle sizes. Therefore, it is necessary to sort out the different calcium carbonate particles based on their sizes. The calcium carbonate particles are separated and classified using for example, a sieve vibrator ( 40) capable of separating particles based on their sizes, sorting them into different grades/groups and classifying them by directing them to corresponding storage tanks (60) comprising a 1 ^st grade/group (61 ) having particle sizes of about 1.4 millimeters to about 3 millimeters (mm) , a 2 ^nd grade/ group (62) having particle sizes of about 0.7 millimeters to less than about 1.4 millimeters, a 3 ^rd grade/group (63) having particle sizes of about 0.5 millimeters to less than about 0.7 millimeters, and a 4 ^th grade/ group (64) having particle sizes of about 150 microns (μπι) to less than about 0.5 millimeters. For particles smaller than 150 microns, an additional sorting stage of such calcium carbonate particles by centrifugation and/or cyclonic suction is required. The separation and classification step of the calcium carbonate particles according to the present invention further comprises calcium carbonate particle separation by centrifugation and/ or cyclonic suction (50) , which are suitable for separating particles smaller than 150 microns into 2 different grades and directing them to corresponding storage tanks (60) comprising a 5 ^th grade/ group (65) having particle sizes of about 75 microns to less than about 150 microns, and a 6 ^th grade/ group (66) having particle sizes of about 0.01 microns to less than about 75 microns. Each grade/ group ( 1 ^st to 6 ^th ) of calcium carbonate particles can be used in various industries, such as the cement production industry, the paint industry, and the construction materials industry. Moreover, the calcium carbonate particles are suitable as animal feed for calcium supplementation because of the comminution step in which the limestone rocks are grounded by centrifugal throw in a vertical shaft impactor, and the particle size separation, sorting and classification step being carried out in a closed system. Consequently, the calcium supplement as an animal feed additive obtained from the process of the present invention comprises high purity calcium carbonate particles that are relatively round-shaped and substantially void of acute angularity and sharpness, with low moisture content.

The present invention also relates to an animal feed formulation comprising:

(a) base materials;

(b) additive materials;

(c) ground calcium carbonate particles to be used as animal feed in an amount of 1-10% by dry weight;

characterized in that the said ground calcium carbonate particles to be used as feed additive contain calcium carbonate of more than 95% by dry weight, moisture content of between 0.01 and 0.05% by dry weight, heavy metals content of 0- 10 part per million ( ppm) and are of rounded shapes with low angularity or sharpness resulting from comminution by vertical shaft impactor and subsequent particle size classifying of said ground calcium carbonate particles by sieving apparatus, centrifugal type and/or cyclonic suction type apparatus. According to another aspect of the present invention, there is an animal feed formulation comprising:

at least one base material; and

an animal calcium supplement comprising calcium carbonate particles in an amount of less than or equal to about 10% by dry weight of the animal feed formulation,

wherein the calcium carbonate particles are obtained by a process according to an aspect of the present invention.

The animal calcium supplement for feed additives according to the present invention preferably contain calcium carbonate of more than about 95% by dry weight. Such a high purity calcium carbonate is suitable for providing a source of calcium in animal diets, which is essential for, for instance, metabolic process and growth. Calcium is also a structural component in bones and can enhance the strength of egg shells. The animal calcium supplement for feed additives according to the present invention are relatively round- shaped particles having low angularity and sharpness, which are suitable to be used as animal feed since a small amount of angularity and sharpness of particles would not affect the animal digestive process. They also provide a source of calcium that is efficiently utilized during the formation phase of avian eggshell walls. In contrast, ground calcium carbonate produced by prior art grinding processes using a roller mill or a ball mill, contain particles with a high degree of angularity and sharpness, which are undesirable for use in animal feed and can adversely causing wear in feed blending machines.

The animal calcium supplement for feed additives according to the present invention has a moisture content of about 0.01% to about 0.05% by dry weight. Controlling the moisture content is advantageous in feed additive use because excessive moisture content would cause agglomeration of calcium carbonate leading to inferior non- uniform dispersion of calcium carbonate during the feed ingredients blending process. For this reason, moisture content is preferably maintained between 0.01 and 0.05% by dry weight. The animal calcium supplement for feed additives according to the present invention is characterized in that its heavy metals (namely arsenic, cadmium, lead and mercury) content, is preferably less than or equal to about 10 parts per million (ppm), and more preferably between about 0. 1 ppm to about 10 ppm in accordance with the food safety standard or Commission directive 2008/128/EC. Heavy metals as described are harmful to growth of animals and humans.

The animal feed formulation according to the present invention comprises a base material which includes but is not limited to corn, rice bran, soybean, steamed soybean, peanut, corn dreg, rice bran dreg, soybean dreg, peanut dreg, sunflower seed dreg, and a combination thereof. Such base materials are useful as nutrition sources of protein, carbohydrate, and fatty acid which are nutrients essential for animal growth and building of muscle, for example.

Preferably, the animal feed formulation according to the present invention also comprises an additive material which includes but is not limited to vitamin substances, mineral substances, growth stimulants, feed preservatives, feed additives, enzymes, probiotic substances, hormone- like (parahormone) substances, semi- hormone substances, insect larvae suppressants, and a combination thereof. A small amount of the additive material is added to the animal feed, for example, to improve nutrient utilization effectiveness, productivity, or disease control and prevention in livestock. The animal feed formulation according to the present invention further comprises feed additives to supplement calcium intake. Optimal proportion of calcium is 3-4% by dry weight, allowing calcium to be beneficial to animal growth performance, bone development, and egg wall formation, for example.

The animal feed formulation according to the present invention can be used as, for example, a broiler chicken feed, egg- laying chicken feed, broiler duck feed, egg- laying duck feed, Muscovy duck feed, quail feed, beef cattle- fattening cattle feed, dairy cattle feed, goat feed, sheep feed, fish feed, shrimp feed, mantis shrimp feed or domestic pet food. Tests on animal feed formulation according to the present invention have been carried out to study effects of using the calcium supplement for feed additives as described herein on the performance response of egg- laying chicken, egg quality, eggshell quality, chicken bone characteristics and calcium and phosphorus contents of eggs. These tests also include an Acid Binding Capacity assay and a Buffering Capacity (BUF) test For the purposes of these tests, the following feed additives were chosen:

• The 1 ^st grade/group having particle sizes between 1.4-3 millimeters;

• The 6 ^th grade having particle sizes between 0.01-75 microns; and

• Commercially available standard calcium carbonate. Effects of the particle sizes of the invention to be used as animal feed raw materials were investigated by comparing against conventional grades available in the market.

Experiments were conducted with Lohmann Brown egg- laying chicken breed at the age of 24 weeks. The total number of 360 hens was divided into 4 treatment groups, each consisting of 6 replication units, and each of which in turn consists of 15 hens. Hens were raised with the same feed formulation until age of 24 weeks, after that the experiments began by feeding 4 different trial feed formulations to these hens for a week.

Each group of egg-laying hens was randomized to receive one of 4 different formulations of trial feeds being corn-soybean dreg based feeds wherein compositions and nutritional amount of which were calculated and optimized according to growth stages. The usable calorie and protein content of every formulation were calculated to have uniform values of 2,750 kcal/kg and 17.5 percent, respectively. Prior to the start of the experiments, calcium level of all sources of calcium carbonate was analyzed and the proper amount of calcium content in each source of nutrition supplement additives was 3-4% of total feed.

Animal feed formulations according to the present invention, which were provided with calcium supplement for feed additives obtained from either the 1 ^st grade/ group having particle sizes between 1.4 and 3 millimeters or the 6 ^th grade/ group having particle sizes between 0.01 and 75 micron, did not affect overall chicken performance nor contribute to a noticeable impact on egg quality, calcium and phosphorus levels in blood, or quality of chicken bones. The results of the study reveal that variation in particle sizes does not have an influence on their use as feed additives. As a consequence, the calcium supplement for feed additives according to the present invention can be used as an alternative supply source of calcium nutrient in animal diets.

Examples of feed additives preparation for calcium supplementation

EXAMPLE 1

The process for preparing calcium supplement for feed additives consists of drying the natural calcium carbonate. To begin with, limestone stored in limestone storage tank (10) is passed through calcium carbonate drying system where convective hot air (20) is applied. Moisture content is controlled between 0.01 and 0.05% by dry weight before entering calcium carbonate grinding system (30) which employs a vertical shaft impactor to shatter calcium carbonate into particles and reduce the particle size by means of centrifugal throwing and breaking up of rocks on vertical anvils. This kind of grinder is able to provide particulate calcium carbonate having diversified range of particle sizes to be further processed in the calcium carbonate particle size separation system using sieve vibrator ( 40) capable of particle separation based on particle sizes into different grades and directing the same to corresponding storage tanks ( 60) . Sorted particle grades obtained herewith consist of the 1 ^st grade (61) having particle sizes between 1.4 and 3 millimeters, the 2 ^nd grade (62) having particle sizes between 0.7 and 1.4 millimeters, the 3 ^rd grade (63) having particle sizes between 0.5 and 0.7 millimeters, and the 4 ^th grade ( 64) having particle sizes between 150 microns and 0. 5 millimeters. The calcium supplement for animal feed additive/calcium supplement for animal feed additive according to EXAMPLE 1 belongs to the 1 ^st grade having particle sizes between 1.4 and 3 millimeters with a mean particle size of 1.7 millimeters, calcium carbonate content 96.8%, solid residue larger than 325 mesh 98.7% by weight, and moisture content 0.01% by weight, to be representative of a feed additive for calcium supplementation having particle sizes between 1.4 and 3 millimeters to undergo feed evaluation test for its suitability of application as animal feed. EXAMPLE 2

The process for preparation of the animal calcium supplement for feed additives starts with a step of drying of natural calcium carbonate. Firstly, limestone is stored in limestone storage tank ( 10) then it is introduced to the calcium carbonate drying system where convective hot air (20) is applied. Moisture content is controlled between 0.01 and 0.05% by dry weight before entering calcium carbonate grinding system (30) which employs a vertical shaft impactor to shatter calcium carbonate into particles and reduce the particle size by means of centrifugal throwing and breaking up of rocks on vertical anvils. This kind of grinder is capable of providing particulate calcium carbonate having diversified range of particle sizes which would require further processing in calcium carbonate particle size separation system using sieve vibrator (40) for separating particle sizes into different grades and directing the same to corresponding storage tanks (60) . Sorted particle grades obtained herewith consist of the 1 ^st grade (61 ) having particle sizes between 1.4 and 3 millimeters, the 2 ^nd grade (62) having particle sizes between 0.7 and 1.4 millimeters, the 3 ^rd grade (63) having particle sizes between 0.5 and 0.7 millimeters, and the 4 ^th grade (64) having particle sizes between 150 microns and 0.5 millimeters. Particles smaller than 1 0 microns proceed next to the calcium carbonate particle sorting system relying on centrifugal type and cyclonic suction type sorting apparatus (50) which can separate fine particles into 2 different grades consisting of the 5 ^th grade (65) having particle sizes between 75 and 150 microns and the 6 ^th grade (66) having particle sizes between 0.01 and 75 microns. The calcium supplement for animal feed additive/calcium supplement for animal feed additive according to EXAMPLE 2 belongs to the 6 ^th grade having particle sizes between 0.01 and 75 microns with a mean particle size of 9.3 microns, calcium carbonate content 96.0%, amount of solid residue larger than 325 mesh 4.6% by weight, and moisture content 0.02% by weight, to be representative of a feed additive for calcium supplementation having particle sizes smaller than 75 microns to undergo feed evaluation tests for suitability of its application as animal feed. Composition evaluation test of feed additive for calcium supplementation

Calcium supplement for feed additives as prepared in EXAMPLE 1 and EXAMPLE 2 are evaluated for composition contents of elements by using an X- ray Fluorescence elemental analysis instrument which is able to determine quantities of calcium carbonate, magnesium carbonate, iron oxide and silica oxide and report the results in weight percent of the compositions.

Particle size measurement test of feed additive for calcium supplementation

Calcium supplement for feed additives according to EXAMPLE 1 and EXAMPLE 2 are analyzed to carry out particle size measurement with a Mastersizer 2000 particle sizing instrument which can perform particle size analysis over the range of 0.02-2000 microns. The instrument's operation relies on the principle of High resolution optical laser diffraction. Mastersizer 2000 instrument conducts particle analysis on samples and reports the observed mean particle size (d50) values.

Solid residue testing of polymeric additive obtained from lime mud Calcium supplement for feed additives according to EXAMPLE 1 and EXAMPLE 2 are sampled for making measurement of solid residue particles larger than 325 Mesh to enable determination the amount of particles with size above 45 microns in test samples by means of Wet Sieving particle size separation procedure. This method requires specimens to be dry and volume as well as shape of particles to remain unchanged in the presence of water. Dry sample is weighted (W ₀ ) and put in a number 325 Mesh sieving screen then clean water is continuously poured into the test sieve. Particles smaller than 45 microns will be flushed out and exit through sieve openings. Rinsing of the sample with clean water is continued until the liquid discharged past screen becomes clear without escaping sample detected. Remaining portion on the sieve is oven- dried at 105 degrees Celsius until its weight stays constant. Solid residue weight (W _re s) is recorded. The solid residue content with particle size above 325 Mesh of the sample is calculated from the following formula; residue = ^ x l00

w ₀

Acidity-basicity test of feed additive for calcium supplementation

Calcium supplement for feed additives according to EXAMPLE 1 and EXAMPLE 2 are dissolved in water to prepare calcium carbonate concentration of 5% by weight. The acidity- basicity measurement is then made using a pH meter which performs analysis and reports pH values of calcium supplement for feed additives.

Moisture content test of feed additive for calcium supplementation

Measurement of moisture content in calcium supplement for feed additives obtained from EXAMPLE 1 and EXAMPLE 2 is made. A sample of polymeric additive from lime mud of each example is weighted for its initial value (W ₀ ) with a weighting scale having 4 decimal places resolution. It is subsequently oven dried at 105 degrees Celsius for 4 hours then allowed to cool down to ambient temperature in a desiccator. Post-drying weight is recorded. The sample's baking and weighting procedures are repeated until dried weight (Wdry) between consecutive runs remains unchanged. Moisture content of the specimen is determined by the following formula;

Moisture = ^^ w ₀ ) X I 00

Test of heavy metals content of feed additive for calcium supplementation

Calcium supplement for feed additives obtained from EXAMPLE 1 and EXAMPLE 2 are initially digested by weighting out 0.3 grams of feed additives into a vessel and dissolving it in 5 mL of HNO3. Heating is applied at 150°C for 2 hours. Each sample is allowed to cool down in a fume hood and quantity adjustment is made using a 10 mL- sized volumetric flask with distilled water to serve as a sample solution.

Measurement of As (arsenic) content is made by transferring the sample solution with pipette into a round bottom flask and filling in 1 mL of Mg(N0 ₃ ) ₂ . The sample is then heated on a Hot Plate to 375 °C and put in a furnace at 450 °C. Following temperature reduction, the sample is dissolved in 2 mL of 8 M HC1 and 0. 1 mL of 20K.I (potassium iodide) . Quantity adjustment is again made using a 10 mL- sized volumetric flask with distilled water. The As content is determined by Atomic Absorption Spectroscopy instrument equipped with As lamp using H ₂ air flame. For each element undergoing analysis, a corresponding Calibration Curve is prepared for determination of the heavy metal content of the sample in accordance with ISO 6869:2000(E) standard.

Property test of feed additive for calcium supplementation

EXAMPLE 3

Animal feed in EXAMPLE 3 is prepared by blending corn- soy basal (control) diet with commercially available conventional grade calcium carbonate to obtain a mixture having 3.7% by weight of calcium.

A 200kg sample of the above- described feed in EXAMPLE 3 is taken for an evaluation of feed properties for egg- laying chickens regarding its effects on egg quality and behavior of egg- laying chicken raised under practical circumstances. A total of 360 Lohmann Brown egg-laying hens near their maximum maturity stage for egg production are allocated into battery cages (3 hens/ cage) located inside a poultry housing which is equipped with an evaporative cooling system. Experiments are conducted for 4 time periods (28 days per each time period) under 16 hours of Lighting Program regulation based on recommendations given by the chicken breeder guidelines. Trial results on egg quality and performance parameters of egg- laying chicken are recorded including such as resulting performance response of egg- laying hens, egg quality, eggshell quality, chicken bone characteristics, calcium and phosphorus contents of eggs, and the outcomes of Acid Binding Capacity assay and Buffering Capacity (BUF) test. EXAMPLE 4

Animal feed in EXAMPLE 4 is prepared by blending corn-soy basal (control) meal with the calcium supplement for animal feed additive/calcium supplement for animal feed additive according to EXAMPLE 1 to obtain a mixture having 3. 7% by weight of calcium. A 200 kg sample of the above-described feed in EXAMPLE 4 is taken for an evaluation of feed properties for egg- laying chickens regarding its effects on egg quality and performance of egg- laying chicken raised under practical circumstances. A total of 360 Lohmann Brown egg-laying hens near their maximum maturity stage for egg production are allocated into battery cages ( 3 hens/ cage) located inside a poultry housing which employs an evaporative cooling system. Experiments are conducted for 4 time periods (28 days per each time period) under 16 hours of Lighting Program regulation based on recommendations given by the chicken breeder guidelines. Trial results on egg quality and performance of egg- laying chicken are recorded including such as resulting behavioral response of egg- laying hens, egg quality, eggshell quality, chicken bone characteristics, calcium and phosphorus contents of eggs, and the outcomes of Acid Binding Capacity assay and Buffering Capacity (BUF) test.

EXAMPLE 5

Animal feed in EXAMPLE 5 is prepared by blending corn- soy basal (control) diet with calcium supplement for animal feed additive/calcium supplement for animal feed additive premixed from 50% by weight of EXAMPLE 1 and another 50% by weight of EXAMPLE 2 to obtain a mixture containing 3.7% by weight of calcium. A 200 kg sample of the above-described feed in EXAMPLE 5 is taken for an evaluation of feed properties for egg- laying chickens regarding its effects on egg quality and performance of egg- laying chicken raised under practical circumstances. Lohmann Brown egg-laying hens, 360 of them in total, near their maximum maturity stage for egg production are allocated into battery cages (3 hens/cage) located inside a poultry housing which is equipped with an evaporative cooling system. Experiments are conducted for 4 time periods ( 28 days per each time period) under 16 hours of Lighting Program regulation based on recommendations given by the chicken breeder guidelines. Trial results on egg quality and performance of egg-laying chicken are recorded including such as resulting performance response of egg- laying hens, egg quality, eggshell quality, chicken bone characteristics, calcium and phosphorus contents of eggs, and the outcomes of Acid Binding Capacity assay and Buffering Capacity (BUF) test. EXAMPLE 6

Animal feed in EXAMPLE 6 is prepared by blending corn-soy basal (control) diet with the calcium supplement for animal feed additive/calcium supplement for animal feed additive according to EXAMPLE 2 to obtain a mixture having 3. 7% by weight of calcium.

A 200 kg sample of the above-described feed in EXAMPLE 6 is taken for an evaluation of feed properties for egg- laying chickens regarding its effects on egg quality and performance of egg- laying chicken raised under practical circumstances. Lohmann Brown egg-laying hens, 360 of them in total, near their maximum maturity stage for egg production are allocated into battery cages (3 hens/cage) located inside a poultry housing which is equipped with an evaporative cooling system. Experiments are conducted for 4 time periods ( 28 days per each time period) under 16 hours of Lighting Program regulation based on recommendations given by the chicken breeder guidelines. Trial results on egg quality and performance of egg-laying chicken are recorded including such as resulting performance response of egg- laying hens, egg quality, eggshell quality, chicken bone characteristics, calcium and phosphorus contents of eggs, and the outcomes of Acid Binding Capacity assay and Buffering Capacity (BUF). Experimental Design and dietary manipulations

Experiments are carried out with Lohmann Brown egg- laying chicken breed at the age of 24 weeks. Total numbers of 360 hens are divided into 4 groups, each consisting of 6 replication units, each of which in turn is composed of 15 hens. Hens were raised with the same feed formulation until age of 24 weeks, after that the experiments began by feeding the different trial feed formulations to these hens for a week.

Each group of egg- laying hens is randomly assigned to receive one of the 4 different formulations of trial feeds being corn- soybean dreg based feeds wherein compositions and nutritional amount of which are calculated and optimized according to growth stages. The calorific energy and protein content of every formulation are calculated to have uniform values of 2,750 kCal/kg and 17.5 percent, respectively. Prior to the start of experiment, calcium level of all sources of calcium carbonate is analyzed and the proper calcium content in each source of dietary supplement additives is ensured to be 3.25% of the total amount.

Chicken feeding procedures

All feed diets are calculated to be isonutritive for the entire feeding cycle (24-40 weeks of chickens age) to meet or exceed the nutrient requirements recommended for the particular breed. Feed and water are offered for freely accessible consumption at all times (ad libitum). Chicken feed are mixture, and they are provided twice daily about 7:00 am in the morning and 3:00 pm in the afternoon.

The feed that has been produced is stored in a cool, dry place until the time of use. Trial feeds produced for the experiment contain a feed quantity of 100 kg for each treatment, all being in a form of blended mixture. Feed for each trial is separately packaged in a 25 kg-sized bag which is clearly labeled with messages indicating information of feed used in the treatment such as bag number, trial code, type of feed, treatment number, etc. One bag for each time period is placed outside of each set of trials which are uniquely identified for individual treatment and replication.

Test of egg-laying hens' performance response

The body weight of all hens is recorded at the beginning and the end of the experiments. Change in body weight is then calculated during a period of each experiment. Egg production, feed intake, and number of hen mortality are recorded. Egg mass and feed conversion per kilogram of egg can be calculated. Data collection is divided into four 28- day periods.

Quantity of daily feed intake = weight of feed intake per hen number of days x number of weighted hen

Quantity of feed intake = feed intake during the period of experiment (kg) per 1 kg of egg weight weight of eggs in each period of experiment (kg) Hen-day egg number of eggs produced in experiment period ^χ 100 production; HD number of days * number of hen at experiment ending (percent)

Hen-house egg number of eggs produced in experiment period x 100 production; HH number of days * number of hen at experiment beginning (percent)

Average egg = total egg weight of the replicate for each period of experiment weight per egg Total number, of eggs weighted

Egg mass per hen-day egg production (HD) ^χ Average egg weight per egg I [en per day 100 Livability rate number of hens at the end of experiment period * 100

(percent) number of hens at the beginning of experiment period

Effects on egg quality test

During the last successive 3 days of each period of experiments, the numbers of dirty eggs and cracked eggs are recorded. Internal quality of an egg is assessed by breakage open the egg to measure egg weight, egg white weight, yolk weight, eggshell weight, albumen level, color of yolk, eggshell thickness, and the Haugh unit. As each experiment period concluding, egg quality measurement is made in the final 3 days of each period by randomly collecting 5 eggs from each replicate, 120 eggs in total. The value of Haugh unit can be determined from the observed values of egg weight and height of the albumen, as follows:

Haugh unit (HU) 100 * log (H + 7.57 - 1.7 W ^{0 37} )

where HU Haugh unit

H average height of the albumen (millimeter) WW = weight of the egg (gram) Effects on eggshell quality test

During the last part of each time period, 3 eggs from each example are randomly chosen to test the eggshell breaking strength and the color of eggshell. The Ca and P contents of the blood and the bone are also measured. At the ends of the 2 ^nd and 4 ^th time periods, 2 egg-laying hens from each experiment are randomly selected for blood sampling (2 cc.) then the Ca and P levels are analyzed. In the final part of the 4 ^th time period, 2 egg-laying hens are slaughtered; both of their left and right tibia bones are collected and submitted for the bone breaking strength test and the Ca and P contents analysis, consecutively. As each experiment period concluding, eggshell quality measurement is made in the final 3 days of each time period by randomly collecting 5 eggs from each replicate, 120 eggs in total. The value of Haugh unit can be determined from the observed values of egg weight and height of the albumen, as follows:

Haugh unit (HU) = 100 log (H + 7.57 1.7W ^{0 37} )

where HU = Haugh unit

H = average height of the albumen (millimeter)

W = weight of the egg (gram)

Acid Binding Capacity assay and Buffering Capacity (BUF)

Acid Binding Capacity ( ABC) of feed is calculated as the amount of acid in milliequivalents (rneq) required to lower the pH of 1 kg feed sample to pH 3. Higher ABC of feed additive materials indicates its greater ability of binding to H ⁺ ions in the alimentary system and increasing pH value. Experimental findings show that such condition will reduce efficiency of digestion and absorption of nutrients. Buffering Capacity (BUF) refers to the effectiveness of buffer solution to resist or neutralize the change in pH when acid or base is added. The BUF value was calculated by dividing the ABC by the total change in pH units (from the measured initial pH to the final pH). Analysis of bone characteristics

To maintain the quality of bone tissues, left tibia bones are preserved in formalin in the concentration of 10 percent (9 parts of water: 1 part of formalin) for 3-4 weeks. A middle portion of the tibia bone is then cut for 1 centimeter long and soaked in decalcified solution for 24-48 hours. Upon completion, the bone specimen is rinsed with flowing water for at least 2 hours. Afterwards, the specimen is dehydrated by using ethanol of different concentrations and hardened by casting into a paraffin block ( paraffin embedding) . Tissue is sliced with a microtome into a 5 microns thick section which is mounted on a glass microscopic slide coated with poly- L- lysine. The microscopic slide is cured in an oven at temperature of 60 Celsius for 60 minutes. Paraffin is eliminated from the bone tissue with twice applications of xylene for a duration of 5 minutes each time and water is also removed with ethanol at different levels of concentrations. The tissue slide is then stained with hematoxylin and eosin dyes for the study of morphological changes in the bone tissues by measuring thickness (from periosteum to endosteum) of the cortical dense bones on 4 randomly selected positions of the bone tissue in each slide through a microscope having 4X magnification objective.

Analysis of Ca and P contents of the blood and the bone

Blood test: At the ends of the 2 ^nd and the 4 ^th periods of experiment, blood samples in the amount of 2 milliliters per each hen are drawn from wing veins of 2 randomly chosen hens per each replicate ( 12 hens per group) or 48 hens in total. Blood samples are collected at 06.00 hour which is 1 hour after chickens' exposure to light then the samples are centrifuged to separate the serum for analyses of calcium content in accordance with the Thermo Fisher Scientific (2008) method and phosphorus content in accordance with the Thermo Electron Corporation (2006) method.

Bone test: The right tibia leg bones are tested for the bone breaking strength with a model LR 5 material testing machine in accordance with the protocol set forth in the ASAE Standards (2005) using a loading speed of 10 mm/minute and distance between supports of 50 millimeters. The remainders of leg bones from the strength test will be used for the analyses of calcium and phosphorus contents according to the A.O.A.C (1990) method. Table 1

Table 2 presents experimental results showing influence on the performance of egg- laying chickens by average values of 1 ^st - 4 ^th periods in response to feed EXAMPLES 3,4,5, and 6.

Treatments SEM P- value

Properties

EXAMPLE EXAMPLE EXAMPLE EXAMPLE

3 4 5 6

Hen day 94.39 95.21 93.85 95.42 0.364 0.355 production (%)

Hen house 94.38 94.91 93.84 95.41 0.365 0.457 production(%)

(kg.feed/kg.egg)

Table 3 shows experimental results showing influence on egg quality by average values of 1 * periods in response to feed EXAMPLES 3,4,5, and 6.

Treatments SEM P- value

Properties

EXAMPLE EXAMPLE EXAMPLE EXAMPLE

3 4 5 6

Eggshell breaking

48.22 49.09 48.16 48.63 0.347 0.744 strength (N)

Mean Albumen

8.1 l ^ab 8.2t ^a 7.88 ^bc 7.84 ^c 0.051 0.016 height (mm)

Haugh Unit

90.00 ^a 90.1 l ^a 88.48 ^b 87.76 ^b 0.305 0.007

Calcium content

42.59 ^a 42.34 ^a 39.12 ^b 39.19 ^b 0.530 0.008 of eggshell (%)

Yolk color 7.10 7.27 7.25 7.26 0.029 0.074

Eggshell weight

13.26 13.06 13.02 12.85 0.002 0.175 (%)

Table 4. Acid Binding Capacity and Buffering Capacity results.

Description ABC (mEq/kg) BUF

EXAMPLE 3 trial feed formulation 572 210

EXAMPLE 4 trial feed formulation 549 199

EXAMPLE 5 trial feed formulation 557 197

EXAMPLE 6 trial feed formulation 653 240

Conventional calcium carbonate in the

8525 2310

market

EXAMPLE 1 6075 1681

EXAMPLE 2 7550 2019

Notes : Acid binding capacity the ability to bind acid of feedstuffs.

Buffering capacity the ability of the buffer solutions to

withstand changes in pH when adding acid or base.