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An overview macroscopic and microscopic of kidneys of mice (Mus musculus) after consuming collagen extract from broiler's bone.

INTRODUCTION

Study related to the production process of collagen extracts lately grown rapidly. Scientists are working to find a production process that efficient and effective, but the impact is very low in human. The production process of collagen extract performed by developed countries more applying chemical processes in order to increase production. However, the use of chemicals in the production process of course feared to affect humans as consumers.

Collagen extracts usually produced from cattle bones and skin. Bone has potential as a raw material source of collagen as a health food anti-osteoporosis and premature aging [1]. Osteoporosis is one of the health problems that experienced by many people, especially the elderly. Osteoporosis cases were more experienced by women than men. The reason related to hormonal problems.

Bone is one by-product of cattle that have not been widely used. Bone of broiler is one that has potential as a source of protein collagen. Bone is structurally rich in bioactive compounds, especially proteins collagen. It is bonded firmly with the minerals calcium and phosphorus. In producing the extract collagen from the bones, it takes demineralization. This process can use HCl 1M for 48 or 96 hours [2]. The use of solvent extractant has affected the quantity and quality of the product. One of them has affected the value of the yield, moisture content, protein content and calcium levels. The application of a solution of 0.5 M C[H.sub.3]COOH extractant has produced a better yield value [3]. In addition, the curing process needed to produce collagen extract optimum. The applications of the curing process for 4 days with a concentration of 9% (v/v) were to produce collagen extract better [4]. The Collagen is compound found in the bones of cattle. In principle, have similarities in terms of chemical composition, morphology, distribution, function and pathology with collagen compound owned by the human body [5]. Collagen is the main structural protein in the extracellular tissues of animal bodies. Collagen is a structural protein by the body tissues of cattle [6]. Recently, it has been widely used as a food supplement to prevent bone disease (osteoporosis), joint pain (osteoarthritis) and premature aging [7-8]. Collagen extract generally produced using chemical processes [9-10]. Structurally, bone is rich in bioactive compounds particularly collagen protein [11-12]. The production process is involving a chemical compound that needs special attention.

Exposure to chemical compounds in the human body will certainly affect the body's metabolism. It is most important that chemical compounds have the potential to damage a particular organ in the human body. One of the organ in the human body that can be damaged due to exposure to the chemical compound is a kidney.

To study the extent of organ damage in humans caused by the consumption of collagen extract is not possible to use human beings as objects of experimentation. Therefore, considered for the use of experimental animals, such as mice (Mus musculus). The researchers have carried out the use of mice (Mus musculus) as experimental material. Mice have many advantages such as short life cycle; mice can live up to the age of 1-3 years [13]. Kidneys are vital organs that function in homeostasis by regulating the volume and composition of the plasma, particularly electrolytes and water. Kidneys regulate electrolytes and non-electrolytes of the body, as well as urine excrete excess [14]. The use of experimental animals in assessing the effect of chemical compounds of Aluminum chloride (Al[Cl.sub.3]) on the tissue structure of testicular has been done by previous researchers. Use of Aluminum chloride was combined with turmeric (Curcuma longa) indicates a change in the structure of the testes form tubular dilation and hypoplasia [15].

The objective of this study was to evaluate the effect of the consumption of collagen extracts processed chemically to the macroscopic and microscopic appearance of the kidneys in mice (Mus musculus).

MATERIALS AND METHODS

Research Materials:

The study was conducted in Laboratory of Animal By-Product Technology, Faculty of Animal Science, Hasanuddin University, Makassar, South Sulawesi, Indonesia. Analysis of tissue structure carried out in the Central Veterinary, Maros regency, South Sulawesi, Indonesia.

A total of 30 mice (Mus musculus) as main materials. Characteristics of mice were: strain of balb/C, 2-3 months age, body weight of 20-30 g. Digital scales, water bath, and oral needles number 14 were used as research equipment. Extract collagen was extracted by broiler's bone was used as research material, distilled water, and commercial feed. Trial cage equipped with a feeding and drinking tools was used as complementary equipment.

Research Procedures:

A total of 0 mg/kg of bw level dose was used as a control (group A); 5 mg/kg of bw as group B; 50 mg/kg of bw as group C; 500 mg/kg of bw as group D and 5000 mg/kg of bw as group E. Experimental animals were place in five groups, namely A, B, C, D and E. Each group consisted of six mice. Feeds and water given ad libitum each day. Before treatment, the mice were fasted for 2-3 hours. Collagen extract given by orally to the mice for once a week (four times for a month).

Histological observations of kidney in mice conducted on the 30th day. A total of two mice were randomly tested. Making preparations have been carried out by fixation technique using formalin, then soaked in 70% alcohol. The next stage dehydrated in alcohol 80%. Liver tissue purification process has been carried out in the next stage of xylol and paraffin embedding. Tissue blocks were cut using a microtome (5 ^m) and a piece of tissue attached to the glass object.

Data Analysis:

Data on the analysis macroscopic and microscopic obtained from this study show of the pictures and then analyzed descriptively [16].

RESULTS AND DISCUSSION

Macroscopic Analysis:

Analysis morphology and macroscopic of this study aimed to compare the shape and surface area of the mice kidney after given collagen extract consumption for 30 days (Table 1 dan Fig. 1)

[FIGURE 1 OMITTED]

Based on Table 1 and Fig 1 shows that the kidneys of mice in group A (control) did not show any real change, associated with the color, texture and consistency. Appearance kidneys of mice in-group B, C, D and E have seen the change in color, but related to the texture and consistency do not change significantly. The kidney showed brownish red color, surface of smooth and consistency of rubbery can be expressed as a normal kidneys [17,18]. Most the organs in the body have a regulation to function normally and optimally. Most of the toxic chemicals have been studied the effect when entered into the human or animal body. Effect of chemicals toxic to several organs has not always the same. There are chemicals that can affect the central nervous system, liver, heart and kidneys. Chemicals can be effect on the one organ or several together [19].

Microscopic Analysis:

The chemical compounds in the kidneys within a certain period can lead to changes in the tissue structure. Chemical compounds of collagen extract during the production process have the potential to affect the structure of the kidney tissue in mice. Overview of the tissue structure of kidney in mice after consuming the collagen extract at various dose levels were presented in Figs. 2-6 [20].

The chemical compounds in each food are expected to affect the structure of the tissues in the body. Collagen extract of group A (0 mg/kg of bw) given to mice showed no change in the structure of the kidney tissue (Fig. 2) This influence can also occur in mammalian organs such as the kidneys. Kidney is the primary target organ of toxic effects due its role in the filtrate concentrated toxicant, bringing toxicant through tubular cells and then activate specific toxicant. Toxicant effects shown can vary, ranging from biochemical changes to cell death, which generally appear as changes in kidney function to kidney failure [21].

[FIGURE 2 OMITTED]

[FIGURE 3 OMITTED]

Consumption extract collagen in mice at a dose level of 5 mg/kg of bw (group B) have shown damage to the initial level in the form of hemorrhage around tubules, tubular necrotic as well as the inflammatory process of cell infiltration (Fig. 3) In the treatment of group C (50 mg/kg of bw) showed a change in the form of congestion (Fig. 4)

[FIGURE 4 OMITTED]

Congestion is a term that indicates an excess volume of blood in a blood vessel section. This is can happen because too much blood into the arteries or too little blood to the veins. Microscopically, congestion is characterized by dilatation of the artery or capillary walls caused by a large volume of blood in that section [22].

Congestion can also occur due to an increase in the liquid is at a place as a passive process by the failure of the flow of fluid out of tissues, for example in the vein damage [23].

Changes in the structure of the kidney tissue of mice in the form of hemorrhagic and necrotic tubules occurred in group D (500 mg/kg of bw) (Fig.5). Necrosis presence on the tubule is the result of circumstances due to ischemia or toxic substances that enter the tubule epithelium. Tubule epithelial cell response followed by necrosis. Epithelium of kidney is the part that is sensitive to toxic substances. Toxic material that has usually enters through the bloodstream to the kidneys. It is can cause changes in the kidneys in the form of cloudy swelling, fatty degeneration and necrosis.

[FIGURE 5 OMITTED]

[FIGURE 6 OMITTED]

Application of collagen extract dose level of 5000 mg/kg of bw (group E) in mice demonstrated the presence of hemorrhage in the tubules or glomeruli as well as the occurrence of necrotic (Fig. 6). One form of damage of the structure of the kidney tissue was necrosis. Necrosis was missing of cell membrane and cytoplasm were broken and subsequently forming particles. The characteristics of necrosis was seen eusinofilik cytoplasm and the nucleus chromatin clotting process. The nucleus of chromatin will look smaller and more basophilic [24]. The glomerular is a capillary complex that the main function in filtration. If there were damage to the glomeruli, filtration process will disrupt. Injury of glomerular can severely disrupt the peritubular vascular system. This is potential to lead the toxic substances that can flow past the tubule. Conversely, severe damage to the tubules due to increased intra-glomerular pressure also causing atrophy of glomerular [25]. One of the factors that could cause damage to the glomeruli were strees factor. Stress factor can be cause damage to the glomeruli [26]. This statement was related to the study, that a narrowing of kidney damage "bowman" can be caused by inflammatory processes or parts of glomerular epithelial cell proliferation [27].

Conclusion:

1. No significant differences, especially the texture and consistency of the kidneys of mice (Mus musculus) during given collagen extract (30 days) at different doses, except for color

2. No significant differences appearance on the kidney tissue structures of mice that consume collagen extract at a dose of 5-50 mg/ kg of bw compared to controls (0 mg/kg of bw), except for the doses of 500-5000 mg/kg of bw found any significant differences compared to control

3. Consumption of collagen extract the maximum recommended for dose of 50 mg/kg of bw is once a week.

ACKNOWLEDGEMENT

This project was supported by the Ministry of Technology Research and Higher Education through funding Research Grants of National Strategic on the second year (2015) with the contracts number: 1764/UN4.20/ PL.09/2015 on March 31, 2015 and then my students Selfin Tala, S.Pt, M.P as colleagues in the laboratory.

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(1) Muhammad Irfan Said, (2) Effendi Abustam, (3) Farida Nur Yuliati, (4) Budi Utomo

(1) Lab. Animal By Product Technology, Faculty of Animal Science, Hasanuddin University, Jl.Perintis Kemerdekaan Km.10 Makassar, South Sulawesi, Indonesia 90245

(2) Lab. Meat and Egg Processing Technology, Faculty of Animal Science, Hasanuddin University, Jl.Perintis Kemerdekaan Km.10Makassar, South Sulawesi, Indonesia 90245

(3) Lab. Microbiology and Animal Health, Faculty of Animal Science, Hasanuddin University, Jl.Perintis Kemerdekaan Km.10 Makassar, South Sulawesi, Indonesia 90245

(4) Undergraduate at Animal Science Study Program, Faculty of Animal Science, Hasanuddin University, Jl.Perintis Kemerdekaan Km.10 Makassar, South Sulawesi, Indonesia 90245

Address For Correspondence:

Muhammad Irfan Said, Lab. Animal By Product Technology, Faculty of Animal Science, Hasanuddin University, Jl.Perintis

Kemerdekaan Km.10 Makassar, South Sulawesi, INDONESIA-90245

Tel: +62-587217, Fax: +62-587217; E-mail: irfanunhas@gmail.com

This work is licensed under the Creative Commons Attribution International License (CC BY).

http://creativecommons.org/licenses/by/4.0/

Received 22 June 2016; Accepted 28 August 2016; Available online 31 August 2016
Table 1: Kidney morphology of mice (Mus musculus) given collagen
extract consumption for 30 days

Group      Kidney
of Doses
           Color          Texture of surface   Consistency

A          Red brownish   Slippery             Chewy
B          Pale red       Slippery             Chewy
C          Dark red       Slippery             Chewy
D          Dark red       Slippery             Chewy
E          Dark red       Slippery             Chewy

Note:

(A) 0 mg/kg of bw ; (B) 5 mg/kg of bw ; (C) 50 mg/kg of bw ; (D) 500
mg/kg of bw ; (E) 5,000 mg/kg of bw bw = body weight
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Author:Said, Muhammad Irfan; Abustam, Effendi; Yuliati, Farida Nur; Utomo, Budi
Publication:Advances in Environmental Biology
Article Type:Report
Date:Aug 1, 2016
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