The Role of Vitamin E to Overcome Tropical Heat Stress Related with Expression of HSP70 Gene in Broiler

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THE ROLE OF VITAMIN E TO OVERCOME TROPICAL

HEAT STRESS RELATED WITH EXPRESSION OF

HSP70 GENE IN BROILER

RAYMUNDUS GENTY LARAS

GRADUATE SCHOOL

BOGOR AGRICULTURAL UNIVERSITY BOGOR

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STATEMENT OF RESEARCH ORIGINALITY

I hereby declare that this thesis entitled with The Role of Vitamin E to Overcome Tropical Heat Stress Related with Expression of HSP70 Gene in Broiler is really and true my erudition under the guidance and direction of the assigned supervising committee and has not been submitted in any form to any college or other universities. Sources of information which is derived or cited either from published or unpublished scientific paper from other writers have mentioned in the script and listed in the Bibliography at the end part of this thesis.

Bogor, July 2014 Raymundus Genty Laras

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RINGKASAN

RAYMUNDUS GENTY LARAS. Peran Vitamin E dalam Mengatasi Cekaman Panas Daerah Tropis melalui Kajian terhadap Ekspresi Gen HSP70 pada Ayam Broiler. Dibimbing oleh DEWI APRI ASTUTI, RITA MUTIA, dan JAKARIA.

Pengaruh cuaca dan iklim terhadap performa ternak broiler sudah lama dikaji oleh industri broiler dari hulu ke hilir, terutama di kawasan-kawasan yang terpapar temperatur dan kelembaban tinggi. Hal ini erat kaitannya dengan isu mengenai pemanasan global yang menjadi perhatian utama berbagai pihak (akademisi, pebisnis dan pemerintah). Cuaca dan iklim Indonesia sebagai negara tropis basah akan memicu stress akibat cekaman panas pada ternak broiler. Menghadapi kondisi ini, dari sisi ilmu nutrisi pakan, sudah banyak dilakukan usaha solutif dan upaya adaptif, salah satunya adalah dengan melakukan penambahan vitamin E ke dalam pakan sebagai antioksidan untuk menekan efek negatif dari pengaruh cekaman panas pada ternak. Pengaruh penambahan vitamin E pada pakan broiler terhadap mekanisme aktivitas metabolis di dalam tubuh perlu dianalisis hingga ke tingkat ekspresi gen, atau yang lebih dikenal sebagai kajian nutrigenom, untuk menggali kemungkinan-kemungkinan pemecahan masalah lebih lanjut terhadap dampak perubahan lingkungan bagi ternak broiler. Melalui pendekatan nutrigenomik kita dapat mengetahui bagaimana suatu nutrien mampu memodulasi gen dan ekspresi protein yang akan mempengaruhi metabolisme sel, status kesehatan, dan performa ternak.

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pada kandang NE dibandingkan dengan kandang AC dan kandang PE. Dengan demikian, dapat disimpulkan bahwa broiler yang diberi suplementasi 225 mg/kg vitamin E pada kandang yang terpapar panas lingkungan tropis sama bagusnya dengan broiler yang dipelihara pada kandang suhu nyaman AC pada performa (tampilan), respon fisiologis, dan ekspresi gen, serta keseimbangan protein. Dosis vitamin E sebanyak 225 mg kg-1 dapat menghambat pengaruh buruk dari paparan panas lingkungan tropis pada ayam broiler.

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SUMMARY

RAYMUNDUS GENTY LARAS. The Role of Vitamin E to Overcome Tropical Heat Stress Related with Expression of HSP70 Gene in Broiler. Supervised by DEWI APRI ASTUTI, RITA MUTIA and JAKARIA.

The influence of weather and climate on the performance of broiler chicken has long been studied by the upstream and downstream industry of broiler chicken, especially in areas which are exposed by high temperatures and high relative humidity. It is closely related to the global warming issue which is become major concern of various parties (academician, businessman, and government). The weather and climate of Indonesia as a tropical wet country will trigger heat stress in broiler chicken. Faced with these conditions, feed and nutrition science has done a lot of solution-adaptive efforts, one of which is: by the addition of vitamin E into diet as an antioxidant agent to suppress the negative effects of heat stress on broiler chicken. Effect of the addition of vitamin E within broilers diet towards the mechanisms of metabolic activity in the broiler's body needs to be analyzed into the level of gene expression, or better known as nutrigenom study, to explore the possibilities for further solution on the impact of environmental changes for broiler chicken. Through the nutrigenomics approach we can determine how a nutrient is able to modulate the expression of genes and the expression of proteins which affect cell metabolism, health status, and broiler performance.

The purpose of this study was to determine the effect of vitamin E in overcoming tropical heat stress based on productivity, hematology, and protein balance related with expression of heat shock protein 70 gene (HSP70 gene) in broiler. Three kinds of environmental pens were designed in this experiment using completely randomized design, 40 chickens in ideal environment (the temperature of Air Conditioner was adjusted to 22˚C), 40 chickens in wet tropical climate pens

(± 30˚C) with supplementation of 225 ppm vitamin E (PE), and other 40 chickens

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The 225 ppm dosage of vitamin E which is supplemented within broiler’s diet can prevent negative effect of tropical heat stress.

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© Hak Cipta Milik IPB, Tahun 2014

Hak Cipta Dilindungi Undang-Undang

Dilarang mengutip sebagian atau seluruh karya tulis ini tanpa mencantumkan atau menyebutkan sumbernya. Pengutipan hanya untuk kepentingan pendidikan, penelitian, penulisan karya ilmiah, penyusunan laporan, penulisan kritik, atau tinjauan suatu masalah; dan pengutipan tersebut tidak merugikan kepentingan IPB

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Thesis

as one requisite to obtain a degree in Master of Science

on

Study Program of Nutrition and Feed Science

THE ROLE OF VITAMIN E TO OVERCOME TROPICAL

HEAT STRESS RELATED WITH EXPRESSION OF

HSP70 GENE IN BROILER

GRADUATE SCHOOL

BOGOR AGRICULTURAL UNIVERSITY BOGOR

2014

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AUTHENTICATION

Title : The Role of Vitamin E to Overcome Tropical Heat Stress Related with Expression of HSP70 Gene in Broiler

Name : Raymundus Genty Laras

NIM : D251120071

Aproved by Advisory Committee

Prof Dr Ir Dewi Apri Astuti, MS Main Supervisor

Dr Ir Rita Mutia, MAgr Co-Supervisor

Dr Jakaria, SPt MSi Co-Supervisor

Acknowledged by

Head of Study Program Nutrition and Feed Science

Dr Ir Dwierra Evvyernie A, MS MSc

Dean of Graduate School

Dr Ir Dahrul Syah, MscAgr

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ACKNOWLEDGEMENT

There is no words higher than noblest expression of gratitude for all things which I can endlessly received from HIS generosity, especially about this

completion form of my thesis entitled with “The Role of Vitamin E to Overcome Tropical Heat Stress Related with Expression of HSP70 Gene in Broiler” as fulfillment of the requisite to obtain Master of Science degree in Bogor Agricultural University.

Also, author thanks are extended to Prof Dr Ir Dewi Astuti Apri MS, Dr Ir Rita Mutia MAgr, and Dr Jakaria SPt MSi as the best supervisors ever who I fully respect, for giving a lots of advanced advice, guidance, time, energy and thought to this thesis so that it can be done. I have also expressed my thanks not only to Dr Ir Dwierra Evvyernie A. MS MSc but also to Dr Ir Sumiati MSc as chairman and vice-chairman of Nutrition and Feed Science Study Program at BAU Graduate School, Dr. Ir. Niken Ulupi, Ms as the wisest examiner ever whom give lots of constructive criticism and useful suggestion during my close exam. Mrs Lanjarsih as a co-assistant researcher in Poultry Nutrition Laboratory, Mr Eryk Andreas SPt MSi as a co-assistant in Animal Molecular Genetics Laboratory, the entire staff, lecturers, technicians, and my best co-researcher Mr Robi Amizar, graduate class mate from generation of 2012, and to all friends who have contributed in the completion process of this thesis, Devide, Reikha, Taryati, Puput, Nanang, Benny, Syafaat, Dedi, etc. My gratitude also goes to my lovely old uncle Mr Dwijawandawa, my beloved parents Mr and Mrs Yohanes, my smartest young brother Raymundus, my dearest Sarinah Monica, Prof. Nahrowi, Mr Suaedi Sunanto, Mr. Niko, Mr Wira Wisnu, Mr. Sigit Widayanto, Mr. Agus Wiyono, Mr. Aryono, Bumiraya Group and Trouw company, as well as the entire of my family,

Perwira 77’s board, especially Lira, Fitri, Rafi, Habib, Julian, Steve, Septi for all the prayers and support.

Author has been working maximally in completion process of this thesis. But, author realizes that it might be, in the process of writing this thesis, still, there will be a shortage of shortcomings. Therefore, constructive, both suggestions and criticism from readers are gladly accepted by me. Hopefully this scientific paper can be useful for all of us and for further development of science and knowledge.

The authors thanks not only to Directorate General of Higher Education of Indonesia for Beasiswa Unggulan, but also BOPTN Program Funding Research (Project ID No.238/IT3.41.2/L2/SPK/2013).

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LIST OF TABLE

1. Composition and nutrient content of the basal diets 4 2. Analysis of diets and vitamin E content (as feed) 5 3. The average of daily ambient temperature and daily relative humidity

during 35 of experimental days. 11

4. The quantity of HSP70 gene expression of hipocampus sample and breast sample of broiler in three kind different treatments. 15 5. Blood profile examination result of broiler in three kind of different

treatments. 19

6. The performance of broiler during 35 experimental days in three kind

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APPENDIX

Appendix 1 ANOVA of Hippocampus 28

Appendix 2 DMRT of Hippocampus 28

Appendix 3 ANOVA of Breast 28

Appendix 4 DMRT of Breast 28

Appendix 5 ANOVA of Hemoglobin 28

Appendix 6 DMRT of Hemoglobin 28

Appendix 7 ANOVA of Hematocrit 29

Appendix 8 DMRT of Hematocrit 29

Appendix 9 ANOVA of Erythrocyte 29

Appendix 10 DMRT of Erythrocyte 29

Appendix 11 ANOVA of Leukocyte 29

Appendix 12 DMRT of Leukocyte 29

Appendix 13 ANOVA of Heterophile 30

Appendix 14 DMRT of Heterophile 30

Appendix 15 ANOVA of Lymphocyte 30

Appendix 16 DMRT of Lymphocyte 30

Appendix 17 ANOVA of H/L Ratio 30

Appendix 18 DMRT of H/L Ratio 30

Appendix 19 ANOVA of Monocyte 31

Appendix 20 DMRT of Monocyte 31

Appendix 21 ANOVA of Eosinophils 31

Appendix 23 ANOVA of Feed Intake 31

Appendix 24 DMRT of Feed Intake 31

Appendix 25 ANOVA of Crude Protein Consumption 32

Appendix 26 DMRT of Crude Protein Consumption 32

Appendix 27 ANOVA of Body Weight 32

Appendix 28 DMRT of Body Weight 32

Appendix 29 ANOVA of Weight Gain 32

Appendix 30 DMRT of Weight Gain 32

Appendix 31 ANOVA of Crude Protein Content of Meat 33 Appendix 32 DMRT of Crude Protein Content of Meat 33

Appendix 33 ANOVA of Crude Protein Efficiency 33

Appendix 34 DMRT of Crude Protein Efficiency 33

Appendix 35 ANOVA of Feed Conversion 33

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1 INTRODUCTION

Background

Weather and climate is one kind of predisposer in the performance of broiler chicken. It has long been studied by livestock industry, up and downstream in all over of the world, especially in wet or dry tropical region with relatively high temperature exposure. Further, it also related with global warming issue which nowadays has long been discussed as a major big problem concern which should been solved by scientist, businessman, and government with the same goal to set several adaptive strategies to overcome it. In its relation to the terms of animal science, one of best solution which can be offered by scientist to deal with this problem is through the manipulation of feed and nutrition.

As a wet tropical country, Indonesia has an average temperature around 27˚C along year. During the daytime, the temperature range is between 29.8 to

34.9˚C (MCGI 2012). Thereby, this situation will lead to stress due to heat (external) exposure from environment to broiler. At the time of broilers challenged by heat stress, the respiratory rate tends to increase so that the broiler experienced hyperthermia which then led to circumstances of panting as a form of respiratory tract evaporation mechanism. According to the North and Bel (1990), three weeks aged broiler chickens and up, will start to do panting at 29˚C of environment temperature or when the body temperature of chicken reaches 42˚C. Panting will effect on respiration intensity which is likely to increase by more than 20 times to its normal condition. Further, it will influence the acid-base balance in the blood. When broilers do panting, a lot of CO2 will lose (alkalosis)

and the degree of blood acidity will become more alkaline which impact on oxygen binding inhibition by red blood cells, which, if unresponded soon, will cause hemolysis. During external heat exposure which cause heat stress, occurs excessive production of free radicals. These free radicals will damage the cell conversion. Its weight average is 200 grams less than the broilers which reared in the comfortable pens use air conditioner adjusted at 22˚C. Thus, the wet tropical environment has an impact on productivity and feed efficiency of broilers.

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was a vital instrument in the formation of red blood cells (Winarno 2008). In Indonesia, broiler meat is highly demanded which is approximately about 1428.8 tonnes (MARI 2012) and 90% of it is fulfilled by local production of domestic farms (Daghir 2007). Thus, the optimization of broiler meat production from broiler strains which comes from temperate region is required and possible through the manipulation of nutrient in feed which has support ability mechanism to overcome heat stress, such as vitamin E. According to Muchtadi (1994), the main function of vitamin E is as an antioxidant of body, in which vitamin E can act as a scavenger (catcher) of free radicals that enter the body or formed in the body from normal metabolic processes. As a protector, vitamin E will continually act as scavenger against free radicals which are formed to malfunctions the cells. The accumulation of free radicals in the body leads to stress (Winarno 1997). The effect of vitamin E supplementation in broilers diet and its relation to the mechanisms of metabolic activity inside the body need to be analyzed down to the level of gene expression, which is better known as nutrigenom, a science that uses molecular technology to uncover gene expression when affected by certain nutrients derived from feed to be more understand the performance of nutrients in modulating gene and protein expression in influencing cellular metabolism, and the health consequences related to performance (De Busk 2009).

Heat stress is triggered by an increase in the temperature of the environment which exceeds the comfort zone range of ideal temperature. Heat stress affects the productivity of broilers which mainly reared on maintenance system with the open enclosure (Daghir 2007). This is due to the broiler strains that exist today are the result of intensive selection. The broilers were characterized by, relatively, not only high at metabolic rate but also fast at growth rate. In order to achieve their optimum potential genetic, then the broiler chicken must be reared at thermoneutral zone. Attempts to relieved stress which caused by tropical environment heat shock, the supplementation of vitamin E is expected to overcome it. In addition, the observation of it through the expression of gene related to heat stress, especially HSP70 gene is very important to note. So that, the modulation of gene and protein expression which will affect the metabolism of the cell, health status and the performance of broilers under tropical heat stress becomes clear.

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Purpose

Through this study, is expected to determine the effect of vitamin E on gene expression changes and heat shock protein 70 gene (HSP70 gene), physiological responses, performance, and blood and immunity profiles Broiler chickens.

Utility

Heat stress is stimulated by an increase of ambient temperature which exceeds the strecthes of comfort zone temperature. Thus, it will influence the productivity of broiler chickens, especially on farms with an open rearing system of maintenance. It is caused by strains of broiler which exist nowadays are the result of intensive selection. The strain as mentioned before was featured with relativity in high metabolic rate and relatively in fast growth rate. Therefore, to achieve optimum genetic potential, the broilers should be kept at the thermoneutral zone. Ways and mean to control heat stress by vitamin E supplementation in the diet is expected to reduce the stress by heat exposure which come from tropical environment. And what is more, the observations towards gene expression, especially HSP70 gene, through vitamin E addition is very important to note so that the modulation of gene and protein expression which would impacted the metabolism of cells, health status, and performance of broilers against tropical heat stress becomes unequivocally clear.

Scope and Limitation of the Study

This study focuses on 225 ppm supplementation of Vitamin E (Lutavit® E 50, BASF, Netherland) as an antioxidant in diet ration of Cobb broiler’s chicken

and its relation to broiler’s performance, blood physiology, and gene expression of

HSP70, under oxidative stress which is emerge after the exposure of tropical heat stress at Dramaga, Bogor, Jawa Barat.

2 RESEARCH METHODOLOGY

Date and Location

The research study was carried out from June 2013 until November 2013.

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Materials Birds, Diet, and Animal House

In this experiment, 120 day old chick Cobb CP707 broiler chicks were individually weighed then randomly allocated to three treatments. Each treatments had four replicates of 10 birds (male and female mixed) and placed in 1.5 x 1.5 m2 pens.

Table 1 Composition and nutrient content of the basal diets

Starter Finisher

Premix provided (in mg/kg premix): vit A 1200000 IU; vit D3 200000 IU; vit E 800; vit K 200; vit B1 200; vit B2 500; vit B6 50; vit B12 1200µg; vit C 2500; Ca-D pantothenate 600; niacin 4000; choline chloride 1000; methionine 3000; lysine 3000; manganese 12000; iron 2000; iodine 20; zinc 10000; cobalt 20; copper 400; santoquin 1000; zinc bacitracin 2100.

b

Calculated value.

c

Calculated based on analyzed ingredient composition.

Experiment began in Grower period. Three treatments in this experiment were: 1) thermoneutral pens, temperature of the environment adjusted by Air

Conditioner at 22˚C (Effendi 2010), no supplementation of vitamin E in their diet

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suppress the detrimental effects of heat stress so that broilers must be able to respond to its properly. The birds were raised to 5 weeks and were fed ad libitum in crumble form with basal starter and basal grower diets which formulated to fit the minimum nutrient requirement of broiler chicken based on balance amounts of energy and crude protein content (Table 1) according to the recommendation of Leeson and Summer (2005).

Further, the grower diets of PE pens were supplemented by vitamin E (Lutavit® E 50, BASF, Netherland). In this experiment, water were provided ad libitum too. In all experiments, broiler chicks of each replicate were weighed weekly until the end of period of feeding. Acurate weekly records were kept of all feed added to each replicate and the amount of residual feed remaining until the end of periods of feeding. Feed conversion ratio (feed/gain, g/g) was calculated and mortality was recorded as it occurred. Table 1 showed the formulation and ingredients of nutrient in diet which used in this research.

Table 2 Analysis of diets and vitamin E content (as feed) Nutrient ingredients*) Starter Diet

*) Proximat analysis was carried out in Laboratorium of Feed Science and Technology, Faculty of Animal Science, Bogor Agricultural University (2013), **) Analyze result of Centre of Agro Industry Laboratorium (BBIA), Bogor (2013), GE: gross energy, Ca: calcium, P: phosphor.

Methods Care and Maintenance Procedure

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measurement was conducted and recorded at 07.00 am, 13.00 pm, and at 17:00 pm.

Statistical Analysis

Three kinds of environmental pens were designed in this experiment using completely randomized design (Steel and Torrie 1993).

Yij= µ + τi+ εij

Note:

Yij : jth observed sample value from the ith population

µ : reference value, usually called the “grand” or overall mean

τi : parameter that measures the effect of an observation being in the ith

population

εij : difference or derivation of the jth observed value from its respective

population mean

Statistical analysis system was performed by using ANOVA procedure, using SAS program (Version 9.3; SAS Inst. Inc, Cary, NC, 2011). Means were

separated by Duncan’s multiple range test (Duncan 1955).

Variables

Several variables were observed in this study, include: a. Broiler Performance

Broiler reared along 35 days of experiment. (five weeks), total daily consumption and weekly body weight were regularly recorded.

1. Feed consumption (g bird-1)

The average of feed consumption was calculated from the quarrel between feed which is given to feed which is remain uneaten. This quarrel divided by total number of chickens which is exist in one plot. Measurement of residual feed recorded once in a week, in the morning.

Feed Consumption = Feed Given – Feed Remain Total Broilers 2. Body weight (g bird-1)

Final body weight was obtained by weighing the whole broilers of each treatment at the end of reared based on research, then averaged in each of plot.

3. Weight gain (g bird-1)

Body weight gain is obtained from the calculation of the final weight of treatment minus the initial weight of treatment. Body weight was measured once in a week.

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4. Feed conversion ratio

Feed conversion was calculated from the ratio between the average feed consumption with an average of body weight gain.

Feed Conversion Ratio = Average Feed Consumption Weight Gain of Broilers 5. Mortality (%)

Mortality was recorded and counted as it happened. 6. Crude protein consumption (g bird-1)

Crude protein consumption was calculated from the percentage value of crude protein content within diet multiplied by feed consumption.

Crude Protein Consumption =

Crude Protein Content of Feed x Feed Consumption 7. Crude protein content of meat (g bird-1)

Crude protein content of meat was obtained from its percentage value multiplied by weight gain.

Crude Protein Content of Meat =

The Percentage of Crude Protein Content of Meat x Weight Gain 8. Crude protein efficiency (%)

Crude protein efficiency was obtained from the value of crude proteint content of meat divided by the value of crude protein consumption, then multiplied by one hundred percent.

Crude Protein Eficiency = Crude Protein Content of Meat x 100% Crude Protein Consumption

b. Evaluation of Biochemical Parameters

Considering to animal welfare, broiler were restrained manually and 2 ml of blood sample was collected from axillary veins from eight chicks per treatment group and placed in a microtube with EDTA for determining hematological value: (haemoglobin (Hb), packed cell volume (PCV), heterophyl, lymphocyte, and heterophyl to lymphocyte (H/L) ratio counts (EDTA, anticoagulant) (Sikar et al. 1984; Sastradipradja et al. 1989).

1. Blood sampling

Blood sampling conducted in broiler chickens aged 32 days. In each pens, one broiler was taken as sample. First step, set up well, tools and materials such as syringes, test tubes, cotton, Ethylene Diamine Tetraacetic Acid (EDTA), ice bucket, ice cubes, and alcohol. Second, cleaned the

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cleaned using a steril tissue. Rees and Ecker diluting solution were sucked up unto the mark reached 101 on the erythrocytes pipette, then put off the aspirator pipe. Blocked not only top part but also bottom part of the pipette using your thumb and your right hand index finger, then the contents

inside the pipette was shaken by forming a figure of “8 motion”, and fluids

which was unshaken will discarded. A drop of fluid entranced into the counting chamber and let it precipitated. Red blood cells counted under a microscope at a magnification of 400 times (a). To measure quantitively erytrocyte inside the hemocytometer neubeur, a total of 5 pieces erytrocyte boxes area from 9 were used by taking part as follows: the upper right corner part of the box, the top left corner part of the box, one box in the middle, the bottom right corner part of the box, and the bottom left corner part of the box. To differentiate erythrocytes box and leukocytes box can be based on the three lines divider on the erythrocytes box. Erythrocyte box is relatively narrower than a box of leukocytes. Once the numbers of erytrocyte were obtained, then the number of blood should be multiplied by 5000, to determine the number of erythrocytes in 1 mm3 of blood. The constant number of 5000 was derived from the multiplication count of the thick of counting chamber 1/10 mm, the length of counting chamber 1/5 mm, the width of counting chamber 1/5 mm and 5 counting chamber represented in mm3, then multiplied by 100 of diluting solution. The total amount of erythrocytes can be calculated with the following formula:

The number of erythrocytes per mm3 of blood = a x 5 x 103 corpuscle 3. The number of leukocytes

The number of leukocytes which was obtained from the counting process using microscope (b) shall be multiplied by 200 to determine the number of leukocytes per 1 mm3 of blood. The figure of 200 is obtained by multiplying the 5 counting chamber, a length of 1 mm, a width of 1 mm, and thick of 1/10 mm, was then made into 1 mm3, after that, the dilution factor multiplied by 100. The total amount of leukocytes can be calculated within the formula below.

The number of leukocytes per mm3 of blood = b x 2 x 102 corpuscle 4. Leukocyte differentiation

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c. Quantification of mRNA HSP70 gene

Considering to animal welfare, two broilers from each replicate were killed by decapitation at 12.00-14.00 p.m (which is a time with high radiation, and high heat exposure) and 3 g samples of hippocampus and muscle tissues of each broiler were collected and stored in a 1.5 ml tube containing 500 mL of RNA Later. Then, stored at -20 _°C which would then be used for gene expression analysis for total RNA extraction and real time RT-PCR.

The amount of or HSP70 mRNA quantification were calculated by determining scheme by plotting the natural log standard curve using a predetermined cycle (Ct) which is paired with the natural log of the number of molecules used in the standard sample of DNA (dilution range between 9.65 x 105 -9,65 x 109 copies per mL), whereas Ct is the cycle in which the increase based on statistic, accounted significantly to the number of signals obtained from the PCR reaction when first detected. Ct was calculated by setting common standards that have been agreed upon prior to sequence detection results of real-time software. Equation which is derived from the graph will be used in the calculation of the value of cDNA molecules per mg, derived from oligo-dT cDNA pratotal. Then the results were tested using the same reaction from the plate as standard (Tamzil et al. 2013).

1. Isolation and Extraction of RNA

Isolation of RNA: RNA isolation from tissue performed by using RNA Purification Kit GeneJET reagent (Thermo Scientific, Lithuania, EU). A total of 30 mg sample of tissue, put in 300 µL lysis buffer.

containing β-mercaptoethanol and then smoothed by using sample micro-pestle. Next step, added 600 µL Proteinase K (10 µL Proteinase K diluted in 300 µL TE buffer), then vortexed and incubated for 10 minutes at room temperature. Furthermore, the solution were centrifuged at 12000 rpm for 5 minutes and the supernatant portion was moved into a new tube which has been added by 450 µL of ethanol before, and mixed by using pipeting method. Forthwith, the solution was transferred into the column tube and centrifuged at 12000 rpm for 1 minute. The solution was discarded , and then added the first type of 700 µL wash buffer (containing ethanol) to the column and centrifuged at 12000 rpm for 1 minute, the solution was discarded and then added the second type of 600 µL washing buffer (containing ethanol) to the column and then centrifuged 12000 rpm for 1 minute. After that, the solution was cast off, added with 250 µL washing buffer type two and centrifuged at 12000 rpm for 2 minutes, then the column was transferred to 1.5 µL tubes. After adding 100 µL of nuclease free water, the samples were centrifuged at 12000 rpm for 1 minute. Pellet RNA (template) which has been obtained were stored at -20 °C until ready for use.

2. Reverse Transcriptase

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oligo (dT) and 9 µL of water. The solution was incubated at 65 °C for 5 minutes. Then, added 4 µL 5xRB (buffer), 1 µL riboblock, 2 µL dNTPs and 1 µL of reverse transcriptase. Furthermore, the solution was incubated using polymerase chain reaction (PCR) (GeneAmp PCR System 9700, AB Applied Biosystems, Singapore) at a temperature of 42 °C for 5 minutes and 78 oC for 5 minutes. Quantification of complementary DNA which is obtained, then analyzed (260 : 280 nm absorbance) using a spectrophotometer (Agilent 8453, USA). As a result, based on the cDNA quantification, concentration standard with the help of DNA / RNA copy number calculator were obtained (http://endmemo.com/bio/dnacopynum.php).

3.Primary HSP70 gene.

Primary which is used to amplify mRNA HSP70 consist of: GADPH: F-5’GTG TTA TCA TCT CAG CTC CCT CAG-3’, R-5’GGT CAT forward and reverse primer (10 pµol); master mix containing 5 µL, 12 : 25 µL forward primer (10 pµol), 12 : 25 µL (10 pµol), 1 µL of cDNA sample and 3.5 µL nuclease-free water. Run the following PCR conditions, 95 °C for 5 min, 39 cycles at 95 °C for 10 seconds, followed by 60 °C for 20 sec and 72 °C for 30 seconds.

qRT - PCR using fluorescent reporter molecules to monitor the production from amplification products in each cycle of the PCR reaction. In qRT-PCR cycle, threshold values will be obtained (CT),

which is the cycle when the intensity of fluorescence emission of the dye passes through the threshold value. The higher the initial amount of target nucleic acid copies, the faster the increase in fluorescence, so that the lower the value of CT (Bustin 2005). HSP70 gene expression

calculated based on approach as it quantified as to relative amount of gene mRNA target quantity (HSP70 gene) to control gene (GAPDH).

Standard curve was created by plotting natural log with cycle threshold (CT) to molecules natural log number used in the standard DNA samples

(the range of dilutions is between 17.0775 x 104 – 17.0775 x 108

copies/μL), in which CT is the cycle where statistic was significantly

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graph will show the equation which is used to calculate the number of cDNA molecules per microgram from oligo-dT premed of total cDNA. With the same reaction, it was tested of plat as a standard.

3 RESULT AND DISCUSSION

Temperature and Humidity

The average of temperature and relative humidity during the 35 experimental days at Dramaga, Bogor is presented in Table 3. Any variation in temperature and relative humidity which was occur during the day of experimental maintenance, it caused by the cloudy and rainy weather at the time of rearing. So, it tends to lower the temperature and increase the humidity.

Low environmental temperatures was required by broilers in the process of releasing theirs body heat into environment which was generated during the process of metabolism. The rate of metabolic processes of nutrients inside broilers body is directly proportional to the increasing age of the broilers. Thus, the heat increment is also likely to increase. Heat release process of broilers is an attempt to maintain balance between the temperature of the body and the temperature of the environment through evaporation mechanism which is characterized by the upsurge of panting activity (breathing motion), so that the chicken could avoid the stress which interfere the productivity and even cause death. Meanwhile, The

average of temperature and relative humidity inside AC’s animal house, and PE’s

animal, and NE showed in Table 3.

Table 3 The average of daily ambient temperature and daily relative humidity during 35 of experimental days.

Weeks

Control Pens (AC) Experimental Pens (NE & PE)

Temperature (oC) Humidity (%) Temperature (oC) Humidity (%) AC = broiler reared in comfortable environment pens without supplementation of vitamin E, NE = broiler reared in tropical environment pens without supplementation of vitamin E, PE = broiler reared in tropical environment pens with supplementation of 225 ppm vitamin E.

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averaged at 26.4 ˚C, meanwhile the relative humidity is averaged at 78.4 %. The several rainfall was take place not only in the morning but also in the evening or night, and cloudy weather at day time which occurs during 35 of experimental day was a main assumption as an affector to the high value of the relative humidity in PE and NE animal house. Meanwhile, the high values of relative humidity which has recorded happened in the AC animal house is due to the availability of exhaust fan which is only one that is used actively throughout the study. According to Ross (2009), a temperature which is comfortly suitable for the preservation of broiler (three weeks of age and above) for the continuance of its optimal life are in the range of 26-30 ˚C. Meanwhile, best moisture levels of relative humidity should be in 60 % to optimalize its growth and production. Thus, when high moisture levels reached, it will affect all aspects of life, growth, production, and reproduction of the broilers. Table 3 represents the temperature and humidity which is assessed can be felt by broiler during the daytime of 35 experimental days. It seems that the temperature during the daytime, which is averaged recorded at 32.9 ˚C has been exceeded the Charoen Pokphand (2005) recommendations, its suspected that the broilers in PE and NE animal house have been experiencing heat exposure. Similarly, the humidity which is averaged at 62.5 % has been exceeded as well. This situation will desolates broilers body heat to the environment, then will make it uncirculate well.

Kuczynki (2002) states that a comfortable temperature for optimal production in broiler chickens is in the range of 19 - 27˚C, while the humidity is at 60-70 % (Ross 2009). Whereas, during daytime, in the AC animal house, the temperature is averaged at 27.6 ˚C, meanwhile its humid is averaged at 75.5 %. This temperature still belong to the ideal range of comfort conditions for the broilers. However, the high relative humidity value will give negative impact on Broilers. Three weeks aged broilers have high sensitiveness on high temperature. The situation becomes very dangerous when the temperature of the broilers body was measured at 47 ˚C broiler chickens (117 ˚F) or equivalent with ambient temperature above 30 ˚C (86 ˚F). In this situation, when thermometer left unchecked, the tendency of broilers to die will be high in numbers due to heart failure. During the heat exposure by humid and temperatures of wet tropical climate and weather which is experienced by broilers, the broilers body temperature uninversely proportional, and as a cooling mechanism the broilers will drink more to replace water losses which was evaporates significantly (breathing rate increased from 20 times in comfortable temperature to 240 times per minute under heat stress exposure, called hyperventilation) exhaled from lung or commonly referred to insensibility of heat loss.

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that water vapor from the lungs could be transferred out to be absorbed by the surrounding environment outside the body. When broiler loose quickly, death can not be avoided (Suprijatna et al. 2005). The frequency of breath start to increase

when the temperature reach 29˚C and each 1˚C up in temperature will impact synergetically to the body temperature arround 1 ˚C until 2˚C (Ain Baziz et al. 1996). The stress which was resulted due to long period of heat exposure at day time can increase mortality (Xin et al. 1994). The percentage figure which is considered as normal mortality in broiler chicken is 4 % (Lacy and Vest 2000).

Broiler’s behaviour which was experiencing heat stress usually characterized by less in activity, more in rest, and tend to be solitary (split off from its group) in purpose to widen its wings in an endeavor to reduce heat insulation, lie down its body through the floor, approach the wall, stood still or close to drinking water and increase the frequency of respiration which is relatively rapid and relatively shallow (Defra 2005). Broiler is homeotherm bird, which has the ability to maintain its body temperature in a relatively stable state in a narrow range despite significant fluctuations of ambient temperature. Endogenous heat and exogenous heat is a form of heat which is felt by broilers chicken. The heat which is resulted along the body's metabolism called endogenous heat, while the heat which is coming from macro climatic environment, in the form of high temperature and high humidity, is called exogenous heat. Exogenous heat can’t be controlled by the broiler. Thus, it is a necessity that the broiler should control the endogenous heat insulation in order to restrain heat accumulation in the body by increasing the rate of respiration, perform dilatation (expanding the surface area of the body by stretching the wings way up from the body). If heat stress is still unwell coped, the reduction of feed consumption will be occured as a form of adaptation in maintaining body temperature to attain the equilibrium with the outside temperature of the body.

The reduction of feed intake intended to minimize the metabolism process, so the body heat increment is also reduced. In addition, drinking water consumption is likely to cool down the body which is experiencing heat exposure (Butcher and Miles 2003). Broiler do not have sweat glands, body’s heat release is generally done in several ways, including radiation, conduction, convection, and evaporation. If stretches of temperature is in the range between 28-35˚C, the release of heat by radiation, convection, and conduction are generally sufficient to maintain and support homeostasis scheme of broilers (Leeson and Summers 2004) while the release of heat through evaporation process is the most obvious clinical symptoms of heat stress. Fast respiration in order to transfer inside heat to the environment will lead the air flowed from the environment by replacing the air inside the body to cool down the blood in the mucosa. This activity requires energy for muscle motion in the respiration system was increased. Thus, efficient usage of energy becomes low. If prolonged, respiratory alkalosis will occurs, a form of compensation of kidneys which secrete bicarbonate because CO2 is

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(2004) stated that the thermoneutral zone for broiler is ranging from 20- 25˚C and humidity in between 50-70%.

The Quantification of Heat Shock Protein 70 Gene (HSP70 Gene) Expression

Ambient temperature, relative humidity, hyperoxia, radiation, UV, and microwave radiation are several causal factors of stress which are included in the form of stress caused by environmental effect. Stress which was resulted by environmental influences will form free radicals. It would triggers lipid peroxidation, protein, and DNA. The body will try not only to revert but also to restore back to a prior state where body still in normal condition, namely homeostasis, when broilers were disturbed its comfort rearing zone. However, if prolonged of heat stress is standing idly, then the stress condition will raising in line, and the body could not handle it, so that the body will use genetic pathways through the expression of HSP70 gene, this gene be liable for the threatening of heat and only active in conditions of stress. Supplementation of vitamin E in the broilers diet which was reared in the tropical climates can reduce gene expression of HSP70. On the contrary, HSP70 gene expression can be high if reared under tropical climates without the addition of vitamin E in its diet. The broilers reared in tropical climates animal house were exposed by heat stress then wheedle HSP70 gene to be expressed.

Supplementation through feed optimization by adding antioxidant agents (synthetic or natural) can be done to hamper the expression of HSP70 gene which occurs when the internal antioxidant inside the body was run out. Besides, it also can be done through environmental conditioning which is adjusted in such a manner that broilers could be at comfortable temperature for basic living, growth - development, production, and reproduction of broilers (Surai 2003), for example by using air conditioners and exhaust fans or a combination of both. Broiler that is under stress will experience elevated levels of HSP70 gene expression. The production of HSP70 gene in the body will use all nutrients which remain in the body. HSP70 gene acts as the last defender in protecting proteins which are sensitive to high temperature. HSP70 gene also covering proteins from post effects of heat exposure such as degradation, and or denaturation process, thus preventing either temporarily, and or permanently damaged which hereinafter affect the survival of these broilers. When heat stress appears, the mRNA will send a message to produce genes that play a role called HSP70 gene, whereas the tRNA will carry the message to the figuration of proteins. By the time the broilers are stressed, these genes will be produced on a large scale of protein production, while others will be relatively in dormant stage, but if the situation is in normal circumstances, it will be back as usual. Vitamin E affects the expression of HSP70 gene, that could evidently be seen from its antioxidant activity which approach directly active in reducing free radicals so that the HSP70 gene unnecessary to be excessively produced and expressed in protects cells from free radical damage in the body (Gabriel et al. 1996; Mahmoud et al. 2004; Zhen et al. 2006; Yu and Endong 2008; Tamzil et al. 2013; Surai 2003; Etches et al. 2008; Noor and Seminar 2009).

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proved that tropical environment at day time had already made broiler under stress which is caused by tropical heat exposure. As stated in Love and Jenner (1999), oxidative stress is the primary cause of cranial nerve disease. According to Youdim (1988), brain tissues are responsive to oxidative damage, perhaps because of high rate of oxygen consumption, which is finite to 20%, the presence of affluent amount of polyunsaturated fatty acids in membranes, high in iron (Fe) content, and low activities of anti-oxidative enzyme. Based on Yuan et al. (2012), hippocampal area in brain tissue are highly susceptible to oxidative stress. There is also inscripted that in breast muscle sample has high in expression of HSP70 gene too (notiwithstanding statistically lower than hippocampus), as a usual consequence body’s reaction towards superoxide radical production (which is charged as normal causal of metabolism process) within mitochondria.

Table 4 The quantity of HSP70 gene expression of hipocampus sample and breast sample of broiler in three kind different treatments.

Organ Treatment (10

Means within the same row with different alphabet are significantly different (P<0.05)

AC = broiler reared in comfortable environment pens without supplementation of vitamin E, NE = broiler reared in tropical environment pens without supplementation of vitamin E, PE = broiler reared in tropical environment pens with supplementation of 225 ppm vitamin E.

The inclination of HSP70 genes to be expressed in broilers inside NE pens tends to be high (Table 5) compared with PE pens, and AC pens. And expression was lower in males than females, and appears to be more expressive in the brain sample than in the breast meat sample. This situation shows that the expressivity of HSP70 genes is dominantly determined by the environment influence, as showed in AC pens and in NE pens. The interesting part is condition of AC pens and PE pens was undifferently significant (P<0.05). Through this method, it is clearly seen that HSP70 gene is actively expressed when broilers experience heat stress, but less active while at thermoneutral zone and or reared on the animal house with supplementation of vitamin E in its diet. Free radicals are molecules that are relatively unstable, having one or more unpaired molecules in outside of its orbit. The molecules are reactive in finding the pairs of its electron. If the activity of free radicals aforementioned before was happened, it will generate chain reaction which popped out the new free radicals that continually to grow in numbers (Valdez et al. 2000).

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membrane lipids, cellular proteins, nucleic acids, and gene expression ordination (Wu and Meydani 1999). Free radical inhibitors mechanism is consist of endogenous and exogenous antioxidants. Endogenous antioxidants could be derived from the deposition of vitamin E in the fat tissues of the body. Exogenous antioxidants are vitamin E which is supplemented to the feed (Surai 2003).

Blood Profile of Broiler

According to Table 4, there were (relative high in) difference (P<0.05) among AC pens and PE pens compared to NE pens for Hemoglobin, Hematocrit, Leukocyte, Erythrocyte, and Lymphocyte as parameters which was observed and studied along this experimental research. Also, there were (relative low in) difference (P<0.05) in either Heterophile, H / L ratio, Monocyte, Eosinophils, or Basophils (as variables studied) among AC pens and PE pens compared to NE pens. Examination towards the broiler’s blood status can be used as a way to detect stress and presume disease, know the delineation of physiological condition, get to see the abnormalities inside, and look for (detect) whether the nutrients metabolic processes occurred or not inside broiler’s body. Blood consists of liquid and blood cells. The composition and its volume depend on nutritional intake, stress levels, activity, strain, sex, and its interaction with its environment. In most of animals, the increase in corticosteroids plasma level is a marker that the animal is experiencing stress.

Hemoglobin (Hb) is a pigment of red blood cells which is derived from complex bond of conjugated protein containing iron (Fe) as its components, so it will emerge the red color of red blood. Hb has a role in transporting oxygen which is derived from the lungs then transported to all tissues of body and thus released and given to the cell. Forthwith, receive carbon dioxide through the process of diffusion into the blood which has been produced by all tissues of body and so brought back to the lungs for disposal process which is occurs via the respiratory scheme of exhale (Guyton and Hall, 2010; Frandson 1992). In blood, hemoglobin is in the form of transferrin and ferritin. Quantity of hemoglobin within the body is related to and depends on the availability of Fe in the form of ferritin and hemosiderin, which are predominantly bound in the liver, spleen, and bone marrow (Piliang and Djojosoebagio 2006b). Hemoglobinity are concerned with the availability of oxygen and total red blood cell. Thus, if the level of erythrocyte were low, so did Hb will directly low in line too. The low levels of Hb trigger

anemia. Anemia will disrupt the supply of oxygen which is needed by body’s

tissues, the viscosity directly drops and the flow rate of blood circulation which then interfere the metabolism of bodies (Frandson 1992; Schalm 2010). Normal average value of hemoglobin according to Mangkoewidjojo and Smith (1998), are in the range of 7.3 g 100 ml-1 and 10.9 g 100 ml-1.

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hemoglobin are low in numbers (Jain 1993). Increased value of hematocrit can affect blood viscosity, the greater the hematocrit in value, the greater the percentage of cells in the blood will be, so the more often friction between different layers of the blood appear, and this friction which shows the viscosity in blood (Guyton and Hall 1997). Environmental temperature not only affects but also altered the value of feedintake (Austic 2000) which then lowers the availability of nutrients, such as iron, vitamin B12, folic acid, vitamin B6 (pyridoxine), and proteins. The deficiency in all of nutrients which is aforementioned will result in the decrease of erythrocytes in value, and will affect Hb and HC (Fauci et al. 2008). Normal average value of hematocrit according to Mangkoewidjojo and Smith (1988), are in the range of 24.0 % and 43 %.

Leukocytes or white blood cells are divided into two major groups, namely granulocytes and agranulocytes. It’s named granulocytes because they contain granules and have an irregular nuclei in their cytoplasm. Included in this group are heterophile (it has specific granules), eosinophils, and basophils. Whereas agranulocytes do not have granules in their cytoplasm but its nuclei has a regular shape. Further, agranulocytes consist of monocytes and lymphocytes. Leukocytes have a nucleus. Leukocytes have the capability of independent motion. Generally, leukocytes in the blood stream are nonfunctional, just transported to tissues when needed, for example in the condition of serious infection, and inflammation inside body (Frandson 1992; Sturkie 1976). Leukocytes also play a role as destroyer foreign cells through phagocytosis scheme and form antibodies. Thus, the white blood cells are the unit of body's defense system. Its quantities will follow in line to the increase of infectious diseases due to pathogenic bacteria invasion inside the body. Therefore, observations through leukocyte will be useful in diagnosing the immune status of broilers in pertinent (Guyton and Hall 2010). Sometimes an increase in white blood cells, exceed the limit, this condition called leukocytosis. In peripheral blood, its amount in circulation system was set within certain limits and strict to regular or normal condition of bodies, and only modified if there were pathogenesis activity detected inside (Price and Wilson 1985). Normal average value of leukocytes to Telabi et al. (2005), are in the range of 20.7 x 103 ml-1 and 24.1 x 103 ml-1.

Red blood cells or erythrocytes have an important role in serves as the carrier of Hb (O2 distributor of the lungs) and nutrients from the digestive tract to

all parts of body tissues. Then vice versa, from all parts of body tissues, carrying the remains of the metabolism process to be secreted by kidneys, and carrying CO2 to be secreted through the lungs. The quantity of erythrocytes is influenced

by individual activity, nutrient intake, altitude of rearing place, and ambient temperature. Erythrocyte contain carbonic anhydrase enzyme which has functions in the regulation of bicarbonate which is catalyze the reaction of carbon dioxide with water (Guyton and Hall 1997) and also plays a role in maintaining the integrity of cells and its permeability. Normal average value of erythrocytes according to Sugito (2007), are in the range of 2.3 x 106 ml-1 and 2.7 x 106 ml-1.

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through inflamation scheme by migrating to areas which is under attack by bacteria , penetrate the blood vessel walls, and destroy it (Frandson 1992). The high quantity of heterophile, indicate stress in chickens (Swenson 1984) and at the time of such stress, heterophile are actively looking for, glut, kill, and prevent bacterial infections. Thus, heterophile was known as the body's first line of defense against the invasion of odd matter. Heterophile become intensely active when the quality of immune decreased (Day and Schultz 2010). Tizard (1988) reported that heterophile have limited energy and can’t be replenished, so after being released from the spinal cord and perform phagocytosis, the cell will then become drained and not being able to outwore. Normal average value of heterophile according to Khan et al. (2007), is equal and or less than 31.95 %.

Lymphocytes are part of white blood cells which are the most significant in numbers of broilers, have varying in sizes, ranging from small to large, similar to mammals (Bacha and Linda 2000). Grouped into three types based on its size, ie, small, medium, and large. However, the small and medium sized of its which is detected excessively relative in the circulatory system (Champbell 1995). Lymphocytes which is rounded in its nuclei with scalloped edges (Samuelson 2007), have a fairly coarse in its chromatin patterns, but fused as well (Bacha and Linda 2000), majoring role to produce antibodies (B lymphocytes) and act as effector of cells specifically with antigen attached to macrophages (T lymphocytes) (Tizard 1982). High temperatures makes the broilers vulnerably susceptible to pathogenesis, then will activated the cell-mediated immunity, a special lymphocytes which are specifically made to destroy foreign substances or odd matters that enter or invade the body (Dellman and Brown 1989). Lymphocytes in the active state, recognized in two forms, that is to say: T lymphocytes as a producer of T cells which was derived from the thymus and B lymphocytes as a producer of B cells which was derived from the Bursa. T cells play a role as detect of proteins or odd objects (substance or matter) which freighted in to host cells (non-specific), adjust to active within the cell which is infected by antigens (cellular immunity) and undergo the differentiation in diverge function as subpopulations at all times to enhance the current body's immune which is experiencing pathogenesis. B cells are responsible for the formation of immunoglobulin, a protein which has specific response towards microbial antigens invasion, act in the surface of cell and blood circulation (humoral immunity) (Tizard 1982; Dellman and Brown 1989). Normal average value of lymphocytes acording to Khan et al. (2007), is equal and or more than 51.20%.

However, the ratio of heterophile to lymphocyte is also valid to use in the measurement of stress. It was reported that when corticosteron added to animal

diets, the content of lymphocytes in animal’s blood will increase, while the

content of heterophile decreased. Thus, at this time, this method is commonly used to measure the level of stress in animals (Akers et al. 2008). Normal range of H / L ratio is from 0.19 to 0.40 (Rahayu 2000).

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When monocytes are produced relatively large in value within the body, indicating the presence of a pathogen infection (Guyton and Hall 1997).

Eosinophils take an active part in adjusting the intensity of the severity of allergy or killed a number of parasites that infect the body. The amount of eosinophils can be bullied or affected by worm infections (Eurell and Frappier 2006). When eosinophils are produced relatively large in valuee within the body, it is indicating the presence of a parasitic infection (Eurell and Frapier 2006).

Basophils are myeloid cells, has the lowest in its quantity of broilers blood. It plays a role in the release of heparin and histamin to blood in reaction to overcome early inflammatory (Sturkie 1976) and also as an immediate act in hypersensitivity reactions (Maxwell and Robertson 1998). If basophils undetecable in the blood, then its condition aforementioned before could be not only a marker of inflammation but also the absence of hypersensitivity reactions inside the body. If there is no basophils inside blood analysis, indicate the absence of inflammation and hypersensitivity reactions inside the body (Sturkie 1976).

Deficiency of vitamin E will bring disease which is called Zanker degeneration by perivascular infiltration and characterized as accumulation of eosinophils, lymphocytes, and histiocyte which had been infiltrated (Leeson and Summers 2001). The addition of its as many as 25000 IU kg-1 to the diet will increase the hematocrit (Yang and Desai 1977).

Table 5 Blood profile examination result of broiler in three kind of different

a,b_{Means within the same row with different alphabet are significantly different (P<0.05)}

Broiler Performance

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and AC pens. This indicates that the presence of 225 ppm of vitamin E as supplement on PE diet, through its role as an antioxidant, can overcome the tropical heat stress during the experimental rearing process. CPE value between AC compared to NE and PE compared to NE which showed highly significant different (P<0.05) in Table 5 was displayed an agio around 3.6% and 3.1%, respectively. This value was highly significant in terms of livestock economic calculation losses of CPCM contribution nationwide. If this value is multiplied by body weight gain value of broilers in AC and in PE animal house, resulted in reduction of crude protein around 47.16 g e-1 and 39.12 g e-1. If there is a broiler assumed around a million in number, then the amount of crude protein meat losses (originated from broiler) level of national reaches 47 tons and 39 tons. The loss values has been proved could be pressed not only by rearing in animal house which its temperature was adjusted at a comfortable zone using Air Conditioner but also by rearing in the tropics animal house with the addition of 225 mg kg-1 of vitamin E in its diet.

Increasing in age and in body weight during the growing up process is a result of the rising need in nutrients. The nutrients aforementioned before, will allocated for basic living, growth, production and reproduction. Hence, this energy allocation requirement are synergetically in line to the level intake of feed. However, at the age of 7 weeks, this increase trend which was mentioned before, reduced (Bell and Weaver 2002). In addition to: the body capacity of broiler, the age of broilers, activity level, quantity of diet, health status, form of diets, nutrient balance, speed of growth and genetic; there is also: environmental temperature and environmental relative humidity which could affect the intake level of feed (NRC 1994; Korgh 2000). High ambient of temperatures decreased feed intake. Similarly, the opposite applies, the conditions under thermoneutral zone, will lead to increase of feed consumption. For each constant stepped-up of 1˚C ambient temperature above 24.5˚C, the rate of feed consumption will decrease by 1.58% (NRC 1994).

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nervous system became aroused. On the other hand, the central controlling of hunger and the secretion of Thyroid Stimulating Hormone (TSH) which have a role in thyroid hormone secretion is being inhibited. Thus, broiler growth becomes stunted. The total consumption of feed is closely related to weight gain, feed conversion, and water consumption. All four were mentioned before is under the same coordination of hypothalamus which is also controls hormone system, thermoregulation, and water consumption (Hafez 1968). Weight gain is one of the criteria for measuring the growth in broiler chickens, weight gain is influenced by several factors, inter-alia: age, breed, gender, growth velocity performance, animal health and quantity of rations, as well as environmental factors which also support (Rasyaf 1999) weight gain which is stated before, including the gaining of meat, bones, and feathers inside the body. Increase which is occurred in the broiler's body is also will bring growth to the structural tissues, such as bone and muscle (Jull 1979).

Uncomfortable temperatures for broilers can divert the use of nutrients, which shall primarily for the production, but then used in homeostasis (body balance) metabolic scheme, this situation and condition is characterized by a decrease in feed consumption, blood flow rate, and energy metabolism which is measured as low in level (Mc Kee et al. 1997).

Table 6 The performance of broiler during 35 experimental days in three kind of different treatments.

Variable1 Experiment

AC NE PE

Feed Intake (g bird-1) 2215±34a 1862±122b 2159±113a Crude Protein

Consumption (g bird-1) 504.51±7.79a 424.15±27.9b 491.82±25.7a Body Weight (g bird-1) 1351±51a 1056±41b 1302±49a Weight Gain (g bird-1) 1310±51a 1014±42b 1262±48a Crude Protein Content

of Meat (g bird-1) 235.83±9.24a 182.52±7.53b 227.17±8.76a Crude Protein

Efficiency (%) 46.73±1.18a 43.13±2.55b 46.23±1.38a Feed Conversion Ratio 1.69±0.04b 1.84±0.11a 1.71±0.05b

Mortality (bird) 2 6 3

a,b

Means within the same row with different alphabet are significantly different (P<0.05)

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diet quality, disease, temperature, sanitation, ventilation, treatment, and animal house management.

4 CONCLUSSION

Broilers supplemented with 225 ppm of vitamin E in tropical climate was not significantly different with broilers in thermoneutral environment in perfomance (feed intake, body weight, weight gain, feed conversion, and mortality), blood physiology response (hemoglobin, hematocrit, heterophile, lymphocyte, and H/L ratio), and expression of HSP70 gene (not only in hippocampal brain area but also in breast muscle of broilers). An 225 ppm of vitamin E is needed to overcome heat stress in tropical environment without any relative costly adjusment using air conditioner and exhaust fan in maintenance animal house of broilers.

5 REFERENCES

Ain Baziz H, Geraert PA, Padilha JCF, Gullaumin S. 1996. Cronic heat exposure enhances fat deposition and modifies muscle and fat partition in broiler carcass. Poult Sci 75 : 505 -513.

Akers, Michael R, Michael Denbow D. 2008. Anatomy and Physiology of Domestic Animals. Blackwell Publishing Professional. 1st Ed. USA. Amrullah IK. 2004. Broiler Nutrition. Bogor: Lembaga Satu Gunungbudi.

Austic RE. 2000. Feeding in Hot and Cold Climates. In: Yousef MK editor. Stress Physiology in Livestock Vol III, Poultry. Florida: CRC Pr. Hlm. 123 – 136.

Bacha WJ, Linda MB. 2000. Color Atlas of Veterinary Histology. 2nd Ed. Philadelphia. Baltimore. New York. London. Buenos Aires. Hongkong. Sydney. Tokyo : Lippincott William & Walkins Co. Pp 27-36.

Bell DD, Weaver WD. 2002. Commercial Chicken Meat and Egg Production. 5th Ed. New York: Springer Science Business, Inc.

Bollengier-Lee S, Williams PEV, Whitehead CC. 1990. Optimal dietary concentration of vitamin E for alleviating the effect of heat stress on egg production in laying hens. J Poult Sci Brit. 40: 102-107.

Borges SA, Da Silva F, Maiorka A, Hooge DM, Cummings KR. 2004. Effects of diet and cyclic daily heat stress on electrolyte, nitrogen and water intake, excretion and retention by colostomized male broiler chickens. Int J Poult Sci. 3:3134321.

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Bustin SA. 2005. Real-time PCR. In: Fuchs J, Podda M, editor. Encyclopedia of Diagnostic Genomics and Proteomics. New York (US): Marcel Dekker. hlm 1117-1125.

Butcher GD, Miles R. 2003. Heat Stress Management in Broilers 1.

http://edis.ifas.ufl.edu. [15 November 2013]

Champbell TW. 1995. Avian Hematology and Cytology. Iowa State University Press.

Charoen Pokphand Indonesia. 2005. Manual manajemen broiler CP 707. http://www.charoenpokphand/org.com/. [15 November 2013].

Daghir NJ. 2007. Present Status and Future of the Poultry Industry in Hot Regions. In: Daghir NJ, editor. Poultry Production in Hot Climates. CAB International, New York: hlm 1-10.

Day MJ, Schultz RD. 2010. Veterinary Immunology: Principles and Practice. Manson Publishing, London.

De Busk RM. 2009. Nutritional Genomics: The Foundation for Personalized Nutrition, What Is Nutritional Genomics. In: Advanced nutrition and human metabolism.

Defra. 2005. Heat Stress.

http://www.thepoultrysite.com/featuredArticle/FATopic. [15 November

2013]

Dellman HD, Brown EM. 1989. Textbook of Veterinary Histology. Ed ke-3 Jakarta. Iowa State University. Ames Iowa. Terjemahan. UI Press.

Duncan DB. 1995. Multiple Range and Multiple F Tests. Biometrics 11:1-42, 1955

Effendi D. 2010. Performance and physiologycal respons of broiler fed with diet containing 1,5% of red fruit dregs in different period of feeding and different temperature of animal house. [Thesis]. Faculty of Animal Science, IPB, Bogor.

Etches RJ John TM, Verrinder Gibbins AM. 2008. Behavioural, Physiological, Neuroendocrine and Molecular Response to Heat Stress. In: Nuhad J. Daghir (ed.). Poultry production in Hot Climates. Pp: 49-69.

Eurell JA, Frappier BL. 2006. Dellmann’s Textbook of Veterinary Histology. Ed ke-6. USA: Blackwell.

Fauci, Hauser BK, Longo, Jameson. 2008. Principles of Internal Medicine. 17th Ed. McGraw Hill Companies, New York.

Frandson RD 1992. Anatomy and Physiology of Livestock. IV Edition. Translator: B. Srigandono dan K. Praseno. Yogyakarta (ID): Gadjah Mada University Press.

Gabriel JE, Ferro JA, Stefani RMP, Ferro MIT, Gomes SL, Macari M. 1996. Effect of acute heat stressor the heat shock protein 70 messenger RNA on the heat shock protein expression in the liver of broilers. Br Poult Sci. 37:443-9.

Gowe RS, Fairfull RW. 1995. Breeding for Resistance to Heat Stress. In: N.J. Daghir, editor. Poultry Production in Hot Climates. CAB International, New York: hlm 11-29.

The Role of Vitamin E to Overcome Tropical Heat Stress Related with Expression of HSP70 Gene in Broiler

THE ROLE OF VITAMIN E TO OVERCOME TROPICAL

HEAT STRESS RELATED WITH EXPRESSION OF

HSP70 GENE IN BROILER

RAYMUNDUS GENTY LARAS

STATEMENT OF RESEARCH ORIGINALITY

RINGKASAN

SUMMARY

© Hak Cipta Milik IPB, Tahun 2014

Hak Cipta Dilindungi Undang-Undang

THE ROLE OF VITAMIN E TO OVERCOME TROPICAL

HEAT STRESS RELATED WITH EXPRESSION OF

HSP70 GENE IN BROILER

AUTHENTICATION

ACKNOWLEDGEMENT

TABLE OF CONTENTS

LIST OF TABLE

APPENDIX

1

INTRODUCTION

2

RESEARCH METHODOLOGY

3

RESULT AND DISCUSSION

4

CONCLUSSION

5

REFERENCES