other hand, freshwater teleost fish, such as common carp, rainbow trout and Atlantic ..... Skeletal muscle is made up of muscle fibers and connective tissues. (tendon ..... present in teleost fish, except the Dipnoi, although it is universal in sharks.
Kurdistan Regional Government-Iraq Ministry of Higher Education and Scientific Research University of Zakho Faculty of Science
Fish Swimming Performances and Metabolism Under Different Nutritional Regimes A Thesis Submitted to the Council of the Faculty of Science/University of Zakho, In Partial Fulfillment of the Requirements for the Degree of Doctor of Philosophy In AQUATIC ANIMAL PHYSIOLOGY By BASIM SALEEM AHMED SLIVANEY B.Sc. Veterinary Medicine / Duhok University-2005 M.Sc. (Animal Physiology)/ Zakho University-2010 Supervised by Prof. Dr. Omar A. M. Al-Habib Prof. Dr. Marco Saroglia
K. 2717
A.H. 1439
45
A.D 2018
الر ِح ِيم من َّ هللا َّ ِبس ِْم ِ الر ْح ِ س َّخ َر ْال َب ْح َر ِلتَأ ْ ُكلُوا ِم ْنهُ لَ ْح ًما َ ط ِريًّا َو ُه َو الَّ ِذي َ سونَ َها َوت َ َرى ْالفُ ْل َك َوت َ ْست َ ْخ ِر ُجوا ِم ْنهُ ِح ْليَةً ت َ ْلبَ ُ ون اخ َر فِي ِه َو ِلت َ ْبتَغُوا ِم ْن فَ ْ ض ِل ِه َولَعَلَّ ُك ْم ت َ ْش ُك ُر َ َم َو ِ صدق هللا العظيم ]سورة النحل[14:
46
Supervisors Certificate We certify that the thesis entitled “Fish Swimming Performances and Metabolism under Different Nutritional Regimes” was prepared under our supervision in the Department of Biology, Faculty of Science, University of Zakho and Department of Biotechnology and Molecular Sciences, University of Insubria, Italy as a partial requirement for the degree of Doctor of Philosophy in Aquatic Animal Physiology.
Signature: Name: Prof. Dr. Omar A. M. Al-Habib Academic rank: Professor Date:
/
/2018
Signature:
Name: Prof. Dr. Marco Saroglia Academic rank: Professor Date:
/ /2018
-----------------------------------------------------------------------------------------
Chairman's Certification In view of recommendations, I forward this thesis for debate by the Examining Committee
Signature: Name: Prof. Dr. Wijdan M. S. Mero Academic rank: Professor Date: / /2018
47
Examination Committee Certification We are “the examination committee” certify that we have read the thesis entitled Fish Swimming Performances and Metabolism under
Different Nutritional Regimes and we have examined the student “Basim Saleem Ahmed Slivaney” in its content and in what is related to it, and in our opinion, it meets the standards of a thesis for the degree of Doctor of Philosophy in Aquatic Animal Physiology. Prof. Dr.
Prof. Dr.
University of
University of Duhok
(Chairman)
(Member)
Date: 00/ 00 /2018
Date: 00/ 00/2018
Prof. Dr.
Dr.
University of
University
(Member)
(Member)
Date: 00/ 00 /2018
Date: 00/00 /2018
Dr. University of (Member) Date:00 / 00 /2018 Prof. Dr. Omar A.M. Al-Habib
Prof. Dr. Marco Saroglia
University of Zakho
University of Insubria/Italy
(Member and Supervisor)
(Member and Supervisor)
Date: 00/ 00 /2018
Date: 00/ 00 /2018
----------------------------------------------------------------------------------------Approved by the College Committee of Graduate Studies Prof. Dr. Maher Kh. Ali Date: 00/00 /2018
48
Dedication I dedicate my research to unending love of family, especially my mother and father who have supported me throughout the process. In addition, I dedicate this work with special thanks to my wife Gulistan, who supported me throughout the years of my study. I also dedicate this research to my brothers Salim, Salar, and sisters Sundus and Sana for their enthusiastic support and my sons Shayan and Keyhan, who made me to forget the tighness throughout the study period.
Basim
49
ACKNOWLEDGMENT Thanks God, the most merciful and compassionate, for giving me the strength and guidance I needed to face today and not worry about tomorrow. First and foremost, I would like to express the utmost gratitude to my supervisors Prof. Dr. Omar A. M. Al-Habib and Prof. Dr. Marco Saroglia (Insubria University, Varese, Italy) for their kind permission to work in their research laboratories and their constant guidance, support, motivation and untiring help during the course of the work. I would like to express my thanks to the Presidency of University of Zakho especially Assist. Prof. Dr. Lazgen A. Jamil (University President) and Prof. Dr. Omar A. M. Al-Habbib (former Vice President for Scientific Affairs) and the Assist. Prof. Dr. Yasin Taha (the Present vice President for Scientific Affairs) for giving me the opportunity to be enrolled in Ph. D program. Also, I would like to thank Prof. Dr. Fathil Esiff (the former Dean / Faculty of Science) and the Assist. Prof. Dr. Maher Kh. Ali (the present Dean / Faculty of Science) for their help and continuous support during the period of the study. It is my great pleasure to acknowledge the support and help of Prof. Dr. Giovanni Bernardini I would like to extend my thanks to Prof. Dr. Genciana Terova for her promote, and support care. My deep appreciation and sincere thanks to Dr. Micaela Antonini for her support and allowing me to use facilities for performing the fish experiments. Furthermore, I want to extend my thanks and appreciation to Dr. Simona Rimoldi, Mudhir S. Shekha for an excellent guidance alongside my research. I would like also to express my gratefulness to Dr. Chiara, Dr. Sarbast, Dr. Saniya, Mr. Kameran, Mr. Mino, Mr. Sardar, Mr.
50
Mohammed, Mr. Sha’aban, Mr. Lina, Mr. Husni, Mr Masuod and Mr. Mariana for their valuable supports, during all the phases of the work. Finally, I have to say that the sincerity, the patience and the encouragement from my spouse Gulistan and our two children, Shayan and Keyhan, have been my inspiration as I hurdled all the obstacles in the completion of all the requirements needed for my doctoral study.
Basim Saleem Ahmed
51
ABSTRACT The present study included two parts which focus on 1- The effect of taurine on antioxidant parameters and some respiratory parameters in Sea bass (Dicentrarchus labrax) under conditions of forced swimming uses. 2- The relaxant effect of taurine on intestinal segments and aortic rings in the common carp (Cyprinus carpio), with a spread emphasis on the rate of ion channels and endothelial derived hyperpolarizing factors.
1. Effect of Taurine on Biometric and Respiratory Parameters in Sea bass This part of the work aimed to study the effect of taurine on biometric parameters, some respiratory parameters, swimming performances, respiratory burst and molecular analysis (mRNA gene expression) in adult Sea bass, from both sexes, weighting (75-110g). The fishes were used in this part collected from Nouva Azzurro
commercial hatchery
(Civitavecchia, Roma, Italy), and transported to the animal house and stocked into indoor tanks 2500L capacity. The fishes were allowed to acclimate to the laboratories conditions for 90 days before starting the experiments. The tanks were connected to a sea water recirculation system. After acclimation of the fishes, they were subdivided into two subgroups and each of which was further subdivided into two time period for feeding and swimming performances (AutoTM Resp, Loligo® Systems, Tjele, Denmark). The fishes of the first group were fed on commercial pellets, whereas in second group, they were fed on commercial pellets plus 1.5% taurine. The time period of feeding and swimming performance in first set of this experiment was thirty four days whereas in the second set, it was sixty four days.
52
The respiratory burst (RB) was measured four times. The first measurement (T0) was done before separating the fishes. The second measurement (T1) was done after fifteen days of feeding in first set of experiment and thirty days in the second set of the experiments. The third measurement (T2) was done after four days of resting the fishes. The last measurement (T3) was done after 15 days of feeding in the 1st part of experiments and 30 days after the 2nd part of feeding. The biometric performances results revealed that fish's fed on commercial pellets plus 1.5% taurine for 15 days did not show any significant changes in body weight. Whereas after 34 days of feeding the fishes fed on commercial pellets plus 1.5% taurine, there was a significant elevation in the BW as compared with that of control groups before and after training the fish in swimming chamber respirometer. Furthermore, the effect of taurine on total Body length did not show any significant changes in all of feeding periods T0, T1, T2 and T3. The level of condition factor was significantly increased in fish's fed commercial pellets plus 1.5% taurine after exercising in swimming chamber T1 and T3 for 15 days and 34 days, respectively. Fish's fed on commercial pellets plus 1.5% taurine for 15 days did not show any changes in specific growth rate, whereas, there was a significant elevation in the growth rate in fishes fed on commercial pellets plus 1.5% taurine. The ration of feed conversion ratio did not changed significantly after feeding the fishes on commercial pellets plus 1.5% taurine for 15 days whereas, after 34 days of feeding there was a significant increase in FCR. After exercising the fishes in the respirometer swimming chamber, the critical swimming speeds (Ucrit-1) and (Ucrit-2) showed significant elevation in fish's fed on commercial pellets plus 1.5% taurine after exercising in swimming chamber for 15 days but after 30 days (Ucrit-1). Did not show any change whereas (Ucrit-2) showed significant elevation
53
in fish feds on commercial pellets plus 1.5% taurine. The rate of oxygen consumption (mg O₂kg-1 hr-1) showed no changes after feeding the fishes with pellets plus 1.5% taurine. The level of standard metabolic rate (SMR), routine metabolic rate (RMR) and active metabolic rate (AMR) were significantly decreased when fish feds commercial pellets plus 1.5% taurine. Antioxidant effect of taurine did not change the energy required for fish to move (Cost of transport) and aerobic scope (mg O₂ kg-1 hr-1) after exercising in respirometer swimming chamber. The results of respiratory burst revealed a significant inhibition in the level of integral relative light unit (IRLU) before and after exercising the fishes in swimming chamber respirometer during times T1, T2 and T3 in fish fed on commercial pellets plus 1.5% taurine as compared with the control group. After exercising, five fish's from each group were taken out from the water and sacrificed by immersion in ice. The tissue samples were taken at the time of death by taking out sections from red muscle and the liver tissues. The total RNA was extracted from the red muscle and liver tissues by using the Maxwell® 16 LEV simply RNA Tissue Kit (Promega, Italy). The absolute number of catalase (CAT), superoxide dismutase (SOD) and glutathione proxidase (GPX) gene transcript copies could be quantified by comparing them with a standard graph constructed using the known copy number of mRNA of this gene The levels of CAT mRNA gene expression in liver and red muscle did not show any significant changes in fish fed on commercial pellets plus 1.5% taurine in both feeding times of 34 days and 64 days. Similarly, fishes fed on diet plus 1.5% taurine did not show any significant change in the level of liver SOD and GPX mRNA gene expression when compared with fishes fed on commercial pellets during both time periods 34 days and 64 days. On the other hands, the levels of red muscle SOD and GPX in
54
fishes fed on control diet plus 1.5% taurine for 34 days showed a significant elevation in mRNA copy number/100ng RNA. However, the level of red muscle SOD and GPX showed no significant change in fishes fed on control diet plus 1.5% taurine.
2. The Relaxant Effect of Taurine The second part of the current work included the relaxant effects of taurine on isolated intestinal segments and aortic rings of common carp. Power Lab Data Acquisition Organ Bath System (ADI), was used to which measured the isometric tension resulted from smooth muscle contractility. Prior to the experiment, the organ bath was set at 20oC for at least one hour, followed by the addition 5ml Ringer’s solution to the glass tissue chamber containing intestinal segments and 10ml for aortic rings. The preparation was aerated continuously with 99.7% oxygen (O2) and 0.3 % carbon dioxide (CO2). The resting tensions for intestinal segments was calibrated at 0.5 gram, whereas for aortic rings was calibrated at 2 gram. This part of the work aimed to study the relaxant effect of taurine on intestinal and aortic smooth muscle and role of potassium (K+) and L- type Ca++ channels in taurine induced relaxation. In addition, it also included the role of endothelium derived hyperpolarizing factors such as L-nitro arginine methyl ester (L-NAME) (NO synthase inhibitor), Indomethacin, (cyclogenase inhibitor) and methylene Blue (cGMP inhibitor) on taurine induced relaxation in the studied tissues. Taurine produced enhanced relaxant responses in intestinal segments as compared to aortic rings. The results also indicated the presence on intra-specific variation in the role of various K+ channel subtypes in taurine induced relaxation. Thus, using different K channel subtype blockers revealed that in intestinal segments, both KATP and Kca channel subtypes played significant roles in taurine induced relaxation, while KV and KIR played no role the relaxation. On the other hands, in aortic rings, both KIR and Kca, but not KATP and KV, played 55
significant roles in induced vasorelaxation. The results also indicated that Nifedipine (L- type Ca++ channels blocker), significantly diminished the relaxation induced by taurine in aortic rings. This reflect the important role of in L-type Ca++ channel in taurine induced relaxation, while a similar effect was not exhibited by intestinal L-type Ca++ channels since Nifedipine enhanced the taurine induced relaxation instead of its inhibition. Furthermore, experimental results also indicated that endothelial derived hyperpolarizing factors such as NO, cyclogenase and cGMP have no effects on taurine induced relaxation in intestinal and aortic tissues; except at the highest taurine concentration used (2x10-2M) which significantly reduced the induced relaxation in aortic rings. From the results of the current study it can be concluded that taurine induced relaxation in intestinal smooth muscle involves the activation of KATP and Kca channel; whereas in aortic smooth muscle, involves the activation of KIR, Kca, and Ca++ Channels with a partial participation of NO.
56
TABLE OF CONTENT ACKNOWLEDGEMENT
I
ABSTRACT
III
TABLE OF CONTENTS
VIII
LIST OF FIGURES
XIV
LIST OF TABLES
XXI
LIST OF APPENDIX
XXIV
LIST OF ABBREVIATIONS
XXV
1. INTRODUCTION
1-5
2. LITERATURE REVIEW
6-44
2.1 Taurine (2-Aminoethanesulfonic Acid)
6
2.1.1 Chemical Properties of Taurine
6
2.1.2 Origin of Taurine
6
2.1.3 Distribution of Taurine
6
2.1.4 Taurine and Nutrition
7
2.1.5 Biosynthesis and Metabolisim of Taurine
7
2.1.6 Physiological Roles of Taurine
8
2.1.6.1Role of Taurine in Cardiovascular System
8
2.1.6.2 Role of Taurine in Intestinal Function
9
2.1.6.3 Role of Taurine in Membrane Stabilization
9
2.1.6.4 Role of Taurine in Immune Cells
10
2.1.6.5 Taurine and Oxidative stress
11
2.2 Aquaculture
12
2.3 European Sea bass (Dicentrarchus labrax,L.)
13
57
2.3.1 Nomenclatures of Sea bass
13
2.3.2 Family Description
14
2.3.2.1 Family Moronidae
14
2.3.3 Morphology of Sea bass
14
2.3.4 Geographical Distribution of Sea bass
15
2.4 Common carp (Cyprinus carpio)
16
2.4.1 Nomenclatures of Common carp
16
2.4.2 Family Description of Common carp
17
2.4.2.1 Family Cyprinidae
17
2.4.3 Morphology of Common carp 2.4.4 Geographical Distribution of Common carp
17 18
2.5 Muscle Tissue
18
2.5.1 Skeletal Muscle Tissue
18
2.5.2 Skeletal Muscle Fibers
19
2.5.3 Types of Skeletal Muscle Fibers in Fishes 2.5.3.1 Slow-Red Muscle Fibers 2.5.3.2 Fast- White Muscle Fibers 2.5.3.3 Intermediate-Pink Muscle Fibers
19 20 21 22
2.5.4 Contraction of the Skeletal Muscle 2.6 Fish Exercises 2.7 The Concept of Swimming Speeds
22 23 25
2.7.1 Sustained Swimming Speed
25
2.7.2 Prolonged Swimming Speed
26
2.7.3 Burst Swimming Speed
26
2.8 Recovery Time and Burst Swimming 2.9 Metabolic Rate (MR) 2.9.1 Standard (SMR) and Basal Metabolic Rate (BMR)
27 27 28
58
2.9.2 Active Metabolic Rate (AMR)
29
2.9.3 Maximum Metabolic Rate (MMR)
30
2.9.4 Routine Metabolic Rate (RMR)
30
2.9.5 Metabolic Scope (Aerobic scope)
30
2.10 Respiratory burst (RB)
31
2.11 Digestive System Organization 2.11.1 Topographical Regions of Digestive System 2.11.2 Functions of Digestive System
33 33 33
2.11.3 Histology of Intestine in Common carp
34
2.11.4 Histology of Ventral aorta in Common carp
35
2.12 Smooth Muscle Cells
36
2.12.1 Structure of Smooth Muscle
36
2.12.2 Types of Smooth Muscles
36
2.12.3 Smooth Muscle Receptors
38
2.12.3.1 Intestinal Cholinoreceptors
38
2.12.3.1.1 Nicotinic ACh Receptors
38
2.12.3.1.2 Muscarinic ACh Receptors
39
2.12.5 Physiology of the Smooth Muscle
40
2.12.5.1 Smooth Muscle Cell Contraction
40
2.12.5.2 Molecular Basis of Relaxation
41
2.13 Smooth Muscle Ion Channels
42
2.13.1 Potassium Channels
42
2.13.1.1 Voltage-dependent K+ Channels (KV channels)
43
2.13.1.2 Ca2+-activated K+ channels (KCa)
43
59
2.13.1.3 ATP-Sensitive K+ Channels (KATP) 44 2.13.1.4 Inward Rectifier K+ Channels (KIR) 3. MATERIALS AND METHODS
44 45-73
3.1 Materials
45
3.1.1 Experimental Animals
45
3.1.1.1 Sea bass (Dicentrarchus labrax, L)
45
3.1.1.2 Common Carp (Cyprinus carpio) 3.1.2 Fish Tagging
46 46
3.1.3 Diet Composition
46
3.1.4 Feeding Experiments
47
3.2 Experimental Designs
48
3.2.1 First Experiment
48
3.2.2 Second Experiment
48
3.3 Measurement of Biometric Parameters 3.3.1 Measurement of Body Length, Width and Height
48 48
3.3.2 Measurement of Body Weight
49
3.3.3 Measurement of Fulton's Condition Factor (K)
49
3.3.4 Measurement of Specific Growth Rate (SGR)
49
3.3.5 Measurement of Feed Conversion Ratio (FCR)
50
3.4 Measurement of IRLU of Respiratory Burst (RB)
50
3.4.1 Blood Sampling
50
3.4.2 Analysis of Reactive Oxygen Species (ROS)
50
3.4.3 Time of Measurement the Respiratory Burst
52
3.4.3.1 First Measurement, Zero time (T0)
52
60
3.4.3.2 Second Measurement (T1)
52
3.4.3.3 Third Measurement (T2)
52
3.4.3.4 Fourth Measurement (T3)
52
3.553 Swim Chamber and Swimming Performances Test
53
3.5.1 Swim Chamber
53
3.5.2 Swimming Performance Test
54
3.5.3 Measurement of Oxygen Consumption (MO2)
55
3.5.3.1 Standard Metabolic Rate (SMR)
55
3.5.3.2 Active Metabolic Rate (AMR)
56
3.5.3.3 Measurement of Aerobic scope (AS)
56
3.5.3.4 Measurement of the Cost of transport (COT)
56
3.5.3.5 Critical Swimming Speed (Ucrit)
56
3.6 Molecular Analysis
57
3.6.1 Fish Sacrification
57
3.6.2 Preparation of Total RNA
58
3.6.2.1 Total RNA Extraction
58
3.6.2.2 Quantitative One-Step Taqman® real-time PCR
58
3.6.2.2.1 Generation of in vitro-transcribed mRNAs for standard curves. 58 3.6.2.2.2 Quantitation of mRNA one-step TaqMan® real-time PCR
59
3.6.2.2.3 Sample Quantification
60
3.7 Physiological Studies
61
3.7.1 Isolation of Intestine
61
3.7.1.1 Measurement of Isometric Force
61
3.7.1.2 Protocol of Experiment
62
61
3.7.1.2.1 Group A
62
3.7.1.2.2 Group A, B, C and D
63
3.7.1.2.3 Group E
63
3.7.1.2.4 Group F
63
3.7.1.2.5 Group G and H
63
3.7.2 Isolated Aorta Preparation
64
3.7.2.1 Protocol of Experiment
64
3.7.2.1.1 Group A
64
3.7.2.1.2 Group A, B, C and D
64
3.7.2.1.3 Group E
65
3.7.2.1.4 Group F
65
3.7.2.1.5 Group G and H
65
3.8 Instruments, Chemicals and Solutions Used
65
3.8.1 Instruments
65
3.8.1.1 Respiratory Burst Experiments
65
3.8.1.2 Swimming Performance Experiments
66
3.8.1.3 Molecular Analysis Experiments
66
3.8.1.3.1 Primers and probes of quantitative real-time RT-PCR.
67
3.8.1.4 Organ Bath Experiments
67
3.8.2 Chemicals
68
3.8.3 Preparation and Buffer of Solutions
69
3.8.3.1 Buffer solutions for Respiratory burst
69
3.8.3.1.1 Hanks’ balanced salt solution (HBSS)
69
3.8.3.1.2 Borate Buffer 0.2M
69
62
3.8.3.1.3 Phorbol Myristate Acetate (PMA) Stock Solution
69
3.8.3.1.4 Preparation of Luminal
69
3.8.3.2 Organ Bath Ringer’s Solution, pH=7.8
70
3.9 Statistical Analysis
70
3.9.1 Statistical Analysis of Biometric Performances, Metabolic Rates and Respiratory Burst. 70 3.9.2 Statistical Analysis of Oxygen Consumption Rates
70
3.9.3 Statistical analysis of Molecular Biology.
71
3.9.4 Statistical analysis of Organ Bath Results.
71
4. RESULTS
72-128
4.1 Effect of Taurine on Some Biometric Parameters
72
4.1.1 Effect of Taurine on Body weight
72
4.1.2 Effect of Taurine on Total Body Length.
73
4.1.3 Effect of Taurine on Fulton's Condition factor.
74
4.1.4 Effect of Taurine on Specific Growth Ratio.
75
4.1.5 Effect of Taurine on Feed Conversion Ratio.
76
4.2 Effect of Taurine on Some Metabolic Rates
80
4.2.1 The Effect of Taurine on Critical Swimming Speed
80
4.2.2 The Effect of Taurine on Oxygen Consumption Rate
81
4.2.3 The Effect of Taurine on Standard Metabolic Rate.
86
4.2.4 The Effect of Taurine on Routine Metabolic Rate
87
4.2.5 Effect of Taurine on Active Metabolic Rate
88
4.2.6 The Effect of Taurine on Cost of Transport
90
4.2.7 Effect of Taurine on Aerobic scope.
93
63
4.3 Effect of Taurine on Respiratory Burst. 4.4 Effect of Taurine on Lipid Peroxidation on Selected Organs.
97 101
4.4.1 Effect of Taurine on Catalase mRNA Gene Expression in Selected Organ. 101 4.4.1.1 Effect of Taurine on Liver Catalase mRNA Gene Expression.
101
4.4.1.2 Effect of Taurine on Red Muscle Catalase (CAT) mRNA Gene Expression
102
4.4.2 Effect of Taurine on Superoxide dismutase mRNA Gene
103
4.4.2.1 Effect of Taurine on Liver Superoxide dismutase mRNA Gene Expression
103
4.4.2.2. Effect of Taurine on Red Muscle Superoxide dismutase mRNA Gene Expression 104 4.4.3 Effect of Taurine on Glutathione Peroxidase mRNA Expression
106
4.4.3.1 Effect of Taurine on Liver Glutathione Peroxidase (GPX) mRNA Gene Expression 106 4.4.3.2 Effect of Taurine on Red Muscle Glutathione Peroxidase mRNA Gene Expression 107 4.5 Physiological Effect of Taurine on Intestinal Segments.
111
4.5.1 Effect of Taurine on KCl- Induced Contraction.
111
4.5.1.1 The Role of K+ Channels in the Intestinal relaxation induced by Taurine. 111 4.5.1.2 The Role of Ca+2 Channels in the Relaxation Induced by Taurine 116 4.5.1.3Inhibitory Effect of Methylene on Taurine Induced Relaxation
117
4.5.1.4 Role of Endogenous NO and PGI2 in the Relaxation Induced by Taurine 118 4.6 Biological Effects of Taurine on Aortic rings.
64
120
4.6.1 Effect of Taurine on KCl- Induced Contraction.
120
4.6.1.1 Role of Potassium Channels in the Vasorelaxation Induced by Taurine.
120
4.6.1.2 The Role of Ca+2Channels in the Vasorelaxation by Taurine.
124
4.6.1.3 Inhibitory Effect of Methylene Blue on Taurine Relaxation.
126
4.6.1.4 Role of Endogenous NO and PGI2 in the Relaxation Induced by Taurine. 127 5. DISCUSSIONS.
129-141
5.1 Effect of Taurine on Growth performance
129
5.2 Effect of Taurine on Some Metabolic Parameters
134
5.3 Effect of Taurine on Respiratory Burst.
135
5.4Effect of Taurine on Lipid Peroxidation on Selected Organs.
136
5.5 Relaxant Effect of Taurine on Fish Intestinal and Aortic Rings
138
5.5.1 Effect of Taurine on the Contractile Activity Induced by KCl
138
5.5.2 The Effect of K+ Channel Subtypes in the Relaxation Induced by taurine 139 5.5.3 The Effect of Ca+2 Channels in the Relaxation Induced by Taurine 140 4.5.4 The Role of NO, PGI2 and Methylene Blue on Taurine Induced Relaxation 140 6. CONCLUSIONS AND RECOMMENDATIONS
142-144
6.1CONCLUSIONS
142
6.2 RECOMMENDATIONS
144
APPENDIX
145-151
REFERENCES
152-174
65
LIST OF FIGURES
Title Figure 2.1 Illustrate different metabolic terminology in fish such as standard (SMR) and routine metabolic rate (RMR). The upper solid line represents the maximum metabolic rate (MMR) and the dashed line represents the active metabolic rate (AMR). The difference between MMR and SMR define as the metabolic scope for activity (MS). Figure 3.1 Show Extraction of leukocytes from blood by lympholyte®- Mammal. Figure 3.2 A photograph showing the parts of swim-chamber respirometer (AutoTMResp, Loligo® Systems, Tjele, Denmark): The parts are mentioned A-G. Figure 3.3 Standard curve for Catalase (A), Superoxide dismutase (B) and Glutathione peroxidase (C) obtained by amplification curves of descending 10-fold dilutions of standard cRNAs. Real-time reaction was carried out using one-step TaqMan technology.
Pages
28
51
54
59
Figure 3.4 Four chamber organ bath systems, AD Instrument (Model LE01046) connected to the PC.
62
Figure 4.1 Shows the body weight in fish's fed either on fish meal (FM) substituted feed, or on the control feed plus 1.5% taurine for 34 days before and after swimming performance. Figure 4.2 Shows the body weight in fish's fed either on FM substituted feed, or on the control food plus 1.5% taurine for 64 days before and after swimming performance. Figure 4.3 Shows the body length in fish's fed either on FM substituted feed, or on the control plus 1.5% taurine for 34 days before and after swimming performance.
66
72
73
74
Figure 4.4 Shows the body length in fish's fed either on FM substituted feed, or on the control diet plus 1.5% taurine for 64 days before and after swimming performance. Figure 4.5 Shows the level of condition factor in fish's fed either on FM substituted feed, or on the control diet plus 1.5% taurine for 34 days before and after swimming performance. Figure 4.6 Shows the level of condition factor in fish's fed either on FM substituted feed, or on the control diet plus 1.5% taurine for 64 days before and after swimming performance. Figure 4.7 Shows the specific growth rate in fish's fed either on FM substituted feed, or on the control diet plus 1.5% taurine for 34days before and after swimming performance. Figure 4.8 Shows the specific growth rate in fish's fed either on FM substituted feed, or on the control diet plus 1.5% taurine for 64 days before and after swimming performance. Figure 4.9 Shows the feed conversion ratio in fish's fed either on FM substituted feed, or on the control diet plus 1.5% taurine for 34 days before and after swimming performance. Figure 4.10 Shows the feed conversion ratio in fish's fed either on FM substituted feed, or on the control diet plus 1.5% taurine for 64 days before and after swimming performance.
74
75
75
76
76
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Figure 4.11 Shows the critical swimming speeds Ucrit-1 and Ucrit-2 in fish's fed either on FM substituted feed, or on the control diet plus 1.5% taurine after 34 days of feeding before and after swimming performance.
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Figure 4.12 Shows the critical swimming speeds Ucrit-1 and Ucrit-2 in fish's fed either on FM substituted feed, or on the control diet plus 1.5% taurine after 64 days of feeding before and after swimming performance.
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Figure 4.13 Shows the rate of oxygen consumption (mg O₂ kg-1 hr-1) in fish fed either on control FM substituted feed, or on the control plus 1.5% taurine after 34 days of feeding before and after swimming performance.
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Figure 4.14 Shows the rate of oxygen consumption as a function of swimming velocity for fish's fed on commercial pellets and those fed on commercial pellets plus 1.5% taurine. The curves were fitted by fish's fed on commercial pellets. y = 50.60x +140.5 (r² = 0.994) and those fed on commercial pellets plus 1.5% taurine. y = 54.29x+ 139.8 (r² = 0.987).
Figure 4.15 Shows the rate of oxygen consumption as a function of swimming velocity for fish's fed on commercial pellets and those fed on commercial pellets plus 1.5% taurine. The curves were fitted by fish's fed on commercial pellets. y= 49.02x + 148.6 (r² = 0.990) and those fed on commercial pellets plus 1.5% taurine. y= 50.60x +140.5 (r² = 0.994). Figure 4.16, Shows the rate of oxygen consumption (mg O₂ kg-1 hr1 ) in fish fed either on control FM substituted feed, or on the control diet plus 1.5% taurine after 64 days of feeding before and after swimming performance. Figure 4.17 Shows the rate of oxygen consumption as a function of swimming velocity for fish's fed on commercial pellets and those fed on commercial pellets plus 1.5% taurine. The curves were fitted by fish's fed on commercial pellets. y = 55.68x + 135.1 (r² = 0.992) and those fed on commercial pellets plus 1.5% taurine. y = 48.04x + 148.6 (r² = 0.997). Figure 4.18 Shows the rate of oxygen consumption as a function of swimming velocity for fish's fed on commercial pellets and those fed on commercial pellets plus 1.5% taurine. The curves were fitted by fish's fed on commercial pellets. y = 41.51 x + 207.8 (r² = 0.992) and those fed on commercial pellets plus 1.5% taurine. y = 39.62x + 186.7 (r² = 0.899). Figure 4.19 Shows the rate of standard metabolism (mg O₂ kg-1 hr-1) in fish fed either on control FM substituted feed, or fed on the control
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diet plus 1.5% taurine after 34 days of feeding before and after swimming performance.
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Figure 4.20 Shows standard metabolic rate (mg O₂ kg-1 hr-1) in fish fed either on control FM substituted feed, or on the control diet plus 1.5% taurine after 64 days of feeding before and after swimming performance.
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Figure4.21 Shows the routine metabolic rate (mg O₂ kg-1 hr-1) in fish fed either on control FM substituted feed, or fed on the control diet plus 1.5% taurine after 34 days of feeding before and after swimming performance.
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Figure4.22. Shows the routine metabolic rate (mg O₂ kg-1 hr-1) in fish's fed either on control FM substituted feed, or fed on the control diet plus 1.5% taurine after 64 days of feeding before and after swimming performance.
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Figure 4.23 Shows the active metabolic rate (mg O₂ kg-1 hr-1) in fish fed either on control FM substituted feed or on the control plus 1.5% taurine after 34 days of feeding before and after swimming performance.
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Figure4.24 Shows the active metabolic rate (mg O₂ kg-1 hr-1) in fish's fed either on control FM substituted feed, or on the control diet plus 1.5% taurine after 64 days of feeding before and after swimming performance.
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Figure 4.25 Shows the cost of transport (J/kg/s) in fish's fed on control FM substituted feed, or fed on control diet plus 1.5% taurine after 64 days of feeding before and after swimming performance.
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Figure 4.26 Shows the cost of transport (J/kg/s) as a function of swimming velocity for fish's fed on commercial pellets and fish's fed on commercial pellets plus 1.5% taurine. The curves were fitted for fish's fed on commercial pellets by; y = -0.170x + 0.953 (r² = 0.740) and those fed on commercial pellets plus 1.5% taurine by y = -0.162x + 0.915(r² = 0.783). Figure 4.27 Shows the cost of transport (J/kg/s) as a function of swimming velocity for fish's fed on commercial pellets and fish's fed on commercial pellets plus 1.5% taurine. The curves were fitted by: fish's fed commercial pellets. y = -0.203x + 1.073 (r² = 0.698) and
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those fed on commercial pellets plus 1.5% taurine. y = -0.173x + 0.955(r² = 0.768). Figure 4.28 Shows the Cost of transport (j/kg/s) in fish's fed on control FM substituted feed, or on the control diet plus 1.5% taurine after 64 days of feeding before and after swimming performance Figure 4.29 Shows the cost of transport (J/kg/s) as a function of swimming velocity for fish's fed commercial pellets and fish's fed on commercial pellets plus 1.5% taurine. The curves were fitted for fish's fed commercial pellets by y = -0.163x + 0.929 (r² = 0.745) and those fed on commercial pellets plus 1.5% taurine by y = -0.176x + 0.959(r² = 0.762). Figure 4.30 Shows the cost of transport (J/kg/s) as a function of swimming velocity for fish's fed commercial pellets and fish's fed on commercial pellets plus 1.5% taurine. The curves were fitted for fish's fed commercial pellets by y = -0.243x + 1.234 (r² = 0.775) and those fed on commercial pellets plus 1.5% taurine by y = -0.214x + 1.104 (r² = 0.811). Figure4.31 Shows the aerobic scope (mg O₂ kg-1 hr-1) in fish fed on control FM substituted feed, or on the control plus 1.5% taurine after 34 days of feeding before and after swimming performance. Figure 4.32 Shows the aerobic scope (mg O₂ kg-1 hr-1) in fish fed on control FM substituted feed, or on the control plus 1.5% taurine after 64 days of feeding before and after swimming performance
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Figure4.33 Shows the levels of relative light units of RB in the blood after PMA stimulation in fish's fed on control FM substituted feed, or those fed on the control plus 1.5% taurine after 34 days of feeding before and after swimming performance.
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Figure4.34 Shows the relative light units of RB in the blood after PMA stimulation in fish's fed on control FM substituted feed, or the control plus 1.5% taurine after 64 days of feeding before and after swimming performance.
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Figure 4.35 Shows the level of Catalase (CAT mRNA copy n°/100ng RNA) in liver of fish fed either on control FM substituted feed, or fed
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on control diet plus 1.5% taurine after 34 days of feeding before and after swimming performance.
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Figure 4.36 Shows the level of Catalase (CAT mRNA copy n°/100ng RNA) in liver of fish fed either on control FM substituted feed, or fed on control diet plus 1.5% taurine after 64 days of feeding before and after swimming performance.
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Figure 4.37 Shows the level of Catalase (CAT mRNA copy n°/ 100ng RNA) in the red muscle of fish fed either on control FM substituted feed, or fed on control diet plus 1.5% taurine after 34 days of feeding before and after swimming performance.
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Figure 4.38 Shows the level Catalase (CAT mRNA copy n°/ 100ng RNA) in the red muscle of fish fed either on control FM substituted feed, or fed on control diet plus 1.5% taurine after 64 days of feeding before and after swimming performance.
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Figure 4.39 Shows the level of Superoxide dismutase (SOD mRNA copy n°/100ng RNA) in the liver of fish fed either on control FM substituted feed, or the control feed plus 1.5% taurine after 34 days of feeding before and after swimming performance.
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Figure 4.40 Shows the level Superoxide dismutase (SOD mRNA copy n°/100ng RNA) in liver of fish fed either on control FM substituted feed, or the control feed plus 1.5% taurine after 64 days of feeding before and after swimming performance.
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Figure 4.41 Shows the level Superoxide dismutase (SOD mRNA copy n°\ 100ng RNA) in the red muscle of fish fed either on control FM substituted feed, or fed on control diet plus 1.5% taurine after 34 days of feeding before and after swimming performance.
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Figure 4.42 Shows the level of Superoxide dismutase (SOD mRNA copy n°\ 100ng RNA) in the red muscle of fish fed either on control FM substituted feed, or fed on control diet plus 1.5% taurine after 64 days of feeding before and after swimming performance. Figure 4.43 Shows the level of Glutathione peroxidase (GPX mRNA copy n°/100ng RNA) in the liver of fish fed either on control FM
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substituted feed, or fed the control diet plus 1.5% taurine after 34 days of feeding before and after swimming performance.
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Figure 4.44 Shows the levels of Glutathione peroxidase (GPX mRNA copy n°/100ng RNA) in the liver of fish fed either on control FM substituted feed, or fed on the control diet plus 1.5% taurine after 64 days of feeding before and after swimming performance.
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Figure 4.45 Shows the level of Glutathione peroxidase (GPX mRNA copy n°/ 100ng RNA) in the liver of fish's fed either on control FM substituted feed, or there fed on the control diet plus 1.5% taurine after 34 days of feeding before and after swimming performance.
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Figure 4.46 Shows the level of Glutathione peroxidase (GPX mRNA copy n°/ 100ng RNA) in the liver of fish fed either on control FM substituted feed, or there fed on the control diet plus 1.5% taurine after 64 days of feeding before and after swimming performance.
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Figure 4.47 (A) A typical chart view trace showing the relaxant effects of different concentrations (4×10-4 to 2×10-2 M) of taurine and (B) Cumulative dose-response curve for the effects of taurine on KCl (50mM) induced contraction in fish's intestinal segments.
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Figure 4.48 (A) Typical traces showing the effects of preincubation with TEA on the relaxation responses to taurine and (B) Taurine Dose-response curves for KCl precontracted intestinal segments preincubated with and without TEA (50mM).
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Figure 4.49 (A) Typical traces showing the effects of preincubation with GLIB on the relaxation responses to taurine and (B) Taurine Dose-response curves for KCl precontracted intestinal segments preincubated with and without GLIB (50mM).
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Figure 4.50 (A) Typical traces showing the effects of BaCl2 on the relaxation responses to taurine and (B) Taurine Dose-response curves for KCl precontracted intestinal segments preincubated with and without BaCl2 (50mM).
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Figure 4.51 (A) Typical traces showing the effects of 4-AP on the relaxation responses to taurine and (B) Taurine Dose-response curves for KCl precontracted intestinal segments preincubated with and without 4-AP (100μM).
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Figure 4.52 (A) Typical traces showing the effects of Nifedipine on the relaxation responses to taurine and (B) Taurine Dose-response curves for KCl precontracted intestinal segments preincubated with and without Nifedipine (1×10-3M ).
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Figure 4.53 (A) Typical traces showing the effects of Methylene blue on taurine induced relaxation and (B) dose-response curves in 117 intestinal segment precontracted with KCl and preincubated with and without Indomethacin (1×10-6M). Figure 4.54 (A) Typical traces showing the effects of L-NAME on taurine induced relaxation and (B) Taurine dose-response curves in intestinal segments precontracted with KCl and preincubated with and without L-NAME (1×10-3 M).
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Figure 4.54 (A) Typical traces showing the effects of L-NAME on taurine induced relaxation and (B) Taurine dose-response curves in intestinal segments precontracted with KCl and preincubated with and without L-NAME (1×10-3 M).
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Figure 4.55 (A) Typical traces showing the effects of Indomethacin on taurine induced relaxation and (B) Taurine dose-response curves for intestinal segments precontracted with KCl and preincubated with and without Indomethacin (3×10-5M).
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Figure 4.56 (A) Typical chart view trace showing the relaxant effects of different concentrations (4x10-4 to 2x10-2) of taurine and (B) Cumulative dose-response curve for the effects of taurine on KCl (50mM) induced contraction in fish's aortic rings.
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Figure 4.57 (A) Typical traces showing the effects of TEA on the relaxation responses to taurine and (B) Taurine Dose-response curves for KCl precontracted aortic rings preincubated with and without TEA (50mM).
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Figure 4.58 (A) Typical traces showing the effects of GLIB on the relaxation responses to taurine and (B) Taurine Dose-response curves for KCl precontracted aortic rings preincubated with and without GLIB (50mM). Figure 4.59 (A) Typical traces of BaCl2 on the relaxation responses to taurine and (B) Taurine Dose-response curves for KCl precontracted aortic rings preincubated with and without BaCl2 (50mM). Figure 4.60 (A) Typical traces of 4-AP on the relaxation responses to taurine and (B) Taurine Dose-response curves for KCl precontracted intestinal segments preincubated with and without 4-AP (100μM).
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Figure 4.61 (A) Typical traces showing the effects of Nifedipine on the relaxation responses to taurine and (B) Taurine Dose-response curves for KCl precontracted aortic rings preincubated with and without Nifedipine (1×10-3M ).
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Figure 4.62 (A) Typical traces showing the effects of Methylene blue on taurine induced relaxation and (B) Taurine dose-response curves in aortic rings precontracted with KCl and preincubated with and without Methylene blue(1×10-6M).
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Figure 4.63 (A) Typical traces showing the effects of L-NAME on taurine induced relaxation and (B) Taurine dose-response curves in fish aortic rings precontracted with KCl and preincubated with and without L-NAME (1×10-3 M).
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Figure 4.64 (A) Typical traces showing the effects of Indomethacin on taurine induced relaxation and (B) Taurine dose-response curves in aortic rings precontracted with KCl and preincubated with and without Indomethacin (3×10-5M). (O; n=6).
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LIST OF TABLES
Title Table 3.1 Show the composition of the experimental diet for Sea bass (Dicentrarchus labrax), and Common Carp (Cyprinus carpio) 1% of the body weight/day. Table 3.2 Sequences of primers used to synthesize DNA template for in vitro transcription of standard RNAs. Table 4.1 Shows some biometric parameters of the sea bass after feeding on commercial pellets with and without 1.5% of taurine for 34 days before and after exercising in the swimming chamber respirometry. Table 4.2 Shows some biometric parameters of the sea bass after feeding on commercial pellets with and without 1.5% of taurine for 64 days before and after exercising in the swimming chamber respirometry.
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Table 4.3 Shows the rates of MO2 (mg O₂ kg-1hr-1) at each of the following swimming speed (0.7, 1.4, 2.1, 2.8, 3.5 BL/s) in 1 st part of experiments after feeding the fish's on control FM substituted feed, or on the control plus 1.5% taurine for fifteen days (T2).
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Table 4.4 Shows the rates of MO2 (mg O₂ kg-1 hr-1) at each of the following swimming speed (0.7, 1.4, 2.1, 2.8, 3.5 BL/s) in 1 st part of experiments after feeding the fish's on control FM substituted feed, or on the control plus 1.5% taurine for thirty four days (T3).
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Table 4.5 Shows the rate of MO2 (mg O₂ kg-1hr-1) at each of the following swimming speed (0.7, 1.4, 2.1, 2.8, 3.5 BL/s) in 2 nd part of experiments after feeding the fish's on control FM substituted feed, or on the control plus 1.5% taurine for thirty days (T2).
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Table 4.6 Shows the rates of MO2 (mg O2/kg/hr) at each of the following swimming speed (0.7, 1.4, 2.1, 2.8, 3.5 BL/s) in 2 nd part of experiments
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after feeding the fish's on control FM substituted feed, or on the control plus 1.5% taurine for sixty four days (T3). Table 4.7 Shows some metabolic parameters of Sea bass fed on commercial pellets with and without 1.5% taurine in 34 days after exercising the fish's in the swimming chamber respirometry. Table 4.8. Shows some metabolic parameters of Sea bass fed on commercial pellets with and without 1.5% taurine in 64 days after exercising the fish's in the swimming chamber respirometry.
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Table 4.9 Show the average values of relative light units of Respiratory Burst (RB) in the blood measured in the four experimental intervals (T0, T1, T2 and T3) in Sea bass feds commercial pellets with and without 1.5% of taurine in 34 days.
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Table 4.10 Show the average values of relative light units of Respiratory Burst (RB) in the blood measured in the four experimental intervals (T0, T1, T2 and T3) in Sea bass fed commercial pellets with and without 1.5% of taurine in 64 days.
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Table 4.11 Shows the level of Catalase (CAT), Superoxide Dismutase (SOD) and Glutathione Peroxidase (GPX) expression levels measured by real-time PCR in the liver of Dicentrarchus labrax fed on commercial diet with and without 1.5% taurine. The mRNA copy number of each gene was normalized as a ratio to 100ng total RNA. Table 4.12 Shows the level of Catalase (CAT), Superoxide Dismutase (SOD) and Glutathione Peroxidase (GPX) expression levels measured by real-time PCR in the liver of Dicentrarchus labrax fed on commercial diet with and without 1.5% taurine. The mRNA copy number of each gene was normalized as a ratio to 100ng total RNA. Table 4.13 The pIC50, percentage of relaxation and Correlation Coefficient (R2), for the effect of taurine on fish's intestinal segments preincubated with K+ channel blocker (TEA blockers). Table 4.14 The pIC50, percentage of relaxation and Correlation Coefficient (R2), for the effect of taurine on fish's intestinal segments perincubated with K+ channel blocker (GLIB blockers).
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Table 4.15 The pIC50, percentage of relaxation and Correlation Coefficient (R2), for the effect of taurine on fish's intestinal segments perincubated with K+ channel blocker (BaCl2 blockers). Table 4.16 The pIC50, percentage of relaxation and Correlation Coefficient (R2), for the effect of taurine on fish's intestinal segments perincubated with K+ channel blocker (4-AP blockers). Table 4.17 The pIC50, percentage of relaxation and Correlation Coefficient (R2), for the effect of taurine on fish's intestinal segments perincubated with Ca++ channel blocker (Nifedipine blockers). Table 4.18 The pIC50, percentage of relaxation and Correlation Coefficient (R2), for the effect of taurine on fish's intestinal segments perincubated with methylene blue. Table 4.19 The pIC50, percentage of relaxation and Correlation Coefficient (R2), for the effect of taurine on fish's intestinal segments perincubated with L-NAME. Table 4.20 The pIC50, percentage of relaxation and Correlation Coefficient (R2), for the effect of taurine on fish's intestinal segments perincubated with Indomethacin.
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Table 4.21 The pIC50, percentage of relaxation and Correlation Coefficient (R2), for the effect of taurine on fish's aortic rings perincubated with K+ 123 channel blocker (TEA blockers). Table 4.22 The pIC50, percentage of relaxation and Correlation Coefficient (R2), for the effect of taurine on fish's aortic rings perincubated with K+ channel blocker (GLIB blockers). Table 4.23 The pIC50, percentage of relaxation and Correlation Coefficient (R2), for the effect of taurine on fish's aortic rings perincubated with K+ channel blocker (BaCl2 blockers). Table 4.24 The pIC50, percentage of relaxation and Correlation Coefficient (R2), for the effect of taurine on fish's aortic rings perincubated with K+ channel blocker (4-AP blockers).
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Table 4.25 The pIC50, percentage of relaxation and Correlation Coefficient (R2), for the effect of taurine on fish's aortic rings perincubated with Ca++ channel blocker (Nifedipine blockers). Table 4.26 The pIC50, percentage of relaxation and Correlation Coefficient (R2), for the effect of taurine on fish's aortic rings perincubated with methylene blue. Table 4.27 The pIC50, percentage of relaxation and Correlation Coefficient (R2), for the effect of taurine on fish's aortic rings perincubated with LNAME. Table 4.28 The pIC50, percentage of relaxation and Correlation Coefficient (R2), for the effect of taurine on fish's aortic rings perincubated with Indomethacin.
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LIST OF APPENDIX Pages Title Figures Figure A1 The simplified representation of DAQ-M instrument, Auto RespTM (Loligo ® Systems setup). Figure A2 Show the diagram of fast set point adjustment of calibrating the oxygen probe (OXY-REG). Figure A3 Show the diagram of fast set point adjustment of calibrating the temperature Analyzer (TMP-REG) (AutoTMResp, Loligo® Systems). Figure A4 Typical chart view trace showing the curve of Oxygen consumption (MO2 mg O2/Kg/hr) after setting the instrument with intermittent respirometry after running at three phases: Measuring period (M), Flushing period (F) and Waiting period (W).
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Tables Table A1 The calibration of luminescence reader parameters (TECAN)
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Table A2 Reveals the default Setting of OXY-REG
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Table A3 Reveals the default Setting of TMP-REG
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Table A4 Settings, Swimming Speeds of swimming chamber respirometry, (F= Flushing. W= Waiting. M= Measurements).
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LIST OF ABBREVIATIONS
4-AP
4-Amino Pyridin
AAT
Aspartate Aminotransferase
Ach
Acetylcholine
ADP
Adenosine Diphosphate
AMR
Active Metabolic Rate
AS
Aerobic Scope
ATP
Adenosine Triphophate
BaCl2
barium chloride
CAD
Cysteine Acid Decarboxylase
cAMP
cyclic Adenosine Monophosphate
CDO
Cysteine Deoxygenase
cGMP
cyclic guanosine monophosphate
CGRP
Calcitonin Gene-Related Peptide
ClO-
Hypo chloride
CNS
Central Nervous System
CO2
Carbon dioxide
COT
Cost of transport
CSD
Cysteine-Sulphinic Acid Decarboxylase
Ct
Cycle threshold
DNA
Deoxyribonucleic acid
DO
Dissolved oxygen
EDTA
Ethylene diamine tetra acetic acid
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FAO
Food and Agriculture Organization
FCR
Feed Conversion Ratio
FL
Fork length
GES
Guanidoethanesulfonate
GLIB
Glibenclamide
GPX
Glutathione Peroxidase
H2O2
Hydrogen Peroxide
HBSS
Hank's Balanced Salt Solution
IP3
Inositol Trisphosphate
J
Jole
K
Condition Factor
KATP
ATP-Sensitive K+ Channels
KCa
Ca2+-activated K+ channels
KCl
Potassium chloride
KIR
Inward Rectifier K+ Channels
Km
Michaeli's Menten equation
KV channels
Voltage-dependent K+ Channels
L-NAME
L-Nitro- L-arginine
mAChRs
Muscarinic Acetylcholine Receptors
MLCK
Myosin Light Chain Kinase
MMR
Maximum Metabolic Rate
MO2
Oxygen Consumption
MPO
Myeloperoxidase
MR
Metabolic Rate
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mRNA
messenger Ribonucleic acid
nAChR
Nicotinic Acetylcholine Receptor
NAD(P)H
Nicotinamide adenine dinucleotide phosphate oxidase
NADPH
Nicotinamide Adenine Dinucleotide Phosphate
NH2Cl
Chloroamines
NMJ
Neuromuscular Junction
NO
Nitric oxide
PAPS
Phosphoadenosine Phosphosulfate
PC
Phosphatidylcholine
PCr
Phosphocreatine
PCR
Polymerase Chain Reaction
PE
Phosphatidylethanolamine
Phox
Phagocyte Oxidase
Pi
Inorganic Phosphate
PLC
Phospholipase C
PMA
Phorbol Myristate Acetate
ppm
Part per Milion
qRT-PCR
One-step real-time PCR
Rap
Ras-Related Protein
RB
Respiratory burst
RLU
Relative light unit
RMR
Routine metabolic rate
RNA
Ribonucleic acid
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ROS
Reactive Oxygen Species
SGR
Specific Growth Rate
SKCa
Small-Conductance KCa channel
SL
Standard length
SMC
Smooth Muscle Cell
SMR
Standard Metabolic Rate
SOD
Superoxide Dismutase
SR
Sarcoplasmic Reticulum
t
Ton
TBL
Total body length
TEA
Tetraethylammonium
TL
Total length
Ucrit
Critical Swimming Speed
VGCCs
Voltage Gated Ca+2 channels
Vit B5
Pantothenic Acid
VSMCs
Vascular Smooth Muscle Cells
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1. INTRODUCTION Taurine (2-amino ethane sulfonic acid) is a sulphur-containing compound characterized as an amino acid (Demarcay, 1938). It is widely distributed in invertebrate as well as vertebrate tissues including fish and usually found free in cytosol and plasma (Abebe & Mozaffari, 2011). The pathway of taurine biosynthesis from sulfur containing amino acids like methionine and cysteine includes the oxidation cysteine to cysteine sulfinic acid by cysteine dioxygenase, its subsequent decarboxylation by cysteine sulfinate decarboxylase and ultimately the oxidation of hypotaurine to taurine by hypotaurine dehydrogenase (Samizu, 1962; Huxtable, 1989). Taurine shows variable body tissue distribution and varies greatly among species (Hayes et al., 1980; Sturman & Hayes, 1980). Taurine plays important roles in different physiological processes including, osmoregulation (Morales et al., 2007), modulation of neurotransmitters (Gao et al., 2011; Haojun et al., 2012), membrane stabilization (Junyent et al., 2011), Calcium homeostasis (Huxtable, 1992), detoxification and anti-oxidation in mammals (Bircan et al., 2011; Silva et al., 2011). Swimming is an important fitness trait in fish because it is closely linked to food intake, predator avoidance, reproduction, migration and schooling (Brett, 1964; Domenici et al., 2013). Some animals move slowly, others move extremely fast whereas others can reach high velocities but show very little stamina (Bennett, 1991). Swimming requires metabolic energy for its muscular work, primarily by the large masses of axial muscles located along both sides of the body, known as myotomes, which are dominant anatomical feature of most fishes. Although active muscle contraction starts by utilization of adenosine triphosphate (ATP) followed by molecular mechanisms as in
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other vertebrate skeletal muscle. Fish have distinct muscle types, each type uses specific metabolic pathways for the primary generation of ATP (aerobic vs. anaerobic) to power different types of swimming (McKenzie, 2011). The process of fish swimming in water is divided into three categories: sustained, prolonged, and burst swimming. It is generally accepted that during sustained exercise, muscle metabolism is largely aerobic and is provided by the well perfused red musculature. Whereas, much of the swimming activity in fish is aerobic in nature, periods when the capacity for this type of swimming is exceeded exist. During these events (e.g. predator- prey interactions and spawning migrations; (Beamish, 1978), whereas burst swimming is performed by anaerobic glycolysis within the white muscle (Milligan & Wood, 1986; Moyes & West, 1995; Milligan, 1996). This burst-type activity which applied to a great number of fish species can only be maintained for brief periods of time (less than 15 second) (Blaxter, 1969). It is usually terminated by exhaustion of intracellular energy supplies (Jones, 1982), or by accumulation of metabolic waste products (Bainbridge, 1960 & Brett, 1964). According to Webb, (1975), bursts may be steady (=sprints) or unsteady (=accelerated). In contrast, fish are able to swim at lower speeds which can sustained for hours, days, or even months (Brett, 1964). On the other hands, the prolonged swimming is the intermediate between the above two types in speed. The energy always supplied by aerobic and anaerobic respiration. This type of swimming can last up to 3 hours and longer bouts can end up in fatigue, and is used occasionally on demands (Hammer, 1995). Swim performance in fish is commonly assessed by determination of the critical swimming speed (Ucrit) which is widely used to evaluate aerobic swimming performance (Reidy et al., 2000; Lee et al., 2003c ),
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with the assistance of a swim tunnel respirometer. The Ucrit assesses the capability for endurance and maximal swim speed in fish through gradual increases in water velocity over time until exhaustion occurs. Therefore, Ucrit and oxygen consumption (MO2) values collected from a swim tunnel respirometer can give an insight into more subtle physiological effects such as resting and active metabolic rates, aerobic capacity and energy utilization (Hammer, 1995; Plaut, 2001). In vitro studies showed that taurine plays an important role in the intestinal functions (Sturman, 1993; O’Flaherty et al., 1997). Indeed, taurine was shown to exert its protective effect on gastric injury and colonic damage through its antioxidant properties (Son et al., 1996, 1998). Taurine is also capable of maintaining small intestinal mucosal thickness and villus height during total parenteral nutrition (Tsuchioka et al., 2006). The antihypertensive effects of taurine have been demonstrated in several experimental models (Harada et al., 2000& 2004). Studies on the effect of taurine on the contractility of vascular smooth muscle in fish are not available. However, there are some studies performed on mammalian blood vessels such as those on isolated arteries, which appear that taurine relaxes rat mesenteric artery (Li et al., 1996), rat aorta (Ristori & Verdetti, 1991), and rabbit ear artery (Franconi et al., 1982). Beta-alanine given chronically to rats to deplete taurine revealed enhanced contractile responses in aortic rings to norepinephrine and high potassium concentrations, and decreased relaxant responses to sodium nitroprusside and acetylcholine (Abebe & Mozaffari, 2003). In the light of above information’s which indicate the availability of limited studies on the physiological effects of taurine, the current study was
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selected to study the effect of taurine on the metabolic activities and smooth muscle contractility of intestinal segments and dorsal aorta in fish. The objectives of the study The present study aimed to investigate the effect of taurine on 1. Growth and biometric performances in sea bass (Dicentrarchus labrax). 2. Swimming performance, measured as critical swimming speed (Ucrit) and oxygen consumption rates (MO2) under routine and exhaustive swimming conditions in sea bass (Dicentrarchus labrax), using a swimming chamber respirometer (AutoTM Resp, Loligo® Systems, Tjele, Denmark). 3. Some metabolic parameters (i.e. standard metabolic rate (SMR), routine metabolic rate (RMR), active metabolic rate (AMR), aerobic scope (AS) and cost of transports (COT) 4. Immunological status assessed through the alternative complement pathway, leukocyte production of reactive oxygen species (ROS). 5. mRNA gene expression levels catalase (CAT), superoxide dismutase (SOD) and glutathione peroxidase (GPX) were calculated in the red muscles and liver tissues of Sea bass by using One-Step Taqman® real-time Polymerase chain reaction (PCR). 6. The relaxant effects of taurine on fish intestinal segments and aortic rings in common carpe (Cyprinus carpio). 7. Role of K+ and Ca++channels in smooth muscle contractility in taurine induced relaxation using K+ and Ca++ channels blockers. 8. The role of endothelium derived hyperpolarizing factors in taurine induced relaxation in fish aorta.
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2. LITERATURE REVIEW 2.1 Taurine (2-Aminoethanesulfonic Acid) 2.1.1 Chemical Properties of Taurine Taurine, 2-aminoethanesulfonic acid is a β-amino acid with a molecular mass of 125 Daltons, colorless and water soluble, crystallizing as tetragonal needles with a melting point 328C° (Ansell, 1959). The dielectric constant at pH 2.8 is 41-53 (Carr & Shull, 1939). The molecular structure of taurine (NH3+ - CH2 - CH2 - SO3) is very similar to that of γ-aminobutyric acid (GABA). Taurine is analogous to βalanine with one carboxyl group which is a typical character for other amino acids, and does contain a sulfonate group (Tachiki & Baxter, 1979). Because it is one of the few amino acids not used in protein synthesis, taurine is often referred to as a “nonessential” amino acid, or more generously as a “conditionally essential” amino acid (Connolly & Goodman, 1980). 2.1.2 Origin of Taurine Taurine is one of the most abundant free amino acid derivatives in invertebrate and vertebrate animals (Knopf et al., 1978). Tiedemann and Gmelin were the first to report the presence of taurine in living material. They isolated it from ox bile, which they named bile- Asparagine (Bostaurus) (Tiedemann and Gmelin, 1827). 2.1.3 Distribution of Taurine Taurine shows a variable tissue distribution and varies greatly among species; the concentration of taurine in the Liver of dog and rat is higher than those of monkey and cats (Hayes et al., 1980; Sturman & Hayes, 1980). A similar variability of taurine was observed in different fish's
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species which is high in rainbow trout (Yokoyama & Nakazoe, 1992) but low in Japanese flounder (Park et al., 2002) and turbot (Wang et al., 2014).
2.1.4 Taurine and Nutrition Endogenous taurine synthesis alone cannot account for the physiological concentrations (Huxtable, 1992). Taurine is present in tissues and exogenous taurine must therefore be obtained from food. As taurine is almost exclusively limited to the animal kingdom, meats (particularly uncooked meat), seafood and fish are the major sources of taurine (Zhao, 1994). Some of the taurine concentrations reported for fish are remarkably high (up to 83µM/g wet wt) in yellowtail (Japanese amberjack). Milk and eggs also have high taurine concentration. Indeed, taurine concentrations in milk have been estimated to about 600µM in gerbil, ~ 300 µM in cat and ~ 40 µM in humans (Hayes & Sturman, 1981a). In contrast, taurine is either absent or present only in trace amounts in vegetables and mushrooms. 2.1.5 Biosynthesis and Metabolisim of Taurine In mammals the endogenous taurine synthesis occurs mainly in the liver and brain by enzymatic oxidation and direct conversion of cysteine, or conversion of methionine into cysteine (Chang et al., 2013). This process involves the actions of cysteinedioxygenase (CDO), which leads to cysteinesulphinate, and cysteinesulphinate decarboxylase (CSD) (Griffith, 1987). Historically, taurine has not been considered as an essential nutrient for fish (Yokoyama & Nakazoe, 1996). Just recently, studies have been indicated that taurine synthesis is widely differs between the fish species, depending on fish habitats, species, size, feeding and CSD activity. Marine fish species, such as Japanese flounder (Paralichthys olivaceus), red sea bream (Pagrus major) and yellowtail (Seriola
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quinqueradiata), show low capacities of taurine biosynthesis due to the absence of or low CSD activities during intermediate metabolism from methionine to cystathionine (Park et al., 2002; Kim et al., 2003). On the other hand, freshwater teleost fish, such as common carp, rainbow trout and Atlantic salmon can synthesize taurine through transsulphuration pathway (Kim et al., 2008a). 2.1.6 Physiological Roles of Taurine 2.1.6.1 Role of Taurine on Cardiovascular System Over 50% of the total amino acid pool in the heart is taurine (Huxtable et al., 1980), taurine has been shown to have positive, chronotropic and inotropic effects, to enhance digitalis inotropy (Sole & Jeejeebhoy, 2000). In addition, several researchers have focused on the positive effects of taurine on cardiovascular physiology such its antihypertensive (Fujito & Sato, 1984; Harada et al., 2004) and myocardial protective effects (Schaffer, et al., 2003). However, little attention has been paid to the vasorelaxant effect of taurine in mammals and almost nothing is known about its effect on the contractility of fish aorta. In vitro studies showed that taurine (10-80mM) produced a strong concentration dependent vasodilation in K-induced contraction in rabbit's ear artery (Franconi et al., 1982). In an in vitro study on rat aorta, taurine induced relaxation in artery segments precontracted with K+ and norepinephrine (Ristori & Verdetti, 1991). Also it has been found that taurine uptake significantly reduced the blood pressure in SHRSP but not WKY rat’s mesenteric arteries (Li et al., 1996). Thoracic aortic rings isolated from rats with chronic administration of taurine showed reduced contractile responses to norepinephrine and high potassium concentrations (Abebe & Mozaffari, 2000).
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2.1.6.2 Role of Taurine in Intestinal Function Taurine play important role in the intestinal functions (O’Flaherty et al., 1997). Indeed, taurine was shown to exert its protective effect on gastric injury and colonic damage through its antioxidant properties (Son et al., 1998). Taurine is also capable of maintaining small intestinal mucosal thickness and villus height during total parenteral nutrition (Tsuchioka et
al., 2006). Additionally, taurine may play a role in
maintaining normal serum IGF-I concentrations (Hu et al., 2000), which promote intestinal development (Park et al., 1999). These observations collectively suggested a possible role for dietary taurine in the maintenance of normal gastrointestinal development and functions. In fish, only limited studies have been carried out on molecular characterization of taurine and almost nothing is known about the mechanism of relaxation induced by taurine in smooth muscle cells isolated from fish organs (Verri et al., 2003; Amberg et al., 2008). 2.1.6.3 Role of Taurine in Membrane Stabilization Huxtable and Bressler, (1973) found that incubation of isolated sarcoplasmic reticulum with phospholipase C damaged the membrane, leading to reductions in Ca+2 transport and ATPase activity that were diminished by adding taurine to the medium. This observation led the authors to propose that taurine acts as a membrane stabilizer. It was subsequently shown that taurine directly interacts with membranes, presumably by forming an electrostatic interaction between the amino and sulfonic acid groups of taurine and the phosphate and amino or quaternary ammonium groups of the phospholipids, respectively (Schaffer et al., 1995). This interaction appears to cause minor changes in the lipid bilayer, allowing more calcium to bind to the phospholipids (Chovan et al., 1979).
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However, based on electron spin resonance, neither polymorphic phase changes nor fluidity changes, and they are induced in the lipid bilayer following acute exposure of isolated membranes with taurine. This led to further characterization of the membrane-linked actions of taurine. One of the most prominent taurine-mediated changes in membrane function was found to be the inhibition of phospholipid -methyltransferase, an enzyme that catalyzes the conversion of phosphatidylethanolamine (PE) to phosphatidylcholine (PC) (Hamaguchi et
al., 1991). This reaction is
important because taurine levels regulate the ethanolamine: choline ratio of some membranes (Huxtable, 1992). In the biological membrane, PE is preferentially localized to the outer membrane leaflet where it assumes a bilayer structure while PC is a hexagonal form and is preferentially localized to the inner leaflet of the membrane. Thus, changes in the PE/PC ratio have a dramatic influence on the structure of biological membranes, which in turn alters both membrane fluidity and the activity of membrane enzymes and transporters (Schaffer et al., 1995). 2.1.6.4 Role of Taurine in Immune Cells High taurine levels in phagocytes and accumulation of taurine in inflammatory lesions indicate its role in biology of the cells innate immunity (Levis, 2004). All these cells are involved in killing of pathogens at a site of inflammation. Activated phagocytes generate a variety of microcidal and toxic oxidants produced by peroxidase system in these cells. On the other hand, taurine, a major scavenger for chlorinated (HOCl) and brominated (HOBr) groups will protect the tissue from oxidative stress. Therefore, taurine will be involved in cytoprotection and regulation of inflammation. As taurine is present at high concentrations in leukocytes, one may suggest that taurine deficiency will affect the immune cell functions (Warskulat et al., 2007).
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2.1.6.5 Taurine and Oxidative stress Oxidative stress is a major factor responsible for tissue damage in conditions such as infection, acute and chronic inflammation, cancer and aging. At the site of inflammation, oxidative stress is mediated by ROS generated to a greater extend primarily by activated leukocytes (neutrophils, macrophages, eosinophils). Reactive oxygen species (ROS) play a beneficial role in host defense against pathogens but they are also responsible for tissue injury (Weiss, 1988; Smith, 1994). A variety of antioxidants are involved to prevent oxidant-induced cell damage and to reduce oxidative modification of self-macromolecules, primarily lipids, proteins, and DNA. Antioxidants (“the antioxidant network”) act through one of three mechanisms shown below: i.
Reducing the generation of ROS;
ii.
Scavenging ROS;
iii.
Interfering with the action of ROS; Taurine can be found particularly at high concentrations in tissues
exposed to elevated levels of oxidants suggesting its role in the alleviation of oxidative stress (Green et al., 1991; Jeon et al., 2009; Oliveira et al., 2010). Indeed, there have been numerous reports indicating taurine as an effective antioxidant, but the mechanism underlying its oxidant activity remains unclear. The best established antioxidant action of taurine is neutralization of hypochlorous acid (HOCl), which is extremely toxic oxidant generated by Myeloperoxidase (MPO-halide system) (Weiss et al., 1982). This action explains anti-inflammatory properties of taurine as its reaction with HOCl results in generation of TauCl, more stable and less toxic anti-inflammatory mediator. Therefore, taurine may be considered the component of innate immunity with a special impact on the
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development of acute inflammation. However, not all of the antioxidant actions of taurine are related to HOCl because they are detected in systems lacking neutrophils. Although taurine is incapable of directly scavenging the classic ROS, it has been suggested that it is an effective inhibitor of ROS generation. 2.2 Aquaculture Fish evolved around 500 million years ago and currently they form the oldest and most diverse vertebrate group which includes more species than all other vertebrate groups combined (Xiaobo et al., 2010). The evolutionary position of fish and their ability to adapt to diverse environmental conditions made them frequently used as models for every biological discipline including neurobiology, physiology, toxicology, endocrinology, developmental biology and environmental research (Powers, 1989). Aquaculture has become the world’s fastest growing sector of food production, increasing nearly 60-fold during the last five decades (FAO, 2011). According to Food and Agriculture Organization statistics (FAOs, 2011) the worldwide production of aquaculture in 1996 was approximately 34x l06 tons (t) compared with total world capture fisheries of approximately 96x106 t, these 34x106 t farmed “fish” consisted of 48.8% fin fish. 24.9% mollusks, 22.7% aquatic plants, 3.4% crustaceans and 0.2% other species (De Silva & Anderson, 1995). In 1996, aquatic meat production of 16.3 x l06 t accounted for approximately 7% of global meat production. It was in the fourth order behind chicken meat (49.5 x 106 t; 21.2% total meat production), beef and calf meat (53.9 x 106 t, 23.1% total meat production) and porker meat (87.2 x 106 t , 38.3% total meat production), which is the fastest growing of all these sectors (Tacon, 1999). In the developing countries, aquatic meat
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constitutes only 1.6% of the total meat production and accounted for 11.2% of a total meat production in developing countries (Uglem et al., 2008). Thus, 91.1% of world aquaculture production occurred in Asia (particularly in China which produced 23.1x106t), 4.7 % in Europe, 1.8 % in North America, 1.6 % in South America and 0.4% in Africa (Tacon, 1999). The expansion in the production of farmed Sea bass (Dicentrarchus labrax) took place particularly in Greece, Turkey, and Italy which has been
slowdown in expansion (FAO, 2016). Total production from all countries decreased from a peak of nearly 71.000 t in 2000 to 57.000 t in 2002. However, Spain and Croatia appear to be bucking this trend (FAO, 2016). The total catch reported for common carp (Cyprinus carpio) species to FAO for 1999 was 75 235 t. The countries with the largest catches were Turkey 17.797 t and Thailand 14.000 t (FAO, 2016). 2.3 European Sea bass (Dicentrarchus labrax, L.) 2.3.1 Nomenclatures of Sea bass The present accepted scientific name for the bass in Europe is Dicentrarchus labrax (Linnaeus) (Pickett & Pawson, 1994). The word”Dicentrarchus” has been derived from the presence of two dorsal fins. Branzino is the name of the fish in Northern Italy, with branzini as the plural; in other parts of Italy, it is called spigola or ragno. It's called karous in Arabic. In Portugal is called lubina or róbalo. In France, the fish called bar commun along the Atlantic coast and loup de mer on the Mediterranean (FAO, 2016).
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2.3.2 Family Description 2.3.2.1 Family Moronidae According to the Moretti et al., (1999); The perciform fish have elongated body, the operculum with two flat spines; the terminal mouth was slightly protractile, end of the maxillary visible, not gliding under the suborbital bone; small teeth was appear on the jaws and vomer, two dorsal fins are separated, the first one with 8-10 spines, whereas the second one with one spine and 11-14 soft rays, anal fin has three spines and 10-12 soft rays; base of the pelvic fins without scales; caudal fin moderately forked, large caudal peduncle, lateral line complete, not continuing on the caudal fin. Small scales, around 55-80 on the lateral line in the Mediterranean species. The color is generally silvery; one species with small black spots; lower fins sometimes are yellowish when fish are a live 2.3.3 Morphology of Sea bass The sea bass has elongated silver body, with two clearly differentiated dorsal fins and long tail. The opercular bone has two flat spines and a range of spines are visible on the lower part of the preopercular bone, pointing toward the direction of the mouth (Froese et al., 2006). The flanks are silver-blue, sometimes pale gold or bronze. The head in young bass appears quite pointed, but it becomes blunter in older fish. Sea bass in their first year tend to be paler in appearance than older fish, and usually have dark spots on the back and upper sides. This species has cycloid scales in inter orbital region. The lateral line is visible as a dark line with 62-80 cycloid scales. The first dorsal fin has 8-10 spiny rays, and the second dorsal fin 12-13 rays of which the first is spiny. The anal fin has 3 spiny rays and 10-12 soft rays. The color is dark grey on the back, passing to grey-silver on the sides, while it is white-silver on the abdomen. Specimens
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from the sea show much clearer color than fish from lagoons and estuarine environments. The maximum size is over 1 m with a weight of over 12 kg (Pickett & Pawson, 1994). 2.3.4 Geographical Distribution of Sea bass The distribution of sea bass was found in coastal waters of the Atlantic Ocean from South of Norway to Western Sahara and throughout the
Mediterranean
and
the
Black
Sea;
its
habitats
include estuaries, lagoons, coastal waters and rivers (Pickett & Pawson, 1994). The European sea bass is typically an inshore species found in the surf zone, around outcrops of rocks, and in shallow coastal waters. Since they are able to survive in estuaries in almost fresh water, it is thought that they could be adapted to live in fresh water (Alwyne, 1975). It has been introduced for culturing purposes in to Israel, and more recently Oman and the United Arab Emirates (Pickett & Pawson, 1994). It is a eurythermic (5-28C°) and euryhaline species, and able to adapt to water salinity lower than that of sea water. Thus, it frequently present in coastal inshore waters, and in estuaries and brackish water lagoons; sometimes it ventures up stream into fresh water (FAO, 2011). Their behavior is gregarious in juvenile age, but solitary during adult age: In nature they mainly feed on fish, cephalopods and crustaceans (Pickett & Pawson, 1994). Sea bass, as the majority of fish, is a gonochoristic species, with female and male gonads residing in separate individuals. However, genders are complex to distinguish due to the absence of external sexual characters (Rocha, 2008). Growth is clearly related to sex, with females growing faster, reaching a larger size at first maturity (Pickett & Pawson, 1994; Carrillo et al., 1995). Females spawn in the Mediterranean Sea during the
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winter (December to March) and up to June in the Atlantic Ocean. They present a high fecundity (on average 200,000 eggs / kg of female), start to reproduce over 2 kg and can reach 6 to 7 years in the wild (FAO, 2016). Eggs and larvae have a great dispersal during the 3 first months of life and adults migrate over several hundreds of kilometers (FAO, 2016). 2.4 Common carp (Cyprinus carpio) The common carp, (Cyprinus carpio L.) can be considered the first fish species that was widely distributed by humans and considered the third most frequently introduced species worldwide (Balon, 1995). The common carp also accounts for the world’s second highest farmed fish production, mainly from poly culture in Asia (Balon, 1995). Fish farming in Iraq beign in 1955 when a little pond in AlZaafaraniya, at about 14 km south of Baghdad city centre was stocked with the common carp (Al-Hamed, 1960). Generally, There are three common subspecies of this type of fish, namely: 1- European carp (Cyprinus carpio carpio) is found in most of Europe (Linnaeus, 1758 ; Zhou et al., 2003). 2- Deniz carp (Cyprinus carpio yilmaz) is found in Anatolia Turkey (Zhou et al., 2003). 3- Amur carp (Cyprinus carpio haematopterus) is native to eastern Asia (Zhou et al., 2003). 2.4.1 Nomenclatures of Common carp The international names of Cyprinus carpio are common carp; European carp; leather carp and mirror carp. In Japan it is called Koi, in Arabic called carp and in Portugal is called sarmão (FAO, 2016).
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2.4.2 Family Description of Common carp Common carp belongs to the family Cyprinidae (minnow and carp family). 2.4.2.1 Family Cyprinidae The family Cyprinidae is the largest of all fish families, since it includes more than 2,000 members of the family which have been already described, representing about 10% of all fish species in the world, or about 25% of freshwater fish species (Hogan, 1998). Members of the family are often referred to as “carp”, although the term is usually applied only to larger species. All members of the family are more accurately termed cyprinids (Hogan, 1998). 2.4.3 Morphology of Common carp The body of common carp is elongated and somewhat laterally compressed, with thick lips. They have two pairs of barbells at the angle of mouth, the shorter ones on the upper lip. Dorsal fin base long with 17-22 branched rays and a strong, toothed spine in front; dorsal fin outline concave anteriorly, the anal fin with 6-7 soft rays; posterior edge of 3rd dorsal and anal fin spines with sharp spinules (Froese & Pauly, 2002). Lateral line with 32 to 38 scales, and the pharyngeal teeth 5:5 and with flattened crowns. Color variable, wild common carps are brownish-green on the back and upper sides, shading to golden yellow ventrally. The fins are dusky, ventrally with a reddish tinge (FAO, 2016). Growth is variable with local conditions. In south-eastern Europe (where conditions are optimum) an average length attained 51-61 cm and weight of 1.8-4.5 kg; and in northern Europe it is rather less, and, a maximum weight of 32 kg is recorded (FAO, 2016).
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2.4.4 Geographical Distribution of Common carp Cyprinus carpio is a widespread fresh water fish of eutrophic lakes and large rivers in Europe and Asia. The wild inhabitance are considered vulnerable to extinction, but also has been domesticated and introduced into environments worldwide. It is often considered a very destructive invasive species, being included in the List of the world's 100 worst invasive species (Farag et al., 2014). The perfect conditions that suitable for carp are lowland lakes and rivers with abundant vegetation to supply food and shelter (FAO, 2016). They are omnivorous feeds mainly on living insect larvae, small snails, crustaceans, and some vegetables. They are active at night, and feed little at low temperatures (FAO, 2016). They naturally live in temperate climates in fresh or slightly brackish water with pH of 6.5–9.0 and salinity up to about 0.5% and temperature range from 3 to 35°C. The ideal temperature is 23 to 30°C, with spawning beginning at 17–18°C. Carp are able to tolerate water with very low oxygen levels, by gulping air at the surface (Farag et al., 2014). 2.5 Muscle Tissue 2.5.1 Skeletal Muscle Tissue Skeletal muscle is made up of muscle fibers and connective tissues (tendon, fascia, adipose tissue and ligaments) (Buckingham et al., 2003). All muscles are surrounded by epimysium which is a thickened layer of moderately dense connective tissue that connects the muscle via its tendons to the bone, skin or cartilage. It protects the muscle, holding fiber bundles together, and separate one muscles from one another for relatively independent contraction (Gordon et al., 2000). Within a muscle, there are bundles of muscle fibers, which are covered by perimysium, a thinner layer
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of connective tissue that separates fiber bundles and permit the blood vessels and nerves supplies to reach into the muscle belly. Finally, each muscle fiber wrapped by a very thin layer of connective tissue, referred to as endomysium (Squire et al., 2005). 2.5.2 Skeletal Muscle Fibers Skeletal muscle cells are long, cylindrical and multi-nucleated cells called muscle fibers due to their thread-like appearance under a microscope (Noguchi et al., 2000). Generally, they are only barely seen to the naked eye. Muscle fibers are formed by the fusion of many immature mononuclear undifferentiated cells called myoblasts (Fluck & Hoppeler, 2003). The plasma membrane and cytoplasm in muscle fibers are called the sarcolemma and sarcoplasm, respectively (Bottinelli & Reggiani, 2000). Similar to other cells, the sarcoplasm contains cell organelles such as nuclei, mitochondria, rough endoplasmic reticulum, Golgi apparatus, unique sarcoplasmic reticulum for skeletal muscle, the sarcoplasm reticulum, also contains T-tubules and myofibrils (Bottinelli, 2001). Myofibrils are bundles of myofilaments, consist mainly from two proteins, actin and myosin. Molecules that form thick and thin filament involved in muscle contraction (Pette & Staron, 2001). They are organized in a very distinctive banding pattern that appear as transverse “stripes” along the length of a fiber, in which the repeating pattern is called a sarcomer. 2.5.3 Types of Skeletal Muscle Fibers in Fishes The main function of fish skeletal muscle is movement, unlike the mammals whose muscles also give important support to the skeleton. The most striking difference between striated fish muscle and that found in higher vertebrates is firstly the separation of fibers types into discrete layers in fish, where the high glycolytic and anaerobic type dominates and
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constituting about 90-95% of all body muscles in most fish species. Secondly, in the majority of fish species growth continues throughout most of the life and the growth of muscles is the combined effect of formation of more muscle cells (fibers) and increase in size of already existing fibers (Dunajaski, 1979). All fishes have two main types of locomotory muscle fibers, red and white, specialized for either low speed cruising or short bursts of maximum speed, respectively (Bone, 1978). The characteristic found in teleost is as follows: the axial muscle consists mainly of fast white fibers, covered by a thin layer of slow-red muscle fibers, with a layer of pink intermediate muscle fibers in between them (Martineze et al., 1993; Kiessling et al., 1995).
2.5.3.1 Slow-Red Muscle Fibers Slow-Red muscle fibers are usually confined to narrow strip, along the lateral line whereas the red muscle fibers are small in diameter (2545μm), usually constitute less than 10 % of the myotomal musculature. The red muscle fibers are also called slow fibers, since they are used usually to supply sustained energy for efficient swimming. They are characterized by good capillary supply, high amount of mitochondria, lipid droplets and glycogen stores. Concentrations of myoglobin and cytochromes are also high. The energy metabolism in red muscle is almost entirely aerobic, based mainly on lipid as fuel complemented with carbohydrates (Sanger & Stoiber, 2001). This red muscle is composed primarily of slow-twitch oxidative (SO; type I) fibers, which produce most of the power at low contraction frequencies, and generate their ATP by mitochondrial oxidative phosphorylation, which produces 36 molecules of ATP for each glucose equivalent. This requires substrates (lipids, proteins, and carbohydrates) to be used as fuels and a supply of oxygen as the terminal electron acceptor.
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The substrates and oxygen are generally provided to the muscle via the blood stream, and red muscles have a rich blood supply and a high density of mitochondria. They also have intracellular myoglobin, a transient oxygen store that, along with many capillaries, gives them their characteristic red color (McKenzie, 2011). 2.5.3.2 Fast- White Muscle Fibers Fast-White muscle fibers, constitute the major part of the skeletal muscle in fish (not less than 70%) (Sanger & Stoiber, 2001). The white fibers exhibit the largest fiber diameter ranging from 50 to 100 μm or even more. The proportion of the cross sectional area of the skeletal muscle that is comprised of white muscles varies along the length of the fish, being greatest in the anterior of the animal body and declining caudally. Generally, the white muscle is used at high swimming speeds e.g., in fast-start burst swimming for prey capture and escape response, though there is an overlap of labour between red and white muscles in most teleosts. White muscle fibers are tightly packed with myofibrils occupying between 75 and 95% of the fiber volume. Organelles such as mitochondria which interrupt the arrays of myofibrils are few, with very low lipid droplets and myoglobin content in most fish species (Zhou et al., 1995). Vascularization in white glycolytic muscle is poor, and the glycogen content is also low with granules mainly located between the myofibrils. However, there seems to be a marked heterogeneity in glycogen content between different sized white fibers, with a significantly higher content in the smaller fiber (Kissling & Ostrowski, 1997). The energy for white muscles, operating in more or less closed system, dominates by anaerobic breakdown of intramuscular glycogen with small contribution from cytosolic phosphocreatine (PCr) and ATP. In addition, in glycolytic based system, energy is likely provided via the slower but more efficient aerobic
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break down of
lipids. Enzymatic activity levels of β-oxidation and
respiratory chain in the range of 10% of that found in red muscle is reported through out the life in white muscle of rainbow trout (Kissling et al., 1991b), Atlantic salmon (Froyland et al., 1998) and migrating sockeye salmon (Kissling et al., 2004a). 2.5.3.3 Intermediate-Pink Muscle Fibers Intermediate or pink fibers are in accordance with their names not only intermediate in position between red and white muscle fibers, but also in many other aspects. In juveniles and adults of most teleost species, a zone of intermediate or pink fibers is inserted between red and white fibers. The mean fiber diameter lies between those of red and white. Pink fibers are characterized as fast contracting with intermediate resistance to fatigue and intermediate speed of shortening between red and white muscles (Martinez et al., 1991). 2.5.4 Contraction of the Skeletal Muscle Sarcomeres are the basic unit of muscle contraction. They are organized between two Z-lines that cross (and attach to) actin filaments at the so-called H-line in the I-band; between two I-bands there is an intervening A-band of myosin filaments (Morgan & Proske, 2004). Myosin and actin filaments in the sarcomere cooperate or interact to make contraction of a muscle fiber by sliding past one over another using crossbridges to pull the myosin filament along actin in the cross-bridge cycle involving calcium ions (Ca+2) and myosin ATPase, using ATP as an energy source for tension generation (Pollard & Borisy, 2003). This sliding filament process shortens the sarcomere between the Z-lines in a concentric contraction, and lengthens the sarcomere in an eccentric contraction.
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The contractile process is regulated by the structures inside a muscle fiber, and calcium-release mechanisms. Each myofibril is surrounded by vesicles of sarcoplasmic reticulum, and those vesicles hold a reserve store of Ca+2 that is used to produce muscle contraction by interaction with troponin, tropomyosins and actin (Chhabra & Higgs, 2007). The sarcoplasmic reticulum branches around fibrils having distal “end sacs” called terminal cisternae. There are also T-tubules that project from the sarcolemma into the core of a muscle fiber (Defranchi et al., 2005). Around the T-tubules as they penetrate between fibrils and at particular sarcomere regions (A/I junction in mammalian muscle), two terminal cisternae surround the T-tubule and the three vesicles that appear by electron microscopy are called a triad. The T tubule is a critically important component of the signaling pathway that transmits a depolarization of the sarcolemma from the action potential in a nerve via the neuromuscular junction (NMJ) into the deeper regions of the muscle fiber. Depolarization which stimulates the sarcoplasmic reticulum to releases Ca+2 and which increases Ca+2 concentration within the sarcoplasm leading to muscle fiber contraction (Bottinelli & Reggiani, 2000). 2.6 Fish Exercises Animals show many locomotory strategies which allow them to survive in various ecological niches. Many factors, such as predator–prey interactions, reproductive behavior and habitat distributions are of profound ecological importance that depends heavily on an animal’s capacity for movement (Baker, 1978). Some animals move slowly, others move extremely fast, whereas others can reach high velocities but show very little stamina (Bennett, 1991). Thus, both the endurance and burst activity capacities of an animal are important determinants of many lifehistory characteristics (Wood, 1991).
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Swimming requires energy for its muscular work, provided primarily by the large blocks of axial muscle arrayed along each side of the body, known as myotomes, which are dominant anatomical feature of most fishes (McKenzie, 2011). Although active contraction occurs by the same ATP consuming molecular mechanisms as in other vertebrate skeletal muscle (McKenzie, 2011). However, stress was originally defined in general terms by Selye (1950) as cited by Wedemeyer et al., (1990) as “the sum of all the physiological responses by which an animal tries to maintain or reestablish a normal metabolism in the face of physical or chemical forces”. About 50 years ago, the study of exercise physiology in fish began with the pioneering works of Brett (1964) and Black (1966). They showed that fish have capacity for both aerobic sustained swimming and anaerobic burst type swimming. Fish are mobile, and can be easily manipulated to swim against the current, which makes them ideal subjects for exercise training (Davison, 1997; Johnston, 2001a). Since the first experiments of Brett and Black, teleost have become one of the most suitable vertebrates model for exercise studies (Kieffer, 2000). Swimming in fish is generally characterized as either aerobic or anaerobic (Beamish, 1978). During sustained exercises, muscle metabolism is generally aerobic, and is supported by the red musculature. During events such as predator-prey interactions and spawning migrations, the capacity of the red muscle is exceeded and burst-type exercise occurs. This burst-type action is largely supported by anaerobic glycolysis within the white muscles (Beamish 1978; Kieffer, 2000). Black (1962) and his colleagues showed that fish possess a large anaerobic capacity, and that the post exercise recovery process is much slower in fish as compared to mammalian species. In nature, fish must swim in order to capture prey, avoid predators and reproduce. The critical swimming speed (Ucrit) (Brett,
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1964) is often used to assess the impact of environmental factors such as temperature, hypoxia, diseases or contaminants on fish performance (Hammer, 1995; Plaut, 2001). 2.7 The Concept of Swimming Speeds Swimming performance is considered a primary determinant of survival in fish and other aquatic animals. According to Beamish (1978) swimming performance is classified into three categories: 1- Sustained swimming speed 2- Prolonged swimming speed 3- Burst swimming speed 2.7.1 Sustained Swimming Speed Sustained performances are those speeds that fish can maintain for long periods (more than 200 minutes) without muscular fatigue (Beamish, 1978). At sustained speeds, energy is supplied to slow oxidative (red) muscle fibers through aerobic processes. These fibers do not get fatigued and do not have high power output (Webb, 1984). Metabolic demand is matched by its supply and waste production is matched by its removal (Jones, 1982). A subcategory of sustained performance is cruising speed; these speeds are used by migrating fish or fish that are negatively buoyant and must swim to maintain their places in the water column. The maximum sustained speed is the highest velocity that a fish can maintain without eventually getting fatigued. Swim speeds above sustained speed fall into the prolonged or burst swimming categories. 2.7.2 Prolonged Swimming Speed Prolonged performances are those speeds that fish can maintain for 20 seconds to 200 minutes and ends with fatigue (Beamish, 1978). The prolonged category spans the swimming speeds between sustained and
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burst. At prolonged speeds, energy is supplied to slow (red) and /or fast glycolytic (white) fibers through aerobic and anaerobic processes, respectively. As speed increases, so does anaerobic metabolism (Webb, 1984). 2.7.3 Burst Swimming Speed Burst performances are the highest speeds attainable by fish and can be maintained for only short periods of time (less than 20 seconds) (Beamish, 1978). At burst speeds, energy is primarily supplied to myotomal (body) white muscle through anaerobic processes (Webb, 1984). The conclusion of short periods of burst swimming occurs as a result of the exhaustion of extracellular energy supplies or accumulation of waste products (Colvavecchia et al., 1998). Fish often use burst speeds to pass through short high velocity areas, such as the inlet or outlet of a culvert. Median and paired fins tend to power slow swimming which is supplemented and then replaced with body and caudal (tail) fin undulation swimming at higher speeds and for acceleration (Webb, 1984). At burst speeds, the caudal fin is expanded and made as rigid as possible (Nursall, 1962). When fish swim at low speeds, they modulate the frequency and amplitude of their body and caudal fin undulation and at a high speed; they only modulate frequency (Webb, 1971). 2.8 Recovery Time and Burst Swimming If fish must swim in burst mode to pass through high velocity areas, it is important to consider their capability to recover and perform multiple bouts of exhaustive swimming. The fish’s ability to swim in a burst mode may be limited to a short term (a few hours to a day) because some fish species require relatively long periods to recover from exhaustive exercise (Black et al., 1962). Additionally, some fish die after performing
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exhaustive exercise, for example, trout subjected to intensive exercise for six minutes had a mortality rate of 40 percent with the majority of death occurring four to eight hours post-exercise (Wood et al., 1983). Recovery from exhaustive exercise have revealed that species differences span several orders of magnitude (Boutilier et al., 1993; Keiffer, et al., 2001), as in Salmonids which have high burst speeds but appear to recover relatively slowly. Paulik et al., (1957) found that Coho salmon recovery from exhaustive exercise was 67 percent after three hours and full recovery occurred after 18 to 24 hours. These fishes were forced to swim until they were unable to maintain their position upstream of an electrical shock organ. On the other hand, Steelheads were able to re-perform their baseline swim speed after six hours (Paulik & DeLacy, 1957). It is thought that species differences in recovery rates reflect the differences in ecological requirements, morphology, and behavioral differences (Keiffer, 2000). 2.9 Metabolic Rate (MR) Metabolism can be refered as the sum of the all physical and chemical processes that occure in the body of the organism by which materials are either produced (anabolism) or degradad (catabolism) to produce energy. All metabolic processes require energy which is delivred from the hydrolysis of ATP, whether a meal is being digested and converted into body tissues as stored energy (lipids, protein or glycogen) or it is being synthesized to produce energy for locomotion (Svendsen et al., 2010). Metabolism is the physiological engine that powers all activities such as swimming, growth and reproduction (Neill et al., 1994). Different terms of metabolism used in fish are shown in Figure (2.1).
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Figure 2.1 Illustrate different metabolic terminology in fish such as standard (SMR) and routine metabolic rate (RMR). The upper solid line represents the maximum metabolic rate (MMR) and the dashed line represents the active metabolic rate (AMR). The difference between MMR and SMR define as the metabolic scope for activity (MS). 2.9.1 Standard (SMR) and Basal Metabolic Rate (BMR) Standard metabolic rate applies to a post-absorptive, in thermally acclimated organism at rest, and may be considered the minimum metabolic rate for organism maintenance (Fry 1971; Brett & Groves 1979). Krogh (1916) subsequently reserved the term of basal metabolic rate for the lowest rate of oxygen consumption of an organ that is not performing any work. Therefore, BMR was impossible to measure for a whole organism, because the circulatory and respiratory functions required to maintain an animal alive involved some expenditure of energy above BMR. Instead, he preferred the term SMR for whole organisms exhibiting minimal functional activity, i.e. in total absence of voluntary muscular movements and when no food was being digested or absorbed (Krogh, 1914).
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Two methods are used for determining SMR: one is indirect, whereas the other is direct. In the first, a power-performance curve that relating the logarithm of oxygen consumption rate with the relative swimming speed which is constructed from data obtained from swim tunnel or annular respirometer. The SMR can be estimated by extrapolating the slope of the curve back to zero activity (Leonard et al., 1999; Lowe, 2001). However, extrapolation does not take into account physiological differences between active and quiescent fish, specifically the induction of anaerobic metabolism during high-velocity swimming (Cech, 1990). The second option for measuring SMR is to confine the fish in a flowthrough box respirometer and measure the decrease in oxygen concentration between the inflow and outflow of water streams (Hopkins & Cech, 1994; Gibb & Ferry, 2001). This method is suitable for sedentary, and quiescent animals (Hopkins & Cech, 1994). 2.9.2 Active Metabolic Rate (AMR) Active metabolic rate (AMR) is defined as the rate of oxygen consumption (MO2) at a maximum sustained swimming speed (a swimming speed, which can be maintained for at least 15 min) and it is measured using a standardized protocol known as a critical swimming test (Ucrit) (Brett, 1972; Brett & Groves, 1979). From energy budget point of view, AMR represents the energy used for swimming related activities independent of the speed (Schurmann & Steffensen, 1997). 2.9.3 Maximum Metabolic Rate (MMR) Maximum metabolic rate is gained by enlarging gill ventilation and blood flow to insure an optimal oxygen delivery to the mitochondria and ATP supply to the energy demanding tissue (Frisk et al., 2012). Maximum metabolic rate can be measured either during exhaustive exercise normally
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within a critical swimming speed test (Ucrit) or immediately following exhaustive exercise (Schurmann & Steffensen, 1997). The relative merits of Ucrit versus exhaustive exercise approach to determining MMR are debatable (Roche et al., 2013). 2.9.4 Routine Metabolic Rate (RMR) Routine metabolic rate (RMR) is the mean metabolic rate observed in an organism performing random physical activity over a given period of time (Fry, 1971; Carlson et al., 1999). A number of studies have reported that RMR values are about 1.5-3 times the basal or standard metabolic rate (Brett & Blackburn, 1978; Lowe, 2002). This increase in metabolic rate over SMR is primarily due to the costs of powering the swimming muscles during routine activity (Fry, 1971).
2.9.5 Metabolic Scope (Aerobic scope) The metabolic scope (MS) is the difference between MMR and SMR and includes the determination of the amount of oxygen available for aerobic activities such as swimming, digestion, growth and reproduction (Fry, 1971). 2.10 Respiratory burst (RB) Respiratory burst describes a sudden rise in oxygen consumption of phagocytes upon contact with microorganisms or stimulation with, e.g. anaphylatoxins, immune complexes or proinflammatory cytokines. It is the physiological equivalent to superoxide anion production by NADPH oxidase and is essential to initiate the killing reaction during phagocytosis (Yang, et al., 2016). The respiratory burst involves the formation of O2through the enzyme Nicotinamide adenine dinucleotide phosphate
112
(NADPH) oxidase system (Babior, 1999). The activation of enzyme NADPH oxidase depends on: Increased oxygen consumption (MO2); Increased production of hydrogen peroxide (H2O2); Formation of superoxide anion (O2-). The mechanism of the RB implies the action of an enzyme NADPH oxidase; which is able to employ the NADPH as a reducing agent for reducing the free oxygen (O2) to the superoxide (O2-), which spontaneously combines with other molecules to produce reactive free radicals (Lopia, 2014). The 'NADPH oxidase, is an enzyme composed of various multimeric subunits: gp91-phox, p22-phox, p40-phox, p47-phox, p67-phox, Rap 1A and Rac2. These subunits are often distributed in the cytosol or
anchored to the cell membrane. The gp91-phox and p22-phox form a complex called cytochrome b558, which is located at the level of the membrane, while the phox (phagocyte oxidase) and accessory proteins Rac2 (a GTPase) and Rap (Ras-related protein: protein linked to the RAS) are found in the cytoplasm (Camilo, et al., 2012). When the cell activates the respiratory burst, oxidase still remain on the membrane of phagosomes and began his work. The oxidase extracts a pair of electrons from' NADPH transforming it into NADP+ and captures them in a chain of electrons (Garcia et al., 2001). This mechanism causes an increase in oxygen consumption and energy by the cell causing a rapid increase in the concentration of protons. These protons are captured by the gp91-phox component that also acts as a channel of the protons (Carcia et al., 2001).
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The peroxide radical reacts with the enzyme superoxide dismutase (SOD) to form hydrogen peroxide (H2O2), which in turn acts as a substrate for the peroxidase enzyme oxidizing the Cl-, Br- and I- to hypo chloride ( ClO-), hypo bromide (brO-) and hypo iodide (IO-), which are highly toxic to microorganisms. The RB in cells is so powerful and violent that most often causes death of the cell, forming free radicals and redox imbalance. The RB is a test to exploit made of tests that exploit the amplified chemiluminescence from luminol. Chemiluminescence is the emission of photons (in particular in the visible and near infrared) that may accompany a chemical reaction. Through this technology, we can evaluate the production of ROS at the level of leukocytes can be evaluated (Kalgraff et al., 2011). The observation of the effects of the RB, in RLU (Relative Light Unit values for the unit of light) can be magnified in animals subjected to controlled effort, like swimming (Bobadilla et al., 2008).
2.11 Digestive System Organization 2.11.1 Topographical Regions of Digestive System The definition of the constitutes and the gut varies somewhat between different authors. Sometimes it is defined as the gastrointestinal tract (i.e. The gut is a tubular structure beginning at the mouth and ending at the anus) (Harder, 1975), whereas other, state that the gut also includes the accessory organs (i.e. liver, salivary glands, biliary system, and pancreas) (Gräns, 2012). The gut is commonly divided into four parts, the most anterior part, the head gut, is most often considered in terms of its two components, the oral (buccal) and pharyngea cavities (Harder, 1975; Kapoor et al., 1975). The foregut begins at the posterior edge of the gills
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and includes the oesophagus, stomach, and the pylorus. In fish, such as the cyprinus caprio, which lack both a stomach and pylorus. The foregut consists of the oesophagus and an intestine anterior to the opening of the bile duct. The midgut includes the intestine posterior to the pylorus, often with no distinct demarcation posteriorly between it and the hindgut. The midgut often includes a variable number of pyloric caecae (pyloric appendages) near the pylorus, although pyloric caecae are always absent in fishes which lack stomachs (Harder, 1975). The midgut is always the longest portion of the gut and may be coiled into complicated loops. The posterior end of the hindgut is the anus. Only rarely there is a hindgut caecum in fish comparable to that found in mammals. A cloaca never present in teleost fish, except the Dipnoi, although it is universal in sharks and rays (Harder, 1975; Kapoor et al., 1975).. 2.11.2 Functions of Digestive System The digestive system is responsable for the digestion of food and absorption of nutreints into the bloodstream, whilst undigested food are transported out of the body (Jutfelt, 2011). This is achieved through controlled digestive secretions, complex motility patterns and regulated perfusion. Secretions of digestive enzymes are essential for the breakdown of complex food as well as preventing the digestion of gut tissues ( Boyle et al., 2006). Gut motility helps to mix, break down and transport the food through the gut (Jutfelt, 2011). 2.11.3 Histology of Intestine in Common carp Despite of the presence of many specialized regions in the gastro intestinal tract, cross-sectional tissue organization remains more or less the same throughout the intestines of vertebrates which consists of four tissue layers: the mucosa, the submucosa, the muscular layers and the serosa
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(Olsson & Holmgren 2001; Olsson 2011). The vertebrate intestine consists of several histologically distinct tissue layers with correspondingly distinct functions. (Gallery, 2014). Epithelium usually lining the lumen, forming a barrier between the exterior and interior media of the gut, which is attached to the connective tissue layer of the basement membrane (Nilsson, 1983). Fish intestinal epithelia are also expanded through folding, but lack the typical crypts of the mammalian villi (Clements & Raubenheimer 2006). The term mucosal folds will hence be used when referring to the fish epithelial folding. The epithelium, together with the underlying lamina propria constitutes the mucosa. Adjacent to the lamina propria is the connective and contractile muscularis mucosa which separates the lamina propria from the submucosa. Within the submucosal layer, the submucosal nerve plexus is found. Further away from the lumen, the circular muscle layer is followed by the myenteric nerve plexus and the longitudinal muscle layer. The perimeter of the intestine is lined by the serosa, a connective tissue layer attached to the mesenteric tissue (Clements & Raubenheimer 2006). 2.11.4 Histology of Ventral Aorta in Common carp The heart of teleost fish consists of four compartments, two contracting muscular chambers, the atrium and the ventricle, which propel blood into the vasculature and two collecting cavities, the sinus venosus and the bulbus arteriosus which connect the heart to the vasculature (Santer, 1985). The sinus venosus receives oxygen-poor venous blood from the body and directs it to the atrium through an opening guarded by the sinoatrial valve, while the bulbus arteriosus provides a blood pressure for stabilizing and compliant exit route for blood from the ventricle to the ventral aorta and further to the gills (Santer 1985; Olson & Farrell 2006).
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The arteries afferent to the gills have normal vertebrate arterial structure made up of three layers: adventitia on the outside, media and intima. These layers differ in development depending on the vessel and its function. The endothelium of the intima is comprised of flattened cells which can usually be distinguished only by their dark-staining nuclei which bulge into the lumen (Olson & Farrell 2006). Contiguous cells interdigitate so that the endothelium forms a continuous surface. There is a fine basement membrane beneath the endothelium, but this is visible only with the electron microscope. The intima is largely elastic tissue and the media is composed of elastic tissue laminae, or fibers, with smooth muscle cells in between. Adventitia is made up of fibroblasts and collagenous fibers (Mumford et al., 2007). 2.12 Smooth Muscle Cells 2.12.1 Structure of Smooth Muscle The smooth muscle fiber is a spindle shaped cell with a diameter ranging from 2 to 10μm, as compared to a range of 10 to 100μm for skeletal- muscle fibers. Smooth-muscle fibers have a single nucleus with the capacity to divide throughout the life (Vander et al., 2001). Numerous small invaginations (caveolae) found in the cell membrane significantly increase the surface area of the cell. The sarcoplasmic reticulum (SR) is poorly developed as compared with the SR found in cardiac myocytes and skeletal muscle cells (Klabunde, 2005). Although both thick and thin filaments are present, they are not aligned with each other and with no microscopic visible striations or sarcomeres. The Z discs are absent; instead, the thin filaments are attached by cytoskeleton to dense bodies, which are small masses of protein scattered throughout the sarcoplasm and on the inner face of the sarcolemma (Saladdin, 2003). Although smooth
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muscle is slower to contract than skeletal muscle, it can sustain prolonged contractions and does not fatigue easily (Mader, 2004). 2.12.2 Types of Smooth Muscles There are considerable variations in the structure and function of smooth muscles in different parts of the body. In general, smooth muscles can be divided into multi-unit smooth muscle and urinary or (visceral) smooth muscle (Ganong, 2003). Multiunit SMC is composed of discrete, separate smooth muscle fiber, each can contract independently of the others and it is often innervated by autonomic nervous system (ANS) (Guyton & Hall, 2006). This type of smooth muscles must be stimulated by their nerve branches to initiate contraction. Therefore, these muscles are referred to as neurogenic (Kelly, 2005). Interestingly, nerve stimulation elicits graded potentials only, without the generation of action potentials. Multiunit SMCs are not electrically coupled, so that stimulation of one cell does not necessarily result in activation of the adjacent smooth muscle cell. The multiunit smooth muscle cells are present in vas deferens of the male genital tract, the iris of the eyes and the piloerector muscles that cause erection of the hairs (Berne et al., 2007). Visceral smooth muscle occurs as large sheets, with low-resistance bridges between individual cells, and they function in a cyncytial fashion. The bridges, like those in the cardiac muscle, are junctions where the membranes of the two adjacent cells fuse to form gap junctions (Ganong, 2003). Some cells of visceral smooth muscles only receive autonomic innervations and they also display intrinsic, or myogenic, electrical activity and contraction in response to stretch (Fox, 2006). Actions potentials generated in single-unit smooth muscles are propagated to other smooth muscle cells via gap junctions. Visceral smooth muscle is found primarily
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in the walls of the hollow visceral organs such as blood vessels, digestive tract, respiratory tract and reproductive tract (Saladin, 2003). Smooth muscle cells which exhibit rhythmic or intermittent activity are termed phasic smooth muscle and they includes smooth muscles in the wall of the gastrointestinal and urogenital tracts, and that correspond to the single-unit, because the smooth muscle cells contract in response to action potentials that propagate from one cell to another. On the other hand, smooth muscle that is continuously active is termed tonic smooth muscle, as those present in sphincters. The continuous partial activation of the tonic smooth muscle is not associated with action potentials generation, although it is proportional to the membrane potential. Tonic smooth muscle would thus correspond to the multiunit smooth muscle (Germann & Stanfield, 2005; Berne et al., 2007). 2.12.3 Smooth Muscle Receptors Cells respond to several signals such as hormones, growth factors drugs...etc, which cannot enter the cell readily. The molecules can produce cellular responses in the target cells by binding to their respective specific receptors (Guytun & Hall, 2006). The signal transduction mechanism of these ligand may include second messenger systems that phosphorylate different protein kinase and change membrane permeability to ions (Vander et al., 2001; Guytun & Hall, 2006). 2.12.3.1 Intestinal Cholinoreceptors Intestinal smooth muscle are similar to those of the vascular smooth muscle cells (VSMCs), but different from them in being mostly under cholinergic
control.
Cholinergic
control
is
performed
by
the
neurotransmitter acetylcholine (ACh). There are two main classes of cholinergic receptors: namely nicotinic and muscarinic receptors (Vladimir
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et al., 2010). Both subtypes share the property of being activated by the endogenous neurotransmitter ACh, and they are expressed by both neuronal and nonneuronal cells throughout the body (Albuquerque et al., 2009). 2.12.3.1.1 Nicotinic ACh Receptors The nicotinic acetylcholine receptor (nAChR), are present at the neuromuscular junction, which is characterized by having Ligand-gated ion channels (Unwin, 2003). It is composed of five subunits, namely: two α1 and one of each β1, δ, and γ subunits. Each subunit has four transmembrane domains (M1-M4). Collectively, these subunits are present in various cell types, which extending from skeletal muscles to other nonneuronal cells in skin, pancreas, and lung to neurons in the central and peripheral nervous systems (Albuquerque et al., 2009). Based on their primary sites of expression, the nAChRs are classified into muscle type and neuronal type. The muscle type nAChR is located in at the neuromuscular junction of somatic muscles; and stimulation of this receptor leads to muscle contraction. The neuronal type nAChR is found in the nervous system and it is involved in neurotransmission and regulation of neuronal activity (Jimenez, 2013). When ACh molecules bind to both α subunits, a conformational change occurs in the receptors, which results in an increase in channel conductance for Na+, leading to depolarization of the postsynaptic membrane. This depolarization is a result of the strong inward electrical and chemical gradient for Na+, which predominated over the outward gradient for K+ ions and results in a net inward current (Rhoades & Bell, 2009).
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2.12.3.1.2 Muscarinic ACh Receptors Muscarinic acetylcholine receptors (mAChRs) are found in the brain, ganglion, smooth muscles, cardiac muscle and the cells of particular glands. Because they are coupled to heterotrimeric G-proteins, the structure and function of these receptors are different from those of nAChR (Fox, 2006). The five muscarinic subtypes (M1-M5) are grouped on the basis of intracellular signaling pathway that is activated by a ligand binding (Strang et al., 2010). Activation of mAChR is relatively slow (milliseconds to seconds) and depending on the segments. They directly alter cellular homeostasis of phospholipase C (PLC), inositol trisphosphate (IP3), cyclic adenosine monophosphate (cAMP), and free Ca+2 (Eglen, 2005). However, in the small intestine, all types of muscarinic receptors are present (Pilija et al., 2010). The most numerous type present in the smooth muscles are the subtype M2 and M3. Since M3 receptors that interact with Gq to trigger phosphoinositide hydrolysis, Ca+2 mobilization and a direct contractile response. In contrast, M2 receptors interact with Gi to inhibit adenylyl cyclase and Ca+2-activated K+ channels to potentiate a Ca+2 dependent, nonselective cation conductance (Ehlert, 2003). Recently, Kishore & Rahman (2012) demonstrated that ACh induced contractions in the rat ileum involve two different mechanisms coupled to muscarinic receptors. One mechanism activates non-selective cation channels in the plasma membrane, which results in membrane depolarization. The depolarization stimulates Ca+2 influx through voltage gated Ca+2 channels (VGCCs). The other mechanism activates contraction by the release of intracellular calcium.
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2.12.5 Physiology of the Smooth Muscle 2.12.5.1 Smooth Muscle Cell Contraction Smooth muscle contraction is controlled by cytopasmic Ca+2 level (Thorneloe & Nelson, 2005), which is initiated by elevation of cytosolic Ca+2concentration (Fox, 2006), in response either to primary change in membrane potential, or to chemical events that is not necessarily involve changes
in
membrane
potential
(Ackermann,
2002).
The
electromechanical excitation is initiated by depolarization of the cell membrane that is sufficient to activate voltage gated Ca+2 channels. On the other hand, pharmacomechanical coupling are chemically induce smooth muscles contraction, which is mediated by plasma membrane receptors and activation of either membrane Ca+2 channels or its action as a second messenger, such as inositol triphosphate (IP3) that opens specific channels that release Ca+2 from SR (Mohrman & Heller, 2007). However elevation of cytosolic Ca+2 concentrations in response to specific stimuli causes Ca+2 to bind to calmodulin forming Ca+2- calmodulin complex. This causes a conformational change in calmodulin, allowing interaction of Ca +2calmodulin complex with myosin light chain kinase (MLCK) (Hilgers & Webb, 2005). This interaction results in a conformational change in the calmodulin- MLCK complex to expose the catalytic site. This enzyme catalyzes the phosphorylation of myocin light chain kinase protein which is a part of the cross-bridge head of myosin, which enables cross-bridge formation and tension development as a result of ATP utilization (Ganong, 2003; Hilgers & Webb, 2005). Hydrolysis of ATP convert the myosin head into high energy state, and produces ADP and inorganic phosphate (Pi), which are bounded to myosin head. Following the interaction of myosin head to actin filaments,
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causing sliding of actin over myosin and subsequent force development. Finally, a new molecule of ATP binds to myosin head. The cross-bridge cycling continues, with the hydrolysis of one ATP per cycle until cytoplasmic Ca+2 concentration falls. Decreases in Ca+2 concentration leads to inactivation of MLCK, and the cross-bridge is dephosphorelation by myosin phosphatase (Berne el al., 2007). The contraction of smooth muscle cells is slow and sustained. Myosin ATPase in smooth muscle is slow in splitting ATP, than in striated muscle. Thus, the cross-bridge of smooth muscles can enter in latch state which allows smooth muscles to maintain prolonged tonic contraction with low energy expenditure (Fox, 2006). 2.12.5.2 Molecular Basis of Relaxation Smooth muscle relaxation occurs either as a result of removal of the stimulus or by the direct action of a substance that inhibits the contractile mechanism. The process of relaxation requires a decreased intracellular Ca+2 concentrations and increased MLC phosphatase activity (Webb, 2003). Removal of Ca+2 from the cytosol to bring about relaxation is achieved by the active transport of Ca+2 back into the SR as well as out of the cell across the plasma membrane. The rate of Ca+2 removal in smooth muscle is much slower than in skeletal muscle, with the result that a single twitch lasts several seconds in smooth muscle (Vander et al., 2001). The opening of K+ channels increase the conductance of K+ across the SMCs membrane, producing net movement of K+ from the cytoplasm to the extracellular space, which is driven by electrochemical and concentration gradients. The loss of the positively charged K+ ions hyperpolarizes the SMCs, which in turn closes voltage gated Ca+2 channels. The closing of
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these channels result in the relaxation of the SMCs due to decreased cytoplasmic Ca+2concentration (Marrelli et al., 1998). 2.13 Smooth Muscle Ion Channels 2.13.1 Potassium Channels Ion channels are integral membrane proteins that span in the lipid bilayers to form a central pore through which selected ions can pass at a limited diffusion rates (ca. 107 ions channel-1 s-1) (Hille, 2001). Potassium channels are considered as a large family of ion channels that share a common property of selectivity for K+ over Na+ ions. As a result of advances in structural and computational biology, K channels provide a paradigm for the study of ion channels and membrane transport proteins (Hille, 2001). 2.13.1.1 Voltage-dependent K+ Channels (KV channels) Voltage-dependent K+ channels belong to the super family of voltagegated channels. The KV channels open when the membrane potential of the cell is depolarized. They are important regulators for smooth muscle membrane potentials, and they are also activated by dilation acting via cAMP signaling pathway such as adenosine (Yellen, 2002). Conversely, the close of KV channels through signaling pathway involving protein kinase C and calcium ions (Pintérová et al., 2011). The channels open to allow an efflux of K+ in response to membrane depolarization, resulting in repolarization and returning the membrane to resting potential (Korovkina & England, 2002). Pharmacological blockers of KV channels are 4aminopyridine (Matsushita et al., 2006).
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2.13.1.2 Ca2+-activated K+ channels (KCa) The history of Ca2+ -activated K+ channels date back to the 1970 s, when Meech first reported that intracellular Ca2+ injection leads to an increase in K+ conductance in nerve cells (Souza et al., 2013). The opening of K+ channels repolarizes the membrane potential and reduces Ca2+ entry into the cell by closing voltage-dependent Ca2+channels, which regulate various physiological processes including neurotransmitter release in synapses, contraction of SMCs in airway and blood vessels (Cui, 2010). Three subtypes of KCa channels exist, differing in both their voltage sensitivity and single-channel conductance: large-conductance KCa (BKCa), intermediate-conductance KCa (IKCa) and small-conductance KCa (SKCa) (Uhiara, 2012). KCa channel may be blocked by external TEA, iberiotoxin, and charybdotoxin, respectively (Park et al., 2007). 2.13.1.3 ATP-Sensitive K+ Channels (KATP) ATP-sensitive K+ channels were first identified in cardiac myocytes using patch clamp technique (Teramoto, 2006). Furthermore, he added that several endogenous agonists (such as calcitonin gene-related peptide (CGRP),
adenosine,
etc.)
activate
KATP
channels
leading
to
hyperpolarization and relaxation. The KATP channels are regulated by other intracellular signals including ADP and Ca2+. Adenosine triphosphatesensitive K+ channels may be activated by protein kinase A and cGMPdependent protein kinase. Conversely, activation of protein kinase C and elevation of intracellular calcium ion concentration (Pintérová et al., 2011). The blocking of KATP channels with sulphonylurea drugs such as glibenclamide routinely revealed in the treatment of Type II diabetes (Uhiara, 2012).
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2.13.1.4 Inward Rectifier K+ Channels (KIR) Inward rectifier K+ channels are present in a variety of excitable and non-excitable cells. The KIR channels are activated by membrane hyper polarization in contrast to the KV and KCa channels, which are activated by membrane depolarization (Nevala, 2001). Smooth muscle KIR channels which served as sensor for increasing extracellular K+ concentration, leading to membrane hyper polarization and vasodilation (Jackson, 2005). Inward rectifier K+ channels currents in VSMCs are blocked externally by Ba2+ blocks in a voltage-dependent fashion (Ko et al., 2008).
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3. MATERIALS AND METHODS 3.1 Materials 3.1.1 Experimental Animals 3.1.1.1 Sea bass (Dicentrarchus labrax, L) Adult sea bass (Dicentrarchus labrax, L), from both sexes, weighting (75-110g) was obtained from Nuova Azzurro commercial hatchery (Civitavecchia, Roma, Italy). The fishes were transported to the animal house of the Department of Biotechnology and Molecular Sciences, Faculty of Science, University of Insubria (Varese), Milan, Italy. The fishes were stocked into indoor tanks (3x1x1m) of 2500 L capacity. They were acclimated to laboratory conditions for 90 days before starting the experimental work. The tanks were connected to a sea water recirculation system, and the water conditions were strictly controlled as follow: - Air Temperature 24 oC.
- Water Temperature: 21.8±0.9 oC.
- PH 7.
-Total Ammonia below 0.2 mg/L.
- Nitrite 0.05) affected by the presence 1.5% taurine in the diets. Also there was none significant interaction between the fish's fed on commercial pellets with and without 1.5% taurine and the 1 st and 2nd swimming exercise on the SGR (F = 0.15. P value = 0.856 and r = 0.17).
75
Chapter Four …...…………….........................………… Results
SGR (% weight/day)
0.6
1 s t Experiment
0.4
Control 0.2
Taurine 1.5%
0.0 T1
T2
T3
Figure 4.7 Shows the specific growth rate in fish's fed either on FM substituted feed, or on the control diet plus 1.5% taurine for 34days before and after swimming performance. The results of Table (4.2) and Figure (4.8) indicate that after 30 days (T1) of feeding the fish's on control diet with 1.5% taurine, there was a significant gradual increase in the SGR with (F=0.9; P value =0.0312 and r =0.33) as compared with fish's fed on commercial pellets alone. On the other hands, the SGR after exercising the fish's in the swimming respirometer (T3) was highly significant (P 0.05) in the level of critical swimming speed Ucrit-1 after feeding the fish's commercial pellets with and without 1.5% taurine for 30 days. However, after 64 days of fish's feeding there was significant increases (P < 0.05) in the level of Ucrit-2 in fish's fed on control diet plus 1.5% taurine as compared with the control group. On the
80
Chapter Four …...…………….........................………… Results other hands, There was no significant interaction between Ucrit-1 and Ucrit-2 (F = 2.26; P value = 0.12 and r = 0.16) 2 nd Experiment 4.9
Ucrit (BL/s)
4.2
a
a
a
b
3.5 2.8
Control
2.1 1.4
Taurine 1.5%
0.7 0.0 T2 (Ucrit1)
T3 (Ucrit2)
Figure 4.12 Shows the critical swimming speeds Ucrit-1 and Ucrit-2 in fish's fed either on FM substituted feed, or on the control diet plus 1.5% taurine after 64 days of feeding before and after swimming performance. 4.2.2 The Effect of Taurine on the Rate of Oxygen Consumption Tables (4.3, 4.4 and 4.7) and Figures (4.13, 4.14 and 4.15) show the rates of MO2 in fish's fed on commercial pellets with and without 1.5% taurine for 15 days. After exercising the fish's in the swim chamber respirometer there was a non-significantly (P>0.05) decrement in the rate of MO2 and it can be described by the linear model as MO2 for the control = 54.29x +139.8. r² = 0.987, and MO2 = 50.60x + 140.5. r² = 0.994 for diet containing 1.5% taurine. After 34 days in (2nd swimming training), the rate of MO2 remained more or less the same without any changes and it can be described by the linear model as MO2 for the control = 50.60x + 140.5. r² = 0.994 and MO2 for fish's fed on normal diets containing 1.5% taurine= 49.02x + 148.6. r² = 0.990. There was no significant interaction in the level of MO2 between the first and second trails of swimming performances (F = 0.051; P value = 0.82 and r = 0.2)
81
Chapter Four …...…………….........................………… Results
MO2 (mg O2kg-1hr-1)
400
1 st Experiment
300
200
Control 100
Taurine 1.5% 0 T2
T3
Figure 4.13 Shows the rate of oxygen consumption (mg O₂ kg-1 hr-1) in fish fed either on control FM substituted feed, or on the control plus 1.5% taurine after 34 days of feeding before and after swimming performance. 400
1st swimming performance/ 2nd part
MO2 (mg O2 kg-1 hr-1)
350 300 250 200 150 100
Control Taurine 1,5%
50 0 0.0
0.7
1.4
2.1
2.8
3.5
4.2
4.9
Speed (BL s -1)
Figure 4.14 Shows the rate of oxygen consumption as a function of swimming velocity for fish's fed on commercial pellets and those fed on commercial pellets plus 1.5% taurine. The curves were fitted by fish's fed on commercial pellets. y = 50.60x +140.5 (r² = 0.994) and those fed on commercial pellets plus 1.5% taurine. y = 54.29x+ 139.8 (r² = 0.987). Table 4.3 Shows the rates of MO2 (mg O₂ kg-1hr-1) at each of the following swimming speed (0.7, 1.4, 2.1, 2.8, 3.5 BL/s) in 1 st part of experiments after feeding the fish's on control FM substituted feed, or on the control plus 1.5% taurine for fifteen days (T2). T2 Velocity (BL/s) 0.7 1.4 2.1 2.8 3.5 4.2
MO2 (mg O₂ kg-1hr-1) Control Taurine 185 170 215 210 249 244 288 288 319 322 380 348
82
Chapter Four …...…………….........................………… Results
400
2nd swimming performance/1st part
MO2(mg O2 kg -1hr -1)
350 300 250 200 150 100
Control Taurine 1,5%
50 0 0.0
0.7
1.4
2.1
2.8
3.5
4.2
4.9
Speed (BL s -1)
Figure 4.15 Shows the rate of oxygen consumption as a function of swimming velocity for fish's fed on commercial pellets and those fed on commercial pellets plus 1.5% taurine. The curves were fitted by fish's fed on commercial pellets. y= 49.02x + 148.6 (r² = 0.990) and those fed on commercial pellets plus 1.5% taurine. y= 50.60x +140.5 (r² = 0.994). Table 4.4 Shows the rates of MO2 (mg O₂ kg-1 hr-1) at each of the following swimming speed (0.7, 1.4, 2.1, 2.8, 3.5 BL/s) in 1 st part of experiments after feeding the fish's on control FM substituted feed, or on the control plus 1.5% taurine for thirty four days (T3). T3 Velocity (BL/s) 0.7 1.4 2.1 2.8 3.5 4.2
MO2 (mg O₂ kg-1hr-1) Control Taurine 174 183 212 215 245 248 289 294 311 326 350 346
Tables (4.5, 4.6 and 4.8) and Figures (4.16, 4.17 and 4.18) demonstrate the rate of MO2 in fish's fed on commercial pellets with and without 1.5% taurine for 64 days after exercising the fish's in the swimming chamber respirometer. Two-way analysis of variance ANOVA revealed that there is no significant differences (P > 0.05) in the rate of MO2 in fish's fed commercial pellets with and without 1.5% taurine and its linear model can be described by MO2 for the control after the 1 st swimming performances (T2), y = 55.68x + 135.1. r² = 0.992 and for fish's fed on 83
Chapter Four …...…………….........................………… Results control diet plus 1.5% taurine was y = 48.04x+148.6. r² = 0.997. The rate of MO2 in the 2nd swimming performance (T3) was diminished nonsignificantly (P>0.05) in fish's fed on 1.5% taurine. The linear models of the fish fed on commercial pellets was y = 41.51x + 207.8. r² = 0.992 and the linear regression of fish's fed control diet plus 1.5% taurine was y = 39.62x + 186.7. r² = 0.899. There was no significant interaction in the rate of MO2 between the two groups (F = 0.37; P value = 0.54 and r = 0.2). 400
MO2 (mg O2 kg-1 hr-1)
2 nd Experiment 300
200
Control
100
Taurine 1.5% 0 T2
T3
Figure 4.16, Shows the rate of oxygen consumption (mg O₂ kg-1 hr-1) in fish fed either on control FM substituted feed, or on the control diet plus 1.5% taurine after 64 days of feeding before and after swimming performance. 1st swimming performance/2nd part
400
MO2 (mg O2 kg-1hr-1)
350 300 250 200 150 100
Control Taurine 1,5%
50 0 0.0
0.7
1.4
2.1
2.8
3.5
4.2
4.9
Speed (BL s -1)
Figure 4.17 Shows the rate of oxygen consumption as a function of swimming velocity for fish's fed on commercial pellets and those fed on commercial pellets plus 1.5% taurine. The curves were fitted by fish's fed on commercial pellets. y = 55.68x + 135.1 (r² = 0.992) and those fed on commercial pellets plus 1.5% taurine. y = 48.04x + 148.6 (r² = 0.997). Table 4.5 Shows the rate of MO2 (mg O₂ kg-1hr-1) at each of the following swimming speed (0.7, 1.4, 2.1, 2.8, 3.5 BL/s) in 2 nd part of
84
Chapter Four …...…………….........................………… Results experiments after feeding the fish's on control FM substituted feed, or on the control plus 1.5% taurine for thirty days (T2). MO2 (mg O₂ kg-1hr-1) Control Taurine 180 181 209 217 252 243 289 286 321 319 378 349
T2 Velocity (BL/s) 0.7 1.4 2.1 2.8 3.5 4.2
2ndswimming performance/2nd part
400
MO2 (mg O2 kg-1hr-1)
350 300 250 200 150 100
Control Taurine 1,5%
50 0 0.0
0.7
1.4
2.1
2.8
3.5
4.2
4.9
Speed (BL s -1)
Figure 4.18 Shows the rate of oxygen consumption as a function of swimming velocity for fish's fed on commercial pellets and those fed on commercial pellets plus 1.5% taurine. The curves were fitted by fish's fed on commercial pellets. y = 41.51 x + 207.8 (r² = 0.992) and those fed on commercial pellets plus 1.5% taurine. y = 39.62x + 186.7 (r² = 0.899). Table 4.6 Shows the rates of MO2 (mg O2/kg/hr) at each of the following swimming speed (0.7, 1.4, 2.1, 2.8, 3.5 BL/s) in 2 nd part of experiments after feeding the fish's on control FM substituted feed, or on the control plus 1.5% taurine for sixty four days (T3). T3 Velocity (BL/s) 0.7 1.4 2.1 2.8 3.5 4.2
MO2 (mg O₂ kg-1hr-1) Control Taurine 235 202 271 274 287 254 327 290 357 327 379 359
85
Chapter Four …...…………….........................………… Results 4.2.3 The Effect of Taurine on Standard Metabolic Rate The results of the effect of taurine on SMR in the 1st part of experiment in Table (4.7) and Figure (4.19) revealed obviously that after feeding the fish's on diet for 15 days (T2) caused a significant elevation, (P 0.05) in the SMR in the 1st swimming exercise (T2) between the fish's fed on commercial pellets and those fish's fed on commercial pellets with 1.5% taurine. On the other hands, after the 2nd swimming exercise (T3) there is a highly significant decrement (P0.05) between the fish's fed on commercial pellets and fish's fed on commercial pellets plus 1.5% taurine. Also there is no significant interaction in the level of RMR between the 1st and 2nd swimming respirometer (F = 0.4, P value 0.52 and r = 0.27). 1 st Part
RMR (mg O2 kg-1 hr-1)
300
200
Control
100
Taurine 1.5%
0
T2
T3
Figure4.21 Shows the routine metabolic rate (mg O₂ kg-1 hr-1) in fish fed either on control FM substituted feed, or fed on the control diet plus 1.5% taurine after 34 days of feeding before and after swimming performance.
87
Chapter Four …...…………….........................………… Results Table (4.8) and Figure (4.22) reveal the RMR in the 2nd experiment, after feeding the fish's with commercial pellets with and without 1.5% taurine for 30 days. The mean RMR after the 1st training exercise T2 shows significant decreases (P>0.05) in fish's fed commercial pellets with 1.5% taurine as compared with the control group which were 245.4 ± 3.187 and 269.4 ± 2.985 respectively, whereas after 34 days of the of the 1 st swimming exercise, the fishes were used for the 2nd exercise (T3) in which the RMR remain unchanged (P>0.05) in the fish's feed with commercial pellets plus 1.5% taurine when compared with the fish's
feed with
commercial pellets alone.
RMR (mg O2 kg-1hr-1)
400
2 nd Part a
300
b
a
c
200
Control 100
Taurine 1.5% 0 T2
T3
Figure4.22. Shows the routine metabolic rate (mg O₂ kg-1 hr-1) in fish's fed either on control FM substituted feed, or fed on the control diet plus 1.5% taurine after 64 days of feeding before and after swimming performance. 4.2.5 Effect of Taurine on Active Metabolic Rate The results of Table (4.7) and Figure (4.23) reveal that AMR in fish's fed on control diet with 1.5% taurine for 15 days significantly decreased (P>0.001) after 1st swimming exercise (T2) when compared with the fish fed on commercial pellets alone. The AMR was 346.0 ± 1.646 for taurine group and 379.8 ± 4.35, for the control group. However after 34 days of feeding, the level of AMR remain low in the fish's fed on control diet with 1.5% taurine, and the differenc was non-significant (P0.001) when compared with the control group. This decrease in the level of AMR remain low (P>0.001) after repeating the measurement of MO2 by the swimming chamber respirometer in the fish's fed on control diet plus 1.5% taurine for 64 days. Also a significant interaction AMR was found between the 1st and 2nd swimming respirometer (F= 0.704; P value 0.0132 and r = 0.11). AMR (mg O2 kg-1hr-1)
500 400
2 nd Part c
a
b
b
300 200
Control
100
Taurine 1.5%
0 T2
T3
Figure4.24 Shows the active metabolic rate (mg O₂ kg-1 hr-1) in fish's fed either on control FM substituted feed, or on the control diet plus 1.5% taurine after 64 days of feeding before and after swimming performance.
4.2.6 The Effect of Taurine on Cost of Transport
89
Chapter Four …...…………….........................………… Results The results of energy required for a fish to move a unit of distance are shown in Table (4.7) and Figures (4.25, 4.26 and4.27). The results reveal that after feeding the fish's with commercial pellets with and without 1.5% taurine for 15 and 34 days in the 1st and 2nd swimming exercise (T2 and T3) there is no significant changes (P0.05) in the fish's fed with control diet plus 1.5% taurine after the 2nd swimming performances. In addition, also there was no significant interaction between the two groups of fish's and the time of the 1st and 2nd exercise on the level of AS (F= 0.239; P value = 0.628 and r = 0.27).
AS (mg O2 kg-1 hr-1)
250 200
1 st part
a
ab
ab
b
150 100
Control
50
Taurine 1,5%
0 T2
T3
Figure4.31 Shows the aerobic scope (mg O₂ kg-1 hr-1) in fish fed on control FM substituted feed, or on the control plus 1.5% taurine after 34 days of feeding before and after swimming performance. As shown in Table (4.8) and Figure (4.32), the extents of changes in AS parameters during 64 days of taurine consumption were not the same after the 1st exercising the fish's in the respirometer swimming chamber respirometer T2 in which there was a highly significant decrease (P 0.05) between the fish's fed on commercial pellets alone and those fed
93
Chapter Four …...…………….........................………… Results on commercial pellets plus 1.5% taurine. There was extremely highly significant differences in the interaction in the rate of AS between the 1st and 2nd swimming respirometer (F= 32.38; P value = 0.0001 and r = 0.16). 250
2 nd Part
AS mg O2 kg-1 hr-1
a 200
c c
150
b
100
50
0 T2
T3
Control
Taurine 1.5%
Figure 4.32 Shows the aerobic scope (mg O₂ kg-1 hr-1) in fish fed on control FM substituted feed, or on the control plus 1.5% taurine after 64 days of feeding before and after swimming performance.
94
Chapter Four …...…………….........................………… Results
Table 4.7 Shows some metabolic parameters of Sea bass fed on commercial pellets with and without 1.5% taurine in 34 days after exercising the fish's in the swimming chamber respirometer. Time of Feeding Group Performances
T0
T1
T2
Control
Taurine
Control
Taurine
Ucrit(BL\s)
-
-
-
-
Oxygen Consumption (mgO2\kg\hr)
-
-
-
-
Standard Metabolic Rate (mgO2\kg\hr)
-
-
-
-
Routine Metabolic Rate (mgO2\kg\hr)
-
-
-
-
Active Metabolic Rate (mgO2\kg\hr)
-
-
-
-
Cost of Transport ( J\kg\s)
-
-
-
-
Aerobic Scope (mgO2\kg\hr)
-
-
-
-
Control
a
Taurine
b
Control
b
Taurine
c
3.93 ±0.11
4.34 ±0.014
4.25 ±0.018
4.6 ±0.053
272.8±29.21
264.5±27.14
264.5±27.14
268.7±26.33
a
179.0 ±2.6
266.3 ±6.1
235.6 ±9.9
c
202.0d±2.5
268.2±2.249
267.0±4.577
276.1±2.412
270.8±2.871
c
b
a
b
b
379.8 ±4.35
346.0 ±1.646
392.8 ±7.001
346.4 ±2.487
0,53±0,10
0,51±0,098
0,57±0,13
0,53±0,1
a
195.6 ±6.99
95
T3
ab
172.8
±3.54
179.4
ab
±9.93
b
163.7 ±6.96
Chapter Four …...…………….........................………… Results Table4.8. Shows some metabolic parameters of Sea bass fed on commercial pellets with and without 1.5% taurine in 64 days after exercising the fish's in the swimming chamber respirometer. T0
Time of Feeding Group Performances
T1
T2
Control
Taurine
Control
Taurine
Ucrit (BL\s)
-
-
-
-
MO2 (mgO2\kg\hr)
-
-
-
-
Standard Metabolic Rate (mgO2\kg\hr)
-
-
-
-
Routine Metabolic Rate (mgO2\kg\hr)
-
-
-
-
Active Metabolic Rate (mgO2\kg\hr)
-
-
-
-
Cost of Transport (J\kg\s)
-
-
-
-
Aerobic scope (mgO2\kg\hr)
-
-
-
-
96
Control
a
T3 Taurine
a
Control
a
Taurine
b
4.03 ±0.12
3.93 ±0.13
4.32 ±0.039
4.61 ±0.06
271.6±29.88
249.8±24.16
309.6±22.28
254.9±19.14
a
184.3 ±3.48
b
269.4 ±2.985
c
a
173.2 ±3.1
c
245.4 ±3.187
b
b
213.4 ±4.85
a
310.2 ±12.39
a
a
182.7 ±5.05
a
285.3 ±11.67
b
382.8 ±3.7
347.4 ±2.01
396.3 ±3.18
337.8 ±7.8
0.53±0.10
0.52±0.107
0.63±0.14
0.57±0.12
a
193.6 ±2.082
b
82.5 ±6.59
c
154 ±6.59
c
138.3 ±7.30
Chapter Four …...…………….........................………… Results 4.3 Effect of Taurine on Respiratory Burst The data in Table (4.9) and Figure (4.33) show the integrative relative light unit (IRLU) of RB during the entire experimental trails T0, T1, T2 and T3. The RB in fish's at the beginning of the experiments didn’t show any significant changes (P>0.05) between fish's fed on commercial pellets and fish's started to eat commercial pellets plus 1.5% taurine. On the other hand, after 15 days of feeding fish's on control diets with 1.5% taurine, there was a significant inhibition (P0.05) as compared with fish's fed on control diet. However, after exposing the fish's to the 2nd swimming exercise, the level of RB reduced significantly in fish fed on commercial pellets plus 1.5% taurine as compared with those fed on control diet only. There is significant interaction between the two groups of fish's and the time of feeding and exercising the fish's T0, T1, T2 and T3 on the level of reactive oxygen species, RB after PMA stimulation (F= 0.912; P value = 0.0001 and r = 0.62). IRLU after PMA stimulation
40000
30000
RB / 1 st Part b
c c
c
c c
a
a
20000
Control 10000
Taurine 1.5% 0 T0
T1
T2
T3
Figure4.33 Shows the levels of relative light units of RB in the blood after PMA stimulation in fish's fed on control FM substituted feed, or those fed on the control plus 1.5% taurine after 34 days of feeding before and after swimming performance.
97
Chapter Four …...…………….........................………… Results Table (4.10) and Figure (4.34) revealed the effect of control diet plus 1.5% taurine on the level of RB after feeding the fish's for 64 days. The level of RB after separating the fish's into two groups didn’t show any significant change (P>0.05) between fish's fed on commercial pellets and fish's fed on commercial pellets plus 1.5% taurine. Whereas, after 15 days of feeding the fish's on commercial pellets plus 1.5% taurine showed a significant reduction in IRLU of RB when compared with those fed on commercial pellets alone. On the other hands, after exercising the fish's in 1st swimming exercise, the IRLU of RB reveals significant decrease (P0.05). In addition, there is a significant interaction between the two groups of fish's and the time of feeding and exercising the fish's T0, T1, T2 and T3 on the level of RB (F= 39.39; P value = 0.0001 and r = 0.52). IRLU after PMA stimulation
40000
RB /2 nd Part 30000
b
bc
c
bc ac
c ac a
20000
Control 10000
Taurine 1.5% 0 T0
T1
T2
T3
Figure4.34 Shows the relative light units of RB in the blood after PMA stimulation in fish's fed on control FM substituted feed, or the control plus 1.5% taurine after 64 days of feeding before and after swimming performance.
98
Chapter Four …...…………….........................………… Results
Table 4.9 Show the average values of relative light units of Respiratory Burst (RB) in the blood measured in the four experimental intervals (T0, T1, T2 and T3) in Sea bass fed commercial pellets with and without 1.5% taurine in 34 days. T0
T1
T2
T3
Time (s)
Control
Taurine
Control
Taurine
Control
Taurine
Control
Taurine
0
873.8654
1837.951
1019.228
646.0535
832.086
1003.12
501.3099
1284.424
180 360 540 720 900 1080 1260 1440 1620 1800 1980 2160 2340 2519.9 2699.9 2879.9 3059.9 3239.9 3419.9 3599.9 Std. Deviation
2114.201 3098.122 4535.608 6181.167 7515.536 8578.769 9969.607 10671.51 11840.34 12770.52 13732.9 14733.54 15868.8 17147.73 19003.07 20648.09 22107.85 22631.75 24541.03 25548 7540 26896c± 440
2552.329 3105.241 4027.054 5469.031 7015.018 8074.119 8854.185 9528.04 10702.57 11634.54 12607.33 13609.94 13760.57 15057.91 16943.4 18611.26 20124.9 21704.52 22681.86 23835 6833 26646c± 553
2285.151 3285.572 4492.558 5963.929 7217.673 8209.407 9485.495 10238.9 11276.86 12135.69 13198.7 14287.46 15368.97 16804.15 18535.74 20012.01 21429.27 22471.92 22919.7 24230 7206 25754c± 490
1573.9 2395.259 3153.009 4244.484 5026.941 5826.656 6987.969 7605.06 8371.313 9111.144 9805.523 10682.52 11495.8 12533.51 13991.74 15446.23 16992.3 17854.64 18468.39 19312 5818 20309a± 281
2175.698 3429.941 4807.201 6375.24 7873.046 9038.052 10403.04 11508.61 12862.68 14120.58 15331.98 16533.45 17806.74 19211.9 21043.2 22729.86 24376.61 25382 27513.76 28375 8513 28920c± 657
2106.498 2909.651 3745.744 4816.358 5749.919 6734.835 8001.02 8762.848 9635.713 10526.1 11221.44 12169.14 13314.52 14404.97 15824.79 17335.26 19168.81 20020.83 20900 21700 6479 23503c± 600
1229.503 2764.162 4557.874 6270.485 7890.148 9467.536 11029.22 11906.41 14086.5 15844.5 17159.6 18842.74 20345.76 21640.51 23498.18 24997.45 26541.33 27482.74 27886.4 28181 9310 30990b± 534
2403.051 3257.706 3853.335 4799.453 5656.683 6164.821 6778.142 7483.437 8112.151 8974.435 9730.103 10424.43 11328.11 12115.94 13073.35 13892.93 15091.57 16595.79 17938.23 22646 5563 21058a± 1458
RB (IRLU)
99
Chapter Four …...…………….........................………… Results Table 4.10 Show the average values of relative light units of Respiratory Burst (RB) in the blood measured in the four experimental intervals (T0, T1, T2 and T3) in Sea bass fed commercial pellets with and without 1.5% taurine in 64 days. T0 Time (s) 0 180 360 540 720 900 1080 1260 1440 1620 1800 1980 2160 2340 2519.9 2699.9 2879.9 3059.9 3239.9 3419.9 3599.9 Std. Deviation
T1
T2
T3
Control 973.8654 2014.201 2898.122 4435.608 5081.167 6415.536 7578.769 9069.607 12671.51 12840.34 12770.52 13732.9 14733.54 15068.8 17147.73 19893.07 21648.09 22107.85 22631.75 24541.03 26896 7877
Taurine 837.951 2252.329 2905.241 3027.054 4469.031 5615.018 6474.119 8954.185 9028.04 10702.57 11634.54 12607.33 13609.94 13660.57 15057.91 16743.4 17611.26 20124.9 21704.52 22681.86 26646 7373
Control 819.2279 1285.151 2285.572 3092.558 4093.929 5217.673 6209.407 7485.495 11238.9 12276.86 12135.69 12998.7 13087.46 15368.97 16804.15 17535.74 20012.01 21429.27 22871.92 23919.7 25754 7892
Taurine 546.0535 1444.9 2005.259 3013.009 4014.484 4826.941 5126.656 6987.969 8605.06 9371.313 10111.14 11805.52 12682.52 13495.8 14533.51 14991.74 15336.23 16992.3 17854.64 18468.39 20309 6156
Control 1573.865 2314.201 2998.122 4435.608 5081.167 6415.536 7578.769 9069.607 12671.51 12840.34 13070.52 13732.9 14733.54 15068.8 16247.73 18893.07 20648.09 22007.85 24931.75 26841.03 28920 8206
Taurine 746.0535 1944.9 2105.259 3013.009 4014.484 4826.941 5126.656 6987.969 8605.06 9371.313 11211.14 12105.52 12682.52 13495.8 14533.51 14991.74 15136.23 16332.3 18854.64 20468.39 23503 6566
Control 999 2711 3011 4435.608 5081.167 6700 7578.769 9069.607 10349 12840.34 13933 14809 17439 20138 22100 21324 23444 25022 26531.75 28841.03 30990 9379
25548bc±1814
23835c±1295
24230c±630
19312a±483
28375b±383
21700ac±439
28181bc±505
RB (IRLU)
100
Taurine 803 2019 2600 3013.009 4014.484 4826.941 5126.656 6987.969 8605.06 9371.313 11211.14 12321 12943 13495.8 14533.51 15221 16555 18944 19854.64 20768.39 21058 6610 22646
ac
±1916
Chapter Four …...…………….........................………… Results 4.4 Effect of Taurine on Lipid Peroxidation in Selected Organs 4.4.1 Effect of Taurine on Catalase mRNA Gene Expression 4.4.1.1 Effect of Taurine on Liver Catalase mRNA Gene Expression The data in Table (4.11) and Figure (4.35) for the fish's fed on commercial pellets with 1.5% taurine for 34 days and after 1 st and 2nd swimming exercises showed physiological enhancement in the level of liver CAT gene expression but it was statistically non-significant (P>0.05) when compared with the fish's fed on commercial pellets alone. CAT mRNAcopy n\ 100ng RNA
5.010 7
CAT in liver \ 1 st Part
4.010 7
3.010 7
2.010 7
1.010 7
0
Control
1.5% Taurine
Figure 4.35 Shows the level of Catalase (CAT mRNA copy n°/100ng RNA) in liver of fish fed either on control FM substituted feed, or fed on control diet plus 1.5% taurine after 34 days of feeding before and after swimming performance. Also the highest number of mRNA copies found in liver CAT gene expression was non-significant (P>0.05) after feeding and exercising the fish's for 64 days on control diet enriched with 1.5% taurine (Table 4.12 and Figure 4.36). The means were 8.89×106 ± 1.35 ×106 for fish fed on diet devoid of taurine and 9.6×107± 2.48×106, for diet supplemented with 1.5% taurine, respectively.
101
CAT mRNAcopy n\ 100ng RNA
Chapter Four …...…………….........................………… Results
1.510 7
CAT in liver \ 2 nd Part 1.010 7
5.010 6
0
Control
1.5% Taurine
Figure 4.36 Shows the level of Catalase (CAT mRNA copy n°/100ng RNA) in liver of fish fed either on control FM substituted feed, or fed on control diet plus 1.5% taurine after 64 days of feeding before and after swimming performance. 4.4.1.2 Effect of Taurine on Red Muscle Catalase (CAT) mRNA Gene Expression After 34 days of feeding and exercising the fish's in the swimming respirometer, the red muscle CAT gene expression (CAT mRNA copy n°\ 100ng RNA) was non significantly (P>0.05) enhanced in the fish's fed on commercial pellets plus 1.5% taurine when compared with the fish's fed on
CAT mRNAcopy n\ 100ng RNA
commercial pellets alone (Table 4.12 and Figure 4.37). 2.010 7
CAT in red muscle \ 1 st Part
1.510 7
1.010 7
5.010 6
0
Control
1.5%Taurine
Figure 4.37 Shows the level of Catalase (CAT mRNA copy n°/ 100ng RNA) in the red muscle of fish fed either on control FM substituted feed, or fed on control diet plus 1.5% taurine after 34 days of feeding before and after swimming performance.
102
Chapter Four …...…………….........................………… Results Figure (4.38) revealed that the level of red muscle CAT gene expression was not significantly increased (P>0.05) in the fish fed on 1.5% taurine for 64 days when compared with the fish fed on commercial pellets alone and the means were 8.71×106 ± 3.64×105 and 8.23×106±5.96×105,
CAT mRNAcopy n\ 100ng RNA
respectively. 1.010 7
CAT in red muscle \ 2 nd Part
8.010 6
6.010 6
4.010 6
2.010 6
0
Control
Taurine
Figure 4.38 Shows the level Catalase (CAT mRNA copy n°/ 100ng RNA) in the red muscle of fish fed either on control FM substituted feed, or fed on control diet plus 1.5% taurine after 64 days of feeding before and after swimming performance. 4.4.2 Effect of Taurine on Superoxide dismutase mRNA Gene Expression 4.4.2.1 Effect of Taurine on Liver Superoxide Dismutase mRNA Gene Expression In fish's fed on diet supplemented with 1.5% taurine for 34 days, the level of liver SOD was non-significantly (P>0.05) increased when compared with that of the fish's fed on commercial pellets alone (Table 4.11 and Figure 4.39).
103
SOD mRNA copy no./ng total RNA
Chapter Four …...…………….........................………… Results
1.510 7
SOD in liver\ 1 st Part
1.010 7
5.010 6
0
Control
Taurine
Figure 4.39 Shows the level of Superoxide dismutase (SOD mRNA copy n°/100ng RNA) in the liver of fish fed either on control FM substituted feed, or the control feed plus 1.5% taurine after 34 days of feeding before and after swimming performance. Furthermore, after 64 days of feeding fish's the level of liver SOD remain non-significant (P>0.05) between fish's fed on commercial pellets plus 1.5% taurine 6.9×106±5.4×105 and those fed on commercial pellets
SOD mRNA copy n\ 100ng RNA
alone 6.48×106±6.6×105 (Table 4.11 and Figure 4.40). 8.010 6
SOD in liver\2 nd Part
6.010 6
4.010 6
2.010 6
0
Control
Taurine
Figure 4.40 Shows the level Superoxide dismutase (SOD mRNA copy n°/100ng RNA) in liver of fish fed either on control FM substituted feed, or the control feed plus 1.5% taurine after 64 days of feeding before and after swimming performance. 4.4.2.2. Effect of Taurine on Red Muscle Superoxide dismutase mRNA Gene Expression: The results of red muscle SOD gene expression in fish's fed on commercial pellets alone with or without 1.5% taurine for 34 days, showed a significant enhancement (P0.05) as compared with fish's fed on commercial pellets alone (Table 4.12 and Figure 4.42). SOD mRNA copy no./ng total RNA
SOD in red muscle\2 nd Part 1.510 7
1.010 7
5.010 6
0
Control
1.5% Taurine
Figure 4.42 Shows the level of Superoxide dismutase (SOD mRNA copy n°\ 100ng RNA) in the red muscle of fish fed either on control FM substituted feed, or fed on control diet plus 1.5% taurine after 64 days of feeding before and after swimming performance.
105
Chapter Four …...…………….........................………… Results 4.4.3 Effect of Taurine on Glutathione Peroxidase mRNA Gene Expression 4.4.3.1 Effect of Taurine on Liver Glutathione Peroxidase (GPX) mRNA Gene Expression: The general pattern of liver glutathione peroxidase gene expression presented in Table 4.11 and Figure 4.43 demonstrate that there is no significant change (P>0.05) in the mRNA copy numbers of liver GPX gene expression in fish's fed on commercial pellets plus 1.5% taurine for 34 days
GPX mRNA copy no./ng total RNA
and fish's fed on commercial pellets alone. 2.510 6
GPX in liver\ 1 st Part
2.010 6
1.510 6
1.010 6
5.010 5
0
Control
Taurine
Figure 4.43 Shows the level of Glutathione peroxidase (GPX mRNA copy n°/100ng RNA) in the liver of fish fed either on control FM substituted feed, or fed the control diet plus 1.5% taurine after 34 days of feeding before and after swimming performance. Furthermore, the Table 4.12 and Figure 4.44 indicate that the mRNA copy numbers of liver GPX gene expression remain unchanged (P>0.05) in fish's fed on commercial diet plus 1.5% taurine and fish fed on commercial diet alone. The mean numbers were 8.8×106±1.11×105 and 7.5×106±4.8×105 mRNA copy no. / ng total RNA, respectively.
106
GPX mRNA copy no./ng total RNA
Chapter Four …...…………….........................………… Results
1.510 6
GPX in liver\ 2 nd Part 1.010 6
5.010 5
0
Control
Taurine
Figure 4.44 Shows the levels of Glutathione peroxidase (GPX mRNA copy n°/100ng RNA) in the liver of fish fed either on control FM substituted feed, or fed on the control diet plus 1.5% taurine after 64 days of feeding before and after swimming performance.
4.4.3.2 Effect of Taurine on Red Muscle Glutathione Peroxidase mRNA Gene Expression The data of Table (4.12) and Figure (4.45), indicate that there are significant elevation (P 0.05) reduced in aortic rings preincubated with Indomethacin (cyclooxygenase inhibitor) when compared with control aortic rings, with IC 50’s of 1.808 M (with IC50 of CI 95% between 0.004 to 0.059) and 1.927M (with IC50 of CI 95% between 0.007492 to 0.01871), and the percentage of relaxation were 39.34±0.109% and 40.08±0.16% respectively. Figure 4.63 (A) Typical traces showing the effects of L-NAME on taurine induced relaxation and (B) Taurine dose-response curves in fish aortic rings precontracted with KCl and preincubated with and without L-NAME (1×10-3 M).
A
150
Taurine T a u r in e
B
x
Tension (%)
2 gr
x
Taurine Taurine
100 75 50
0 0
KCL
5 min
25
KCl
Indomethacin Indomethacin
KC l
KCL
125
Control Indomethacine
0 10 -4
10 -3
10 -2
10 -1
Taurine (log M)
Figure 4.64 (A) Typical traces showing the effects of Indomethacin on taurine induced relaxation and (B) Taurine dose-response curves in aortic rings precontracted with KCl and preincubated with and without Indomethacin (3×10-5M). (O; n=6). 145
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Table 4.27 The pIC50, percentage of relaxation and Correlation Coefficient (R2), for the effect of taurine on fish's aortic rings perincubated with LNAME. Treatment Number of Segments pIC 50 IC50 95% CI IC50 Relaxation (%) ± SEM R2
Taurine Control 8 1.648 0.02251 0.0005 to 0.96 66.17±0.096 0.75
L-NAME(10-3M) 8 1.808 0.01555 0.004 to 0.059 39.34±0.109 0.78
Table 4.28 The pIC50, percentage of relaxation and Correlation Coefficient (R2), for the effect of taurine on fish's aortic rings perincubated with Indomethacin. Taurine Control Indomethacin (3*10Treatment 5 M) 8 6 Number of Segmentss 2.069 1.927 pIC 50 0.008532 0.01184 IC50 0.005569 to 0.01307 0.007492 to 0.01871 95% CI IC50 30.2±0.06 40.08±0.16 Relaxation (%) ± SEM 2 0.76 0.75 R
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5. DISCUSSION Aquaculture continues to expand, with a gradual shift from traditional, low cost semi intensive systems to more costly intensive systems, where processed feed is a major component. As a result, total industrial processed aqua feed production has been increased from 7.6 million tons in 1995 to 35 million tons in 2010, with an average annual growth rate of about 11%. It is also expected that this growth rate will continue, and aqua feed production will reach 70 million tons by 2020 (Tacon, 2012). Taurine is a dietary supplement used to promote growth in aquaculture, and involved numerous important biological functions, including membrane stabilization, detoxification, anti-oxidation, calcium transport, retina development, bile acid metabolism, osmotic regulation, endocrine functions and modulation of the immune response (El-Sayed, 2013). Dietary taurine supplementation to farmed finfish has been shown to reduce nutritional diseases, such as Green liver, regulate hematocrit levels (Rhodes & Davis, 2011), reduce body lipid content (Kim et al., 2008b), and is considered as an essential nutrient for promotion of growth performance in several fish species (Park et al., 2002), including rock bream (Lim et al., 2013). The present study aimed to study the effect of taurine on sea bass and common crap which represents the first qualitatively and quantitative detailed documentation study using different method for the determination of the effects of taurine. 5.1 Effect of Taurine on Growth performance Since some free amino acids have an appetite stimulating effect (Kasumyan & Doving, 2003), and if taurine had such an effect, an increase in feed consumption would explain the observed increase in growth. On 145
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the other hand, taurine had only limited stimulating effect on red sea bream (Fuke et al., 1981) has a depressant effect in marbled rockfish Sebasticus marmoratus (Takaoka et al., 1990). Thus, taurine must promote growth by another unknown mechanism. Also, other α-amino acids such as glycine and arginine can also participate in cellular osmoregulation in fish (Li et al., 2009), it could be hypothesized that taurine spares these amino acid which then become available for protein synthesis or energy production. However, it has been indicated that growth depression was observed during taurine deficiency, reduced feed and protein efficiencies are generally reported (Matsunari et al., 2008b). The production of fish meal remained relatively stable over the last 15 years, and it is unlikely to be improved (Lunger et al., 2007). Indeed, it has been suggested that the stable fish meal will decline in the future and it can no longer be considered as a sustainable source of protein for aqua feeds (Craig & McLean, 2006). Accordingly, alternate in proteins are needed to replace the fish meal especially for carnivorous species diets. Plant proteins are probably the most widely used as an alternative to fish meal, but they express a number of disadvantages such as containing lower crude protein levels, palatability issues, amino acid deficiencies and the occurrence of anti-nutritional factors such as trypsin inhibitors (Francis et al., 2001). Taurine is not considered to be an essential amino acid for fish only, but it is a free amino acid present in large quantities in various tissues of marine fish (Park et al., 2002). Wild carnivorous fish consume relatively large quantities of taurine since it is highly abundant in animal tissues, but this is not the case when the diets contain large amounts of plant protein sources, which are naturally low in taurine. Therefore, it may be necessary to supplement these diets with taurine and other amino acids to enhance its nutritional values.
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In addition, taurine as an amino acid also possesses the major characteristics of a feeding stimulant for fish such as low molecular weight, nitrogen content, water solubility, acid and base properties….etc (Carr, 1982). Indeed, taurine as well as other amino acids (e.g. glycine, methionine, glutamine and leucine) have been used and promoted as an attractant in fish baits (Martinez et al., 2004). Scientific evidence also documents the stimulatory effect of taurine on sensory organs of fish, which conjugates to form bile salts, can stimulate the olfactory system of Arctic char Salvelinus alpinus and grailing Thymallus (Doving, 1980), as well as trout Onchorynchus mykiss (Hara et
al., 1984). Taurine might
have an increased daily feeding rate of sea bass fry (Martinez et al., 2004), it is possible that a part of the growth-promoting action of taurine is due to the stimulation of feed consumption. From the results of the current study after the sea bass fed on feed supplemented with 1.5% taurine produced a positive impact on body weight, body length, and specific growth rate before and after exercising in swimming chamber respirometer during the 1 st trail experiment. These results agree with those of Japanese flounder fed with taurine supplement feed which improved BW, BL and SGR (Park et al., 2002; Kim et al., 2003) and BW of European sea bass (Martinez et al., 2004), yellowtail Japanese amberjack (Matsunari et al., 2005) and rainbow trout (Gaylord et al., 2006). In the second part of the experiments, BW, BL, SGR and FCR were also improved with taurine supplementation, but all measurements tended to increase with increasing the number of period of feeding with 1.5% taurine before and after exercising. This outcome was analogous to the findings of Martinez et al., (2004) and Matsunari et al., (2005) are reveales that yellowtail Japanese amberjack was investigated by feeding on diets containing various taurine levels (0, 0.5, 1.0, 1.5 and 2.0%)
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significantly improved their growth performance over the initial 3-weeks period. However, some reports has also indicated that taurine (0.4 mg/day) in diet of gourami (Osprhonemus goramy) and tilapia (Oreochromis niloticus), produced a positive effect on body length of juveniles or adults stages (Widiastuti, 2015). The ration of feed conversion and condition factor are not changed in the 1st experiment after feeding the fish's on diet with 1.5% taurine before and after exercising in the swimming chamber respirometer. On the other hand, the rate of FC revealed non-significant, and, whereas CF significantly increased after feeding the fish on feed supplement with 1.5% taurine before and after exercising in swimming chamber respirometer. These results, are also in agreement with those of Chatzifotis, et al (2008) who reported that common dentex (Dentex dentex)
Fed on taurine supplement feed (2 g kg−1 on a dry weight basis) for 12weeks reduced FCR. However, taurine added diet (0.25 to 3.0%) significantly increased the condition factor in Juvenile Rock Bream Oplegnathus fasciatus. As well as the growth performance (Bai et al., 2014). The dietary taurine level for the optimum growth of olive flounder ranges from 14-16 mg taurine/g diet (Park et al., 2002; Kim et al., 2005a, 2005b). Flounder fed on diets supplemented with 10 mg taurine/g diet exhibited superior growth, not only relative to a basal diet, but also to those diets supplemented with gamma- aminobutyric acid (GABA) and β-alanine (Kim et al., 2003; Qi et al., 2012). Also it has been observed that 10 mg taurine/g diet as the dietary taurine requirement for normal growth and feed efficiency in turbot, the growth performance increased until reached a plateau, with no further effect of taurine supplementation greater than the required level (Matsunari et al., 2008). Additionally, taurine requirement for growth and feed efficiency enhancement in red sea bream, Pagrus major, was 145
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estimated to be 5 mg taurine/g diet (Matsunari et al., 2008). Furthermore, taurine supplementation also showed to be indispensable in diets containing high levels of plant-based proteins (Lunger et al., 2007; Espe et al., 2012). Two speculative explanations on the mode of action of taurine have been made. First, taurine cannot be broken down for energy (Huxtable, 1992) nor utilized for protein synthesis, but it is rather found as a free amino acid or as simple peptides (Conceinçao et al., 1997). It is thus plausible that any growth-promoting effect was due to its secondary or auxiliary actions on other biological functions 5.2 Effect of Taurine on Some Metabolic Parameters The results of the current study on the effect of taurine on some metabolic parameters demonstrated that after exposure of fish's to progressive increased swimming speed, between 0.7 to 4.2 BLs -1, the Ucrit-1 for adults sea bass fed on diets augmented with 1.5% taurine for 15 days significantly elevated after exercising in the 1st swimming chamber respirometer (T2) which remains higher even after 34 days of feeding (T3). The level of Ucrit-2 in the second trail after 34 days of feeding fish's diets with and without 1.5% taurine remain more or less the same the after exercising in the swimming chamber respirometer. Whereas, after 64 days the level of Ucrit elevated positively in fish fed on control diet supplemented with taurine. It has been explained that at higher exercise loads, the demand for energy production could exceed the capacity of muscle to produce ATP, leading to further impairment of muscle performance (Ito et al., 2014). It has been reported that taurine released from muscle during exercise, is associated with increased muscle fiber osmolality related to the accumulation of metabolic by products, such as lactate (Maitra et al., 2009) 145
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Due to the availability of limited information on the subject, it is difficult to compare the results. However, the primary reasons for critical swimming speed to increase may be due to oxygen transport across the gills or increased the capacity of erythrocytes to transport oxygen to tissues. In the repeated swimming trial, a significant reduction was observed in the active metabolic rate of the fish fed on diet with taurine when compared with control group. This indicates that the maximum sustainable aerobic capacity of fish may be impacted by taurine and is attributable to increased oxygen uptake, transport or utilization (Hammer, 1995). The differences in Ucrit among Sea bass could be due to differences in acclimation time and Ucrit protocol adopted in the present study as compared with those reported by Thomas & Janz, (2011). For example, differences in increased velocity and time interval between velocity and increments in Ucrit tests which have been shown to alter swimming performance of fish (Hammer, 1995). The results of the current study on the rate of oxygen consumption (MO2) revealed a non-significant elevation in both experimental trails after feeding the fish's on diet with and without 1.5% taurine after exercising in the swimming chamber respirometer. However, the rates for SM, RM, AM, AS and COT in fish fed diet with 1.5% taurine indicated lesser requirements for energy, during both 1st and 2nd swimming exercise. Furthermore, the metabolic rate in 2nd experimental trails was reduced after feeding and exercising the fish's in the swimming chamber respirometer (T2 and T3). Since the results are considered a novel one, on the effect of taurine the metabolic rate of sea bass, it is difficult to compare the results.
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5.3 Effect of Taurine on Respiratory Burst The results on the effect of taurine on stress induced by forced swimming in sea bass (Dicentrachus labrax) indicated that taurine produced an important compensatory response to free radicals produced during swimming activities. The relative light units (RLU) of integral luminescence in fish's fed on diet with and without 1.5% taurine at rest didn’t differ in both experimental trails, whereas, it is dropped by a factor, slightly less than 0.25, in the groups treated for 15 days (1st experimental trail), and 30 days (2nd experimental trail) with taurine (T1). On the other hand, in the control group, monitored at the end of the swimming test, the respiratory burst is significantly increased, in the group treated with taurine in which the respiratory burst was less than a factor near 0.26 (T2). The level of RLU was reduced to 0.4 in fish's fed on diet with 1.5% taurine when compared with the control group after exercising in swimming respirometer (T3). The level of RLU in the second experimental trails revealed the same effect of taurine in all group (T0, T1, T2 and T3) after 64 days when compared with the control group. Thus, the fish's fed on control diet plus 1.5% taurine my, to some extent, due to suppressed some immune functions. These inconsistent results concerning the effect of taurine on RB may be due to the type of cells and the method of assessment. In our case, RB was assayed on whole blood, which avoids the mechanic impact of the isolation procedures on the cells. Furthermore, luminol-enhanced chemilumiscent is thought to measure intracellular as well as extracellular ROS, whereas, other methods measure only one type of ROS production (Vila et al., 2009). Since most of the results of the current study are novel due to lack of information the comparison of the data is not possible.
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5.4 Effect of Taurine on Lipid Peroxidation on Selected Organs One of the most common benefits of taurine is its antioxidative action, often associated with the cytoprotective characteristics. In addition to the fore mentioned reaction with HOCl, taurine has been shown to reduce lipid peroxidation levels (Aydogdu et al., 2007). However, the data on direct measurements of scavenging activity indicated that taurine is a weak scavenger for various radicals such as hydrogen peroxide, oxygen superoxide, or peroxynitrite (Mehta and Dawson, 2001). Rather, taurine likely modulates the production of reactive oxygen species, whether indirectly such as with Tau-Cl. An example of the latter is seen in mitochondria: when cultured in taurine-poor medium, cardiomyocytes suffer from oxidative stress caused by a disruption of the mitochondria electron transport chain, leading to the generation of superoxide anions (Parvez et al., 2008). Reintroduction of taurine in the medium restores the electron transport chain integrity, thereby decreasing the production of superoxide anions (Jong et al., 2012). A likely mechanism for this is the modification of mitochondrial tRNA with taurine at the wobble anticodon; without the taurine modification, translation of mitochondrial proteins (including subunits of enzymes from the electron transport chain) is severely impaired (Umeda et al., 2005). In fish, the relationship between taurine and oxidative stress was hypothesized in jaundiced Seriola quinqueradiata (Sakai et al., 1998). Taurine supplementation also led to a restored catalase activity and reduced lipid peroxidation levels in Totoaba macdonaldi (Bañuelos-Vargas et al., 2014). Finally, it has also been shown in Trachinotus carolinus maintained on a taurine-deficient diet for 16 days: a significant decrease in hepatic mitochondrial protein content and mitochondrial activity was reported to
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be strongly correlated with a decrease in taurine content (Salze et al., 2014b). The potentially deleterious effects of ROS are counteracted by suitable antioxidant enzymes, including radical-scavenging enzymes such as catalase (CAT), superoxide dismutase (SOD) and peroxidases such as glutathione peroxidase (GPX). Super oxide dismutase is a metalloprotein that represents the first enzyme involved in the antioxidant defense by lowering the steady-state level of O2- by its conversion into H2O2 and water. Catalase which decomposes H2O2 to water and O2- is a widely distributed enzyme and an important member of the cellular defense system against oxidative stress. Even if it is not strictly essential, the lack or malfunction of catalases may lead to severe defects, such as an increased susceptibility to thermal injury (Leff, 1993), high rate of mutations (Halliwell & Aruoma, 1991) and inflammation in higher organisms (Halliwell & Gutteridge, 1990). Reduction in the activities of these enzymes lead to the accumulation of O2- and H2O2, which in turn can form hydroxyl radical (OH٠) and bring about a number of reactions harmful to the cellular and subcellular membrane systems (Kalra et al., 1988). Catalase gene expression was elevated in liver and red muscle of Sea bass fed on commercial pellets with 1.5% taurine for 34 days. Whereas, after 64 days of feeding, the copies number of liver and red muscle CAT gene expression remain high but did not changed significantly. The SOD mRNA expression was non-significantly increased in liver and red muscle of fish's feds commercial pellets plus 1.5% taurine when compared with the control group in both experiment trails, Glutathione peroxidase gene expression was quite strong in fish's fed on diets
145
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containing 1.5% taurine as compared to fish's fed on commercial pellets only. As antioxidant properties of taurine, showed in the present study is represented by marked reduction in MDA formation in the fish's fed on 1.5% taurine. The potent antioxidant properties of taurine are associated with increased antioxidant enzyme activities such as catalase, SOD, and GPX which are the key cellular antioxidant enzymes that defend against oxidative stress (Izquierdo et al., 2012). Accordingly, evidences showed that the activities of these antioxidant enzymes are decreased when cells and tissues are subjected to oxidative stress (Ghyasi et al., 2012; Ramesh et al., 2012). Furthermore, it has been suggested that the antioxidant effects of taurine may be associated with its sulfur moiety and the modulation of glutathione levels by taurine which is critical in the cellular defense against oxidative stress (Huxtable, 1992; Woo, et al., 2003). However, in the current study, it has been demonstrated for the first time that taurine has beneficial roles on subtle aspects of fish performance such as swimming endurance, aerobic capacity and removing free radicals. 5.5 Relaxant Effect of Taurine on Fish Intestinal Segments and Aortic Rings 5.5.1 Effect of Taurine on the Contractile Activity Induced by KCl The results of the current study on the effect of taurine on the relaxation of intestinal and aortic smooth muscle cell of fish represent the first record, in which taurine showed a very high relaxant effect on intestinal segments (98.48%) as compared with aortic rings (60.66%). This variation reflects the activation of different K channel subtype during taurine induced relaxation in intestine and aorta. In addition, it may also be
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resulted from the quantitative difference between the intestinal and aortic smooth muscle cells. In spite of the unavailability of data on fish, more or less a similar vasorelaxant effect of taurine have been observed in other vertebrates such as the relaxation induced by taurine in KCl and thromboxane A2 analogue precontracted porcine coronary artery (Liu et al., 2009). Taurine also induced endothelium independent relaxation in rat’s aortic rings (Li et al., 2009). Niu et al., (2008) reported that taurine relaxes rat’s aorta and inhibited phenylephrine induced contraction of renal and mesenteric arteries and suggested that it may involves a mechanism related to the activation of K channel. However, a rare bidirectional regulatory effect of taurine had been reported in jejunal segments since it induced stimulatory effects at low contractile states, and inhibitory effect at high contractile states (Yao et al., 2014). 5.5.2 The Effect of K+ Channel Subtypes in the Relaxation Induced by taurine The novel results of the current study showed the presence of intraspecific variation in the relaxation induced by taurine in the presence of different K channel subtype inhibitors clearly indicate that the mechanism involved in taurine induced relaxation is related to the activation of different sets of K channel subtypes in different tissues. Thus, Kca and KATP channels are activated and play important role in the taurine induced relaxation of the intestinal segment but not Kv and Kir channel subtypes. On the other hand, in aortic rings, both Kca and Kir subtypes were activated, but not KATP and Kv channel subtypes.
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5.5.3 The Effect of Ca+2 Channels in the Relaxation Induced by Taurine Intraspecific variation was also observed in the role of L-type Ca++ channel in taurine induced relaxation in intestinal and aortic smooth muscle since its preincubation with L-type Ca++ channel blocker diminished taurine induced relaxation in fish’s aorta. This clearly indicates the role of L-type Ca++ channel in taurine induced relaxation in aortic rings. On the other hand, intestinal relaxation was enhanced in the presence of the Nifedipine, which may be due to the direct effect of the blocker on intestinal smooth muscle cells. These indicate that taurine induced relaxation in intestinal segments, didn’t depends on L-type Ca++ channel. Since no data are available for fish to compare the results. However, in general these aorta results are more or less coincided with those reported for other vertebrates such as rat’s aorta (Li et al., 2009). They indicated that the relaxation induced mechanism involve the inhibition of Ca ++ influx and release as well as the activation of KATP and Kca++ , but not Kir channels subtype. Similarly, in procine contracted artery, it had been suggested that taurine induced relaxation involved the activation of K ir, KATP and Kca++ channel subtypes (Liu et al., 2009). 4.5.4 The Role of NO, PGI2 and Methylene Blue on Taurine Induced Relaxation The results of the current study indicated that the endothelial derived hyperpolarizing factors such as nitric oxide synthase inhibitor, cyclogenase inhibitor and cGMP inhibitor have no role in taurine induced relaxation in intestinal tissues and aortic tissues; except at a high concentration of taurine (2x10-2M) which reduced significantly the induced relaxation in aortic rings. Since no data available on the taurine induced relaxation in the studied tissues, it is difficult to compare the results. 145
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145
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6. CONCLUSIONS AND RECOMMENDATIONS 6.1 Conclusions From the respiratory parameters and relaxant effect results, the followings conclusions were reached. 1- The results of present research work, indicate that after feeding the Sea bass on commercial pellets plus 1.5% taurine for 15days did not changed the weight, length, SGR and FCR. Whereas, after 34 days of feeding, there was enhanced in the weight, CF and FCR. On the other hand, during 64 days of feeding the weight and SGR was elevated in fish's fed on commercial pellets plus 1.5% taurine as compared with fish's fed on commercial pellets alone. 2- The results revealed that feeding the fish's on commercial pellets plus 1.5% taurine and after exercising in the respirometer swimming chamber for 34 and 64 days, showed enhanced in the Ucrit as compared with fish's fed on commercial pellets alone. 3- The rate of oxygen consumption showed no changed whereas the rate of standard metabolic (SM), routine metabolic (RM) and active metabolic (AM) were significantly decreased when fish fed commercial pellets plus 1.5% taurine. 4- Fish fed on commercial pellets with and without taurine did not show any changed in the COT and aerobic scope after exercising in the swimming chamber respirometer for 34 and 64 days. 5- The level of integrative relative light unit (IRLU) of respiratory burst revealed significant inhibition before and after exercising the fish's in swimming chamber respirometer in fish fed on commercial pellets plus 1.5% taurine as compared with those fed on commercial pellets only.
145
…...…..................................................................................… Appendix
6- The level of catalase mRNA gene expression in liver and red muscle did not changed in fish fed on commercial pellets plus 1.5% taurine during both feeding periods 34 and 64 days. Similarly, the level of liver SOD and GPX mRNA gene expression show no changed. The levels of red muscle SOD and GPX in fishes fed on diet plus 1.5% taurine for 34 days showed elevation in mRNA copy number. However, the level of red muscle SOD and GPX showed no change in fishes fed on diet plus 1.5% taurine. 7- The results revealed that taurine induced enhanced relaxation in intestinal smooth muscles as compared to aortic smooth muscle of the Common carp. 8- Using different K channel subtype blockers revealed that in intestinal segments, both KATP and Kca channel subtypes played significant roles in taurine induced relaxation, while KV and KIR played no role the relaxation. On the other hands, in aortic rings, both KIR and Kca, but not KATP and KV, played significant roles in induced vasorelaxation. 9- The results also indicated that Nifedipine (L- type Ca++ channels blocker), diminished the relaxation induced by taurine in aortic rings while a similar effect was not exhibited by intestinal L-type Ca++ channels since Nifedipine enhanced the taurine induced relaxation instead of its inhibition. 10-
The endothelial derived hyperpolarizing factors such as NO, cyclogenase and cGMP
have no effects on taurine induced
relaxation in intestinal tissues and aortic tissues; except at the highest taurine concentration used (2x10-2M) which reduced the relaxation in aortic rings.
145
…...…..................................................................................… Appendix
6.2 Recommendations Based on the results obtained from the present study, the following points can be recommended for the future are: 1- Study the pharmacological effect of taurine, could be useful source in pharmacology. 2- Since the results of taurine induced relaxation on intestinal segments and aortic rings of fish have been studied for the first time, further studies are required to confirm the mechanism of its actions on different tissues. 3- Further studies on the effect of taurine on voltage gated Ca++ channels using Patch Clamp Record. 4- Expansion of this project to include the use of different concentrations of Taurine and other available antioxidants.
145
…...…..................................................................................… Appendix
Figure A1 The simplified representation of DAQ-M instrument, Auto RespTM (Loligo ® Systems setup).
145
…...…..................................................................................… Appendix
Figure A2 Show the diagram of fast set point adjustment of calibrating the oxygen probe (OXY-REG). 146
…...…..................................................................................… Appendix
Figure A3 Show the diagram of fast set point adjustment of calibrating the temperature Analyzer (TMP-REG) (AutoTMResp, Loligo® Systems).
147
…...…..................................................................................… Appendix
Figure A4 Typical chart view trace showing the curve of Oxygen consumption (MO2 mg O2/Kg/hr) after setting the instrument with intermittent respiromet after running at three phases: Measuring period (M), Flushing period (F) and Waiting period (W).
Table A1 The calibration of luminescence reader parameters (TECAN)
Calibration of Luminescence Reader
Integration time
100 ms
Attenuation
Automatic
Agitation
5s before the first reading of 2 s between each reading
Incubation time
0
# of Cycles
20
Range
3 min
Control
No 148
…...…..................................................................................… Appendix
Temperature range
20-30 ° C
Values once they leave, you should switch to a spreadsheet excel
Table A2 Reveals the default Setting of OXY-REG.
INPUT TYPE
POTMETER
DECIMAL POINT
111.1
DISPLAY LOW VALUE
0
DISPLAY HIGH VALUE
100
RELAY 1 UNITS
DISP
REL1 SETP
50
ACT
DECR
HYS
1
ON.DE
0
OF.DE
0
SETP
50
ACT
INCR
HYS
1
ON.DE
0
OF.DE
0
REL2
149
…...…..................................................................................… Appendix ANALOG OUTPUT
4-20 (converted into a 0-5V instrument output)
RESP
0.4
E.PASS
YES
N PASS
1234
Table A3 Reveals the default Setting of TMP-REG.
IN
TEMP
TYPE
PT
PT.TY
100
CONN
4W
DEC.P
111.1
UNIT
C
REL1 SETP
25
ACT
DECR
HYS
0.5
ERR
HOLD
ON.DE
0
OF.DE
0
SETP
25
ACT
INCR
RELL2
150
…...…..................................................................................… Appendix
ANALOG OUTPUT
HYS
0.5
ERR
HOLD
ON.DE
0
OF.DE
0
4-20 (converted to a 0-5 instrument output)
0.LO
0
0.HI
100
0.ERR
3.5Ma
RESP
1
E.PASS
YES
N.PASS
1234
151
…...…..................................................................................… Appendix
Table A4 Settings, Swimming Speeds of swimming chamber respirometer, (F= Flushing. W= Waiting. M= Measurements).
Time (min)
30 min
1st MO2
60 min
2nd MO2
1st MO2
90 min
2nd MO2
1st MO2
120 min
2nd MO2
1st MO2
150 min
2nd MO2
1st MO2
180 min
2nd MO2
1st MO2
2nd MO2
Intermettent Respirometer
F
W
M
F
W
M
F
W
M
F
W
M
F
W
M
F
W
M
F
W
M
F
W
M
F
W
M
F
W
M
F
W
M
F
W
M
Time (min)
7
1
7
7
1
7
7
1
7
7
1
7
7
1
7
7
1
7
7
1
7
7
1
7
7
1
7
7
1
7
7
1
7
7
1
7
Swim Spees (cm\s)
20
40
60
80
100
120
Swim Speed (Bl\s)
0.7
1.4
2.1
2.8
3.5
4.2
152
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