Blood chemistry parameters in the African sharptooth catfish (Clarias gariepinus)

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CHAPTER 4: JUSTIFICATION

South Africa is facing a freshwater crisis, both from a quantitative and qualitative perspective. Most of the environmental research in southern Africa has been focussed on the terrestrial ecosystems and the conservation of terrestrial animals. It is generally accepted that freshwater ecosystems in South Africa are severely threatened (more than terrestrial ecosystems) and are in urgent need of more scientific attention (e.g. ecotoxicology).
South Africa is a water scarce (quantity) country with a rapidly expanding human population, and a very poor track record of freshwater conservation. Attempts are made to address the quantity problem by trans-basin pumping of freshwater (e.g. from Sterkfontein Dam to the Vaal River to supply Gauteng Province). Unfortunately, it is not only the quantity of water that is a problem; the on-going deterioration of water quality is slowly becoming a major constraint for a healthy and a sustainable future. Water of poor quality becomes useless to humans, animals and plants and may also affect their health. Freshwater is a finite resource, any water source that has become useless (due to pollution), decreases the quantity of potable water, even further.
“We never know the worth of water until the well runs dry….” Thomas Fuller
The poor maintenance of sewage purification works by most municipalities, uncontrolled release of acid mine drainage, industrial pollution and agricultural mismanagement of irrigation water are some of the most important contributors to freshwater quality deterioration. In addition, the non-enforcement of the South African National Water Act (Act 36 of 1998) to stop or prosecute polluters is also not contributing to a sustainable solution for this looming crisis.
“….and the river flows short of life.” Mike Cuyugan
I was entrusted with the responsibility to develop the ecotoxicology research programme in the Department of Paraclinical Sciences. Thus, the programme was initiated by asking how we can we start with on-going research projects that will contribute to a better understanding of the current freshwater quality dilemma, stimulate research initiatives, and establish aquatic animal diagnostic capabilities at the Faculty of Veterinary Science.
“….I was looking at a river bed. And the story it told of a river that flowed made me sad to think it was dead.”
From the song: A horse with no name by Dewey Bunnell
4.1 The potential benefits of this PhD project are:

1. Stimulation and establishment of basic aquatic animal research at the Faculty of Veterinary Science. The basic anatomical research projects (Nile crocodile cloaca and spinal venous sinus) will be to support safe and efficient sample collection. Considering that most crocodilians are listed as endangered or threatened, it is nearly impossible to obtain ethical permission to kill any wild crocodilians for research purposes. However, the large numbers of Nile crocodiles on commercial farms in South Africa afforded us the opportunity to do basic research on slaughtered animals without influencing wild animals. Samples will be collected by using the established techniques for biochemical laboratory investigations.
2. Samples collected from the sharptooth catfish will be used to test specific blood biochemical parameters. Some of the biochemical parameters have never been evaluated in the sharptooth catfish before, and others only occasionally for clinical diagnostic purposes (no survey). The clinical chemistry results would not only give us some insight into the physiological functioning of these aquatic species (stimulate new research questions and future research projects), but will also serve as normal reference values for the specific species and parameters.
3. Development and validation of specific tissue culture techniques for the African sharptooth catfish. These in vitro tissue culture techniques are innovative (never been established for the sharptooth catfish before) with numerous diagnostic and research advantages.
4. On-going monitoring of aquatic animal health could become a reality by applying the established techniques and methods.
5. All of the planned research projects will involve a number of national (mostly from the University of Pretoria) and international collaborators. Through these different research projects the Department of Paraclinical Sciences will build capacity and also strengthen its international network (United States of America, Japan, Norway, Tanzania and Mozambique). In addition, the collaboration with all the international researchers could potentially stimulate new research questions for future projects, as well as the exchange of expertise and post graduate students.
6. The development and validation of sampling techniques and selected biomarkers will open the door for future research projects for postgraduate students in our Department, and eventually also more scientific publications.

Blood chemistry parameters in the African sharptooth catfish (Clarias gariepinus)

Preface

Blood is a very convenient sample for routine health status monitoring of animals and humans. Although the collection method is minimally invasive, it is not a very difficult procedure, and samples can be collected without being harmfull to the patient. Blood samples are, unfortunately, not as often used for diagnostic purposes in fish, especially not in the case of the African sharptooth catfish, if compared to other animals and humans.
The head of the sharptooth catfish is flattened horizontally and the elongated body is flattened dorso-ventrally, making it easy to work with the catfish lying on its side (in lateral recumbency) (Fig 5.1). Although the method of blood collection from the sharptooth catfish is relatively well known, the anatomy of the caudal vein, running just below the vertebral column, has not been described in any detail for the sharptooth catfish. No detailed blood collection technique investigation was deemed necessary, since this clinical procedure has been used successfully before by the main author to sample large numbers of African sharptooth catfish.
A hypodermic needle is inserted perpendicular over the lateral line, in the region behind the cloacal opening (Fig 5.1), until the tip of the needle touches a vertebral bone. The lateral line is directly over the vertebral column. As the sharptooth catfish has no scales, it is much easier to find the lateral line on the side of the body of the catfish, if compared with scaled fish. The bloodvessel is directly underneath the vertebral bones; therefore, after touching a verterbral bone, the tip of the needle is adjusted slightly ventrally to access the area just underneath the vertebral column. Regular aspiration is needed to detect if the tip of the needle has entered the bloodvessel. The caudal vein was used for the collection of the blood samples.
It was decided to collect blood samples from apparently healthy looking African sharptooth catfish to determine reference ranges for blood parameters, and to investigate if a standard clinical pathology profile, usually used for companion animals and livestock, would be of any diagnostic value (see: aim and objectives). For this investigation, blood was collected directly from the caudal vein using a hypodermic needle and syringe (Fig 5.1). The inside surfaces of all the syringes were pre-coated with heparin and the collected blood samples were immediately transferred to heparin containing blood tubes to prevent coagulation before being centrifuged. Plasma samples were submitted to the laboratory for testing. During this investigation larger internal diameter hypodermic needles (20G; Terumo Corporation, Tokyo, Japan), were also tried to reduce unnecessary red blood cell (RBC) damage due to turbulence when the blood was aspirated through the needle (Fig 5.1).
It is also important to take note that the mucous layer on the skin of the African sharptooth catfish is easily disturbed when a catfish is handled – predisposing the fish to a fungal infection (e.g. Saprolegnia spp.) of the skin afterwards. As far as possible, wet hands or wet gloves must be used, as well as wet towels for the handling of the catfish so that the mucous layer is not unnecessary disturbed or removed. A Saprolegnia skin infection would seriously affect the survival of the catfish that was handled, as well as other fish in the same closed aquatic system.

Aim

Blood samples collected from catfish were screened by the Clinical Pathology Laboratory at the Faculty of Veterinary Science of the University of Pretoria. Standard parameters were used to determine possible biomarkers of health

Objective(s)

1. To analyse blood samples, collected from male and female sharptooth catfish, for several clinical chemistry parameters
2. To determine blood plasma enzyme (ALT, ALP, AST, CK, GGT, LD, HBD, GLD) activities
3. To determine plasma protein (TPP, albumin, globulin, A/G ration) concentrations
4. To determine total bilirubin, urea and creatinine concentrations
5. To determine thyroxine (T4) concentrations
6. To define baseline blood chemistry parameters and propose provisional reference ranges

THE OLD SAILOR
TABLE OF CONTENTS
ACKNOWLEDGEMENTS 
DEDICATION
DECLARATION
PUBLICATIONS THAT EMANATED, DIRECTLY OR INDIRECTLY, FROM THE AFRICAN SHARPTOOTH CATFISH AND NILE CROCODILE RESEARCH PROJECTS
FORMAT OF THIS THESIS
LIST OF FIGURES
LIST OF TABLES
LIST OF ABREVIATIONS 
ABSTRACT
CHAPTER 1: INTRODUCTION 
CHAPTER 2: AIM AND OBJECTIVES
2.1 Aim
2.2 Objectives
CHAPTER 3: LITERATURE REVIEW
3.1 INTRODUCTION
3.1.1. Aquatic ecosystems – the South African situation
3.1.2 Aquatic ecosystem health
3.1.3 Aquatic ecosystem monitoring
3.1.4 Aquatic animal health assessment
3.2 AQUATIC SPECIES
3.2.1 Fish and crocodilians as indicators of ecosystem health
3.2.2 African sharptooth catfish (Clarias gariepinus)
3.2.3 Nile crocodile (Crocodylus niloticus).
3.3 CONCLUSION
CHAPTER 4: JUSTIFICATION
4.1 The potential benefits of this PhD project are:
CHAPTER 5: Blood chemistry parameters in the African sharptooth catfish (Clarias gariepinus)
5.1 Preface
5.2 Aim.
5.3 Objective(s)
5.4 Collaborators (alphabetical), affiliation and their respective contributions
5.5 Publication
CHAPTER 6: Evaluation of the gill filament-based EROD assay in the African sharptooth catfish (Clarias gariepinus) as a monitoring tool for waterborne PAH-type contaminants
6.1 Preface
6.2 Aim.
6.3 Objective(s)
6.4 Collaborators (alphabetical), affiliation and their respective contributions.
6.5 Publication
CHAPTER 7: Establishment and validation of a primary hepatocyte system for the African sharptooth catfish (Clarias gariepinus) 
7.1 Preface
7.2 Aim..
7.3 Objective(s)
7.4 Collaborators (alphabetical), affiliation and their respective contributions
7.5 Publication.
CHAPTER 8: The post-occipital sinus of the spinal vein of the Nile crocodile (Crocodylus niloticus): anatomy and its use for blood sample collection and intravenous infusions
8.1 Preface
8.2 Aim
8.3 Objective(s)
8.4 Collaborators (alphabetical), affiliation and their respective contributions
8.5 Publication
CHAPTER 9: Technique for the collection of clear urine from the Nile crocodile (Crocodylus niloticus) 
9.1 Preface
9.2 Aim
9.3 Objective(s)
9.4 Collaborators (alphabetical), affiliation and their respective contributions.
9.5 Publication
CHAPTER 10: GENERAL DISCUSSION AND CONCLUSIONS
CHAPTER 11: REFERENCES
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