eChapters

3.1. Introduction

Various procedures performed on reptiles often necessitate chemical restraint or general anesthesia. Even routine tasks like physical examination and transportation can pose challenges, particularly with large or uncooperative reptilian patients. For safety reasons, venomous species are typically evaluated under chemical restraint. The respiratory anatomy and physiology of reptiles differ significantly from those of more familiar mammals, influencing their response to commonly used anesthetic agents. Historically, anesthetics recommended for reptiles had notable metabolic effects, but inhalant anesthesia has become more popular in recent times. Nonetheless, in species capable of anaerobic metabolism, induction with these agents may not be feasible (Girling 2013).

2.1. Classification of Fish

Fish are incredibly diverse aquatic vertebrates. There are over 32,000 de scribed species of bony fish, over 1,100 species of cartilaginous fish, and over 100 hagfish and lampreys. Fish can be broadly classified into three groups:

2.1.1. Superclass Agnatha (Jawless fish): These are the most primitive fish, lacking jaws and paired fins. They have existed for over 500 million years. Examples include lampreys and hagfish.

2.1.2. Class Chondrichthyes (Cartilaginous fish): These fish have skeletons made of cartilage instead of bone. They have jaws and paired fins. Examples include sharks, skates, and rays.

2.1.3. Superclass Osteichthyes (Bony fish): This is the largest and most diverse group of fish. They have skeletons made of bone and have jaws and paired fins. Examples include tuna, salmon, goldfish, and many more.

1.1. Introduction

Wildlife management often requires interventions to safely handle and transport animals. Chemical restraint, also known as immobilization or tranquilization, is a technique widely used to facilitate these processes (Hernandez, 2014). It involves the administration of drugs to temporarily immobilize wild animals, allowing for various procedures such as veterinary medical examinations, tagging, relocation, or rehabilitation. This chapter provides an overview of the principles, methods, drugs, and considerations involved in the chemical restraint of wild animals.

It has been often stated that the most important techniques required by the zoo veterinarian are those used in the capture and restraint of the patient. These techniques have developed into a highly technical science (Clarke et al., 2014). In the early days of zoos, the only practical method was to manually catch and hold the patient. Modern techniques, however, permit a veterinarian to shoot a CO2 powered gun, which discharges a projectile syringe at an animal resulting in complete immobilization of the animal (Jacques et al., 2009). This is accomplished without any physical contact between the patient and his captors.

Vitamins are natural and indispensable nutrients that are essential for our bodies in small quantities. Vitamins are indispensable for a multitude of biological functions, including growth, development, wound healing, maintaining healthy bones and tissues, supporting the immune system, and more. These vital organic compounds exhibit diverse biochemical functions and are essential for metabolic processes. The discovery of vitamins dates back to 1912 when Casimir Funk, a Polish American biochemist, initiated research on their sources, functions, and deficiency disorders. Because of his groundbreaking work, Funk is widely recognized as the pioneer of vitamins.

Types of vitamins

The classification of vitamins is determined by their solubility, resulting in two distinct groups: fat-soluble vitamins and water-soluble vitamins.

Fat-soluble vitamins

Fat-soluble vitamins, as their name implies, need fat for absorption and are stored in the fat cells. These vitamins include Vitamin A, D, E, and K.

Enzymes are biocatalysts that play a crucial role in accelerating chemical reactions without undergoing any changes themselves. Enzymes enhance the rate of a reaction by reducing its activation energy without affecting the equilibrium position of the reaction. These catalysts specifically target molecules called substrates and convert them into different molecules known as products. Most enzymes have a proteinous nature, and some have a ribonucleic acid nature (like ribozymes and peptidyl-transferase), showing high substrate and stereo-specificity. Enzyme catalysis is indispensable for the majority of metabolic processes in cells, as it ensures that these processes occur at a rate that is sufficient to sustain life. The progression of metabolic pathways heavily relies on the catalytic activity of enzymes to facilitate individual steps.

The proteinous enzymes are classified into simple and conjugated enzymes.

1. Simple enzymes: Enzymes are made up of only protein components.

2. Conjugated enzymes: Enzymes comprise both protein (apoenzyme) and non-protein components (cofactor).

Nucleic acids are complex, chain-like polymers consisting of nucleotides as their monomers. Due to this characteristic, they are often referred to as polynucleotides. The largest molecules present in the biological system are nucleic acids. The two main types of nucleic acids are DNA (deoxyribonucleic acid) and RNA (ribonucleic acid). The bulk of DNA is confined to the nucleus and some cell organelles like mitochondria and chloroplast. RNA is found in both the nucleus and cytoplasm. In 1869, Johannes Friedrich Miescher isolated a substance from the nuclei of the pus cell (leukocytes). The extracted material was named nuclein, which contained an unusually large amount of phosphorus; in addition to proteins, and devoid of sulfur. Richard Altman later named the nuclein as nucleic acid. These organic molecules are found in living cells and play a crucial role in transmitting genetic information from one generation to another.

Nucleotides

Nucleotides serve as the fundamental constituents of nucleic acids. Nucleotides have flat planar and heterocyclic origin. Nucleotides are small molecules composed of three components:

1. Nitrogenous base,

2. Pentose sugar and

3. Phosphate group.

Proteins are composed of amino acids and are large molecules that play a crucial role in various biological processes. They are considered the fundamental building blocks of life due to their abundance in the body, constituting approximately 60% of the dry weight of cells. These complex biomolecules are intricately folded and twisted strands of amino acids, actively participating in vital life processes, including metabolism, locomotion, immune response intercellular signaling, and molecular identification. As macronutrients proteins are present in all living organisms and directly contribute to numerous metabolic pathways. It is important to note that proteins are unique to each organism, exhibit species specificity, and vary within different organs of the same organism. With 22 different amino acids, the properties of a protein molecule are determined by the specific arrangement of these amino acids. While plants can synthesize all the necessary amino acids for protein production, animals rely on external sources for certain amino acids. Proteins consist of amino acids connected through peptide bonds, formed by the linkage between the amino (NH2 ) group of one amino acid and the carboxyl (COOH) group of another amino acid.

Amino acids are organic molecules made up of carbon (C), hydrogen (H), oxygen (O), and nitrogen (N). However, sulfur (S) and selenium (Se) are also found in some cases. Amino acids are essential organic compounds that come together to create proteins, thus making them the fundamental building blocks of proteins. These biomolecules play vital roles in various biological and chemical processes within the human body and are essential for the growth and development of an individual.

Amino acids consist of an acidic group (-COOH), a basic group (-NH2 ), and a side chain (R) or a hydrogen (H) atom, all bonded to the carbon atom. The features of amino acids are due to the side chain because they differ only in their side chain. All amino acids have the same basic structure except proline, which has a cyclic structure.

Various forms of amino acids like alpha (α), beta (β), gamma (γ) or delta (σ) form are found. The position of the amino group (-NH2 ) determines the form of the amino acid, i.e., if the NH2 group is attached to the α-carbon, it is called α-amino acid; if β-carbon, it is called β-amino acid, and so on. Mostly, α-Amino acids are found as the numbering of amino acids is done from the carboxylic group (-COOH).

Lipids are a class of organic compounds composed of hydrogen (H), carbon (C), and oxygen (O) atoms. These classes of biomolecules come together to create the fundamental building blocks that shape and govern the structure and functionality of living cells. The term lipid was used by the German biochemist Wilhem Bloor in 1943. Lipids are heterogeneous groups of substances that are insoluble in polar (water) and soluble in non-polar (ether, benzene, and chloroform) solvents. Lipids are the esters of fatty acids and their derivatives. Lipids play a crucial role in both the plant and animal domains. They are widely distributed in plants, but lipids are primarily stored in animal adipose tissues.

Classification of lipids

Lipids are classified into four different groups, as shown below:

1. Simple lipids

2. Complex lipids

3. Derived lipids and

4. Miscellaneous lipids

Carbohydrates are the macronutrients consisting of carbon (C), hydrogen (H), and oxygen (O) atoms. In simpler terms, carbohydrates can be defined as polyhydroxy aldehydes or ketones or substances, which yield them on hydrolysis. The general formula of carbohydrates is Cn (H2 O)n , where n is the number of carbon atoms. In nature, carbohydrates are the most abundant biological molecules. In the human diet, carbohydrates are one of the main nutrients that provide energy to the body. The green plants, through the process of photosynthesis, synthesize the carbohydrates.

Classification of carbohydrates

Carbohydrates are also known as saccharides, originating from the Greek word sakcharon, which means sugar. However, not all saccharides have a sweet taste. Carbohydrates can be classified into three distinct groups: monosaccharides, oligosaccharides, and polysaccharides. The number of sugar units determines this classification.

Monosaccharides

Monosaccharides, or simple sugars, are the most basic carbohydrates. They are the monomers that serve as the building blocks for forming polymers or complex carbohydrates. Monosaccharides are the most simple carbohydrates possessing carbonyl groups, i.e., aldehyde (CHO) and ketone (C=O) groups with two or more hydroxyl (OH) groups. Monosaccharide has the generic formula Cn (H2 O)n, where n is the number of carbon atoms. Monosaccharides are sugars that cannot be hydrolyzed further to form simple carbohydrates. These crystalline solids without color dissolve in water but remain insoluble in non-polar solvents.

Introduction and Scope of Biochemistry

Biochemistry is the field of study that focuses on studying chemical reactions occurring within living organisms. It is a specialized branch that combines the principles of both chemistry and biology. In order to comprehend the molecular mechanisms behind every biological event, this multidisciplinary f ield incorporates elements of biology, chemistry, and physiology. The scope of biochemistry extends from the study of small molecules and their interactions within cells to the analysis of complex metabolic pathways, enzyme kinetics, and the structure and function of biomolecules.

One of the main goals of biochemistry is to elucidate the chemical reactions that take place in living systems and understand how they regulate key biological processes. Biochemists investigate the structure, function, and properties of various biomolecules, such as proteins, carbohydrates, lipids, and nucleic acids. It helps us to study how these biomolecules interact and contribute to the overall functioning of cells, tissues, and organisms.

Biochemistry also plays a vital role in various areas of research and practical applications. For instance, in medical biochemistry, scientists study the molecular basis of diseases to develop diagnostic tests and therapeutic interventions. They investigate the biochemical mechanisms underlying genetic disorders, cancer, metabolic, and infectious diseases. Thus, understanding the biochemical basis of diseases can lead to the development of targeted and personalized treatments.

Principle

Cell viability refers to the quantity of healthy cells in a sample, while cell proliferation is a crucial indicator for comprehending the mechanisms and pathways involved in the survival of cells or death following exposure to toxic substances. The methods used to assess viability are typically the same as those used to detect cell proliferation. Cell cytotoxicity and proliferation assays are commonly employed in drug evaluations to determine if the test compounds impact cell proliferation or exhibit direct cytotoxic effects. Cell viability can be determined by calculating the ratio of total live cells to the total number of cells (live and dead). Staining also helps to observe the general cell structure. Cell proliferation assay by MTT. Metabolically active cells reduce the yellow tetrazolium MTT (3-(4, 5-dimethylthiazolyl-2)-2, 5-diphenyltetrazolium bromide) through dehydrogenase enzymes to produce NADH and NADPH. The purple formazan produced inside the cell can be dissolved and measured using spectrophotometry. The MTT Cell Proliferation Assay quantifies cell proliferation rate and detects decreases in cell viability caused by metabolic events leading to apoptosis or necrosis. The MTT Reagent produces minimal absorbance values when no cells are present.

Principle

Adenoviral, oncoretroviral, and lentiviral vectors are commonly utilised for gene delivery in mammalian cell culture and in vivo. Additional important examples of viral gene transfer involve baculovirus and vaccinia virus vectors. Stable cell lines produced with lentivirus technology provide consistent, long-term protein expression compared to the short-term expression achieved through transient transfection. This chapter focuses on cell transformation by lentiviruses.

Equipment

a) Biosafety cabinet level 2 (B2)

b) Carbon Dioxide (CO2 ) Incubator (set to 37°C with a 5% CO2 in air atmosphere)

c) Refrigerated centrifuge

d) Refrigerator

e) Inverted Microscope

f) Micro pipettes (1-10 μl, 10-200 μl, 100-1000 μl)

g) -86°C/-80°C deep freezer h) Autoclave

i) Hot air oven

j) Filtration Assembly

Principle

The majority of cell lines are cultivated as monolayer adherent cells, meaning they only grow on the surfaces of culture vessels. However, there are particular cells that are non-adhesive and do not rely on external support for their growth, referred to as suspension cells. A suspension culture, is a cell culture where individual cells or small cell clusters grow and multiply in a stirred growth medium, forming a suspension. Subculturing, also known as passaging cells, involves transferring cells from a previous culture into fresh growth medium to allow the cell line to continue growing. Suspension cultures involve the suspension of cells in a liquid medium and agitation at a specific speed to ensure that the cells are uniformly exposed to nutrient media from every angle. This method prevents cell aggregation and enhances nutrient absorption, thereby promoting cell growth. The maintenance of these cultures is achieved by transferring the cells from the early stationary phase to a fresh medium through subculturing. During the incubation period, there is an increase in both cell division and cell enlargement, leading to rapid cellular growth. The viability of cells in the suspension decreases after the stationary phase due to the exhaustion of certain factors or the accumulation of toxic substances in the medium. This decrease in the viability of the cells further reduces the growth rate of the entire culture. Next, a portion of the cell suspension is added to the freshly prepared medium with the same composition.

Principle

Neutralization refers to the process of reducing or nullifying the effect of a specific component. Neutralization is a process in which the effects of viral antigens or viruses reduce through Ag-Ab reactions. Neutralizing antibodies are a specific type of antibodies that play a role in neutralization. Not all antibodies generated within our body possess the capability to impede the effects of antigens. Only antibodies with the ability to neutralize can prevent the uptake of viral particles by cells and counteract their effects. The virus neutralization assay is a serological test utilized to identify the existence and extent of functional systemic antibodies that hinder the ability of a virus to cause infection.

Media and Materials

Equipment

a) Biosafety cabinet level 2 (B2)

b) Carbon Dioxide (CO2 ) Incubator (set to 37°C with a 5% CO2 in air atmosphere)

c) Refrigerated centrifuge

d) Refrigerator e) Inverted Microscope

f) Micro pipettes (1-10 μl, 10-200 μl, 100-1000 μl)

g) -86°C/-80°C deep freezer

h) Autoclave

i) Hot air oven

j) Filtration Assembly

Principle

The plaque assay was developed in order to enumerate and quantify the infectivity of bacteriophages. This technique has been subsequently adapted for application in animal virology and has proven to be a dependable method for quantifying the concentration of various viruses that have been adapted for growth in cell cultures. This assay is designed to quantify the capacity of a solitary infectious virus to create a visible area of cell death on a layer of cultured cells. A plaque forms during the viral infection cycle, in which the host cell dies after viral replication. Some viruses cause cell lysis during replication, while others cause minimal damage to the cells. The plaque assay is a technique used to measure the quantity of viruses that cause the destruction of their host cells, resulting in cytopathic effects (CPE). During a plaque assay, the host cells and virus are briefly co-cultured to enhance the virus's attachment and entry into the host cell. Subsequently, the concoction is introduced into a partially solidified agar medium. The thick agar is poured onto a layer of "bottom agar" which supplies ample nutrients for the host cell. After one round of virus replication, an infected cell will undergo lysis, leading to the release of multiple new viruses. Within the gelatinous substance, these newly released viruses can only infect neighbouring cells. After a subsequent round of replication, these neighbouring cells will undergo lysis. The cells that manage to avoid infection will continue to proliferate. After a period of 24 to 48 hours, plates that were not contaminated with a virus will display a consistent layer of cells. The plates infected with a multitude of viruses exhibit plaques. Quantifying viral titer is essential for assessing viral infectivity. The determination can be made either by conducting a plaque assay or by calculating the infectious dose.

Principle

Red blood cell sticking (hemadsorption) can reveal the presence of certain viruses. This happens because these viruses, like influenza or measles, sprout proteins (hemagglutinin) on their infected host cells. These proteins act like Velcro, attracting red blood cells and causing them to cling to the surface. A hemadsorption assay uses this natural process to check for viruses. It's faster than waiting for the cells to show damage (cytopathic effect), allowing for earlier detection. Haemadsorbing viruses can be detected several days prior to the appearance of a cytopathic effect.

Equipment

a) Biosafety cabinet level 2 (B2)

b) Carbon Dioxide (CO2 ) Incubator (set to 37°C with a 5% CO2 in air atmosphere)

c) Refrigerated centrifuge

d) Refrigerator

e) Inverted Microscope

f) Micro pipettes (1-10 μl, 10-200 μl, 100-1000 μl)

g) -86°C/-80°C deep freezer h) Autoclave

i) Hot air oven

j) Filtration Assembly

Principle

The quantification of infectious virus particles is commonly achieved through the utilisation of the Median Tissue Culture Infectious Dose (TCID50) assay. The assay functions through the introduction of a serial dilution of the virus sample into cells in a 96 well plate format. The chosen cell type is specifically intended to exhibit a cytopathic effect (CPE), which refers to observable changes in morphology or cell death resulting from viral infection. Following a period of incubation, the cells are examined for cytopathic effects or cell death, and each well is categorised as either infected or uninfected. The concentration at which 50% of the wells exhibit a cytopathic effect (CPE) is employed to determine the 50% tissue culture infectious dose (TCID50 ) of the viral sample. The end point is the dose or dilution that infects or kills or shows CPE of the cells. The calculation can be performed using various mathematical techniques, such as the Spearman-Karber method or the Reed Muench method. The virus titer is quantified as the number of tissue culture infectious dose 50 (TCID50 ) per millilitre (ml). The non-cytopathic virus can be quantified by employing the fluorescent antibody test, immune-peroxidase assay, or other appropriate methodologies.

Principle

Unlike bacteria, which can be cultivated on an artificial nutrient medium, viruses rely on a living host cell for replication. Eukaryotic or prokaryotic host cells that have been infected with viruses can be used to produce viruses. Subsequently, the medium and cells can be collected and used as a virus source. The production of viruses usually consists of a two-step procedure: f irst, the growth of host cells, and then the production phase. Understanding the complex relationship between the host cell and virus is essential. Various crucial factors have been recognised, such as the concentration of host cells during infection and the multiplicity of infection (MOI).

Materials required

Equipment

a) Biosafety cabinet level 2 (B2)

b) Carbon Dioxide (CO2 ) Incubator (set to 37°C with a 5% CO2 in air atmosphere)

c) Refrigerated centrifuge

d) Refrigerator

e) Inverted Microscope

f) Micro pipettes(1-10 μl, 10-200 μl, 100-1000 μl)

g) -86°C/-80°C deep freezer

h) Autoclave

i) Hot air oven

j) Filtration Assembly

Principle

The one-step growth experiment refers to a scientific methodology. A study aimed at observing the molecular events that take place during viral reproduction. This elucidates the fundamental features of the virus replication process and determines the optimal multiplicity of infection and timing for virus harvest. The initiation of modern bacteriophage research can be traced back to the development of the one-step growth curve by Max Delbrück and Emory Ellis in 1939, which utilised the Escherichia coli-T4 bacteriophage system. One step growth curve consists of three clearly defined phases 1. Latent period refers to the initial phase of a process where no significant changes or activities are observed. 2. Burst or Rise period is the subsequent phase characterised by a sudden increase virus particle. 3. Plateau period is the following phase where a stable and constant level virus replication.

Materials required