eChapters

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.

I. WALL COMPONENTS – CHEMISTRY

The main ingredient in cell walls are polysaccharides (or complex carbohydrates or complex sugars) which are built from monosaccharides (or simple sugars). Eleven sugars are common in these polysaccharides including like glucose and galactose. Carbohydrates are good building blocks because they can produce a nearly infinite variety of structures. There are a variety of other components in the wall including protein, and lignin. Let’s look at these wall components in more detail:

A. Cellulose: β1,4-glucan (structure provided in class). Made of as many as 25,000 individual glucose molecules. Every other molecule (called residues) is “upside down”. Cellobiose (glucose-glucose disaccharide) is the basic building block. Cellulose readily forms hydrogen bonds with itself (intra-molecular H-bonds) and with other cellulose chains (inter-molecular H-bonds). A cellulose chain will form hydrogen bonds with about 36 other chains to yield a microfibril. This is somewhat analogous to the formation of a thick rope from thin fibers. Microfibrils are 5-12 nm wide and give the wall strength - they have a tensile strength equivalent to steel. Some regions of the microfibrils are highly crystalline while others are more “amorphous”.

Introduction

Robert Brown discovered the nucleus in some plant cells in 1831. After analyzing tissues from a range of animals and plants, T. Schwann (1839) declared, “All living organisms are composed of cells.”

The fundamental elements of Prokaryotic and Eukaryotic cells

The simplest cells, called prokaryotic cells, have a straightforward structural arrangement. There is only one membrane system in it. They contain rickettsia, spirochete, mycoplasma, virus, blue-green algae, and bacteriablue-green algae, or cyanobacteria are the largest and most intricate prokaryotes, where higher plant photosynthesis occurs and have changed throughout time.

Prokaryotic cells

Prokaryotic cells range in size from 1 to 10 µm. They take place in a range of formats. A bacterial cell is made up of three basic parts:

I. External layer: It is composed of the inner cell or plasma membrane, middle cell wall and outer slimy capsule.

a. Cell membrane: The thin, flexible membrane of the cell, composed of proteins and lipids, regulates the flow of chemicals throughout the cell. It carries respiratory enzymes for reactions that release energy. Respiratory enzymes are found in mesosomes, which are folded regions of the plasma membrane that are compared to the mitochondria in eukaryotic cells. Similarly, the pigments and enzyme molecules found in photosynthetic cells are linked to the in-folds of the plasma membrane known as photosynthetic lamella, which is responsible for absorbing light and converting it into chemical energy.

b. The cell wall: It is the non-living, semi-rigid layer that encloses the cell membrane, with a thickness that varies from 1.5 to 100 µm. It is chemically made up of peptidoglycans. Certain bacteria, such as mycoplasma, don’t have cell walls.

Introduction

The smallest, most basic, most primitive organisms are known as prokaryotic (Greek: pro= primitive or prior, and karyon = nucleus). Likely, they originated around 3.5 billion years ago. Bacteria, such as Mycoplasma and Cyanobacteria, are known to contain these cells. A prokaryotic cell is a deeply organized single-enveloped system. Its constituent nuclear parts are encased in a plasma membrane, with the cytoplasmic ground substance encircling the center. Prokaryotic cells’ cytoplasm lacks distinct cytoplasmic organelles, the nuclear envelope, and any other cytoplasmic membrane.

Despite being incredibly thin and delicate structures, in Eukaryotes plasma membranes are essential to many of a cell’s most vital processes. Membranes are lipid-protein assemblages where non-covalent linkages hold the constituents in place in a thin sheet. Both integral protein and phospholipid lateral mobility are significantly impacted by the physical condition of the lipid bilayer. The plasma membrane is a selectively permeable barrier that permits solute passage via assisted diffusion, active transport, and simple diffusion via membrane channels or the lipid bilayer. A system of membrane organelles, such as the endoplasmic reticulum, Golgi complex, and lysosomes, is found in the cytoplasm of eukaryotic cells. These organelles are physically and functionally connected to the plasma membrane and one another. The cytoplasm is separated into a luminal region inside the ER membranes and a cytosolic space outside the membranes by the endoplasmic reticulum (ER), a network of tubules, cisternae, and vesicles. Additionally, the ER is where the majority of a cell’s membrane lipids are generated and transported to different locations. Proteins’ asparagine residues are added with sugars (a process known as glycosylation), which starts in the rough ER and continues in the Golgi complex.

Brain

The brain, a command centre of the body, is a complex organ. The substance of the brain is made of the grey matter and the white matter. The grey matter is often identified as cerebral cortex but other parts of the brain also contain the grey matter. Inside the brain, the grey matter is organized into large accumulations of neurons which are called the nuclei of the brain. Each nucleus is associated with a specific function. In addition to neurons, there is other type of cells the glial cells called neuroglia. The neurons are responsible for sending and receiving impulses or signals. The glial cells are nonneuronal and provide support and nutrition to neuronal cells, form myelin and facilitate signal transmission in the nervous system. The white matter is made of millions of nerve fibres that are coming from neurons and are going to establish communications with different parts of the brain and spinal cord. Since these nerve fibres are coated with myelin sheath around them these appear white and the area occupied by these nerve fibres become the white matter of the brain.

The brain has three basic components, the forebrain, midbrain and the hindbrain.

Fore brain: The forebrain (Prosencephalon) is the rostral portion of the brain. It consists of cerebrum (Telencephalon) and the thalamus, hypothalamus and pineal gland together constituting the interbrain (Diencephalon).

Mid brain: The midbrain (Mesencephalon) is the middle portion of the brain. It is in the centre lying between the interbrain and hindbrain. It consists of a cranial part of brain stem.

Hind brain: The hindbrain (Rhombencephalon) is the caudal most portion of the brain. It consists of remaining (caudal) part of the brain stem, the medulla oblongata (Myelencephalon) and the cerebellum and pons (Metencephalon).

Large Animals The collection and evaluation of cerebrospinal fluid (CSF) in farm animals with signs of diseases of nervous system provides very valuable diagnostic and prognostic information. Routinely, CSF is evaluated for total protein level, erythrocyte and leukocyte counts, and a differential cell count. Additional tests are available for specific neurologic conditions. CSF is primarily produced by the choroid plexuses in the lateral, third, and fourth ventricles through plasma ultra filtration and active transport across the blood-brain barrier. Smaller amounts of CSF are generated by the ependymal lining, pia arachnoid, meningeal blood vessels, and spinal cord’s central canal. The turnover rate is about 1% per minute, meaning systemic electrolyte or acid-base changes take several minutes to affect CSF

Animal body contains several fluids other than blood and lymph which have specialized functions for specific organs. Two of these fluids are cerebrospinal fluid and synovial fluid. The cerebrospinal fluids protects and supports brain and spinal cord function. Synovial fluid protects the functioning of the movement of the body by bone-muscular joints.

Life is fundamentally a complex interplay of chemical interactions and reactions. Chemistry examines the composition and properties of matter, the interactions between chemical components, and the mechanisms by which complex structures are formed. Biological chemistry, or Biochemistry, delves into these principles within the context of living systems, highlighting the intricate relationships between molecular structure and function. Understanding fundamental chemistry and its application to anatomy and physiology is crucial because the human body is composed entirely of chemical substances. Basic chemistry encompasses the study of matter, its various states, and energy in all its forms. In order to understand the basic chemistry of living let us start from the scratch of learning and remembering everything that we have learnt so far. Matter and Its Properties Anything with mass and occupying space is referred to as matter. Solid, liquid, and gas are its three main states. Mass, distinct from weight, represents the quantity of matter in an object and remains constant regardless of location. In contrast, weight depends on gravitational force. 

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.

4.1. Classification

The class Aves, also known as birds, is a group of warm-blooded vertebrates characterized by feathers, toothless beaked jaws, laying hard-shelled eggs, a high metabolic rate, a four-chambered heart, and a strong yet lightweight skeleton. Birds are found worldwide and come in a variety of sizes, from the tiny humming bird to the massive ostrich.

The classification of Aves can be broken down into two major subgroups: Palaeognathae and Neognathae.

4.1.1. Palaeognathae: Also known as ratites, these are flightless birds with f lat sterna (breastbones) that lack keels, which are important for flight muscle attachment. Examples of palaeognathae include ostriches, emus, rheas, and kiwis.

Carnivora is an order of placental mammals that have specialized in primarily eating flesh, whose members are formally referred to as carnivorans. The order Carnivora is the fifth largest order of mammals, comprising at least 279 species. They come in a very large array of different body plans with a wide diversity of shapes and sizes. The common characteristics of carnivores are given below:

• They are meat eaters.

• There is the presence of canine teeth.

• The intestinal tract is short and is adapted to rapid digestion as well as the assimilation of meat.

• All species have anal glands. Some species, like the striped skunk (Mephitis mephitis), may eject the contents of the anal gland as a defensive maneuver.

• Os penis is present in males.

• There is a lack of a clavicle which helps in the freedom of movement of the forelimb.

• Ulna is well developed.

• Toes end in claws.

9.1. Classification: Order Perissodactyla

The order Perissodactyla, also known as odd-toed ungulates, is a group of herbivorous mammals characterized by having one or three toes on each foot. They are distinguished from even-toed ungulates (Artiodactyla) by this anatomical feature. Even-toed ungulates, such as pigs, cows, and deer, have an even number of toes (two or four) on each foot. Some of the well-known members of this order include horses, zebras, asses, tapirs, and rhinoceroses.

The name “Perissodactyla” comes from the Greek words “perissos,” meaning “odd,” and “daktylos,” meaning “finger or toe,” referring to their unique foot structure. Unlike even-toed ungulates (Artiodactyla), which bear weight on their third and fourth toes, perissodactyls primarily bear weight on their third toe.

7.1. Introduction

Chemical restraint of insectivores is a technique used by veterinarians and wildlife biologists to immobilize small mammals that primarily eat insects, such as shrews, moles, and hedgehogs. It is essential for performing examinations, administering medications, and conducting research on these animals.

There are several factors to consider when chemically restraining insectivores:

• Species: Different species of insectivores may have varying degrees of sensitivity to different drugs.

• Age and health: Younger and healthier animals generally tolerate chemical restraint better than older or sick animals.

• Temperature: Insectivores are sensitive to temperature regulation, so it’s crucial to maintain a stable body temperature during restraint.

10.1. Classification of Primates

The classification of primates is a complex topic that has been revised over time as our understanding of these fascinating creatures has evolved. Here’s a breakdown of the current classification system:

10.1.1. Order: Primates

Primates are an order of mammals that includes lemurs, lorises, tarsiers, monkeys, apes, and humans. They are characterized by a number of shared features, including:

• Opposable thumbs: This allows primates to grasp objects and manipulate their environment.

• Forward-facing eyes: This provides primates with depth perception, which is important for arboreal locomotion and foraging.

• Enlarged brains: Primates have relatively large brains for their body size, which is associated with their intelligence and complex social behaviors.

• Reduced sense of smell:Compared to other mammals, primates have a weaker sense of smell. This is thought to be due to their reliance on vision for foraging and social interaction.

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).

Rodents and lagomorphs are two distinct groups of mammals, often mistaken for each other due to some shared physical characteristics. However, they belong to different taxonomic orders and possess several key differences

5.1. Rodents

They belong to the order Rodentia and the example species include rats, mice, squirrels, hamsters, guinea pigs, beavers, and porcupines. Order Rodentia is the largest and most diversified mammalian group, differing widely in size, behavior, feeding habits, anatomy, and physiology. Based on the anatomy and functional differences of masseter muscles, it is divided into 3 suborders viz., Caviomorpha, Myomorpha, and Sciuromorpha. Caviomorpha is also called hystrichomorpha and includes guinea pigs, chinchillas and degu. The suborder Myomorpha includes mice, rats, hamsters and gerbils. These animals have the strongest ability to gnaw among animals in all 3 suborders. Sciuridae is a family that includes small or medium size rodents. This family consists of tree squirrels, ground or medium size squirrels, and large size squirrels also known as marmots or prairie dogs (Cynomys ludovicianus). Smaller and less bushy tailed squirrels are known as chipmunks (Tamias striatus) and flying squirrels (Glaucomys volans).

8.1. Classification

The ruminants belong to the Order Artiodactyla. The families in order of Artiodactyla include Camelidae, Tragulidae, Cervidae, Giraffidae, Antliocapridae, and Bovidae.

The family Camelidae includesGuanaco (Lama guanicoe), Llama (Lama glama), Alpaca or South American camelid mammal (Lama pacos), Dromedary camel (Camelus dromedarius), and Bactrian or two-humped camels (Camelus bactrianus).

The family Tragulidae includes Chevrotains originate from Africa (Hyumo schusaquaticus) and Chevrotains that originate from Asia (Tragulus species).

The family CervidaeincludesWhite tailed deer(Odococlius virginianus), Follow deer (Dama dama), Roe-deer (Capreolus capreolus), Axis deer (Axis axis), Reindeer (Rangiter tarandus), Musk deer (Moschus species) and Chinese water deer (Hydropotes species).

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.

The constitution of different solutions plays a significant role in successful demonstration of anatomical techniques especially the special stains. The sincerety begins at the time of fixation of the tissues and deviation at any step may lead to failure of procedure. The chemical reagents required for a particular method have been enlisted at their sites however; the commonly used chemicals are comprehended at one place for convenience of the users.

Carbohydrates are the most abundant class of organic compounds found in living organisms. Carbohydrates are also called sugars and are found in plants and animals. Plants synthesize them through photosynthesis (endothermic) by fixing carbon, hydrogen, and oxygen in the presence of solar energy and chlorophyll pigments.
nCO2 + nH2O + energy → CnH2nOn + nO2
Carbohydrates are stored as starch in plants as a source of energy. They are also stored in animals as glycogen in muscles and the liver. They are also found in skeletal structures of cell walls in plants (cellulose), bacteria (peptidoglycans), insects, and crustaceans. This stored food is consumed by animals and man, and upon their oxidation in the body, energy is released for various metabolic activities (work, growth, and various productive functions). Compositionwise, plants are more prosperous in carbohydrates (30%) than animals (1%), with water at 60% and ash at 4%. Plants are poor in proteins (5%) and lipids (1%) as compared to proteins (20%) and lipids (15%) in animals.
2.1 Introduction to Carbohydrates
Carbohydrates or saccharides (sakcharon=sugar or sweetness) are a chemical term applied to a large group of substances, which are organic compounds containing elements Carbon, Hydrogen, and Oxygen in a ratio of 1:2:1. Hydrogen and Oxygen atoms are in the ratio of two to one as in water molecules. As the ratio of hydrogen to oxygen is like H2O, they are also known as “hydrates of carbon” and are represented as (CH2O)n. But there is an exception to this general formula. Deoxyribose (C5H1OO4) and rhamnose (C6H12O5) do not have the required ratio of 2:1 for hydrogen to oxygen. On the other side, some compounds possess this required ratio but are not carbohydrates, e.g., formaldehyde (CH2O), acetic acid (C2H4O2), and lactic acid (C3H6O3). Further, some carbohydrates have nitrogen(glucosamine), phosphorus, and sulfur but do not satisfy the above general formula. The simplest sugars are called monosugars or monosaccharides. There are two main functional groups in