eChapters

Working Principle: An autoclave is a sealed device that creates a different environment from the outside world under extraordinarily high pressure (Auto + clave = automatic locking). It uses moist heat or saturated steam under pressure to kill microorganisms and heat-resistant endospores. Its function is compared to that of a pressure cooker, i.e., steam is used to create a high pressure environment. However temperature of autoclave or steam sterilizer is generally maintained at 121°C for 15-20 minutes that is achieved by raising the pressure inside the device. This high pressure increases the boiling point of water and therefore raises the temperature that is too high for the microbes to survive, so used for sterilization.

Types of Autoclave cycle

1. Liquid cycle: Liquids take time to sterilize as they have high heat capacity. It is based on the principle of pressurized steam at 15 psi, 121°C temperature for 20 minutes. It has latent heat, which is 7× heat 100°C. Liquids and biological waste require slow exhaust to prevent the boiling over of super-heated liquids. This excess temperature is enough to hydrolyze the proteins of bacteria, viruses, or any other contaminant and therefore kill it. However, wet heat is seven times more intense than dry heat, so it requires less temperature than the dry heat cycle of the autoclave.

2. Dry Cycle: Here, steam is the sterilization medium, which penetrates inside the load and makes contact with all the surfaces, so a fast exhaust cycle is used. It has a high temperature of 160°C for 60 minutes.

What is primary cell culture?

In vitro culture of cells freshly obtained from the tissue is called primary cell culture. Unlike that of cell line, primary cell culture grows for a limited period of time. Examples, are primary mammary epithelial cells, primary hepatocyte culture.

Aim: To prepare the primary cell culture from chicken embryos.

Materials required for the primary cell culture

• 70% alcohol

• Cotton swabs

• Small beaker 20-50 ml

• Forceps

• Straight and required curved,

• 9 cm Petri dishes

• 11 day embryonated eggs

• Balanced Salt solution (BSS)

• 250 ml conical flask

• Magnetic centrifuge tubes

• Haemocytometer

• Growth medium with serum

• Culture flasks

What is keratinocyte?

The epidermal layer consists of keratinocytes, melanocytes, Langerhans cells, and Merkel cells. Their major function is to produce Keratinocytes which are the most abundant cells in the epidermis, keratin protein. Keratinocyte synthesizes major structural components of the epidermal barrier through a programmed process of differentiation. They are present in the innermost layer of the epidermis (Eckert et al?, 1989). The epidermis is composed of several layers, stratum corneum (horny layer), stratum lucidum (clear layer), stratum granulosum (granular layer), stratum spinosum (prickle cell layer), and stratum basale (basal layer), from the outermost to the deepest layer respectively. The process of maturation and movement of the keratinocytes from the stratum basale to the stratum corneum is what we call epidermal differentiation.

Procedures for isolation of canine karatinocytes

When studying skin cells, isolating keratinocytes from tissue samples, such as skin biopsies, is a critical initial step. Here is a general guide on how to isolate keratinocytes:

What is dermal fibroblast?

Skin fibroblasts are a type of cell found in the dermis, the middle layer of the skin. They are the most common cells in connective tissue and play a crucial role in maintaining the structural integrity of the skin. Fibroblasts are responsible for synthesizing and secreting extracellular matrix proteins such as collagen, elastin, and fibronectin, which provide support and elasticity to the skin. Additionally, fibroblasts play a role in wound healing by proliferating and migrating to the site of injury, where they aid in tissue repair and scar formation.

Aim: To prepare the primary cell culture of fibroblasts from canine skin.

Tools and instrument required for the procedure

1. Tissue collection box with ice cubes - To prevent contamination and maintain the stable temperature

2. Surgical Gloves - To prevent contamination

3. Tissue collection tube with 1X PBS - To collect sample

4. Laminar flow hood - To work in a sterile environment and prevent contamination

5. Cell culture dish - To digest the skin

6. Sterile 1000 μL and 200 μL pipette and tips-For accurate measurement and transfer of cell suspension and culture media.

7. Forceps - Fine-tipped forceps for handling and manipulating tissue.

8. Scalpel and blade holder - Scalpel for the dissection of skin tissue.

9. Surgical scissors - Chopping of the skin tissue

10. Falcon 70 μm cell strainer - To filter out undigested tissue debris and obtain a homogeneous cell suspension.

11. 15 ml and 50 ml Centrifuge tubes - To collect and centrifuge the digested cell suspension.

What is subculture?

In cell culture, a process called subculturing, or passaging, keeps cells alive and growing. It involves taking cells from an existing container, removing the used medium, and placing them in a new container with fresh nutrients. This allows the cells to continue multiplying and creates more cells for research.

Adherent subculture protocol Cells nearing 80% coverage need splitting!

1. Warm things up: Get your media, detachment solution, and incubator ready at 98.6°F (37°C).

2. Safety first! Disinfect your work area and supplies inside a biosafety cabinet.

3. Gently rinse: Remove the old media and wash the cells with a salt solution at room temperature. Be careful not to dislodge the cells.

4. Detach the cells: Add a pre-warmed enzyme solution to detach the cells from the flask surface. Keep an eye on them to make sure it works (usually 2 minutes). Note: Not all cells need this enzyme, and it can be harmful to some.

5. Stop the enzyme: Once all the cells are detached, add a culture medium with serum to neutralize the enzyme.

6. Collect the cells: Transfer the cell suspension to a tube, rinse the flask with fresh medium to get all the cells, and add it to the same tube.

7. Spin it down: Centrifuge the cell suspension for 5 minutes at room temperature (1000 rpm).

8. Get ready for counting: Remove the liquid, loosen the cell clump, and resuspend the cells in a fresh medium at a good volume for counting.

9. Count your cells: Use a hemocytometer to count the cells and determine their health.

Introduction

The protocol below describes the use of cell freezing methods involving an electric -80°C freezer for the cryopreservation of cell lines. ECACC routinely use a programmable rate-controlled freezer. This is the most reliable and reproducible way to freeze cells but as the cost of such equipment is beyond the majority of research laboratories the methods below are described in detail. If large numbers of cell cultures are regularly being frozen then a programmable rate-controlled freezer is recommended.

Materials

• Freeze medium (commonly 90% FBS, 10% DMSO or glycerol

• 70% (v/v) alcohol in sterile water

• PBS without Ca2+/Mg2+ • 0.05% trypsin/EDTA in HBSS, without Ca2+/Mg2+

• DMSO

Principle

Isolating the virus in a laboratory is the gold standard for diagnosing many viral infections. Virus isolation is a highly specialised technique used to diagnose viral infections and produce a stock of viruses for research purposes. The isolation of the virus relies on the suitability of the sample, the ability of the cells to allow entry, the successful replication of the virus, and the release of viral particles from the cells. The choice of cell lines is dependent upon the suspected viral disease and the availability of such cell lines in the laboratory.

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) Water bath

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

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

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 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

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 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

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

In order to evaluate the correct status of the organs their correct disposition within the body becomes very essential. Although one cannot see the organs beyond the skin, but appropriate knowledge of the topography of the organs can act as a glass covered watch wherein one can see from outside the arms and the dial of the watch.

Horse

Heart: The heart is placed in the middle mediastinum of the thoracic cavity with its long axis directed caudo-ventrally. Three fifth of the heart lies on the left of median plane while two fifth is on the right of the median plane. The base is placed dorsally, with its highest point at the junction of the dorsal and middle thirds of dorso-ventral diameter of the thorax. As a whole it lies opposite to second intercostal space/third rib to sixth rib/sixth intercostal space. The apex of the heart lies in the center of the last segment of the sternum about half inch from the sternal part of the diaphragm. Heart is directly related to the lateral thoracic wall at the cardiac notches formed in the lungs. The left surface of the heart extends from third to sixth rib in the lower third of chest while the right surface extends from third to fourth intercostal space.

Ultrasound is mainly used for reproductive purposes in bovine, now days it is also used for other applications. Ultrasound is an excellent tool for diagnostic imaging in large animals because it is portable, easy to use in the field, produces results at the time of the examination. It is totally non-invasive. Palpation of a gland can only detect certain levels of abnormality. Ultrasound can give an actual perception of mammary health, a picture of the inside anatomy and pathology. Tumors, scarring, stones, trauma, hematoma formation and level of damage due to mastitis can all be evaluated by this technique. Other diagnostic tools are still primary diagnostic tools, ultrasonogram gives yet another dimension to the hard to diagnose case. It will also allow a better estimate of treatment prognosis to aid in deciding whether to cull or treat a dairy animal.

Congenital anomalies at initial stages of the mammary gland development during gestation may result into presence of supernumerary teats or less number of external teats, in place of usual presence of four teats in buffaloes. Hence, cases with two / three / five or six teats are often encountered in the f ield.

Congenital anomaly of newborn buffalo calves is quite common in new born calves. Most common encountered are atresia ani, rectovaginal fistula etc. Sometimes, developmental defects interfere with physiological processes viz. in urination and defecation. Therefore, such cases, if not corrected well in time can cause death of the patient. In the present report, few newborn calves were brought to the veterinary clinic with the following abnormalities:

Agenesis of Urogenital System Without Anus

Five newborn buffalo calves were brought to the veterinary clinic with the complain of not passing urine and feces. There was no visible external genitalia and anal opening. At the site of vulva, there was only an impression of vulvar lips and below it, a cord like structure was hanging.

There was an impression of round structure below the skin; in some cases it was outside giving appearance of scrotum which was located in between the two thighs. On ventral side of abdomen, preputial sheath was not present, therefore, it was very difficult to decide about the sex of the calves and site of external preputial-orifice. There was an indication of both sexes, but organs were not developed, therefore it was difficult to decide about the sex of the calves. In some cases, impression of the anal sphincter was visible but there was no visible external anal opening. There was slight deviation of the tail from its base also. Intake of milk was normal. Abdomen was distended bilaterally. Respiratory rate was higher than the normal with slight discomfort .Heart rate was elevated. Blood picture revealed increased total blood cell count with neutrophilia. Radiography of the abdomen showed distended urinary bladder at the floor of pelvis indicating formation of the urogenital system without development of the external opening. Barium meal study revealed marking of gastrointestinal tract and stagnation of the barium up to the rectum.

Ultrasonography in sheep is becoming popular in the field of reproduction and replacing the other old-age techniques, viz. abdominal ballotment, recto- abdominal probes, X-rays and radioimmuno-assays. The reasons are numerous such as unsatisfactory results, hazardous effects to animal, operator or environment, high cost, long delay in diagnostic results and requirement of sophisticated labs. The ultrasound system has low operating cost and does not need special labs as it can be done at farm itself. Ultrasonography is considered safe to sheep and the operator and provides quick and reliable results. The popular opinion about ultrasonography is that it is noninvasive imaging system and is rapid, safe and practical means of diagnosis of pregnancy in sheep. The B-mode real time ultrasonographic system particularly portable system is popular among sheep breeders. In sheep, B-mode real time ultrasonography was used in 1971, using 5 MHz frequency transducer. A transducer of switchable frequency between 7.5 to 5.0 is more popular in sheep practice.

Transrectal Procedure in Sheep

The procedure is similar to goat, but sheep remain quite during transrectal ultrasonography than goat. Following are some of the steps to obtain good quality image:

Corpus luteum, one of the biological clock of the estrous cycle and pregnancy, is known foremost for its production of progesterone that blocks the pituitary release of gonadotrophins and prepare the uterus for pregnancy. The ovarian follicle wall consists of granulosa cell and theca cells vascularised and luteinize after ovulation to from a corpus luteum. Corpus luteum is temporary steroid- producing gland undergoes marked structural and functional changes far a short time span during its development functional life and regression. During estrous cycle, the corpus luteum undergoes an initial period of growth (metestrus), followed by a period of maximal function (diestus) and finally by a period of regression (proestrus) preceding the next ovulation (Robert, 1986). The most prominent cell in the CL is the large luteal steroidogenic cell.

Ultrasound imaging has been used to detect and measure the corpus luteum throughout the estrous cycle and corpus luteum has been characterized during pregnancy and non pregnancy. Changes in corpus luteum diameter as detected by ultrasonography are highly correlated with progesterone production. A high correlation has been found in the ultrasonographic detection of bovine corpora lutea (Lean et al., 1992) and between luteal tissue area measured ultrasonographically and plasma progesterone concentration (Bergfelt et al., 1989).

Environmental agents (xenobiotics) that are injurious to cells trigger a spectrum of responses that can range from cell death to adaptation, repair, and proliferation. Cell death, in turn, can take the form of apoptosis (a programmed form of cell death). Xenobiotics have been implicated in toxic cell injury, among them free radicals (nitrogen, oxygen, and carbon derivatives) are known to interact with cellular constituents including membrane lipids and chromagen materials.

Cytotoxicity

Cytotoxic: Toxic to cells, cell-toxic, cell-killing. Any agent or process that kills cells. The prefix cyto- denotes a cell. It comes from the Greek “kytos” meaning hollow, as a cell or container. Toxic is from the Greek “toxikon” = arrow poison.

In nutshell, cell cytotoxicity refers to the ability of certain chemicals or mediator cells to destroy living cells. By using a cytotoxic compound, healthy living cells can either be induced to undergo necrosis (accidental cell death) or apoptosis (programmed cell death).

Measuring cytotoxicity can prove to be very valuable tool in identifying compounds that might pose certain health risks in humans and animals. Cytotoxicity assays are widely used by pharmaceutical industry to screen for cytotoxicity in compound libraries from initial high-throughput drug screens for unwanted cytotoxicity. This can be of vital importance and prove to be quite indispensable during research phase of developing therapeutic new pharmaceutical products to ensure the safety of end-users. Additionally, understanding the mechanisms involved in cytotoxicity can likewise give researchers a more in-depth knowledge on the biological processes (both normal and abnormal) governing cell growth, cell proliferation and death. Compounds that have cytotoxic effects often compromise cell membrane integrity. Therefore, assessing cell membrane integrity is one of the most common ways to measure cell viability and cytotoxic effects. There are three commonly used methods which can be employed to assess in vitro cytotoxicity (disruption of cell integrity) in cell cultures:

Safety is a prime concern for humans and animals. Without evaluating the safety of a chemical, no regulatory authority in the world allows the chemical to be used by humans. There are stipulated guidelines for toxicity evaluations. Among the toxicity assays genotoxicity assays are important and have occupied centre stage due to increase in understanding of the genome.

Genotoxicity describes the property of chemical agents to damage the genetic information within a cell to cause mutations, which may even lead to cancer. While genotoxicity is often confused with mutagenicity, all mutagens are genotoxic; however, not all genotoxic substances are mutagenic. Generally and depending on the agent, a cytotoxic agent may be genotoxic (causing DNA damage), mutagenic (causing gene mutation) or it may be harmful for cell organelles (e.g. cell membrane) or it may even be teratogenic. Therefore, every cytotoxic agent is not genotoxic.

Why we Need to Evaluate Genotoxicity?

Toxicity can be at any level according to the nature of chemicals and type of exposure. Duration of toxicity can also be short lived or long lived within a generation or continue to next generation. Genotoxicity effects are able to continue in next generation(s), in which the exposed population may not suffer seriously but the next generation may suffer without being exposed to the same chemicals. Therefore, genotoxicity evaluations need serious attention.

The concept of single cell micro-gel electrophoresis (SCGE)/comet assay is f irst introduced by Ostling and Johanson (1984) as a method to measure DNA single-strand breaks under neutral conditions. Singh et al. (1988) modified this technique by using alkaline conditions (>13 pH) of electrophoresis, which enabled detection of not only frank strands breaks but also alkali labile sites, DNA cross- linking and incomplete excision repair sites. Now-a-days, this technique is extensively used to study the genotoxic effects of various chemicals. The advantages of SCGE technique include: (1) collection of data at the level of individual cell, allowing for more robust types of statistical analysis; (2) need for small number of cells per sample (<10,000); (3) its sensitivity for detecting DNA damage; and (4) that virtually any eukaryotic cell population is amenable to analysis. The idea is to combine DNA gel electrophoresis with fluorescence microscopy to visualize migration of DNA strands from individual agarose-embedded cells. If the negatively charged DNA contained breaks, DNA supercoils are relaxed and broken ends are able to migrate toward the anode during a brief electrophoresis. If the DNA is undamaged, the lack of free ends and large size of the fragments prevented migration. Determination of the relative amount of DNA that migrated provided a simple way to measure the number of DNA breaks in an individual cell.

Genotoxic effects of any xenobiotics in PMNC and splenocytes of Wistar rats can be studied according to the working protocol of Dhawan et al.

Terminal deoxynucleotidyl transferase (dUTP) nick end labeling (TUNEL) is a method for detecting DNA fragmentation by labeling the terminal end of nucleic acids. TUNEL is a common method for detecting DNA fragmentation that results from apoptotic signaling cascades. The assay relies on the presence of nicks in DNA which can be identified by terminal deoxynucleotidyl transferase or TdT, an enzyme that will catalyze the addition of dUTPs that are secondarily labeled with a marker. Here we perform the apoptotic assay test by TUNEL assay kit to identify the apoptotic property of any agent or chemical after treatment of primary cells culture at different concentration as per protocol provided with TUNEL Assay Kit and apoptotic cells are identified by using fluorescent microscope. Detailed procedure of TUNEL assay is as follows:

1. Preparation of primary cells culture as described in earlier methods.

2. Induction of apoptosis in primary cells culture is done by treatment of primary cells culture with different concentration of xenobiotics and incubation in CO2 incubator for 12 h as mentioned in micronuclei assay method.

3. The positive and negative control cells provided with the kit are already f ixed. However, negative control cells are prepared from control sample with absence of inducing agent.

4. Cell Fixation: Cell fixation is a required step in the TUNEL assay and it is done according to the procedure mentioned in the supplied kit by using paraformaldehyde solution. Suspend 1–2 × 106 cells (treated) in 0.5 ml of phosphate-buffered saline (PBS) and the cell suspension into 5 ml of 1% (w/v) paraformaldehyde in PBS and place on ice for 15 minutes.

5. Centrifuge the cells for 5 minutes at 300 × g and discard the supernatant.

Injudicious and indiscriminate use of several agrochemicals viz. pesticides, herbicides, insecticides, dyes and medicaments in the field of agriculture and veterinary practices threatens the human and animal population for the toxicity of these environmental deterrents. These agrochemicals have been associated with several episodes of accidental poisoning and mortality among human and animal population. The episode of Mass ethion poisoning in Gujarat is one of the commonest example of inclusion of pesticides or agrochemicals in the environmental pollutants. There are modern tools to evaluate and analyze these chemicals in the environmental pollutants of human and animal use. The sensitivity of these modern highly sophisticated tools has now immensely changed the analytical environmental pollutants industry. Gas chromatography and Mass Spectrophotometry i.e. (GC-MS) technique is employed to detect the volatile residues from environmental pollutants. Whereas, High performance liquid chromatography i.e. HPLC is employed to analyse and detect non volatile drug residues. Gas Chromatography/Mass Spectrometry (GC/MS) instrument separates chemical mixtures (the GC component) and identifies the components at a molecular level (the MS component). It is one of the most accurate tools for analyzing environmental samples.

Flow Cytometry is a tool used to detect and measure chemicophysical characteristics of a population of cellular particles. Modern day biomedical research needs the molecular assembly of tools to proceed in the advent of biomedical research with real time diagnostic, prognostic and experimental detection of markers. For the same, a naïve tool is flow cytometry. In this technique, a sample having cells or particles is suspended into a fluid and injected into instrument. The ability to perform these measurements in a very rapid time span is one of the key advantages of the flow cytometry process. A single-cell suspension must first be prepared in order to analyse solid Tissues. A flow cytometer is look alike to microscope, except that, instead of producing an image of the cell, it generally offers a cell-by-cell basis high-throughput, automated quantification of specified optical parameters.

They can quantify up to three to six properties or components are quantitated in a single sample, cell by cell, for about 10,000 cells, in less than one minute. Sample is focused to ideally flow one cell at a time through a laser beam, where the light scattered is characteristic to the cells and their components. These cells are tagged with fluorescent markers in order to absorb the light and then emitted them in the form of band of wavelengths. Average ten thousand cells can be quickly examined and the data gathered are processed by a computer.

Isolated tissue preparations are widely used in physiological or pharmaco logical research to delineate the mechanism of action of drugs or any active biomolecules. Traditionally it is used as a tool for bioassay of drugs. The tissue used for such pharmacodynamics studies may be isolated from different animal models viz. frog, rodents, rabbits, sheep, goat, buffalo, chicken etc. Evaluation of drug-receptor interaction and underlying mechanism of action is then performed using isolated organ bath or myograph where the isolated tissues are mounted under optimum resting tension and changes in the tension following exposure of drug is recorded with the help of recording device. With the advancement of science and technology, the recording devices are also gradually upgraded from smoked drum to student’s physiograph to the most advanced Data Acquisition System (DAS) based polyphysiograph.

The isolated tissue preparations (uterine strips, arterial rings, tracheal chains etc.) need essential and appropriate nutrients and environment during experimental which is generally provided by using suitable salt solutions (Physiological Salt Solutions) which provide ionic balance, energy supply, maintain pH and tissue tonicity. The composition of physiological salt solutions is such that it provides an artificial media simulating with the inorganic composition of blood plasma together with a buffer mechanism to maintain an optimum pH of 7.0 to 7.4 and glucose to facilitate tissue metabolism. Commonly used PSS are Frog ringers, tyrode, Kreb’s, de Jalon, Ringer-Locke, modified Kreb’s Henseleit solution etc.

Isolation of Peripheral Blood Lymphocytes

Chemicals

1. Heparin

2. Sterile PBS (pH 7.2)

3. Histopaque-1077

4. RPMI-1640 Media

Protocol

Collect 5 ml of blood from the animal in 1 ml (20 IU/ml of blood) of diluted heparin and add 2 ml of PBS. Layer over the diluted blood on 5 ml of histopaque-1077 in 15 ml centrifuge tube and centrifuge at 1500 rpm for 40 min. Collect the buffy coat at the interface of plasma and histopaque in separate centrifuge tubes. Wash the cells thrice with PBS and suspended in 2 ml of RPMI-1640 media supplemented with 10% fetal calf serum. Determine the percentage cell viability by 0.1% trypan blue exclusion test, using hemocytometer. Finally dilute the cell suspension to 106 cells/ml in RPMI 1640 medium.

Epididymal Total Sperm Count

The epididymal sperm is obtained as described above, incubated at 35°C, which is the optimum temperature of rat epididymal sperm. The epididymal f luid is then diluted to a volume of 5 mL of pre-warmed (32°C) the spermatozoa are counted by hemocytometer using the Improved Neubaur (Deep 1/10mm. LABART, Germany) chamber as described by Pant and Srivastava (2003).

Total viable cell/ml = (Average number of sperm per chamber) x 103 x (Dilution Factor)

Liveability and abnormal sperm

Per cent live spermatozoa is estimated by differential staining technique using Eosin-Nigrosin stain (NE) (Campbell et al., 1953). These slides are also used for estimating the per cent abnormal sperm morphologically on the basis of observable abnormalities of head, neck, mid-piece and trail region of the spermatozoa.

The percent live spermatozoa are determined adopting the differential staining technique using Eosin-Nigrosin stain.

• A drop of epididymal semen is taken on a clean, grease free pre warmed glass slide.

• 4-5 drops of Eosin-Nigrosine stain (1% Eosin and 5% Nigrosine in 3% sodium citrate dehydrate solution) is placed near the semen drop.

• Epididymal semen and stain are mixed gently using a blunt fine glass rod.

• After 30 seconds to 1 minute a thin smear is made on a clean, grease free glass slide.

• The smear is examined under oil immersion objective.

Bugs
Class: Insecta
Subclass: Pterygota 
Division: Exopterygota
Order: Hemiptera (Bugs)
Characters
1. These are true bugs.
2. Mouth parts adapted for piercing and sucking. Proboscis kept folded in head when not in use.
3. Two pairs of wings usually present, anterior harder pair called hemelytra.Posterior pair is membranous.
4. Both male and female are blood suckers.
5. Metamorphosis is incomplete

Class- Arachnida
 •Class Arachnida includes scorpions, spiders, ticks and mites.
 •Antennae, wings and compound eyes are absent.
 •Body divided into head, thorax and abdomen.
 •Mouth is small and adapted for suck blood or tissue fluids of host.
 •Most of the members have poison glands or claws for paralyzing the host before sucking.
 •The first and second pair of appendages is known as chelicerae and pedipalps, respectively.
 •The first six segments of body are termed as prosoma and rest segments as opisthosoma.

ICAR-NDRI, Karnal is running a Kisan Seva Kendra at village Lalukheri of district Muzaffarnagar (UP) to cater the requirement of the farmers for frozen semen doses, crops seed and mineral mixture of villages around the center. This centre was established in year 2005; it started fully functioning in 2015. This centre is providing service to the farmers in ~25 villages around the centre. It provides frozen semen doses of high genetic merit bulls with the aim for making improvement in genetic potential for higher milk production of dairy animals of farmers in villages around the centre. The centre is also making available mineral mixture for improving fertility of dairy animals and also making available fodder crop seed to the farmers for growing quality fodder for their dairy animals. Kisan gosthis and farmers awareness camps are also being organized to increase farmers’ awareness regarding benefits of rearing high producing dairy animals and feeding them balance ration for improving their production and reproduction performance.

Since 2015, center has organised ~35 meetings for farmers awareness for adopting scientific techniques/practicesfor dairy husbandry and animal health checkup camps in ~25 villages for improving production and fertility of dairy animals. The maximum numbers of progenies so far, through the use of frozen semen doses from the centre, have been produced of dairy cows and buffaloes in the village- Bhorakhurd (300), Sonjanikheri (440), Alipur (300), Sotta (250) and Sallakheri (250), total 1540 calves.

Jaggery: Raw brown / unrefined brown sugar in lump made from palm sap (Plate 365).

Jam: A preserved product prepared on boiling pectin contained in fruit pulp with required amount of sugar, water and acid to a reasonably thick consistency, firm enough to hold the fruit tissues in position (Plate 366).

Jam pack: The packing of the commodity tightly or in excess is called Jam pack.

Jamaica rum: Heavy bodied rum made by slow fermentation and marked by a pungent bouquet.

Jamboline: A type of glucose found in the jamun fruit help in cure of the diabetes in the patients.

Jelly: A product prepared by boiling clear extract of fruits with sugar. It should be transparent, well set, and tender enough to quiver but not flower and when cut; it should retain its shape and show a clean cut surface (Plate 367).

Q10 value: The number of times a reaction rate change with a 10oC change in temperature is called Q10 value. If reaction rate doubles with 10oC change in temperature, Q10 = 2. It is also called temperature quotient.

Q-enzyme: It is a branching enzyme responsible for formation of 1-6 bonds in amylopectin.

QHV process: It stands for Quality Has Value Process.

QO2 : It is a unit for measurement of respiration. Or It is the oxygen uptake in microlitres per milligram on dry weight per hour.

Quailing: In this practice the oranges are kept in the baskets in which they are harvested to allow the fruit to loose little moisture. It is done for a couple of hours before transport to packhouse. This practice would allow the fruit to lose a little moisture which is called quailing. This operation is done for oranges to prevent Oleocellosis.

Qualitative measurement: It is a subjective / objective measurement of the quality of the product for presence of certain compound. It gives the idea of the presence or absence of the particular compound. It deals with the nature of the substance and not its amount or quantity.

Zeatin: Zeatin is a cytokinin C10 H13 N5 O first isolated from the endosperm of Indian corn.

Zeaxanthin: Zeaxanthin is an isomer of lutein occurring in fruits and vegetables (spinach and corn) (Plate 682, 683 and 684).

Zein: Zein is a protein from Indian corn that lacks lysine and tryptophan and is used in making textile fibers, plastics, printing inks, edible coatings, adhesives and sizes.

Zeolite: It is clay embedded in the plastic film to adsorb ethylene. It is used in Japan. Zeolite is anhydrous silicates that is analogous in composition to the feldspars and used in water softening, adsorbents and catalysts. Or Zeolite is one of various hydrous silicates that are analogous in composition to the feldspars, occur as secondary minerals in cavities of lavas, and can act as ion-exchangers. These are used especially in water softening and as adsorbents and catalysts (Plate 685).

Zero 2: It is Australian oxygen scavenging plastic packaging materials.

Definition

Paracelsus saying– Nothing is devoid of poison it is the dose that differentiate between a drug and a poison.

? Poison- Any chemical substance either naturally present or Man made produces deleterious effect on the health of living organism are termed as Poisons.

? Toxicology term derived from Greek Word Toxicon which means Poison.

Definitions

? Toxicology: It is the science or study of poisons on biologic systems, including their properties, actions and effects. Also their detection and identification, the treatment and prevention of the conditions produced by them.

? Toxicant: Any poisonous agent/ Professional Term used for Poison.

? Xenobiotic: Derived from Greek Word Xenos means – alien or foreign.

? Xenobiotics are those substances which are foreign to the body and are biologically active but do not take part in the process of energy production.

? Dioscorides- Classified Toxicants on the basis of their origin into Plant, Animal, and Mineral Sources.

? Ibn Hazer- Differentiated between a drug or a poison on the basis of matter of dose.

? Dementhes – died by swallowing an unknown poison in his pen.

? Socrates- was poisoned by a cup of Hemlock (Greek State Poison) obtained from Conium maculatum having active principle coniine causes paralysis, coma followed by death.

? Cleopatra- Egyptian lady use strychinine poisoning and she died of Egyptian Asp.

? Lady Toffana- Arsenic containing perfumes known as Aqua Toffana.

1. Acute    Toxicity    
 ? Duration-exposure of the toxicant for 24 hrs either single dose or 
multiple doses
 ? Species selection-Rat preferred rodent species
 ? weight variation should not exceed ± 20% of mean weight
 ? Number and sex -Atleast 5 rodents at each dose level of same sex
 ? If female-nulliparous and non pregnant
 Housing and feeding    
? Temperature of experimental room-22( ±3 °C)
 ? Relative humidity (R.H.)-30-70%
 ? Animals in cage should be properly spaced
 ? Individual caging for morbid and excited animal
 ? Artificial lightining-12 hrs light,12 hrs dark
 ? Conventional laboratory diet & ad lib water

SOURCES OF POISON

1. Malicious poisoning

Unlawful or criminal killing of the animals by certain toxicants as arsenic, Insecticide, datura etc.

 2.Accidental    poisoning    
A) Natural sources 
 ? Plants - e.g. Lantana, datura etc.
 ? Animals - e.g. snake bite etc.
 ? Microbes - fungi (aflatoxicoses etc.), bacteria (Botulinum toxin) etc.
 ? Minerals - Se, As, fluoride etc.
 B)  Man made sources
 ? Agrochemicals - Insecticides, Herbicides etc.
 ? Industrial wastes - Mercury, Copper, Lead etc.
 ? Household products - disinfectants (phenol), Napthelene etc.
 ? Therapeutic agents - paracetamol in cats be lethal
 ? Food & feed - Genetically modified food (GM)
 ? Environmental pollutants & managemental practices

? Cyanogenetic plants contain hydrocyanic acid or cyanogenic glycosides.

? Two forms of cyanide in plants- HCN( free form) and cyanogenic glycoside(bound form).HCN released from glycosides by physical disruption (mastication, trampling) , stress (drought, frost) or the action of an Hydrolyzing enzyme present in ruminal microflora beta glycosides and hydroxynitrile lyase.

? One of the most common plants poisoning among the grazing livestock.

? Cattle and buffaloes are most susceptible. Sheep and goats are less susceptible.

? Monogastric animals (pig, Horse) are relatively resistant due to destruction of the glycosides by the gastric activity.

? Poisoning occurs when HCN content in plants is high and ingestion of large amount of the plants in short period of time.

? Starved or underfed animal also ingest large amount of plants. Poisoning may also occur due to accidental ingestion of potassium cyanide.

INTRODUCTION

? Nitrate poisoning is generally caused when animals eat too much forage that is high in nitrates not changed to protein in the plant.

? Poisoning can also happen when animals eat too much urea or nitrogen fertilizer spilled in the field or left where the animals can find it.

? Nitrate fertilizer is palatable, especially to cattle.

? Nitrates are relatively non-toxic.

? Reduction of nitrates depends on the supply of fermentable energy as carbohydrates supply hydrogen ions for reduction of nitrates and production of nitrite in rumen.

Nerium oleander

? (Oleander, kaner, rose laurel)

? An ornamental plant and grown in lawns and gardens.

? Other species are Nerium indicum (kaner) & Thevetia peruviana (yellow oleander).

Sources

? Almost all animal species are poisoned.

? Ingestion of branches, stem or leaves during drought or periods of food scarcity or consumption of contaminated water by ruminants or horses.

? Chewing of leaves may be responsible for toxicity in dogs.

? Ingestion of 30-60 gram fresh leaves of oleander is fatal for horses, cattle and sheep.

? All parts of plant are toxic, the leaves and flowers may be less toxic than the seeds.

Key Points

• Sudden severe bleeding or hemorrhage can cause hemorrhagic shock and death.

• Chronic and or occult bleeding or hemorrhage can cause anemia and even death.

• Common causes of bleeding include trauma, surgical incisions, extreme temperatures, vitamin K deficiency, medicines, and toxins.

Introduction

Bleeding is the extravasation (arterial, venous, or capillary) of blood caused by trauma or a surgical incision.

Key Points

• Frostbite occurs when exposed to freezing temperatures. Animals that are weak and malnourished are more likely to be injured.

• It is a prevalent condition throughout the winter, particularly in animals who do not have shelter or have inadequate protection to protect them from the cold.

• Excessive cold can harm skin and subcutaneous tissue. In severe circumstances, injuries may be irreversible.

Introduction

Frostbite, also known as freezing cold damage, is caused by prolonged exposure to temperatures below 0º C. Exposure to freezing temperatures causes excessive heat loss, disrupting the body's heat balance and resulting in systemic or local acute freezing injury. Frost bites cause superficial tissue injury, but in extreme cases, gangrene develops in the joint or synovial sheath, and the animal dies from toxemia. However, all animal species are susceptible. It occurs rarely in healthy and well-nourished animals. Animals that have lost their coats or are malnourished are particularly vulnerable to harm. Prolonged exposure to colder temperatures increases the risk of frostbite and the severity of the injury. In the majority of cases, frostbite occurs in animals that are left in winter.

Key Points

• Animals can experience varying levels of electric shock. Lightning typically kills animals instantaneously and causes them to fall to the ground without struggle.Most electric shock deaths are caused by cardiac or respiratory arrest.

• Animals often recover from minutes to several hours, rarely going unconscious. Residual nervous system effects, including sadness, paraplegia, and increased cutaneous sensitivity to painless stimuli, can last for days, weeks, or even months.

Introduction

Lightning strikes, broken transmission lines, malfunctioning electrical circuits, or an animal chewing on an electrical cable can all cause an animal to be injured or killed by high-voltage electrical currents. Lightning strikes are seasonal and occur in thunderstorm-prone regions. Caution should always be maintained when investigating probable electrocution situations because the source of the electrification, such as broken transmission wires, may still be present.

Key Points

• Physical cooling is more effective in managing hyperthermia.

• Heat stroke occurs when the cause is solely physical.

Introduction

Hyperthermia is an unusually high body temperature (also known as overheating) caused by the body absorbing or producing more heat than it can discharge. Simply said, it is the increase in temperature above the critical point. Heat stroke is most common in animals when they lose control of their body temperature.

Key Points

• Hypothermia occurs when an individual's body temperature drops below its critical level.

• Excessive heat loss or inadequate thermoregulation can lead to this condition.

• It can be minor or severe. Mild hypothermia is harmless, while moderate to severe hypothermia is an emergency.

Key Points

• Constipation can be caused by a variety of clinical disorders or as a subsequent consequence.

• Rehydration and gentle lubrication of the GIT tract with oral paraffin or linseed oil will alleviate constipation caused by feed and water.

• Small animals, especially cats, are more prone to constipation.

• Common treatments for constipation include dietary changes, enemas, and mechanical removal under anesthesia.

Introduction

Constipation occurs when the ingesta moves slowly or not at all. It is a sign of several intestinal disorders. Although not all extra-gastrointestinal illnesses cause constipation, some do. A systemic illness is characterized by parasympathetic nervous system depression and constipation. Many diseases can lead to constipation as a complication. Chronic constipation in dogs leads to obstipation, which is characterized by drier, firmer, and more compact stools. Dogs cannot defecate and hence require medical intervention, such as surgery or manual feces evacuation. When the colon becomes irreversibly swollen and hypomotile (megacolon), surgery may be necessary. As a result, corrective actions for constipation should be implemented as soon as they are discovered. This will aid in a quick recovery from disease. The current chapter provides an overview of constipation in general and its management.

Key Points

• Neonatal diarrhea can be lethal without treatment.

• Administer aggressive fluid and electrolyte therapy to address newborn diarrhea.

• Fecal examination and/or culture findings can help control diarrhea.

• Ringer lactate is the preferred fluid.

• There is a high association between animal age and the presence of the causal factor for diarrhea.

Introduction

Diarrhea is one of the symptoms of a variety of medical problems. Diarrhea is characterized by an increased frequency of defecation and stools of varied consistency, which may have an increased concentration of water or a decrease in dry matter content. Increased peristaltic movement causes an increase in caudal flow of ingesta/GIT content, resulting in a decrease in intestinal transit time and thus diarrhea. Diarrhea is lethal in children; therefore, preventing f luid and electrolyte losses prior to diagnosis is critical to saving lives.

Key Points

• Fluid therapy is crucial for saving dehydrated animals.

• Fluid therapy should be administered within the first 4-6 hours of treatment.

• Ringer lactate is the recommended fluid for diarrhea and enteritis.

• Fluid loss of 4–6 % does not require therapy and does not show any clinical indications.

Introduction

Dehydration is defined as an excessive loss of water and electrolytes from the body (usually due to vomiting and/or diarrhea) or an insufficient intake of water. The body loses water through respiration; however, the skin, face, and/or urine can also cause dehydration. Diarrhea (the loss of fluid and electrolytes from the gastrointestinal tract) is invariably accompanied by dehydration. Chapter 13 provides a brief overview of diarrhea and how it is managed. Dehydration causes lethargy in the patient and, if handled, can have catastrophic consequences for the kidneys and/or other internal organs. Death may occur if the body loses 40% of its water.

Key Points

• Vomiting is a common sign of various conditions, including irritants and toxins, kidney or liver failure, pancreatitis, neurological disorders, and digestive issues. Common in dogs and cats.

• Can be fatal in horses with acute stomach dilatation.

• Ruminants (cattle, buffalo, sheep, and goats) vomit by regurgitating ruminal material through their mouths.

Introduction

Vomiting is the violent ejection of stomach and, on occasion, proximal small intestine contents through the mouth caused by fierce contractions of the diaphragm and abdominal muscles. It is the most prevalent protective disruption in the gastrointestinal system. Vomiting has three distinct phases: nausea, retching, and vomiting. A sense of nausea may or may not cause vomiting. Nausea is scientifically defined as sadness, salivation, lip-licking, and increased swallowing motions in animals. Retching is caused by a series of spasmodic respiratory movements involving the closed glottis. Vomiting is characterized by repeated thoracic invaginations of the abdominal esophagus and cardia. Projectile vomiting often occurs when an animal begins to vomit without going through three stages.