eChapters

Key Points

• With the aid of microorganisms, ruminants typically use NPN to synthesis proteins for nutritional needs.

• When there is an excess of urea in the body, ammonia is produced excessively, which can be harmful.

• The majority of clinical indicators are neurological in nature, and their severity varies depending on a number of variables.

• Ruminal environment acidification to stop excessive ammonia generation and absorption; symptomatic therapy is advised.

Introduction

In addition to being a widely utilized fertilizer for crops, urea is frequently used in animal feeds as a low-cost protein substitute [1]. The previously mentioned explanations explain the reports of urea toxicity in ruminants. These animals may use non-protein nitrogen (NPN) like urea to create ammonia, which is subsequently used to synthesize proteins that partially meet their nutritional needs. This is made possible by the urease enzyme and ruminal microorganisms. On the other hand, rapid overconsumption of NPN leads to excessive ammonia generation and toxicity. This chapter aims to provide an overview of some of the most frequent causes of urea poisoning, clinical symptoms to watch out for, and associated treatments.

Key Points

• The most commonly cystitis and pyelonephritis occurs in farm and companion animals after parturition.

• Hematuria, pyuria, and stranguria are common presenting clinical symptoms;

• In acute situations, a prolonged course of treatment (two to four weeks) with the administration of the proper antimicrobials is usually successful.

Introduction

Both farm animals and companion animals frequently suffer from urinary infections, which are difficult to diagnose. In cows, urine testing is more helpful in excluding the illness if it shows any pus or germs (in females), and f inding the urethral calculus that is causing the urinary obstruction can be done by palpating the bladder using a rectum examination and palpating the urethral tube (in males). Because of the shorter urethra in females and the associated uterine infection that occurs during or after parturition, females are more likely to get urinary tract infections.

Urinnalysis An essential laboratory test that is easily conducted in veterinary settings and is included in a basic database is urinalysis. Documenting diverse urogenital tract conditions is helpful, because it could reveal details about other systemic conditions including hemolysis and liver failure. Cystocentesis, urethral catheterization, or simply voiding can all be used to collect urine. Urine should be examined within 30 minutes, but if this is not possible, it can be refrigerated for up to 24 hours or sent to a outside diagnostic lab; this, however, may cause crystal precipitation. The pH or specific gravity of urine are unaffected by refrigeration. Preservative should be added if cytologic analysis is necessary and a delay in slide preparation is anticipated. A few drops of 10% formalin (exact concentration not necessary) are better than boric acid

Different basic as well as advanced laboratory equipment need to be used in a laboratory while conducting experiments. Many of them like pH meter, laminar air-flow hood, viscometer, thermometer, magnetic stirrer, microwave ovens, incubators, sand-bath, water-bath, oil-bath, etc. are the basic and simple equipment which require to be used routinely while conducting experiments in a laboratory. However, basic knowledge of their operation, handling procedure, when to be used, how to be used, and more importantly what not to be done with it, etc. are essential for keeping them in serviceable condition as well as for the accuracy/precision in their operation. In this chapter, we will discuss about some of the routinely used laboratory equipment along, while some other sophisticated equipment will be discussed in other chapters.

pH Meter

A pH meter measures exactly the H+ concentration of a solution; thus indicates acidity or alkalinity of a solution with greater precision. Actually, it measures the difference in electrical potential between a pH electrode and a reference electrode, and display the results converted into the corresponding pH value. Electrode is the key part (rod-like structure, usually made of glass) of a pH meter, having a bulb at the bottom containing a sensor. The pH meters range from simple and inexpensive pen-like devices (portable/pocket pH meter) to complex and expensive laboratory instruments with computer interface (Figure 5.1).

Frequent calibration of a pH meter using the solutions of known pH (standard solution of pH 4.01, 7.00, 10.01, Figure 5.2), generally before each use, ensures the accuracy of the instrument. For very precise work the pH meter should be calibrated before each measurement. For normal use calibration should be performed at the beginning of each day. This is necessary because the glass electrode does not give a reproducible measurement over a longer period of time.

Introduction

To accomplish experimentations in laboratories, sometimes we need microscope to test/validate our ideas/findings. A microscope is needed to examine objects which are very small which cannot be visualised by naked eyes. However, microscopic study requires some expertise in operating the microscope, particularly the sophisticated microscopes, as well as to prepare (peeling, sections, cutting, fixation, staining, dehydration, mounting, etc.) the sample, so that it can be appropriately examined for the anticipated details.

According to the scientific requirements different types of microscopes are used in the laboratories for research purpose. In this chapter, our aim is to acquaint you with the basics of using and handling of microscopes, particularly a compound microscope that is commonly used by the graduate and post graduate students for basic research.

Microscopy is often designated as the scientific investigation that is employed to review and analyse minute structures and objects using a microscope. The term “microscope” has been derived from ancient Greek words “micros” meaning “small” and “skopeîn” meaning “to look at or see,”.

Microscope

A microscope is a laboratory instrument used to examine objects (biological specimens) which are too small to be seen by the naked eye. The microscope must accomplish three tasks:

• Magnification- to provide a magnified image of the specimen.

• Resolution- to separate the details in the image.

• Contrast- to render the details clearly visible to the eyes.

Although we will tnroduce to you the different types of microscopes used for different speciments, our main focus is to discuss about compound microscope, which is commonly used in the laboratories. Compound microscope was developed around the beginning of 17th century.

A variety of glassware like burettes, pipettes, measuring cylinders, flasks, separating funnel etc. are used for experimental purposes in a laboratory. All of these need to be used and handled correctly, otherwise the chances of experimental error and damage to the glassware increase significantly. Being made of glass, they need special care while washing and using in experiments because even a minor mistake in handling of glassware may result in breakage of the apparatus. With this precautionary note, let us discuss about some of the specific glassware used in the laboratories.

Burette

Burette is a long graduated glass tube with a stopcock at one end for delivering a known/desired volume of a liquid, especially in titration experiments. It contains a stopcock at the lower end and a tapered capillary tube as the stopcock’s outlet. The flow rate of liquid from the tub stopcock’s outlet can be controlled with the help of stopcock valve. Burette is used for measuring the volume of a solution/chemical added in a quantitative (titrimetric) estimation experiment.

Procedure of using a Burette

The burette reading must be noted before and after delivering the chemical/ solution from the burette. The difference in these two readings is the volume of the chemical/solution released out of the burette. The solution should be released slowly/drop-by-drop to avoid any experimental error/mistake. If the solution/chemical is allowed to pour out too fast, the wall of the burette will not drain properly and some liquid may remain sticking to the surface of the walls. Moreover, excessive amount of chemical may be released/dropped during the experiment, particularly during the titration experiment wherein we need to know the exact amount of acid/base needed to get neutralization point. Thus, any minor mistake may lead to get a faulty reading. The measuring capacity of a burette usually used in a laboratory is 50 mL.

Since mouth pipetting of liquid reagents in a laboratory is not advisable, different tools are needed for safe, easier, and precise dispensing of liquid reagents. This chapter is devoted to make you aware of the tools like vacupette and micropipette which can be used for the handling of liquid reagents in a safe and precise way.

Vacupette

Glass pipettes have been traditionally used by sucking liquid solvent/solution directly using mouth. For safety reasons, mouth pipetting is no longer permitted in a laboratory. Hence, to fill a pipette vacupette is used by placing it on the top of glass or plastic pipette. It acts as a device to create vacuum for filling reagent in the pipette as well as for releasing the reagent with mechanical regulation on the flow out of the liquid. Vacupette can be of different types.

1. Rubber Bulb Vacupette

Rubber bulb vacupette is often used to transfer (pump up and discharge) liquid using a pipette when a larger quantity of chemicals is to be transferred. Small/tiny rubber bulb is attached to glass/plastic pipette for drawing little amount of reagents. Small vacupette is used to precisely discharge smaller volume/ drops.

Handling of Vacupette

• Insert the top of a pipette in the bottom of the rubber bulb vacupette (Figure 6.1).

• Remove air from the pipette by squeezing valve “A” at the top of the vacupette while simultaneously squeezing the rubber bulb. The amount of air released depends on the size of the rubber bulb.

Ultrasonography in goat 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 the animal 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 and reproductive disorders in sheep and goat. The B-mode real time ultrasonographic system particularly portable system is popular among researcher and practitioners. In goat, B-mode real time ultrasonography was used in 1971, using 5 MHz frequency transducer. A transducer of switchable frequency from 7.5 to 5.0 is more popular in small ruminant research and practice.

Real time ultrasonography provides means to directly image the reproductive tract. An extensive amount of information can be gained from even a single examination, including determination of ovarian (luteal or follicle detection) and uterine status, detection and monitoring of ovarian and uterine pathology, monitoring the post-partum mare, detection of twins, prediction of ovulation, fetal growth and development and determination of sex of fetus. When first introduced the use of ultrasonography was primarily limited to early pregnancy diagnosis only, with advancement of knowledge and interpretive skills, the diagnostic ultrasonography has a much more application in reproduction. Some of the applications are discussed in this lecture.

Procedure

The procedure and precautions for intrarectal ultrasonographic examination are similar to those for rectal palpation, and no additional restraint is required. The transducer should be protected by the examiner’s hand to prevent trauma to the rectal wall, and the transducer should be well-lubricated. Care should be taken to avoid fecal material attaching to the transducer. After evacuating fecal material from the rectum, the probe is introduced and moved across the reproductive tract in the following pattern: uterine body, right uterine horn, right ovary, right uterine horn, uterine body, left uterine horn, left ovary, left uterine horn, uterine body then cervix. Good contact must exist between the transducer and rectal wall. Air in the rectum or a gas or fluid-filled loop of bowel will result in a distorted image. To minimize scanning errors, principally those of omission, it is recommended to conduct the same scanning procedure during each examination.

Uterine prolapse also called casting of the “Calf Bed” in cow is a condition in which there is eversion of whole or part of uterus outside the vulva after its movement through dilated cervix and vagina. It occurs in cow after the birth of calf and mostly in dairy cows having given birth many times to a calf. It occurs due to hypocalcaemia, defective involution, inadequate exercise, excessive force applied during removal of calf etc.

Uterine torsion in cow is actually the rotation of the pregnant uterus on its longitudinal axis and usually occurs before onset or during late first stage of parturition but rarely encountered during early second stage of parturition. In cattle, uterine torsion involves both gravid and non gravid horns because of strong intercornual ligament and distension of uterine horn and body with placenta and fluid. The remarkable feature of uterine torsion is its association with advanced pregnancy and process of parturition in cow.

One of the most promising areas of veterinary research these days is cancer immunotherapy and the development of CMT vaccines. DNA vaccines, also known as gene vaccines, are designed to transfer functional genes to target cells, allowing them to express functional proteins. Because endonucleases may easily access bare DNA, it requires a reliable and safe delivery route, which might be biological or non-biological. Different delivery strategies have been tried in human breast tumors to transport tumor antigens such Mucin-1 (MUC-1), Twist, HER-2, mammaglobin-1 and p53, as well as immunostimulatory molecules like IL-12 and IL-6. There have been various studies/ trials in canine animals using different molecules like IL-12, HER-2 and IFN-γ as vaccine candidates, with no clinically relevant outcomes or longlasting cell mediated and humoral immune response outcomes

Vaginal prolapse in cow is a condition in which there is protrusion of whole or part of vagina including sometimes the part of cervix through the vulva. It occurs in cow mostly during last 2-3 months of gestation. It occurs due to excess secretion of estrogen from placenta, hypocalcaemia, inadequate exercise, bulky food like roughages etc. Sometimes occurs in non pregnant cows due to cystic ovaries, severe straining due to vaginal irritation etc.

• General considerations

 • Steps in validation

 • Step1- Feasibility studies

 • Step 2- Assay development and standardization

 • Step 3- Determining assay performance characteristics

 • Step 4- Monitoring validity of assay performance

 • Step 5- Maintenance and enhancement of validation criteria

 • Measurement of uncertainity of a diagnostic test

• These are the bad habits of the male animal due to which ultimately their maximum reproductive potential cannot be utilized. • There are so many vices seen in male animals like stall walking, weaving, cribbing, wind sucking, crib biting, viciousness etc. These behavioural disorders are mostly due to improper managemental practices like abusive handling, poor exercise, irregular semen collection and poor exposure to female etc. These may be also due to hormonal imbalance or nervous disorders. - Some important vices which are more related to reproductive behaviour are: 1. Masturbation (Onanism) • Bulls masturbate by rubbing their prepuce and penis against any object. • Boar masturbate and ejaculate by inserting the penis into the preputial diverticulum called Balling up • Boar may show rectal copulation and leads to ejaculate : Pederasty

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.

Introduction

A variety of glassware, plasticware, and other apparatus are used for experimental purposes in a laboratory. They need to be washed, cleaned, dried, and sometimes sterilized before they can be used in the next set of experiments. Glassware needs special care of handling during washing, drying, sterilization, etc. because even a minor mistake in handling of glassware may result in its cracking/breakage and it may become unserviceable. All of these may result in a delay in experimentation if spare piece of the glassware is not available or it may affect serviceability of the associated apparatus.

A large number of glassware such as beaker, flask, measuring cylinder, burette, test tube, etc. are regularly used in a laboratory, which requires careful handling during the different stages of experiment as well as after completion of the experiment while preparing them for another set of experimentation.

Glass being a complex silicate, its properties depends on the type of silicate anions in the structure and the cations formed. Thermal properties of glass changes by the addition of boron oxide (B2 O3 ); hence, borosilicate glass is extensively used in the laboratories. With this brief background information, let us discuss about the necessity, procedures, and precautions during washing, cleaning, drying and sterilization of laboratory-wares. Figure 3.1 shows various advantages of using borosilicate glass in a laboratory.

Good laboratory practices require clean glassware, because an experiment may give erroneous result if dirty glassware is used at any step of the experiment. Hence, the glassware must be physically and chemically clean; and in many cases, it must be microbiologically clean or sterile. The commonly used glassware in a laboratory include test tube, measuring cylinder, pipettes, beakers, flasks, microscopic slides, etc.

Introduction

In a research laboratory, we frequently need weigh chemicals and prepare different solutions of varying strength, as per the requirements. Accuracy in weighing and preparation of solution, with respect to weight/volume and strength, determines the success of experiment. In turn, the accuracy depends on weighing of solid chemical and/or measuring the volume of liquid chemical/making up the volume. In this Chapter, weighing of chemicals using appropriate weighing balance, preparation of solutions of different strength, their dilution, and precautions while preparing will be discuss in brief.

Weighing of Chemical

The primary method to determine the amount of chemicals to be used is weighing the chemical by using a balance.

In general, there are two types of balances to weigh chemicals in a laboratory:

1. Standard laboratory balance

2. Analytical balance

Standard Laboratory Balance

Generally, a standard laboratory balance is used for weighing a chemical when an error of ±0.02 g is acceptable. It is also used for pre-weighing, where approximate mass needs to be determined. Precise mass of the chemical is then determined using an analytical balance. Following are the steps in using a weighing balance in a laboratory.