eChapters

The action of the sex hormone is not confined to the growth and development of the sex organ alone, but the normal body growth as well as the growth of the skeleton are to a great extent influenced by these hormones. It is well documented that following oopherectomy, female experience a “rapid phase” of bone loss for the next few years. Of the female gonadial hormones, the action of estrogen on skeletal growth has been studies by various workers and shows that estrogen replacement therapy can help to prevent the postmenopausal stage of accelerated rate of bone loss. The effects of progesterone on bone healing or bone growth has not yet been studies in detail. The objective in this chapter is thus undertaken to investigate the effects of progesterone supplement on cortical bone healing in spayed rabbits.

The study is conducted on ten clinically healthy female adult New Zealand White rabbits divided into two equal groups (A and B) consisting of five animals each. All animals are spayed and kept in standard diet and management throughout the experiment. In all animals, midshaft simple transverse fractures are created in both the ulnae under thiopental (2.5%) anaesthesia. In test group (gr A), 1mg (=1ml) of progesterone is injected subcutaneously in alternate days for upto 35 post fracture days. Lateral and anterio-posterior radiographs are taken on days 0, 7, 14, 21, 28 and 35 post-fracture. These radiographs are observed for progress of fracture healing and complications, if any, at different intervals.

For a researcher, laboratory is another home where he/she spends much time every day. As we take care of cleanliness, safety, and design of our home, we must take care on these aspects of laboratory too. It is the responsibility of the laboratory personnel to ensure cleanliness/safety of the laboratory. Since every laboratory chemical and equipment have the potential to cause harms if not used with appropriate precautions. Hence, as a laboratory user, it is our responsibility to follow the basic safety/biosafety rules and regulations set up by the Principal Investigator of the laboratory, Institutional Biosafety Committee, as well as by other regulatory authorities, if any. We must be aware of the general/personal protective measures, cleanliness and laboratory waste disposal prior to joining a research laboratory. In this chapter we will briefly discuss about personal safety, chemical safety, cleanliness, waste disposal, etc.

A. Personal Safety

1. Personal Protective Equipment (PPE)

Personal protective equipment (PPE) is special arrangement to protect the worker from specific hazards because of using a hazardous substance. PPE does not reduce or eliminate the hazard, but protects the wearer from exposure to hazardous material. PPE should not be reused or brought outside the laboratory as these might increase the risk of spread hazardous material or contamination. Used PPE must be kept inside the laboratory in a designated waste storage area adjacent to the work area.

Scales are having imperfectly keratinized superficial layers of the epidermis characterized by cracks or thin flakes.

The scapula is more regularly triangular than in horse, relatively, wider at the vertebral end and narrower at the distal end. The scapular index is about T.0.6. The spine is more prominent and is placed further forward, so that the supras- pinous fossa is narrow and does not extend to the lower part of the bone. The spine is sinuous, bent backward in its middle, forward below. Its free border is somewhat thickened in its middle, but bears no distinct tuber. Instead of little more prominent, and is prolonged by a pointed production, the acromion, from which part of the deltoid muscle arises. The sub scapular fossa is shallow. The areas for the attachment of the stratus muscle are not very distinct. The nutri- ent foramen is usually in the lower third of the posterior border. The glenoid cavity is almost circular and without any distinct notch. The tuberosity is small and close to the glenoid cavity. The coracoid process is short and rounded. The cartilage resembles that of the horse. The tuberosity unites with the rest.

It is one of the common epithelial skin tumors. It is more common in head but also recorded in other sites such as back, tail, limb, trunk and eyelids. It is a tumor of sebaceous cells. In sebaceous gland adenoma, it has predominantly sebocytes with less of basaloid reserve cells while in sebaceous epithelioma variant, more of basaloid reserve cells are seen with less of sebocytes.

The drug conventionally used for sedation is xylazine, which is an alpha-2 agonist (Dugdale, 2010; Rankin, 2015). Dexmedetomidine is the latest drug of the same class used for sedation, and is more specific to alpha2 receptors than xylazine (Ishyama et al., 1995). Doses of 1-2 mg per kg body weight of xylazine (Hall and Clarke, 2001) and upto 20 mcg per kg body weight of dexmedetomidine (Ko, 2013) are required for sedation when used alone (Hall and Clarke, 2001; Rankin, 2015). Similar degrees of sedation could still be achieved at lower doses of xylazine (0.2 to 0.4 mg per kg body weight) and dexmedetomidine (2.5 to 5 mcg per kg) if administered in combination with any opioid drug like hydromorphone / oxymorphone / fentanyl / buprenorphine / butorphanol / nalbuphine / tramadol at their usual doses (Hunt et al., 2013). Another drug - acepromazine, which is a phenothiazine tranquiliser, is used at its doses of 0.01 to 0.05 mg/kg combined with an opioid for achieving sedation (Smith et al., 2001; Dugdale, 2020; Ko, 2013). Combination of an opioid with either a phenothiazine tranquiliser or an alpha2 agonist sedative drug provides neurolept anaesthesia/analgesia (Dugdale, 2020; Rankin, 2015). An anticholinergic drug - glycopyrrolate is administered in combination with acepromazine and opioid to prevent bradycardia (Dyson and James-Davies, 1999). Anticholinergics are generally not recommended with sedative cocktails containing dexmedetomidine, but preferred with sedative cocktails containing xylazine (Congdon et al., 2011; Billah et al., 2017). If sedation is to be achieved with acepromazine alone, higher doses need to be administered where there will be an increase in its dose dependent side effects, and hence not preferred (Simon and Steagall, 2020). Benzodiazepines are another class of sedative drugs, which generally doesn’t provide predictable sedation in animals. Paradoxical excitement is caused in healthy adults when these drugs are used alone (Herron et al., 2008). Predictable sedation is achieved when these drugs are administered in combination with any opioids (KuKanich and Wiese, 2015). Also, benzodiazepines can be combined with an alpha2-opioid combination to enhance the overall sedation (Maze et al., 1992). Various balanced sedative cocktails prepared with the drugs available for a veterinary practitioner in India, are presented. The balanced sedative cocktails are prepared with either

Key Points

• Seizures are common in dogs and cats but uncommon in larger animals.

• Animal seizures can be induced by several circumstances.

• Idiopathic epilepsy is the leading cause of seizures.

• Rapidly rising core body temperature and metabolic alterations lead to fatal outcomes.

Introduction

A seizure is defined as any rapid and uncontrollable movement of the body induced by aberrant neuronal activity in the brain. Seizures are common in dogs and cats, but uncommon in larger animals. Seizures can be severe, affecting the entire body, or moderate, affecting only a section of the body. The pet may or may not be conscious or responsive, and it may urinate.

Seizures are usually brief (1-2 minutes) and self-limiting; however, some animals may experience protracted activity (more than 5 minutes). Seizures that persist longer than 3 to 5 minutes might cause serious complications such as pulmonary or brain edema. Death is caused by a sudden rise in body temperature and metabolic alterations that reach a life-threatening threshold.

All animals with cataract are not suitable candidates for surgery. Pre-surgical assessment of a candidate is essential to make a safe judgement for surgical procedure to perform lensectomy. Failure in canine cataract surgery can be associated with poor case selection. A thorough ocular and physical examination should be performed to rule out other ocular diseases prior to cataract surgery. The cataractous lenses in both eyes must be mature to cause functional loss of vision. (24).

The following prerequisites should be fulfilled before cataract extraction is recommended (6):

1. The affected eye should have a significant visual deficit.
2. For the patient to regain vision, its retina must be healthy and functional. if the fundus cannot be examined thoroughly (because of the cataract), retinal function should be evaluated with electroretinography (ERG) to ensure that retinal degeneration is not present as the patient history, signalment, and the speed of pupil contraction

3.1 Selection of Breeding Bulls

A sire is called half the herd and the breeding bulls to be used for semen production should be carefully selected. Selection of superior sire for breeding purpose is very important for the success any livestock breeding programme. Therefore, breeding sire must be carefully selected and maintained in scientific way so that its breeding life is prolonged and maximum number of frozen semen doses can be produced from it. 
The selection criteria for future bulls should be meticulously designed keeping into account all the factors/parameters which affects the breeding programme. The selection of breeding bulls is based on three broad parameters-

3.1.1 Pedigree Examination
3.1.2 Gross Physical Examination
3.1.3 Laboratory Examination

Semen is admixture of secretions of testes containing spermatozoa and accessory sex glands. Spermatozoa are formed in the seminiferous tubules of the testes. The tubules contain a complex series of developing germ cells that ultimately form the male gametes. The spermatozoa are unique type of cells containing nucleus (head portion) and a tail. The acrosome is a double walled structure situated between the plasma membrane and the anterior portion of the sperm head and neck connects the sperm head with its tail.

Semen collection is the most important component of any frozen semen production programme as a high quality end product is not possible unless the raw material is of excellent quality. Therefore, all care should be taken to collect semen aseptically with the aim to harvest the best quality neat semen for processing. The person collecting semen should understand bull’s behaviour and should be friendly with him. Professional bull management results in increasing sperm output and enhancing productive life of the bulls. One must know potentials and limitations of the reproductive capacity of bulls. A highly professional and dedicated team of bull handlers and semen collectors is required for this purpose. The persons involved in bull management should be familiar with bull’s sexual behavior and male reproductive physiology. Preferably a veterinarian should collect semen.

Semen collection in bulls and buffalo bulls

Semen collection from domestic animals is an art and skilled job to get maximum quantity and quality of an ejaculate. Ejaculation of semen in a male is a complex process and consists of several events sexual arousal, courtship, erection, penile protrusion, mounting, intromission, ejaculation, orgasm like reaction and dismounting. Ejaculation is the result of neuro-muscular stimulation that starts in the vas differentia and accessory sex glands. The semen ejaculation is finally brought about by the rhythmical contractions of penile musculature and urethra. The muscular contractions proceed progressively in waves, forcing the semen out of the external urethral orifice. To get good quality semen, the bulls must undergo all the related events of ejaculation. The semen collectors should have knowledge of inhibitory and stimulatory factors for the act of ejaculation in male domestic animals. The stimulatory factors are:

• Semen cryopreservation – It is the process of preserving the semen in a suitable medium at an ultra-low temperature which reduces spermatozoa metabolism with the least production of metabolic waste products of sperm cells to prolong their life span. • Every fall of 100 C temperature decreases the metabolic rate of spermatozoa to half. Principle of various components involved in cryopreservation 1. Low temperature • Reduce metabolism (negligible) and least metabolic waste product • Decrease solute solubility leads to increased viscosity and physical resistance • Reduce motility • Prolong life span 2. Protection from cold shock and cryoinjury • When sperm cells are subjected to a reduction in temperature during the freezing process, there are: • Sperm membrane damages and lead to leakage of intracellular components like enzymes, ATP, K+ , cholesterol, lipoproteins etc. • Irreversible abnormality in motility pattern (bending of sperm tail)

Semen dilution/ extension is done to meet the following objectives

1.    To preserve the fertilizing potential of semen for a long period of time

2.    To increase the number of services per ejaculate. Ideally approximately 500 cows/ buffaloes can be inseminated from diluted/ extended semen of  a single ejaculate.

3.    Extenders provide the spermatozoa with a medium in which they are able to remain viable and fertile for a prolonged time period.

In order to be an ideal extender, it should be able to provide conducive environment to prolong sperm survival and modify the sperm structure to prepare them for freezing through the following properties:

1.    It should be isotonic with blood and should maintain this during preservation.

The idea behind semen dilution is to extend its volume without affecting its fertility so that many females can be bred from an ejaculate. The basic components of semen extender are Tris buffer, fructose, glycerol, egg yolk and antibiotics.

Semen evaluation alone should not be interpreted as the sole indicator of the fertility of bulls but it will provide significant information of the sexual apparatus. After receipt of the semen sample in the laboratory, it is kept in a hot water bath at 350C until the evaluation is complete. One has to prevent cold shock to the living sperm cells during the evaluation process. All the glassware and other items coming directly or indirectly in contact with the sperm cells should be sterilized properly and kept ready in a thermostatically controlled cabinet at body temperature. Sudden changes in exposure of semen to cold or hot temperature results in higher dead spermatozoa or sperm agglutination. Semen should not be exposed to sunlight, wind, chemicals, smoke, vibration and water. A single laboratory test is not sufficient to assess the semen quality and semen should be examined within a shortest possible time after ejaculation as mentioned below (Table 9.1).

Microscopic examination 

A phase contract microscope with biotherm (37o C) is necessary for correct evaluation of semen because the refractive index of spermatozoa differs slightly from that of the surrounding medium. The ordinary bright field microscope will not provide sufficient contrast between the spermatozoa and the medium. Under phase contrast microscope, the object will appear dark on a bright background. This is achieved by reducing the light passing through the object by half a wavelength compared to the light passing through the medium. The eyepiece has a magnifying capacity of 10X while the objectives have 10, 40 and 100X. This will provide the microscope the magnifications of 100, 400 and 1000. The lenses and eyepiece should be kept clean by wiping with tissue paper and distilled water. Xylol can also be used to remove greasy material. The microscope has to be kept in one place and covered with Perspex cover when not in use to avoid dust accumulation. Personnel familiar with the operation of a particular microscope alone should be allowed to handle the microscope, since lot of differences exist between different makes of microscopes.