eChapters

A laboratory is essential for research work in science and development. Most of the research institutes have dedicated laboratories for researchers and students to conduct experiments, perform research work, as well as to provide hands-on learning/training to students. There are certain rules and regulations laid down by the laboratory in-charge which need to be followed strictly for safety purposes. Some of the laboratories keep ‘safety manual’ for researchers to read before entering into the laboratory to conduct experiments. Whenever safety is compromised in a laboratory, there is increase in risk of unwanted incidents in the laboratory. Storing food/beverages in the same refrigerator where laboratory chemicals are kept may lead to disastrous effects on health. This chapter aims to provide instructions in brief on the things to be performed (Do’s) before and after entering onto a laboratory as well as the things never to be done (Don’ts) in a laboratory for the safety reasons.

Good Laboratory Practices (Do’s)

• P Wear apron/labcoat while working in the laboratory.
• P Use gloves/respiratory mask/fumehood while handling toxic/volatile/inflammable chemicals/reagents.
• P Use pippet or pippetor for safety in transfer of solutions.
• P Protect eyes, face and naked parts of the body from UV radiations. Use safety goggles or face mask/apron.
• P Keep your eyes away from Liquid Nitrogen and use cryogloves while handling it.
• P Arrange all the necessary materials/solutions before starting the experiment.
• P Periodically fumigate/ozonize culture and inoculation rooms.
• P Autoclave all contaminated materials before discarding.
• P Pre-treat gel or solution containing Ethidium Bromide (EtBr) before discarding into laboratory waste-disposal bin, and use gloves while handling them.
• P Burn all the transgenic plant materials in an incinerator after taking necessary observations.
• P Make entry in the designated log-book and clean the instrument after using it properly.
• P Switch-off the instrument after use or when not in use.
• P Wash hands with soap and remove the labcoat before leaving/going out of the laboratory7.
• P Ensure that switches for fans/lights, and water taps are off while leaving the laboratory.
• P Follow the biosafety guidelines suggested for handling and disposal of the hazardous chemical.

India is bestowed with an enormous wealth of medicinal plants and has a rich heritage of treating human and animal ailments by using herbal medicines. However, there is need for a systemic, scientific approach to validate the authenticity of claims on plant-based medicaments. This approach has paid rich dividends in developing cheaper, safer, effective and eco-friendly remedies, which recently has gained worldwide attention. Cissus quadrangularis, Linn (Syn. Vitis quadrangularis, Wall; Heliotropium indicum, Linn) is a tender climber with fleshy quadrangular stems and grows in the hotter parts of Indian subcontinent, Sri Lanka and East Africa. In Hindi it is known as hardjore; in English as adamant creeper, bonesetter; in Ceylon as hiressa; in China as di xanh voung; in Burma as shazam-lese and in Bangladesh as harbhanga. Considering the advantage of herbal medicines, it is planned in this chapter to investigate the efficacy of Cissus quadrangularis, Linn, in one of the most commonly encountered problem of fractures in small animals.

Dried ethanolic extract of Cissus quadrangularis (CQ) is obtained from 500g of dried finely powered stems using soxhlet apparatus. It is dissolved in a vanishing base to make a semi-solid paste for local application. For parental use, this extract is reconstituted with distilled water (100 mg/ml) and filtered.

Twelve clinically healthy mongrel adult dogs aged 2-3 years and weighing 10-15 kg is randomly divided into two equal groups (A and B). In all animals operative site is prepared for aseptic surgery. Premedication is done with atropine sulphate @ 0.06 mg/ kg body weight subcutaneously and anaesthesia is achieved with a combination of Xylazine hydrochloride @ 1 mg/kg body wt. and Ketamine hydrochloride @ 10 mg/kg body wt. intravenously. Unilateral comminuted diaphyseal femoral osteotomy is created under general anaesthesia and the bone defect is immobilized with neutralization bone plating (AO/ASIF technique). All the animals are administered 5 mg/kg b wt Enrofloxacin intramuscularly for 5 d and 1 mg/ kg b wt Diclofenac sodium injection intramuscularly for 5 d postoperatively. Skin sutures are removed after 10 days. In the animals of group B, in addition to the postoperative treatment, the ethanolic extract of the herb, Cissus quadrangularis is applied on the skin twice daily along with subcutaneous injection (50 mg/ kg body wt.) on every alternate day for 20 d postoperatively.

Introduction
Assisted reproductive technology (ART) refers to treatments and procedures in the field of reproductive biology that are utilised to achieve pregnancy by artificial or partially artificial means.
Artificial insemination
Artificial insemination (AI) is the earliest form of assisted reproductive technology. The technology has been of immense relevance in enhancing production and health features through the appropriate use of better male animals, as well as in eliminating certain venereal illnesses.
• Semen collection: The man is typically allowed to mount a phantom during the process. In cattle, an artificial vagina is used)
• Semen evaluation: Evaluating semen is crucial before processing. The most basic semen evaluation involves examining the concentration, progressive motility, and morphological normality of the spermatozoa)
• Semen dilution, packaging and freezing: Following examination, the semen is ready for dilution and packaging. Spermatozoa require storage mediums that are particular to each species. After packaging, the semen dosages must be properly maintained until insemination. Cryopreservation is typically used for cattle.
• Insemination: Insemination should occur during the female’s oestrus, when she is receptive to copulation.
Multiple Ovulation and Embryo Transfer (MOET)
The main processes are to stimulate a donor female to create suitable embryos, then remove the embryos from her and transfer them to unmated recipient females at equivalent post-ovulatory reproductive phases, ensuring that the donor’s embryos are carried to term in the recipients’ uteri. Multiple ovulations are caused by superovulation or superstimulation, a procedure that increases the number of ovulations in a donor beyond what is normal for a given species. Embryos are recovered 6-8 days following the start of oestrus. Most embryos are transferred during the late morula or blastocyst stage.

Bone healing can be modified by variety of endogenous or exogenous factors that affect the metabolic function of bone cells. The Food and Drug Administration is approved the use of low-intensity ultrasound for the accelerated healing of fresh fractures in October 1994 and for the treatment of established nonunion in February 2002. The first regulatory approval is based primarily upon two rigorous, double - blind, placebo controlled clinical trials, which shows that the rate of healing of fresh fracture is accelerated by treatment with ultrasound. Therapeutic ultrasound has been able to improve functional activity, reduce post-ambulatory period, alleviate suffering and restore the animal’s health early. It has been used frequently for acceleration of healing in various musculotendinous injuries in animal. However, the role of ultrasound therapy in different dose levels in fracture healing has not been elucidated properly. In view of the above fact, this chapter is undertaken to investigate the effects of therapeutic ultrasound in two different doses, on cortical bone healing in rabbits.

Twelve clinically healthy adult New Zealand white rabbits of either sex are divided equally into three groups A, B and C, and kept on standard diet and uniform management. In all animals, mid-shaft simple transverse fracture is created in both the ulnae under thiopental (2.5%) anaesthesia. All the animals are administered broad-spectrum antibiotic and analgesic and surgical wounds are dressed daily with povidone iodine and antibiotic cream for 4 days postoperatively. Group A animal is received pulsed ultrasound therapy @ lW/ cm² (1:1) whereas, group C animals is received continuous ultrasound therapy @ 3 W/cm², starting on day 4 after the creation of fracture. Ten treatments, each consisting of 5 min duration, are given on alternate days. Group B animals are served as control. In both the test groups (A and C), the transducer head of the ultrasound machine is moved in a linear fashion along the length of the bone after applying ultrasound gel.

Among the epiphyseal and metaphyseal fractures of the different long bones, fractures of the proximal tibia are quite commonly reported and require special attention due to their proximity to the compound stifle joint. Hence, a standard fixation technique is needed to treat such fractures, which also permit early ambulation and preserve range of joint motion in addition to providing rigid fracture fixation. Different treatment modalities have been tried for fixation of metaphyseal fractures with variable results. No single method is appropriate in all circumstances and treatment indication often overlaps. Therefore, in this present chapter, an attempt has been made to evolve a standard technique for the management of such fractures.

Ten clinically healthy mongrel dogs are maintained under uniform housing, feeding and management conditions, acclimatized, immunized against rabies, dewormed and randomly divided into two groups (A and B) of five animals each. In all the animals unilateral metaphyseal fracture is created in the proximal tibia under general anaesthesia with intravenous thiopental sodium (5%). In the animals of group A, the fracture is immobilized with cross intramedullary Steinmann pinning with interfragmentary wiring, whereas, single intramedullary pinning along with interfragmentary wiring is performed in the animals of group B. Antiseptic dressing of the wounds is done daily for 1 week with povidone-iodine. All the animals are administered Enrofloxacin at the dose rate of 5 mg/ kg b.wt intramuscularly daily for 5 days posoperatively. Skin sutures are removed on day 10 post-operation

Clinical Significance The primary aim of laboratory diagnosis is to assess the carbohydrate metabolism impairment in diabetes mellitus, inducing the following: 1. Determination of glucose concentration in fasting blood and demonstration of postprandial glycosuria. 2. Assessment of glucose tolerance

The liver produces the entire albumin and most of the globulin; a small amount of gamma globulin is produced by retculoendothelial tissue. The production of albumin is subject to a mechanism regulating colloidal osmotic pressure and the secondary to alterations in globulin concentration. The liver synthesizes lipoproteins, glycoproteins, mucoproteins, fibrinogen, prothrombin and clotting factors VII, VIII, IX and X.

Fracture of meta-carpals and meta-tarsals comes under lower level fracture. Padded cloth and netted bamboo splint along with conjoint U plate has been considered as standard method of repair.

The fractufe is a lower level fracture involving the meta-carpal and meta- tarsal bones. (Fig.1 & Fig.2)

The fracture site is immobilized extending joint below and joint above level initially with cloth or cotton padding.(Fig.3)

Netted bamboo splint Is applied over it for support involving joint above and joint below which should not be too tight or too loose.(Flg.4)

Splints are retained in position by thread with knots. The ends of bamboo splint kept padded to avoid skin damage.(Fig.4)

More strength is given to immobilisation by application of conjoint U- plate.Conjoint U-plate is a U shaped metallic plate used for lower level fracture immobilisation. (Fig.5 & Fig.6)

At the end the entire bandage is covered with cloth or a bandage roll.The hoof should also be bandaged to avoid damage due to improper blood circulation.(Flg.6)

Fracture of radius-ulna & tibia-fibula comes under middle level fracture. Pop caste application or bandage along with Thomas splint has been considered as standard method of repair.

The fracture site is immobilized extending between joint below and joint above level with either gum bandage or pop caste bandage (Fig.1).

Pop caste bandage is generally applied after 7 days. Hence gum bandage is preferred than pop caste bandage because of immediate application after occurrence of fracture (Fig.2).

For gum bandage application, a 8-16 layer thick cloth is wrapped over the fracture region covering the joint above & joint below of the site after liberal application of fevicol gum. The cotton cloth Is placed in position with few cotton thread knots (Fig.3).

4 bamboo splints are kept on the 4 sites of the limb over cotton padding. The end of bamboo splints kept padded to avoid skin damage. Splints are retained in position by thread with knots (Fig.4).

A 2^nd layer fevicol gum is applied over entire bandage & finally the entire bandage cloth is covered with bandage roll (Fig.5).

At the end the immobilized limb is over strengthened with Thomas splint (Fig. 6).

• In male animals, endocrine regulation of reproduction is mainly controlled by pituitary gonadotrophins and gonadal androgens. It is not the same as in females as there is only negative feedback controls in case of male while it is positively as well as negatively feedback control in females. In other words, the synchrony of pituitary gonadotrophins with gonads in male is non cyclic while in female it is cyclic i.e. even after castration (absence of androgen) the pulse frequency of LH and FSH is retained. • Sex Hormones are sex specific e.g. estrogen and progesterone work primarily in females and testosterone in males but sex specificity is limited as far as sexual behaviour is concerned. As estrogen injection in castrated ram recovers the maleness while testosterone injection in ovariectomized female maintain the receptivity for male up to a certain extent.

• To identity the normal fertile, infertile (Temporary loss of fertility), subfertile (reduced fertility), or sterile (no fertility) male animals makes the requirement of breeding soundness evaluation necessary. Infertile and sterile animals prove to be harmful for the economic survival of the dairy industry and should be eliminated from the breeding management system. • The breeding soundness examination is very necessary for the breeding management system as the ultimate goal is the achievement of healthy pregnancies. It is also useful for successful artificial insemination (AI) programs that involve the collection and cryo-processing of quality semen.

Semen • Semen, a fluid released from the male reproductive system during ejaculation, is made up of a liquid phase called seminal plasma and a cellular component called spermatozoa. • The term “spermatocrit” refers to the percentage of spermatozoa(v/v) in semen, which varies from less than 2% in pigs to more than 30% in sheep. It is about 10% in bull. Semen= Spermatozoa + seminal plasma Spermatozoon • In 1677, the scientist Van Leeuwenhoek first discovered the spermatozoa and called them ‘‘animalacula’’. • Spermatozoa or sperm cells or male gametes, develop within the wall of the seminiferous tubules as a result of a complex process known as spermatogenesis. • The spermatozoon is a highly specialized cell that performs the sole function of fertilizing an oocyte.

Sperm abnormalities may be genetical or morphological. They are directly related to the male infertility. • The wet mount method and phase contrast microscopy facilitate sperm visualization for abnormalities. Semen stained with vital stain can also be used for evaluating sperm abnormalities. Sperm morphology or related abnormalities can be evaluated by using eosin-nigrosine stain, carbolfuschin, aniline blue-eosine etc. • Generally, for head and tail abnormalities rose Bengal and William’s stain can be used while for acrosome and head abnormalities Giemsa stain can be used. Categorization of Sperm Abnormalities 1. On the basis of site of abnormality on the spermatozoa • Head abnormalities e.g. microcephalic head, macrocephalic/giant head, narrow or short sperm head, pyriform head, detached glea capitis (acrosomal cap), detached head and double head etc

• 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)

A) Hypothyroidism

Etiology

It is one of the common endocrine disorders of skin of dogs mainly due to deficiency of thyroid hormone followed by cutaneous and non cutaneous signs. It is caused by lymphocytic thyroiditis or idiopathic thyroid necrosis and atrophy. Deficiency of thyroid hormone hinders the metabolic processes throughout the body.

Combination of an opioid (butorphanol/buprenorphine) and an alpha-2 agonist (xylazine/dexmedetomidine) along with an NMDA antagonist and benzodiazepine (tiletamine-zolazepam or ketamine-midazolam) will make up a balanced anaesthetic cocktail. Examples for balanced anaesthetic cocktails are xylazine/dexmedetomidine-butorphanol-midazolam-ketamine and butorphanol/ buprenorphine-tiletamine zolazepam-dexmedetomidine/xylazine (Ko, 2013; Cagle et al., 2017; Sooryadas et al., 2019; Verma et al., 2020). The latter ones are costly due to the tiletamine-zolazepam and dexmedetomidine in them, while the former ones are cost effective alternatives.
Xylazine/Dexmedetomidine-Butorphanol-Midazolam-Ketamine (XBMK/DBMK) Anaesthetic Cocktail
The preparation can be made by drawing 0.1 ml/kg of ketamine (50 mg/ml presentation) along with 0.1 to 0.2 ml/kg of the xylazine-butorphanol-midazolam (XBM) or dexmedetomidine-butorphanol-midazolam (DBM) sedative cocktail in a single syringe. For intravenous administration, 0.1 ml/kg each of ketamine and the sedative cocktail is to be chosen, while for intramuscular administration 0.1 ml/kg of ketamine and 0.2 ml/kg of the sedative cocktail is preferred. The volume of the cocktail that needs to be administered intramuscularly will be larger by 0.1 ml/kg when compared to the tiletamine-zolazepam based anaesthetic cocktails. Anticholinergic premedication is preferred prior to administration of anaesthetic cocktail containing xylazine (Nesgash et al., 2016), particularly if given intravenously. The XBMK/DBMK anaesthetic cocktails will induce anaesthesia in a few minutes, which last for 20 to 40 minutes (Ko, 2013; Verma et al., 2019; Verma et al., 2020). Intravenous administration of the anaesthetic cocktail provides shorter duration of surgical plane of anaesthesia. When administered as said above, the combination provides xylazine @ 0.2 - 0.4 mg/kg or dexmedetomidine @ 2.5 - 5 mcg/kg, butorphanol @ 0.1 - 0.2 mg/kg, midazolam @ 0.125 - 0.25 mg/kg and ketamine @ 5 mg/kg body weight which is as per their recommended doses when given in combination (Verma et al., 2019; Verma et al., 2020).

Total Intravenous Anaesthesia
Maintenance of anaesthesia, conventionally, is done either with inhalants, or as intermittent boli of injectable anaesthetics. The pharmacological complexity of the uptake and elimination of inhalant anaesthetic agents, and the need of technical and functional knowhow, of inhalant anaesthesia machine, either restricts its use in majority clinical practice, or is not used in an ideal manner. Injectable anaesthetic drugs as intermittent boli “as and when needed” is the practice by majority practitioners for maintenance of anaesthesia. Intermittent bolus injections of anaesthetic drugs creates intermittent peaks of anaesthesia and thereby increased side effects due to high doses. Instead, a continuous rate infusion of the anaesthetic drugs, in smaller quantities is employed in TIVA, providing balanced anaesthetic maintenance. These drugs act through different pathways, providing analgesia, muscle relaxation and unconsciousness, in a synergistic manner. TIVA is thus a technique in which anaesthesia is maintained solely through the use of intravenous drugs, without reliance on inhaled anaesthetics (Kennedy, 2016). Total intravenous anaesthesia is commonly achieved using a combination of sedative-hypnotics, analgesics and even muscle relaxation drugs, often administered via continuous rate infusion (CRI) to maintain stable drug levels (Verma et al., 2020). The key advantage of TIVA is that it provide stable haemodynamics because it maintains the sympathetic tone well. Also, this procedure promises less environmental contamination avoiding exposure to anaesthetic gas in the operating rooms. TIVA is emerging as a popular option in a range of surgical procedures due to its benefits in patient comfort, recovery, and control over anaesthesia depth. Few examples of anaesthetic cocktails for total intravenous anaesthesia in dogs through constant rate infusion are mentioned below. Easy understanding on how to calculate and prepare CRI is mentioned later.
1. Dexmedetomidine (2 mcg/kg/hr), midazolam (3 mcg/kg/min), lignocaine (50 mcg/kg/min) and ketamine (40 mcg/kg/min) as a cocktail intravenous infusion in normal saline. Anaesthesia is induced by intramuscular administration of a cocktail of DBMK mentioned in earlier chapter, followed by an intravenous loading dose of lignocaine at the rate of 2 mg/kg prior to the cocktail intravenous infusion (Verma et al., 2020).
2. Xylazine (1 mg/kg/hr), midazolam (3 mcg/kg/min), lignocaine (50 mcg/kg/ min) and ketamine (40 mcg/kg/min) as a cocktail intravenous infusion in

To know the method of immobilization of the limb after repair of the transected gastrocnemius tendon (tendo achiles).

Procedure

The immobilization procedure of limb after gastrocnemius tendon repair is done in 2 stages.

1^ST Stage

Immobilization of limb for avoiding movement at tendon suture site.

It is done by placing the sutured area within a pre- fabricated metal frame so as to keep the limb undisturbed at the suture site.

The frame is given in Figure 1.

The purpose of addition of the metal frame is to immobilize the limb keeping the fetlock and foot away from in a flexed manner to avoid weight bearing during healing period.

The limb is immobilized with netted bamboo splint (Fig. 2).

The immobilized limb is placed within the metal frame keeping the fetlock and foot in a flexed manner

Estrous cycle

After puberty the female enters a period of reproductive cyclicity, which continues throughout most of her productive life.  Estrous cycle is the rhythmic sexual behavioral pattern exhibited by the female, which begins at one estrus (heat) and ends at subsequent estrus.   It is the period between two estruses of the regular cycle.  The reproductive activity of the female is maintained by the co- ordinated functioning of different organs of reproduction (Table: 1.1). Estrous cycle provides repeated opportunities for the females to become pregnant. When conception does occur, the female enters a period of anestrous during pregnancy, which ends after parturition (giving birth) and uterine involution (repair and returning to normal size).

After the Second World War, the 1940’s and 1950’s was the wide spread establishment of artificial insemination and the introduction of frozen semen in cattle breeding.  In the late 1960s came revolution in measurement of hormones in the body fluid with the introduction of sensitive radio-immuno-assay (RIA) techniques, which provided a new approach to early pregnancy diagnosis, emerging around the same time were early forms of the ultrasonic which could also employed in the detection of pregnancy. The 1970’s were to see embryo transfer and freezing of embryos in cattle becoming commercial realities and their incorporation into increasingly effective breeding improvement programme with the availability of prostaglandins (PGs) and Gonadotrophin releasing hormones (GnRH) that decade also witnessed new possibilities for estrum control in cattle and horses.  Few years later came the ability to produce the cattle embryos in the laboratories in large numbers and at low cost.