eChapters

Artificial Insemination (AI) is one of the most popular first generation assisted reproductive technology as well as a breeding tool, ever evolved for the genetic improvement of cattle and buffalo industry throughout the world. The advent of frozen semen has further revolutionized this technique. Presently, use of sex- sorted-semen in cattle breeding is also coming into vogue. Contrary to cattle and buffalo, use of AI in small ruminants has not gained such impetus.

It has been reported that lower pregnancy rates were obtained when ewes and does are bred with frozen or extended liquid semen using intra-vaginal or intracervical technique of insemination. The reason being anatomy of the cervix of sheep and goats; cervix is long and tortuous with presence of 4-7 cervical rings. Furthermore, these cervical rings are directed caudally hindering the lumen of the cervix thereby causing hindrance (more prominent in sheep) while passing the AI gun during intra-cervical or trans-cervical AI (TCAI). There are several reports which support the fact that, as the depth of semen deposition into the cervix increases, the pregnancy rates also improve due to availability of a greater number of motile sperms for fertilization.

Musculoskeletal system consists of muscles, cartilage, bones, joints ligaments and tendons. Primary function of the musculoskeletal system is to support the body, helps in mobility, and protects vital organs of the body. Diseases of these organs have clinical manifestations such as lameness, failure to support weight, inefficient joint movement and deformity. Examination of musculoskeletal system includes general as well as special examination. General examination consists of examination of posture in sitting/recumbency, standing position and while rising; examination of gait; and any other abnormality. General Examination The animal is observed for conformation (outline and shape), symmetry and posture in standing position from the cranial, caudal and lateral positions. Asymmetrical shape of the body or limbs may be due to swelling that should be examined by palpation,

An early and accurate diagnosis of pregnancy is a crucial factor for successful management and maintaining cost-effective production in scientific goat production for better economic returns. In addition, accurate information on the stage of gestation would also be useful to dry off lactating females at an adequate period. The techniques for pregnancy diagnosis in goats are mostly depend upon visualization of the conceptus or determination of its secretory products in the maternal blood or the milk are the most defined and specific methods for pregnancy. The choice of pregnancy diagnosis method in goats depends on the stage of gestation. Some methods have a higher degree of accuracy at the early gestation while others do at the late stage of gestation. Assessment of pregnancy in terms of hormonal assay of blood plasma, serum, or milk, and estimation of pregnancy-specific antigens or proteins give a higher level of accuracy within 30 days of pregnancy. There are many methods that are currently being used for pregnancy diagnosis in goats. Among those, the method applied should be safe for both offspring and mother and need to be low-cost and easy to apply. Various techniques have different kinds of limitations to their wide-scale use. These techniques are classified into 3 classes, which are as follows:

The major requirement that distinguishes frozen semen/IVF laboratory from other laboratories is the need to maintain aseptic conditions. The wash up and sterilization of media and other equipments should be located at one end of the room and clean area for sterilize handling at the other end, and preparation, storage and incubation in between. The storage and incubation area should be near to the sterile working area.

In recent years, the introduction of laminar flow cabinets has been a great boon in providing a sterile working area and allows the use of unspecialized laboratory accommodation for culture work. For sterile work, area should be in a quiet part of the laboratory preferably in a cubicle where a laminar flow cabinet is suitably fitted. The area should be free from dust or draughts. Nothing should be stored on the laminar flow cabinet or bench used for sterile handling except most necessary equipments like holding pipettes etc. The laminar flow cabinets provide sterile air blown over the work surface. For laboratory, a horizontal laminar flow is more preferable as opposed to a vertical one because of ease of handling the ovaries etc. If an air curtain is provided at the door of the cabinet, introduction of dust etc. can be avoided. In India laminar flow are manufactured by companies like Clenzoids, Yorco etc, among others. The laminar flow size should be a minimum of 300 X 300 or 450 X 450 mm with a 12-18 in, square filter size).

The udder is responsible for milk production. It is composed of four mammary glands each with its own teat (Fig. 167). Swelling of udder or teats is an indication of udder disease warranting clinical exploration and palpation. Visual examination the udder (before and after milking) and palpation is of great significance in detecting abnormalities. The udder is visually inspected from back and sides for symmetry and also palpated thoroughly. Palpation is a simple, quick and easy routine diagnostic tool for mastitis detection. Teats and supra mammary lymph nodes should also be palpated. These examinations aid in detecting fibrosis, inflammation or atrophy. Diffuse fibrosis gives the quarter a firm feeling. Areas of local fibrosis may vary in size and shape. Hot, painful and diffuse swelling with marked abnormality of mammary secretion is seen in acute mammary inflammation (acute mastitis). Atrophy of the gland is the terminal stage of chronic mastitis. With the development of gangrene, the area becomes cold and bluish. Five point palpation scores (Petrovski et al., 2011; Hogan et al., 1995; Scaletti and Harmon, 2012)

The successful AI program depends on the health of the semen donors followed by hygiene in semen collection, processing, storage and AI methods as well as reproductive health of the females. However, the microbial contamination may not be able to avoid completely but it can be reduced to the minimum threshold level by adopting hygienic procedures.

The hygienic condition of the neat / frozen semen samples can be assessed periodically by enumerating the bacterial load apart from the periodical screening of bucks for the presence of various causative agents in animals and its transfer to the females via semen samples.

Though both viable and total (viable and nonviable) bacterial counts can be done, the viable techniques are more commonly used in diagnostic and food hygiene procedures. The viable bacteria are capable of multiplication with the production of visible colonies on or in agar media. In viable techniques, the assumption is made that one well-spaced bacterial cell gives rise to one colony. The bacterial colonies, rather than bacterial cells, are counted in these techniques. Among the viable counting techniques like Spread plate, Pour plate, Miles, Misra, Filtration are available and each has its own inherent errors, the pour plate counting method is explained as follows:

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).

Good quality semen is more acidic than semen with lower sperm concentration. In contrast, bacterial contaminated semen and semen with more dead spermatozoa evolve ammonia that will increase pH of semen. In orchitis, seminal vesiculitis and epididymitis the pH of semen will be alkaline due to presence of pus. In azoospermic or necrozoospermic semen, the pH may be neutral or alkaline. pH variation may be due to change in season/climate and frequency of successive ejaculates. Depending on the concentration and activity of spermatozoa, the pH of semen varies. The narrow range pH (BDH) paper are more convenient in routine work. The first change in color is matched with color on the cover of booklet. Use of BDH capillary set is time consuming whereas digital pH meters are also being used. The normal pH range is depicted in Table 11.1.

The semen storage technology and glass ampoules were earlier developed in USA and French, Danish and West Germany introduced straw technique and in the mean time Japanese scientists developed pellet freezing method. 

1. Ampoule method 

The diluted semen is filled in ampoules after labeling, sealed, frozen and preserved in liquid nitrogen. For insemination, the ampoule is thawed in warm water of 37^ºC until liquidation then ampoule is cut and semen is drawn into glass catheter with the help of a sterile syringe and used for insemination. In this method, semen may not be contaminated and identification of semen is easy. However, the semen is frozen in a larger volume so the freezability and fertility are less and it requires more storage space in cryocan. About 8-10% of semen is lost while thawing, loading and insemination.  Use of glass catheter for insemination is risky because chances of breaking and chances of injury to cow or inseminator and passing of catheter in the cervix.

1. Preparation of diluents 

The diluent is prepared one hour before semen collection so that it is stabilized. The diluent is prepared by mixing the ingredients and kept ready in water bath at 30oC. One of the diluents is detailed below

The reproductive efficiency in dairy herd is one of the important factor in determining total production and profitability. To remain profitable, dairy cows must reproduce at greatest efficiency and should calve every year (12-13 months). The measure of fertility in a herd is the calving rate (percentage of live, normal calves born). However, it is also necessary to measure fertility based on services per conception. In a healthy herd in which fertile bulls are used, an average of about 1.6 to 1.7 services is required per conception. The number of services per conception is particularly useful for analyzing costs and in comprising the A.I. quite similar to that obtained through natural service in well managed herds. Another measure of fertility is the non-return rate that is calculated from insemination records. Non-return rate is the percentage of cows that are not subsequently rebred within a defined period (60 to 90 days) following an insemination. On an average, about 70% percent of cows inseminated fail to return for service after first service on a 60 to 90 days non-return basis.

Introduction
Cleavage is the process of cellular division without growth. It is also called segmentation division and it follows immediately after fertilization. By the end of this process, single cell zygote is transformed into multi-cellular stage called blastula. Daughter cells are called blastomeres. All divisions are mitotic and occur consequently. Ratio of nucleus to cytoplasm is very low at beginning but later they resemble somatic cell. Cleavage division always start at animal pole. Cleavage can be classified as:
1. Holoblastic/Total cleavage: Whole fertilized ovum is involved in production of blastomere. It is also called complete cleavage.
a) Equal: All blastomeres are of equal size. E.g. mammals
b) Unequal: Some blastomeres at vegetal pole are larger. E.g. amphibians
2. Meroblastic/Partial cleavage: When deutoplasm is abundant and only a part of fertilized ovum is involved in cleavage, it is called meroblastic cleavage.
a) Discoidal: Cleavage is confined to the germinal disc i.e. at animal pole. E.g. birds and reptiles.
b) Superficial: Cleavage is confined to the peripheral margin of cytoplasm. E.g. arthropods.
Dividing continuously, embryo attains a stage of 16-32 cells. 16-celled stage is called early/uncompact morula and 32-celled stage is called compact morula. This stage is called morula due to its close resemblance to mulberry fruit. Structure is surrounded by zona pellucida.
After morula stage, fluid accumulation begins and blastomeres are gradually pushed to the periphery. As fluid accumulation progresses further, it leads to formation of large cavity in central part of embryo known as blastocoels and morula stage is now transformed into blastula/blastocyst stage. Blastomeres now arrange themselves as single celled outer layer called trophoblast and small group of cells at animal pole called inner cell mass (ICM). Trophoblasts give rise to fetal membranes and will help embryo in its attachment to uterine wall (implantation). ICM gives rise to all organs of body. Development of blastula is called blastulation.

Introduction

Use of acid/base is common in laboratories for experiments like titration and proper safety in their handling laboratory is necessary. In this chapter we will briefly discuss about the safety measures necessary to be adopted while using acids/bases in laboratory for any experiment.

Handling of Strong Acid/Base

There are four major points that need to be taken care of while working with strong acids or bases that might be quite harmful if any accident happens.

1. Protect Yourself

Wear a lab-coat and acid-resistant gloves: Always use lab-coat/apron and make it sure that the sleeves properly cover your arms. More importantly, use gloves made of acid-resistant materials and also wear shoes to protect your feet while wearing long-pants/jeans. Female with long hair, must tie her hair properly (with rubber band) to avoid any disturbance caused by appearance in between.

Wear safety goggles: It is important to wear safety goggles to protect your eyes using larger and fitting goggles that cover front as well as sides of your eyes. Always use properly fitting and adjustable goggles because smaller/ loose goggles may not protect the areas around your eyes against accidental acid spills and it may fall down from your face, if it is too loose.

Locate emergency wash area: There must be an emergency washing area nearby the work place or laboratory containing lab spill management kits. All the workers in the laboratory must be aware of using the kits and emergency plans.

Neutralize acid: Don’t panic even if an acid or chemical spill happens. Stay calm and act as quickly as possible. In case an acid spills, neutralize it by using sodium bicarbonate so that they can be cleaned and disposed safely. If acid comes in contact of skin, wash it off under running tap water but do not use/ apply sodium bicarbonate on skin.

Artificial insemination in horses is being practiced since many years in Russia and China for genetic up-gradation. The equine artificial insemination industry has evolved a system in which mares are inseminated with fresh or cooled semen (500 million motile sperm) every 48 hours until estrus is no longer detected or until ovulation is detected by trans-rectal palpation or ultrasonography of ovaries respectively. The advantages of AI include:

Artificial insemination (AI) technique in pigs is being performed since several decades in many countries but not widely accepted technology as in cattle due to reasons like sub-standard method of semen cryopreservation and lower fertility rate. The main idea is to increase the breeding efficiency by utilization of superior boars. The number of boars needed for breeding purposes can be reduced by use of AI and reduced transmission of infectious diseases. A minimum of 15 to 20 sows can be inseminated after dilution over a 2 days period with an ejaculate from a boar. Artificial insemination had considerable impact worldwide in its application to commercial pig production. The main advantage of using fresh liquid boar semen is that fertility is maintained even with low number of spermatozoa in the inseminate. AI provides unique opportunity for the genetic improvement of pig industry. Using fewer than 1 million sperm cells per breeding unit can achieve conception rates with liquid semen similar to those with frozen thawed semen containing approximately 15 million sperm and reduced fertility makes cryopreservation an uneconomical option in boars.

Artificial insemination technology is a valuable tool to inseminate 2000 to 3000 does by frozen semen instead of breeding 40 to 50 does by natural service in a year. Goat semen may be stored and frozen more successfully than ram semen. The buck will be sexually mature at 6-7 months of age.

Castration means removal of the testicles and there are two methods of castration in domestic animals viz. closed and open method.

1. Closed method

In closed method, without incision, the spermatic cord is crushed where thrombus is formed which arrest blood flow to the testicles resulting in gradual atrophy of the testicles. ex. Burdizzo method

FEMUR

The femur has a relatively small shaft, which is cylindrical in its middle. The trochanter minor has the form of a rough tuberosity.

The trochanteric ridge connects it with the trochanter major. The third trochanter is absent. The supracondyloid fossa is shallow. The proximal extremity is very wide.

The fovea capitis is a small depression on the middle of the heacKfor the attachment of the round ligament.

The trochanter major is very massive and is undivided; its lateral surface is very rough. The trochanteric fossa is deep. The ridges of the trochlea are less oblique.

The formation of the mammalian body plan is regulated by highly conserved gene regulatory networks. During this developmental stage, the notochord and primitive node play critical roles in establishing the cranial-caudal axis and left-right asymmetry. Ectoderm, the outer germ layer, differentiates into neuroectoderm and surface ectoderm. Neuroectodermal cells move from the developing neural tube’s lateral edges to a dorsolateral position. These neuroectodermal cells are known as neural crest cells. The central nervous system originates from the neural tube, whereas the peripheral nervous system originates from both the neural tube and the neural crest. Mesoderm cells proliferate and develops into three separate components: the paraxial, intermediate, and lateral plate of mesoderm. The paraxial mesoderm cells form whorl-like aggregations known as somitomeres on both sides of the neural plate. Paraxial mesoderm organises into separate blocks known as somites caudal to the seventh somitomere. Somatic cells make up the vast majority of the axial skeleton, accompanying muscle, and covering dermis. The embryonic gut tube, developing respiratory system, and accompanying glands all develop from endoderm, the inner germ layer. Endoderm is also responsible for the epithelial linings of the urinary bladder, middle ear, and auditory tube. Endoderm also gives rise to the parenchymal cells of the thyroid, parathyroid glands, pancreas, and liver.

Introduction
The nervous system is an intricate network that plays a crucial role in coordinating and regulating bodily functions, allowing organisms to interact with their environment. This complex system is fundamental for the existence, enabling to sense the world, process information, and respond to stimuli. Comprised of billions of specialized cells called neurons. The nervous system is responsible for various functions, from reflex actions to complex thought processes. The nervous system is a highly organized and complex network that serves as the primary means of communication within the body of all animals, including domesticated species. Understanding the anatomy and function of the nervous system is critical in veterinary medicine, as it governs essential processes such as locomotion, sensation, cognition, and homeostasis. Comprising the central nervous system (CNS) and peripheral nervous system (PNS), the nervous system enables the coordination of reflexes and voluntary movements, facilitating interactions with the environment. Structural Organization: the CNS is composed of the brain and spinal cord, which are protected by the bony encasement of the skull and vertebral column, respectively. The brain varies significantly across species, reflecting adaptations to specific ecological niches and behaviors. For instance, the canine brain shows notable development in areas associated with olfaction, whereas the equine brain has adaptations for vision and balance, crucial for flight responses. The spinal cord serves as a conduit for neural signals between the brain and the peripheral nerves. It is organized segmentally, with spinal nerves emerging from specific regions. This segmentation corresponds to the vertebral column and is vital for diagnosing spinal cord injuries and diseases in animals. Each spinal nerve innervates specific muscle groups and areas of the skin, forming a somatic map essential for clinical examination and treatment. Neurons and Neuroglia: Neurons are the functional units of the nervous system, specialized for the transmission of electrochemical signals. In veterinary anatomy, it is essential to understand the structure of neurons, which includes the cell body, dendrites, and axon. Neurons can be classified into various types—sensory (afferent), motor (efferent), and interneurons—each playing distinct roles in reflex arcs and neural circuits. Neuroglia, or glial cells, provide support, nourishment, and protection for neurons. In veterinary practice, the role of glial cells in pathology is increasingly recognized, particularly in conditions such as neuroinflammation and neurodegeneration. For example, astrocytes play a crucial