Preface Ample literature covering various aspects of veterinary andrology and frozen semen technology is available but need for a book incorporating practical aspects of the subject was always felt for the students of andrology and semen biology and scientists working in the area. This book is aimed to fill this void in literature by providing insight into various applied aspects of veterinary andrology, frozen semen technology and artificial insemination with the help of relevant illustrations based on author’s experience and research in the subject. Theoretical aspects of the subject have been deliberately omitted as ample literature on the topic has already been published. This book has been written to supplement the requirements of the scientists and Managers working in frozen semen production station, Semen Quality Control Laboratories, Andrological diagnosis Laboratories and students of Andrology and Artificial Insemination. It incorporates topics mentioned in syllabus for Practical course of Andrology and Artificial Insemination of undergraduate students of Veterinary Science. This will also be helpful to the graduate students of Animal Reproduction or Veterinary Andrology as a teaching aid.

Knowledge of male reproductive system and its physiological functions is essential for understanding male reproductive behavior and for effective reproductive management of breeding bulls. Therefore, personnel involved in management of breeding bulls, semen collection, handling and artificial insemination must have sound knowledge of male reproductive anatomy and physiology of breeding bulls. This section provides a recapitulation of the basic physiology and anatomy of male genitalia.   Functions of Male Genital System     (a)    Primary function of the male genital system is to monitor reproduction by providing cutaneous and glandular system of male gonads     (b)    Secondary function of male genital organs is to provide glandular, ductular, copulatory and auxillary system associated with male reproduction.

Success of AI programme depends on fertility of bulls used for semen production. If infertile bulls are selected in semen station for production of semen doses, large population of cows inseminated with such doses is affected. Therefore, the breeding bulls or bulls to be used for frozen semen production should be selected carefully keeping in view genetic potential, disease condition, reproductive health, libido and semen production potential of the bull. The emphasis should not only be on elimination of bulls with questionable or unsatisfactory potential, but also on induction of bulls with good semen production potential into the semen station.  The following aspects should be taken into consideration while performing a breeding soundness examination of bulls:

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 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.

For the diagnosis of clinical reproductive problems it is essential to collect blood, penile swab, Prepucial swab, prepucial washing, semen, smegma etc from breeding bulls. These samples are dispatched to the nearest laboratory for clinical diagnosis.  5.1 Collection of Blood Serum The bull is properly restrained and jugular vein area is cleaned and sterilized with 70% alcohol. An sterile hypodermic needle is used to collect 5-10 ml blood aseptically in a sterile test tube. This test tube is then kept in a slanting position. Serum is separated after the formation of blood clot and dispatched to the laboratory. 5.2 Collection of Seminal Plasma Semen sample is collected as per the standard procedure. Carbolic acid or 1 % sodium acid is added to preserve seminal plasma at the rate of 2 drops in 1 ml ejaculate in the centrifuge tube. It is then centrifuged at 3000 rpm for 20 minutes. The supernatant is collected and stored in deep freeze till further analysis.

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 Tris citric acid egg yolk glycerol extender is the most frequently used extender in the cryopreservation of cattle and buffalo semen. Preparation of the extender plays a very important role in determining the quality of the processed semen. Therefore, care must be taken to ensure that the extender is prepared aseptically and it contains all the ingredients in appropriate concentration. Extender should always be prepared under a laminar air flow. It is also important to check that the pH and osmolarity of the semen extender for a particular species corresponds to the prescribed limits for that particular species. The Tris citric acid egg yolk glycerol contains the following ingredients:

Deep freezing or cryopreservation of semen refers to freezing and storage of semen at –79ºC using dry ice and alcohol or at -196ºC using LN2 as refrigerant. Cryopreservation of semen was first attempted by Parkes in 1945. But cryopreservation of semen received a boost by discovery of glycerol a cryoprotectant by Polge in 1949.  Bull semen was first successfully frozen by Stewart in 1951. Cooling rate is a vital factor determining the post thaw survival of the sperm cells. Cell death occurs in rapid freezing due to formation of intracellular and possibly extracellular ice. In contrast slow cooling rates cause cell death due to diffusion of intracellular fluid into the extender, which then become increasingly hypertonic as the extender’s water freezes causing intracellular osmotic damage as the cell dehydrates. Hence, the cooling rate needs to be balanced in a way that it is neither too fast to cause cell death due to cold shock nor too slow to result the same due to osmotic shock. Ultra rapid freezing gives excellent morphological preservation but with poor retention of functions on thawing due to large number of minute crystal formation.

Early research in field of semen storage and delivery systems was conducted in USA. The straw technique for the storage of frozen semen was evolved by work of French, Danish and German workers whereas the pellet system of storage of semen was devised by Japanese scientists. The different packing systems of frozen semen are:     9.1    Ampoule method     9.2    Pellet method     9.3    Straw method 9.1 Ampoule Method In this method the semen is placed in a glass ampoule and is subsequently sealed and frozen in liquid nitrogen. The ampoule is thawed just before insemination and is then cut open. The semen is withdrawn into glass catheter and used for AI. Since the ampoule is sealed there is no risk of contamination during storage. The relevant details can be marked on the ampoule.

While collecting and processing semen the following points needs to be considered: 1.    Good quality Artificial Vagina (AV) should be used for semen collection. Strict hygiene should be maintained while collecting semen. 2.    Highly scientific and flawless collection technique should be used for semen collection.    3.    Neat semen should be maintained at close to body temperature. 4.    Semen should not come in contact with any contaminant such as lubricant, dust, infection etc. in the collection yard or in any stage of semen processing. 5.    Direct exposure of semen to sunlight or cool white fluorescent light should be avoided as it may cause chromosomal damage. 6.    Enclose all cool white light sources e.g. laminar flow hood, cold room etc. in gold or orange sleeves that absorb ultraviolet and low wavelength visible light (< 475 to 500 nm).

Gross evaluation is generally done to get a rough idea of the quality of semen and to assess the semen producing capacity of the bull. This also gives a fairly good idea about the reproductive health and any pathological conditions of the semen donor. The following are recorded under gross evaluation of semen:     11.1    Color and appearance of semen     11.2     Semen Volume     11.3    pH of semen     11.4     Semen consistency/ Semen density     11.5     Presence of foreign matter     11.6     Gross motility

The following tests are used for microscopic evaluation of semen:       12.1    Sperm motility 12.2     Per cent Live sperm 12.3     Sperm concentration 12.1 Sperm Motility The following two methods of estimation of sperm motility are followed in routine practice:   12.1.1    Mass activity 12.1.2    Progressive sperm motility 12.1.1 Mass Activity Mass activity can be defined as ‘en-mass activity of the spermatozoa in the semen, immediately after collection’. Mass activity is generally assessed by vigour of swirls and waves motion in undiluted semen under 20 X magnification and expressed on 0 to 5 scale.

13.1 The Normal Sperm Morphology A mature spermatozoa is an elongated cell consisting of a flattened head containing the nucleus, a neck and a tail containing the apparatus necessary for cell motility. The sperm cell consists of the following parts covered by the plasmalemma or plasma membrane:       13.1.1    Head      13.1.2    Neck      13.1.3    Tail / flagellum – The sperm tail consist of           a.    Mid piece      b.     Main piece / Principal piece      c.     End piece Sperm Plasma Membrane The sperm plasma membrane is three layered membrane. It lies in close contact with the underlying cytoplasm and attached at three places i.e. over anterior outline of the sperm head, around the base of the head, in circle around the distal end of the midpiece (Jensen’s ring). This attachment causes creation of three compartments with probably no communication with each other. The sperm plasma membrane is invisible under normal light or phase contrast microscopy but can be visualized after addition of substances with light refractory index e.g. bovine plasma albumin or synthetic polysaccharides. It is very easily permeable to water but there is low permeability for inorganic cations (Na, K, Ca etc.) and even lower permeability for anions (NO3, Cl). Exposure of sperm cells to hypertonic media will cause the water to go from sperm to the surrounding media leading to shrinkage of sperm cell whereas exposure of sperm cells to hypotonic media will induce influx of water causing swelling of the sperm cell. The more water that goes into the tail the more it changes its shape and becomes rounded to give room for increasing amount of water. As the length of the tail decreases as it becomes round, the tail fibrils have less space and tail bends and coils within the membrane vesicle. Tail fibrils are not damaged initially and will continue to contract till the critical volume of bull sperm is 3.8-3.9 times of its volume in an isotonic solution. The swelling and change in shape of the spermatozoa are momentary processes and can be corrected if hypotonicity of the media is corrected before the sperm cells reach the critical volume for rupture. Sperm membrane is responsible for maintenance of the intracellular biochemical environment. Damaged plasma membrane is responsible for the leakage of electrolytes and organic substances such as enzymes and may also lead to damage of sperm cells by foreign substances from the surrounding media.

Only few single sperm viability parameters show a significant correlation with fertility of semen samples. Therefore, need to explore functional in vitro fertility tests having capability to disclose the ability of spermatozoa to undergo complicated processes such as capacitation, binding of zona pellucida, acrosome reaction, fertilization of Oocyte and induction of embryo development invitro have been designed and explored for their correlation with fertility achieved after artificial insemination. In vitro fertility tests used in evaluation of semen includes:  14.1 Estimation of membrane integrity              14.1.1 Morphological integrity              14.1.2 Functional integrity              14.1.2.1 Hypoosmotic swelling test              14.1.2.2 Acrosome integrity 14.2     Cervical Mucus Penetration Test

Semen metabolic activity can be assessed with the help of the following tests:      15.1    Incubation test      15.2    Methylene Blue Reduction Test      15.3    Resazurine Reduction Test      15.4    Millovonoo’s Resistance Test (‘r’ test)      15.5    Fructolysis Index      15.6    Oxygen utilization test      15.7     Glutamic oxaloacetic transaminase activity 15.1 Incubation Test The longevity and viability of spermatozoa during storage is a good index of fertilizing ability of sperm. In this test assessment of motility is carried out over a period time to obtain an idea about the length of time over which acceptable level of motility is maintained in the semen sample. Therefore, this test serves as a mean of determining storage probability within a fairly short period of time.

The following tests are discussed under this section:     16.1    Estimation of ascorbic acid concentration     16.2    Effect of cold shock     16.3    Mitochondrial activity     16.4    Capacitation of spermatozoa and calcium influx      16.5    Morphometric analysis     16.6    Flow cytometry 16.1 Estimation of Ascorbic Acid Concentration Ascorbic acid concentration in semen is beneficial for its antioxidant action. It protects the spermatozoa against oxidative damage of free radical produced in the semen. Materials Required     1.    Test tubes with stand     2.    Burrette with stand     3.    Centrifuge tube with stand

The bacterial load in the semen samples is estimated by standard plate count method. The bacterial contaminants of semen have been a major concern for most of the frozen semen production laboratories as it adversely affects semen quality and hence subsequent fertility. There exists a significant negative correlation of bacterial load and sperm livability, motility and in vitro fertility tests. Macrophages and polymorphonuclear granulocytes, which form the first line of defense against these microorganisms, produce reactive oxygen species (ROS) to kill these microorganisms. ROS, however, is also released outside these cells and may react with molecules and cells such as spermatozoa in their vicinity. The cellular antioxidants, present mostly in the cytoplasm, are scanty and inadequate to counteract this ROS as the sperm cell cytoplasm is very small (20 µm3) and mostly distributed in the midpiece. Therefore, semen samples with minimum bacterial load are expect to give better results on insemination.

18.1 Estrus Detection 18.1.1 Estrus Detection on the Basis of External Signs The following signs are suggestive estrous period in cattle and buffaloes 1.    Partial inappetance due to excitement 2.    Drop in milk yield 3.    Restless ness of varying degree 4.    Frequent micturition with raised tail and crouching the back and lumbar region 5.    Marked bellowing 6.    Swollen vulvar lips- Due to swelling the wrinkles on vulvar lips disappear. The vulvar lips appear turgid and stand prominently. 7.    Congestion of vulvar lips  8.    A clear, shiny mucus discharge seen at the vulvar lips extending down to perineal region and smearing the hind quarters. The discharge is stingy, having good consistency and no odour.

The process of converting the frozen semen to liquid state while retaining maximum semen quality is called thawing. The process of thawing is as important as freezing as there is equal probability of loss of semen quality in freezing and thawing. Therefore, great care should be taken while thawing to insure that it is done with minimum loss of sperm quality. There are two methods of thawing: 1.    Rapid thawing 2.    Slow thawing The effectiveness of each method of thawing depends upon the extender composition and freezing procedure. With any thawing procedure, the recommendations of the semen supplier should be followed. The thumb rule is that rapid freezing rates require rapid thawing.

Artificial insemination (AI) is the technique of placing sperm into a female’s uterus (intrauterine), or cervix (intracervical) using artificial means rather than by natural copulation. Modern techniques for artificial insemination were first developed for the dairy cattle industry to allow many cows to be impregnated with the sperm of a bull with traits for improved milk production. It is the first generation reproductive biotechnology which has revolutionized the animal productivity. It has now been established as cheapest and fasted mode of genetic improvement of livestock. AI can be done using fresh semen, semen preserved at room temperature, refrigerated (4ºC) and cryopreserved (-196ºC) semen. The use of AI with frozen semen has become very popular and is being successfully used throughout the country.

21.1 Liquid Nitrogen Container/ Cryocan To store liquid nitrogen a special type of container is used and is known as a LIQUID NITROGEN CONTAI NER OR CRYOCAN. Frozen semen doses are always preserved in liquid nitrogen which is an inert liquid having a boiling point of -1960C and a freezing point of -212°C. Liquid Nitrogen Containers are doubled walled metal (Stainless Steel/Aluminium) vacuum vessels with a very efficient insulation system. Due to highly efficient insulation system the temperature inside the container can remain -196 oC as long as atleast two inches of Liquid Nitrogen is present in the container. It has got an inner chamber which is suspended in outer chamber through neck tube which is a non metal and bad conductor of heat. Walls of the inner chamber are coated with a high quality of insulation material. The space between the inner and outer chamber is also filled with high quality insulation material. Vacuum is created in between inner and outer chamber. Loss of vacuum will lead to the boiling of the liquid nitrogen gas and hence rapid loss of this gas from the inner chamber.

The equipments used in artificial insemination should be clean and sterilized. The use of unsterilized equipments in AI causes the spread of infection in the herd and infertility. Rinse the equipments with tap water immediately after use and then lukewarm detergent solution. The detergent used for washing the AI equipments should be non corrosive. Use of corrosive detergents will reduce half-life of the plastic and rubber wares. Wash thoroughly with tap water till the equipments become grease free and perfectly clean. Then rinse in distilled water at least thrice. Use of distilled water does not leave any salt residues on articles after drying.

Systematic recording is vital for smooth running of any artificial insemination enterprise. All the information in the center should be so arranged so that it can be readily be traced out and used for analysis of performance of the center. The records should be critically analyzed for selecting better animals / personnel and for improving reproductive management. The objective of record keeping is as follows.

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