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There is no rapid direct test to determine fertility of semen sample. During freezing and thawing of semen, 20-80% of the spermatozoa become immotile or dies. In addition to this, there may be damage to spermatozoa by disintegration of sperm cell membrane and acrosome leading to loss of intracellular enzymes. The only reliable method to evaluate the semen is to inseminate a cow and get a calf at the end of gestation period. Now-a-days new methods have been developed for in-vitro assessment of fertility of spermatozoa. Some of these methods are exhaustive, time consuming, need for a full-fledged analysis and difficult under field conditions.

Liquid nitrogen and cryocan

Several cryogenic agents like solid carbon-di-oxide (-79^ºC), liquid air (-183^ºC), liquid helium  (-263^ºC) and liquid nitrogen (-196^ºC) have been used in many countries for freezing and preservation of bovine semen. Liquid nitrogen is fourth coldest substance known and its small amount is converted into a very large amount (near about 700 times) of its volume when exposed to atmosphere. It is a colourless, odorless, non-irritant, non-toxic, non-caustic, non-corrosive, non-inflammatory and inert gas having boiling point of -196^ºC. Being a liquid it comes into good contact with the surface of the semen straws, constant storage temperature is maintained throughout the straw, and due to these properties LN2 is most widely used refrigerant for semen cryopreservation in the world. 

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 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.

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 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.

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.

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. 

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).

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:

Advantages and limitations of artificial insemination

Artificial insemination is the first great biotechnology applied to improve reproduction and genetics of domestic animals. Artificial insemination includes the collection of semen from the male, semen assessment for quantity and quality, dilution with extender, preservation and then insemination. Artificial insemination (A.I.) is a breeding technique where semen with living sperm is deposited into the reproductive tract of a female who is in estrus by use of instruments. Artificial insemination is the most important single technique widely used worldwide to upgrade the genetic makeup of the farm animals. Artificial insemination can be performed in most of the domestic animals like cow, buffalo, mare, sow, bitch, queen cat, lambs, does etc. Due to invention of frozen semen technology, the extended semen doses can be preserved for years together and permit wide spread use of superior germplasm. Artificial insemination is a powerful tool mostly employed for livestock improvement. The advantages and limitations of frozen semen technology are enlisted below:

Miscellaneous causes of male infertility 

1. Failure of proper delivery of semen into the vagina

It may be secondary to lacerations of the ventral caudal portions of the glans penis with a fistulous opening of the urethra ventrally. In such case, at the time of ejaculation, neither semen is deposited in the middle portion of the vagina nor sprayed over the cervix. In addition, it is observed in rams following necrosis of most of the urethral process secondary to urinary calculi or lacerations or injury.

Reduced capacity or incapacity to fertilize by males/Impotentia generandi: Fertility is the normal functioning of the testes, accessory sex glands and ducts to deliver sperm of normal quantity and quality. Infertility or sterility in males is usually characterized by normal sexual desire and the ability to copulate and ejaculate but a complete or abnormally high percentage of failure of fertilization or conception. It may be seen when semen is normal on examination and on the other hand, the semen is abnormal in sperm morphology, concentration, motility and other qualities.

Infertility or reduced fertility in male animals particularly in bulls had considered being a rare occurrence, but now infertility in males is being considered a common and severe problem. Still there is lack of information concerning the male’s infertility.

Sexual behavior

The pattern of courtship, display, motor activities and postures are directed to bring the male and female gametes together to ensure fertilization and pregnancy. The components of copulatory patterns are sexual arousal, courtship (sexual display), erection, penile protrusion, mounting, intromission, ejaculation, dismounting and refractoriness. The duration of courtship and copulation varies with the species both events are shorter in cattle and sheep than in swine and horses. The males sniff and lick the genitalia of female are more common and an important way of chemical communication through olfaction. Except in swine, the males smell the female’s urine and then raise head, with lips curled, in the ritualized “Flehmen’s reaction”. In sheep, goat and cattle, tactile stimulation of the female is made by nuzzling and licking the perineal region, whereas with the horse, the stallion often bites the female’s neck and in swine, the boar noses her flanks.

Puberty and sexual maturity

Puberty is defined as the age at which reproductive function is initiated in animals. This corresponds to changes in hormone levels and the ability to function sexually in order to reproduce. The puberty is the period when the sexual organs are functionally developed, libido is prominent and the male is able to produce the spermatozoa and is able to copulate resulting in pregnancy. The onset of puberty is a gradual process and occurs after achieving optimum body weight, which is influenced by plane of nutrition, genetic differences, management practices, breed, diseases and individual differences.

Andrology is a branch of reproductive physiology that deals specifically with the study and treatment of male animals including humans. The study of reproductive physiology started with Aristotle around 350 B.C. in his book entitled Generation of animals, who believed that, the fetus arose from menstrual blood. He had no way of observing spermatozoa in the ejaculate or the beginning of embryo development. He assumed that, menstruation did not occur during pregnancy and the fetus was derived from menstrual blood. He also proposed that, the conversion of menstrual blood to a fetus was initiated by seminal fluid deposited in the male during copulation. Aristotle also assumed that, the testes were just like pendulum weights that kept the transport ducts from becoming kinked or plugged with seminal fluid. He has no research tools and his speculations were not very unreasonable. Later, a Dutch scientist named van Leeuwenhoek who developed a simple microscope made a major breakthrough in the study of reproductive physiology. Leeuwenhoek observed semen and discovered that it contained small particles that moved about and he named moving particles as ‘Animalcules’ and published a paper in 1677. The speculation was that the animalcule contained a fully formed individual within their cellular confines. Sperm head was thought to contain a microscopic yet fully formed individual. Later, Dumas concluded that, the animalcules now called spermatozoa were responsible for uniting with the ovum and producing an embryo. Over 2000 years elapsed from the speculations of Aristotle until it was understood that the spermatozoa from the male required for fertilizing ova from the female.

1    Suggested fodder species for different regions

2    Package of practices for cultivated fodder crops

3    Estimation of agricultural by-products

1    Consent form for surgery

2    Consent form for euthanesia

3    Slaughter certificate

4    Wound certificate

5    Proforma for export certificate of dogs

Laboratory investigation of the samples assist in diagnosing, confirming the cases including veterolegal cases. The type of material, quantity of specimens & preservatives used and the method of dispatch to the laboratory  is tabulated in this chapter.