artificial insemination in cattle, two, evaluation of semen. Biological examination of semen is essential to the assessment of fertility. Normal ejaculates are white

to ivory in color and have a creamy consistency. Low magnification microscopy is followed by examination at higher magnification. First the wave motion of the semen is graded,

the highest grade being a strong swirling movement like schools of fish rapidly changing directions. The individual spermatozoa are not visible, however the movement is graded from no wave motion to very fast moving waves. This specimen shows an average wave motion.

This is the lowest grade of wave motion. Since the wave motion depends on the sperm density, further tests are needed to establish if such ejaculates are acceptable. This contaminated semen specimen has to be rejected for hygienic reasons

in spite of its good wave motion. The thin preparation under the cover slip is now tested for individual sperm motility. The microscope stage is heated to 38 degrees centigrade. Magnified about 200 times, the spermatozoa exhibit

individual motility. In a dense sample like this one about 90 percent of the sperm are estimated to be moving forward vigorously. The movement pattern is more clearly seen in a diluted preparation and the slow motion effect renders it even more obvious.

Semen samples showing about 70 percent forward movement of spermatozoa comply with the minimum requirements for fertile semen.

Only about half the spermatozoa show forward movement, the rest being non progressively motile or immotile. The fertility of this ejaculate would be considerably impaired. The microscopic analysis indicates a disorder in sperm production, maturation or storage within the male. The slow motion effect

emphasizes the problem. Diluted semen as well as undiluted semen is examined. The motility of spermatozoa in a freshly diluted sample is assessed and the same sample is examined after 72 hours at refrigerated storage. If 70 percent of the spermatozoa show forward

motility, this fulfills the minimum requirements. The dye absorption test with eosin establishes the proportion of live to dead spermatozoa. Dead sperm take up the dye thus becoming stained. The number of unstained corresponds roughly

with the percentage of motile spermatozoa. With the aid of a counting chamber the sperm concentration of a specimen can be accurately determined after it is diluted 200 fold. The spermatozoa contained in 10 large

squares are counted. Bull semen should have a concentration of at least 600,000 spermatozoa per microlitre. The type and number of morphologically

abnormal spermatozoa can be determined from the stained smears. The preferred method is that devised by Karas. The morphology of normal bull spermatozoa is particularly evident for the cells shown in top view.

The dark colored acrosome covers the anterior half of the head and shows a contoured apical ridge. The slender flagellum like tail is inserted centrally at the base of the head. This sperm has a giant head and a protoplasmic droplet at the neck region as well as a bent tail.

Oversized heads generally indicate a duplicate set of chromosomes. Abnormal head shapes such as one which tapers towards the base might possibly be indicative of abnormal chromatin content. Such anomalies occur in the testis during spermatogenesis. Secondary changes

such as detached acrosomes and tailless heads, top left and bottom right, occur during passage through the epididymis or perhaps during semen collection. The chief causes are inflammatory processes and cell aging.

This preparation contains 70 percent abnormal spermatozoa. In addition to capless heads with tapered bases, there are also tailless heads with persistent acrosomic granules. Abnormal variations of the tail such as coiled and retroaxial tails are also evident. In the center

two sperm with the remains of proximal protoplasmic droplets can be seen. Occurrence of droplets indicate a disorder of spermiogenesis or maturation. More recently, phase contrast microscopy has been used for evaluating sperm morphology after immobilization by wet

fixation. These intact spermatozoas show an acrosome with a smooth apical border. The upper spermatasone shows an acrosome in the process of detachment. To the left is a narrow head with the apical edge of the acrosome bent back

in a tongue-like fashion. Another acrosome becoming detached. To the left is a spermatasone showing vacuolization of the caudal head region known as the diadem defect. Acrosome defects like these cause malfunctions which can be visualized by means of a gelatin lysis

test. Spermatozoa placed on a gelatin membrane exhibit either a clear lysed area, reduced lysis or an absence of lytic activity depending upon the condition of the acrosome. Abnormal spermatozoa can also

be detected in fresh undiluted samples. The reason for the agglutination of sperm cells may be the presence of foreign bodies which in this case are epithelial cells. Traces of blood may be due to injury. Round erythrocytes and to the right of the center a leukocyte can be seen in this view. Large accumulations of leukocytes

are indicative of genital infections. Contamination such as feces leads to bacterial growth and thus to a deterioration in fertility.

An increased count of non-progressively motile or backward motile spermatozoa also results in decreased fertility. These spermatozoa shown in slow motion exhibit circular movements.

This spermatasone has an acrosome defect. In the center a spermatasone with the tail reflected backwards is moving in reverse direction and several detached tails exhibit active motility. The tail defect is now seen under higher magnification.

This detached active flagellum has an adherent plasma droplet. Again an intact spermatasone with a protoplasmic droplet attached. The following scenes show further malformations of the head region.

Spermatozoa having primary acrosome defects are infertile in spite of their motility. A motile spermatasone with a giant head. Now a twin deformity,

a double head form. Only semen samples resistant to stress are suitable for insemination. Freezing provides the test for resistance. Before freezing

the semen is cooled to render the spermatozoa immotile. The water crystallizes in a thin layer beneath the coverslip. At this stage the cells are exposed

to considerable osmotic stresses. These occur again during thawing. The minimum requirement for frozen semen is that fifty percent or more of the spermatozoa show forward movement after thawing. Further conclusions about sperm resistance

can be inferred following thawing by further incubation. The proportion of motile and morphologically intact spermatozoa surviving incubation is an indicator of the resistance to stress.

Only semen samples that conform to all requirements in the fresh undiluted state and after thawing can be qualified as fertile.