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Semen Evaluation, Concentration of Sperm Cells, Dilution of Semen

It is economically and biologically important that only semen with a high probability of successfully impregnating  cows be processed and distributed. Immediately after collection, each ejaculate should be examined routinely by the best methods available. It has generally been assumed that, following this examination, there should be as little delay as possible before semen is properly and completely processed. Recent evidence indicated, however, that it may be advantageous to hold semen at room temperature for a short while before processing.

Gross Examination :

Immediately following collection, a gross examination of the semen ejaculate in the collection tube should be made. This examination includes observing the color and opacity of the semen and checking carefully for the presence of foreign material such as dust or pus. The volume  should be recorded not only to serve as a record of the semen production of the bull, but to be used later for extension purposes. Although isolated reports of a positive relation between the volume of the ejaculate and its subsequent fertility have been published (Lasley and Bogart 1944), these results seem to be on spurious correlation.

Morphology of Semen Cells:

Procedures for examining the morphology of sperm cells are changing. British (Morris 1950) and Japanses (Hideo and Otsuki 1966) worker have emphasized that examining unstained specimens with the phase-contrast microscope is useful in eliminating the danger of artifacts. Saacke (1970), however, has pointed out that condition of the acrosome is best observe through a differential inference contrast microscope and that a phase-contrast microscope produce halos that interfere with the interpretation of acrosomal morphology.

Concentration of Sperm Cells:

The concentration of spermatozoa in an ejaculate can be determined by five general methods:

  1. A direct visual count, under the microscope, of  the number of cells found in a standard volume when the semen is diluted at a constant rate;
  2. Determination, in a nephelometer, of the light-absorbing capacity of semen diluted at a standard rate and comparison of its optical density with that of semen whose concentration has been established by a direct count, as above;
  3. Counting the cells in an electronic particle size analyzer (Glover and Phipps 1962);
  4.  Comparison of the visual density of semen diluted at a standard rate with the visual density of barium sulfate or other density standards calibrated against a direct count (Salisbury et al 1943);
  5. Determination of cell volume calibrated against a direct count (Shaffner and Andrews 1943)

Assessment of Spermatozoon Motility:

Current methods for assessing sperm motility are primary visual, and the  results are usually expressed in comparative rather then absolute terms. No means is readily available for characterizing the distribution of motility of individual sperm cells in a semen sample.

Observation of Swirls in Undiluted Semen:

Some investigators and AI Units depend entirely on graded estimates of the vigor or swirls and wave. Blom (1946) has developed a comparing chamber for this purpose, which can also be used for making a rough estimate of cell concentration and for observing motility of individual cells.

Dilution of Semen:

Most American investigators, including the authors, have depended on dilution of the sample in order that the individual cells can be readily seen at 200 to 400 x or higher magnification. Diluents of many kinds have been used, including isotonic citrate or phosphate buffers, glucose containing media, and physiological saline.

Hemocytometer Method:

A thoroughly objective method, based on use of the hemocytometer, was first described by Brady and Gildow (1939), who diluted semen 1:100 with physiological saline in one dilution pipette, and with alcohol to fill chambers of a hemocytometer; counts were made of the non-motile spermatozoa in each; and by difference the number of motile cells were calculated.

Differential Staining of Live and Dead Spermatozoa:

In 1942 Lasely, Easley, and McKenzie reported that live and dead ram spermatozoa could be differentiated by their reaction to certain stains; non-motile, apparently dead sperm being colored by the dye, and live, motile sperm not being stained. Their stain consisted of 2 parts of 2-percent solution of water-solution (a commercial Breslau solution obtained by then from the Dr. Grubler and Co; Germany); and 1 part of M/8 phosphate buffer. Although the phosphate buffer used has a pH of 7.4, the final mixture had a pH of about 6.7. a drop of the stain was placed on a clean glass slide, and a smaller drop of the semen was mixed with the stain. A smear was made by placing another clean slide on the first and drawing them apart without pressure. The smear was then dried at 40°C  and examined under the microscope. The percentage of dead spermatozoa was examined by counting 500 cells and recording the number of stained cells in the total.

Significance of Semen Quality

  1. The goal of all attempts to measure semen quality is to ensure that the subsequent fertility of the semen will be in the normal range  or, in other words, to make maximum use of proven bull while maintaining optimum fertility. The terms “semen quality” and “fertility” are sometimes confused. They are not synonymous, although many efforts have been made over the years, with some success, to make the two more nearly coincide.
  2. Within each AI unit, management must decide which of several criteria to use to supplement determinations of ejaculate volume and sperm cell concentration as well as their product (i.e.; the total cells produced) and the pro-portion of cells exhibiting vigorous progressive motility. These determination are required in any efficiently operated AI unit.

At present there is no single test that offers a high probability of predicting successfully the multiplicity of conditions that obtain in the many AI units throughout the world. Van Duijn (1965) proposes a mathematical theory of fertility, based on “Kinetic” considerations, which expresses the probability of fertilization in terms of number, mean velocity, and halflife of the spermatozoa, as well as in terms of the relative fecundity of the male and female populations.

Expressions deduced from this theory indicate remarkable agreement with major published results in the literature. However, general corroboration and acceptance of his work have been prevented by the sophisticated equipment and the substantial time and labor required.

Server items tend to reduce the relationship between a specific semen evaluation measure and fertility. For example, an optimum measure for a semen characteristic relating to fertility may be reached which is considerably less than the maximum for that measure. Our experience indicates an asymptotic approach to the maximum fertility level for the semen, consistent with the optimum fertility of the cow population, with increasing value of certain individual semen characteristic. Once an optimum semen evaluation value is reached, further increasing values of the characteristic do not result in corresponding increases in fertility. Obviously, if all semen selected for use in artificial insemination is at or above the optimum level for that characteristic, no measurable relationship between the characteristic and fertility would result.

  1. Cell Morphology: The pioneer work of Williams in 1920 marks the first systematic attempt to relate the fertility of bulls to the evaluation of semen. Williams and Savage (1925) found that when the percentage of abnormal sperm cells reached 18 percent or more, the fertility of the bulls was impaired. Lagerlof (1936) obtained similar results. Since publication of these classic works, a large number of papers have appeared on this subject. Bulls that are to be used in AI should produce semen that is proved not to contain excessive abnormal spermatozoa.
  2. Energy Source: The most obvious energy requirement of the sperm cells is for motility, but additional energy is presumably required for all cell maintenance. Spermatozoa are capable of both aerobic and anaerobic metabolism. A simple source of energy such as glucose or fructose should be supplied in the extender to protect intracellular reserves and cell components. Fructose, present in the seminal plasma, will be diluted considerably by semen extension. Egg yolk contains some glucose and other compounds utilizable by bull spermatozoa.
  3. Buffering and pH: Spermatozoa need protection from autotoxcation due to acid products of metabolism, particularly when they are stored without refrigeration. Bull sperm motility and fertility are well preserved in egg yolk and milk extenders near nautral pH, to 6.5 may even be beneficial (Salisbury and Mercier 1945). The optimum pH probably varies storage temperature and other component of the extender (Chang 1943). Saturation of bicarbonate media with CO2 causes metabolic inhibition and reduces the pH to about 6.3 (Salisbury et al 1943). This inhibition may be in part to a reduction in pH (Salisbury and Mercier 1945)

 Semen Evaluation, Concentration of Sperm Cells, Dilution of Semen

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