Biochemical analysis

Biochemical analysis of semen may give indirect information on the quality of the sample. The total number of spermatozoa present within a sample is reflected in the concentration of enzymes such as aspartate amino transferase, glutamic oxaloacetic transaminase (GOT), lactate dehydrogenase (LDH), hyaluronidase, acid and alkaline phosphatase and acrosin (Castro et al., 1991). Adenosine triphosphate (ATP) may also be used as an indicator. Many of the enzymes such as GOT, LDH, acrosin and...

General Semen Handling

In all procedures involving the handling of live semen samples it is imperative that a constant temperature of 37-38 C is maintained throughout. Variation from this temperature will result in cold or heat shock, both of which will significantly affect the viability of the sample and produce erroneous results. The effects of cold and heat shock are discussed in more detail in Chapter 7 (section 7.4.1). When the sample has been brought to the laboratory (ideally immediately post collection or...

Bacteriology

Nearly all semen samples will contain bacteria. These may be non-pathogenic (Burns et al., 1975 Kenney, 1975 Kenney et al., 1975) or may have the potential to cause infection within mares (Bain, 1966 Hughes et al., 1967 Merkt et al., 1975a). The presence of bacteria within a semen sample is not normally evident by gross examination unless the condition has been allowed to develop to such a severe extent that pus is present. Bacteriospermia is the term given to the presence of a high number of...

Appearance

Once the filtered sample is in the measuring cylinder, its colour and consistency can be assessed. This is normally done by eye but requires microscopic examination for confirmation. A good sample should appear milky white in colour, though it may range from watery to creamy, depending upon the spermatozoan concentration within the sample (Kuklin, 1983). The sample should also have a neutral smell and be evenly turbid with no evidence of clots, and should be the consistency of thin single cream...

Flow cytometry

Flow cytometry tests have been mentioned previously in connection with biochemical analysis. Recent work has investigated the use of flow cytometry, with both single and double staining, to assess the functional capabilities of spermatozoan mitochondria (Wilhelm et al., 1996 Magistrini et al., 1997 Papaioannou et al., 1997). The results are encouraging and suggest a significant positive correlation between spermatozoa with optimally functioning mitochondria and viable spermatozoa (Papaioannou...

Microscopic Evaluation

Cellsoft Sperm

Once gross evaluation of the sample has been completed, microscopic examination is required to enable spermatozoan characteristics to be assessed and evaluated. During microscopic evaluation, as with gross evaluation, it is of paramount importance that the sample is maintained at 38 C, especially as microscopic examination may take some time. Prior to microscopic evaluation the sample is invariably extended, though an indication of motility is often assessed microscopically using a raw sample....

Longevity

Assessment of motility over a period of time may be carried out to give an indication of the length of time over which acceptable levels of motility are maintained. The test, using either raw or extended semen, is normally conducted at either 37-38 C, 22 C or 5 C. As the temperature of the test and the dilution rates are known to affect longevity Samper, 1995b , it is important that they are standardized. Semen extender dilution rates between 1 1 and 1 4 are recommended. For a standard...

Cervical mucus penetration test

The ability of spermatozoa to penetrate cervical mucus, the first major biological fluid that the spermatozoa naturally come in contact with, has been used to indicate the viability of spermatozoa from several species. A highly significant, positive correlation between penetration and acrosome integrity and also between penetration and total spermatozoan integrity has been reported Galli et al., 1991 . However, no correlation between penetration and fertility was proved, and the test added...

Filtration assay

As discussed in Chapter 4, a filter of Sephadex beads over glass wool has been quite successfully used in various animals, including the stallion, to give an assessment of spermatozoan viability. The passing of frozen thawed semen through such a filter causes the less viable spermatozoa to be held within the filter, resulting in a filtrate of highly viable spermatozoa. A positive correlation exists, therefore, between the number of spermatozoa in the filtrate and the fertilizing potential of...

Morphology

Spermatozoan morphology is most commonly assessed by microscopic examination either of an unstained semen sample fixed in buffered formal saline, or of a stained semen sample. The most common stain used is an eosin-nigrosin stain Dott and Foster, 1972 , the components of which are given in Table 6.4. Table 6.4. Components of eosin-nigrosin stain used to assess spermatozoan morphology Dott and Foster, 1972 . The use of a stain allows the spermatozoa to be highlighted, allowing easier...