LIVE AND DEAD COUNT OF SPERM

INTRODUCTION :The assessment of sperm vitality is one of the basic elements of semen analysis. Vital stains can help to differentiate live from dead sperm. Results of vitality test are linked to the integrity of the plasma membrane of viable sperm; an intact plasma membrane is able to exclude certain stains either for bright-field microscopy or fluorescence microscopy. Sperm vitality evaluated with the combined use of fluorescent dyes correlates with that determined by eosin-nigrosin stains (bright-field microscopy) but opens a new range of possibilities for automatic counting using flow citometry or fluorescence microscopy and image analysis data collection.

Visual Examination of Semen Sample

The gross appearance of freshly collected semen is usually the first measure of semen quality. Neat (unaltered) semen appears as a thick whitish to slightly yellowish fluid. The thickness of the semen sample is a reflection of the number of sperm present. There should be no odor associated with the semen sample. Potential odors are suggestive of an infection or the presence of urine, which could be detrimental to fertility of the sample. Other problems considered to be detrimental can also be detected in the color of the semen such as blood, urine, and feces, which cause the semen to be pink to brownish. White clumps or flakes indicate pus and the presence of an infection in the reproductive tract of the male. Sometimes debris from the semen collection site might also be found in the semen sample such as sand, dirt, straw or other bedding material. Ejaculates that are abnormal in color or appearance should be discarded at this point in the processing.

Viability Components 1. Motility

Motility of semen is one of the simplest viability characteristics to evaluate. Semen is diluted with an isotonic buffer and then examined either under a bright field microscope or a phase contrast microscope using a heated stage (35 - 39C). Slides and coverslips are prewarmed on a slide warmer prior to adding warm semen (35 - 39C). Several fields of view are examined and the percentage of motile cells estimated to the nearest 5 or 10%. Although it is important to look for progressively motile sperm (sperm moving in a nearly

straight line) it may be just as relevant in evaluating viability to determine if sperm are just motile at all (total motility, sperm being able to propel themselves with a beating tail). For the first time evaluators, the process of estimating percentages of motile sperm often appears difficult and inaccurate. Critical in this is the standardization of the procedures for diluting the semen and making the slides. During the observations on the microscope it is also important to use the fine focus and continually focus on sperm in different planes. Semen samples kept for processing should have greater than 60% motile sperm.

Gross Motility- gross swirling pattern of undiluted semen looks like a large school of fish.

Individual Motility
An estimate of individual sperm movement. Sperm should move progressively from one point to another in a relatively straight line. Dilute raw semen with physiological saline (0.9% NaCl). Count ten sperm cells in ten different areas of the slide. The number of sperm that are progressively moving will be the estimate of percent motile.

2. Morphology
The structure, or morphology, of sperm cells has been studied extensively using light and electron microscopy techniques. The sperm is a highly structured cell that is stream-lined to deliver DNA to the oocyte. To achieve this goal, the DNA is highly condensed and packaged on the nuclear matrix in a unique and specific species manner. Alterations in the packaging or DNA content of sperm results in changes in the morphology of the sperm head. The tail of the sperm contains the locomotion apparatus and without its proper function can not deliver the DNA in the sperm head to the oocyte.

3. Acrosome integrity
The acrosome is a secretory vesicle that is a sac-like structure below the plasma membrane and covering the anterior nucleus of the sperm head. The integrity of the acrosome is very closely associated with sperm viability because damage to the plasma membrane can trigger a disintegration of the acrosome that can be observed on a phase contrast or differential interference contrast (DIC) microscope. While unfixed sperm can be examined to determine the percentage of sperm with intact acrosomes, generally sperm are fixed with glutaraldhyde or other fixatives before examination. Semen samples with less than 70% sperm with intact

acrosomes should be discarded before processing.

Figure 1. Morphology of bull spermatozoa primary abnormalities of the head.

4. Live-Dead
In addition to the viability measures such as motility and acrosome integrity it is also possible to determine the percentage of live sperm using a differential vital stain. Early staining procedures used histological stains such as eosin that would only penetrate sperm with a compromised plasma membrane (Figure 3). Sperm were then dried on a slide and counted as viable (unstained) or dead (stained). Other procedures use a combination of fluorescent dyes such as sybr-14 which stains viable sperm green and propidium iodide which stains dead sperm red (Figure 3: Please view this figure on the web page to see the colors). Semen samples should have more than 70% viable sperm by a vital stain assay prior to processing.

Use a live-dead smear to arrest motility & assess the percent of live/dead sperm. Based on the premise that as sperm die, their membranes degenerate.

Percent live/dead sperm - identifies the percentage of sperm that have intact membranes, which is indicative of the number of live sperm in a semen sample. An eosin/nigrosin stain is commonly used for this purpose.

Figure 3. Illustration of sperm which have been treated with the dual-staining technique. A) Live sperm with intact acrosome. B) Live sperm without acrosome (true acrosome reacted). C) Dead sperm with intact acrosome. D) Dead sperm without acrosome (false acrosome reacted). Picture below using fluorescent dyes to mark viable (green) and dead sperm (dead).

5. Concentration
The accurately determine the number of sperm per unit volume is a central job of freezing semen. Concentration is usually determined using specialized equipment containing a spectrophotometer. The concentration of sperm in the neat semen is

needed to determine how to dilute semen and provide adequate sperm in each breeding dose. The number of sperm ejaculated varies greatly among males but is dependent on age, size of the testes, and the efficiency of spermatogenesis/gram of tissue. The sperm production on any day is also dependent on the collection frequency and the intensity of the sexual stimulation employed. While young bulls produce from 2 to 5 ml of semen, older mature bulls might produce 6 to 15 ml. The concentration of sperm in neat semen is 500 - 2,000 million sperm/ml. Semen that is below 500 million sperm/ml will not yield large numbers of insemination doses and might be considered a poor choice to continue on in processing.

REFERENCE PURPOSES,
1) Dr. John Parishs web site University of Wisconsin (http://www.wisc.edu/ansci_repro/), Department of Animal Science for his Animal Sciences Reproductive Physiology class (2003). 2) The Artificial Insemination and Embryo Transfer of Dairy and Beef Cattle, H.A. Herman, J.R. Mitchell and G.A. Doak, Interstate Publishers, Inc., (1994)