Routine examination of seminal fluid is often advised while investigating cases of sterility. The cause of male infertility may be either in the quality of spermatozoa or in quantity.
Collection of Semen:
Collect semen in a clean and dry condom/in a wide mouth test tube after abstinence for 4 to 5 days. The time of emission should be recorded.
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Physical Properties:
Volume:
Measure volume in a small graduated cylinder. The amount varies from few drops to 10 ml. Samples less than 1.5 ml are considered below normal.
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Viscosity:
Freshly ejaculated semen is highly viscous. Self-liquefaction takes place and should be completed in 15 to 30 min. The absence of liquefaction may inhibit the movements of spermatozoa.
Colour and Odour:
The normal semen is whitish with the typical seminal smell; suppurative infection imparts a foul smell and yellow colour.
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Reaction:
It is alkaline with pH 7.2 to 8.0.
Motility of Spermatozon:
Sperms are motile with flagella. To observe the motility, place a drop of semen on a slide and put a cover-slip over it, examine with low and high objective. A hanging drop preparation with a ring by Vaseline can also be made.
Results:
1. Complete absence of spermatozoa (Azoospermia). It should be confirmed after centrifugation of the specimen.
2. Only a few motile spermatozoa are present (Oligozo- ospermia).
3. Spermatozoa are present, but immobile (Necrozoospermia)
4. The proportion of motile to immobile sperms should be noted. Insert black paper with a small square cut in the middle in the eyepiece of microscope, and if motility is very low, repeat the examination. Count at least 500 spermatozoa and give average percentage. Immobile spermatozoa are counted after the motile ones.
Duration of motility should also be studied. For this, a thick smear, covered with coveraglass, is prepared and this preparation is sealed with Vaseline. It should be examined hourly. A normal specimen shows little cessation of motility at the third hour after emission and cellular activity is noted at fifth or sixth hour also.
Total Spermatozoa Count:
Counting of spermatozoa is made in the same manner as WBC is counted using the WBC hematocrit pipette except diluting fluid. The semen is drawn upto 0.5 marks and draws semen diluting fluid upto 11 mark.
The total count of spermatozoa and calculation of their numbers is done as in case of leucocytes. Count the numbers of spermatozoa in 2 sq mm and add five zeros across to obtain numbers of sperms per ml.
Example:
Number of cells counted in 2 sq. mm = 1149.
Number of spermatozoa/ml = 114900000/ml.
Normal semen contains on average of low to 150 million spermatozoa/mm3. The lower the counts below 60 million, the less likelihood of fertility.
Morphological Examination of Spermatozoa
Preparation of Smear and Staining:
A thin smear of semen on a glass slide is dried in air and fixed by heat. Add 1% chloramine for few minutes and remove excess of mucus. Wash by blotting on filter paper stain for 3 to 5 mints with:
Ziehl-Neelsen’s carbon fuschsin— 2 part
Cone, alcohol solution of eosin — 1 part
Alcohol 95% — 1 part
Wash with water and counter stain with Loeffler’s methylene blue for 30 to 40 sec. Wash, dry and examine under oil immersion lens.
The heads of spermatozoa are stained purple while tail and middle pieces take red or pink colour depending on duration of staining.
Count the number of spermatozoa in microscopic field. The same field is searched for immature forms of spermatozoa.
Examine 100 to 500 spermatozoa for following abnormalities:
Heads:
Too large or too small, pointed, ragged edges atypical distribution of chromatin, presence of acidophilic vacuoles, double hands.
Middle Pieces:
Absent, swollen, bifurcated, etc.
Tails:
Double, curled, rudimentary or absent.
Semen containing upto 20% abnormal spermatozoa are still considered fertile.
Finally examine for cell other than spermatozoa, i.e. pus cell, RBCs, epithelial cells, testicular cells and crystals. Abundant crystals may be found in the smear, especially when the semen is left standing for some time.