Semen Analysis: Sperm Counting Test Procedure and Results

Semen analysis provides essential information on the individual’s clinical status. Clearly, the collection and analysis of semen must be carried out using properly standardized procedures if the results are to provide valid information.Normal semen is a mixture of sperm suspended in testis secretions and epididymis which, at the time of ejaculation, are combined with prostate secretions, seminal vesicles and bulbourethral glands. The final composition is a viscous fluid that includes the ejaculate

Semen analysis is divided into macroscopic and microscopic sample examination

Macroscopic examination

This comprises of the following steps :

  • liquefaction
  • Appearance
  • Volume
  • Viscosity
  • pH

Microscopic examination

  • Assessment of sperm motility
  • Assessment of sperm concentration
  • Sperm vitality by dye exclusion (eosin test)
  • Assessment of sperm morphology
  • Assessment of cellular elements other than spermatozoa

Macroscopic examination of semen samples

Liquefaction

A normal semen sample liquefies at room temperature within 60 minutes, although this usually happens within 15 minutes. In some cases, complete liquefaction does not occur within 60 minutes and should be recorded.

During liquefaction, continuous gentle mixing or rotation of the sample container may reduce errors in the determination of sperm concentration.

Appearance

A normal liquefied semen sample has a homogenous, gray- opalescent appearance.If the sperm concentration is very low, it may appear less opaque; the color may also be different, i.e.Red- brown when red blood cells are present( hemospermia) or yellow in a jaundice patient, or when some vitamins or medicines are taken.

Volume

The volume is best measured by weighing the sample in a vessel in which it is collected

  • Collect the sample in a pre-weighted, disposable container
  • Weight the vessel with semen in it
  • Subtract the weight of the container
  • Calculate the volume from the sample weight, assuming the density of the semen to be 1g/ml

Or you can just read the volume directly from the graduate tube.

Viscosity

The viscosity of the liquefied sample can be estimated by gently aspiring it into a wide- bore( approximately 1.5 mm diameter) plastic pipette that allows the semen to drop by gravity and to observe the length of any thread.A normal sample leaves the pipette in small discreet drops.If viscosity is abnormal, the drop forms a thread longer than 2 cm.

Alternatively, the viscosity can be evaluated by inserting a glass rod into the sample and by observing the length of the thread that forms when the rod is removed. The thread shouldn’t exceed 2 cm.

pH

A drop of semen is spread evenly onto the pH paper( range: pH 6.l to l0.0 or 6.4 to 8.0), or the pH paper is dipped into the semen sample for a moment. After 30 seconds, the color of the impregnated zone should be uniform and compared to the pH calibration strip.

Microscopic examination of semen sample

Semen wet mount analysis

Before removing an aliquot of semen for evaluation, mix the sample well in the original container, but not so vigorously that air bubbles are created. This can be achieved by aspiring the sample 10 times into a wide( approximately 1.5 mm diameter) plastic pipette.

  1. Add 10 µl of semen onto a clean glass slide with an automatic pipette and covered with a 22 mm x 22mm coverslip.
  2. Allow the  freshly made wet preparation is allowed to stabilize. Evaluate the wet preparation as soon as the contents stop drifting.
  3. Initial evaluation at 100x total magnification( i.e. 10x objective and 10x ocular) provides an overview of the formation of the mucus strand, the agglutination and aggregation of the sperm and the evenness of the slide spread.
  4. Examine the preparation using the total magnification of 400x( i.e. 40x objective and 10x ocular).

Preliminary estimation of sperm concentration

Scanning the slide and estimating the sperm count per field is used to decide the dilution by haemocytometer to determine the sperm concentration:

Spermatozoa number per x400 fieldDilutionDilution factorµl of semenµl of fixative
< 21:2 (1 + 1)25050
2-15 1:2 (1 + 1)25050
16-1001:5 (1 + 4)550200
> 1011:20 (1 + 19)2050950
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If the number of sperm cells per visual field varies considerably, the sample is not homogeneous. In such cases, the semen sample should be thoroughly mixed again.

How to dilute semen with fixative

The semen sample is diluted with the fixative in two dilution tubes( eppendorf tubes) as follows

  1. Using an automatic pipette, first dispense the appropriate amount of fixative in both dilution tubes
  2. Mix well the semen sample
  3. aspirate the appropriate volume of semen immediately after mixing, allowing no time for the spermatozoa to settle out of suspension
  4. wipe the semen out the outside of the pipette tip
  5. Dispense the semen into a fixative in one of the dilution tubes and rinse the pipette tip by aspirating and expressing the fixative.
  6. Mix the semen sample well again and prepare the replicate dilution following the above steps

Evaluation of sperm concentration

The sperm concentration should be determined using the haemocytometer method in two separate preparations of the semen sample. The dilution is determined from the preliminary estimate of the sperm concentration as shown on the table above.

Preparation and use of haemocytometer for semen analysis

  1. Secure the cover on the counting chambers of the improved Neubauer haemocytometer by slightly wetting either side of the wells( use a drop of water on the finger). Press the cover firmly onto the chambers so that iridescence( Newton’s rings) is observed between the two glass surfaces.
  2. Transfer approximately 10 μl of the thoroughly mixed diluted specimen from each duplicate dilution to each counting chamber of the haemocytometer. The chambers should not be overfilled or underfilled, and the cover glass should not be moved.
  3. The hemocytometer is allowed to stand in a humid chamber for about 5 minutes to prevent drying out . The cells are then counted at 200 to 400 X objectives. The count should be comprehensive sperm( tops with tails).
  4. The count should be made of complete spermatozoa (heads with tails).

How to count using a haemocytometer chambers

The procedure for counting spermatozoa in the haemocytometer chamber is as follows.

  1. Examine the hemocytometer at x200 or x400 magnification
  2. Count at least 200 spermatozoa in each replicate to achieve an acceptable sampling error
  3. Assess the central grid( no. 5) on one side of the improved Neubauer chamber, row by row as seen below
improved Neubauer haemocytometer

improved Neubauer haemocytometer
  1. Continue counting until at least 200 spermatozoa have been observed and a complete row( of 5 large squares) examined.Counting must be done by complete rows; do not stop in the middle of a row.If no 200 spermatozoa in the 5 rows of the central grid are observed( no.5), continue to count the two adjacent grids in rows( of 4 large squares)( no.4 and 6, as shown above)
  2. Note the number of rows assessed to reach at least 200 spermatozoa. The same number of rows is counted from the other chamber of the hemocytometer
  3. Switch to the second chamber of the haemocytometer and count the replicate on the same number of rows as the first replicate, even if it yields less than 200 spermatozoa
  4. Calculate the sum and difference of both numbers( counts)
  5. Determine the acceptability of the difference from the table below( shows the maximum difference between the counts expected to occur in 95 percent of the sample alone because of the sampling error)
  1. Calculate the concentration if the difference is acceptable. If the difference is too high, prepare two new dilutions, as described above, and repeat replicate counts
  2. Report the average concentration of sperm to two significant figures
  3. Calculate the total sperm count per ejaculate( see below)
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How to identify which spermatozoa to count on the hemocytometer grids

To start,Look at the  center of the three lines defines the boundary of the square( black line left panel).

  • Take note that all spermatozoa in the central square are counted.
  • Spermatozoa with their heads are counted between the two inner lines( white circles, left panel)
  • Do not count spermatozoa when their heads lie between the two outer lines( black circles, left panel)
  • A spermatozoa with most of its head lying on the central line is only counted if that line is the lower or left line of the square( white circles, middle panel)
  • Spermatozoa with most of its head lying on the central line is not counted if that line is the upper or right line of the square( black circles, right panel)
improved Neubauer haemocytometer
improved Neubauer haemocytometer

How to calculate spermatozoa concentration in millions / ml

The concentration of spermatozoa (C) in semen is their number (N) divided by the volume in which they were found, i.e. the volume of the total number (n) of rows examined for the replicate (20 nl each for girds 4,5 and 6), multiplied by the dilution factor.

These is express as follow

C=(N/n) x (1/20) x dilution factor

How to carry out an accurate evaluation of low sperm counts

This sperm counting procedure is carried out when < 2 spermatozoa per x400 field are present in the preliminary estimation of sperm concentration and is done as follow :

  1. Dilute two semen aliquots 1 + 1( 1:2) with fixative, as described above
  2. Fill each haemocytometer chamber with replicate dilutions, one replicate per chamber
  3. examine the haemocytometer at x200 or x400 magnification
  4. Count at least 200 spermatozoa in each replicate to achieve an acceptable sampling error( as seen on the table above)
  5. Examine each grid- by- grid chamber( no. 1,2,3,4,5 etc) and continue to count until at least 200 spermatozoa have been observed and a complete grid has been examined. Counting must be done by complete grids; do not stop in the middle of the grid.
  6. Note the number of grids assessed to reach at least 200 spermatozoa. The same number of grids will be counted from the other chamber of the hemocytometer
  7. Switch to the second chamber of the haemocytometer and count the replicate on the same number of grids as the first replicate, even if it yields less than 200 spermatozoa.
  8. calculate the sum and difference of the two numbers (counts)
  9. Determine the acceptability of the difference ( shows the maximum difference between the counts expected to occur in 95 percent of the sample alone because of the sampling error)
  10. Calculate the concentration if the difference is acceptable. If the difference is too high, prepare two new dilutions, as described above, and repeat replicate counts
  11. Report the average concentration of sperm to two significant figures
  12. calculate the total number of spermatozoa per ejaculate (see below)

The concentration of sperm( C) in semen is their number( N) divided by the volume in which they were found, i.e. the volume of the total number( n) of the grids examined for the replicate( where the volume of the grid is 100 nl), multiplied by the dilution factor.

That is,

C=(N/n) x (1/100) x dilution factor

How to determination of Sperm vitality using Dye exclusion – Eosin test

Sperm vitality is reflected in the proportion of sperm that are  alive as determined by dye exclusion( eosin test)

Procedure

  • mix the semen sample well
  • Remove the aliquot of 5 ul semen and combine on a microscopic slide with 5 ul eosin solution, cover with coverslip and leave for 30 s.( Prepare replicate)
  • examine each slide at x400 magnification
  • tally the number of stained (dead) and unstained (vital) spermatozoa with the aid of a laboratory counter
  • calculate the average and difference of the two percentages of vital cells from replicate
  • determine the acceptability of the difference (table below)
  • Calculate the concentration if the difference is acceptable. If the difference is too high, prepare two new dilutions, as described above, and repeat replicate counts
  • report the average percentage of vital and dead spermatozoa to the nearest whole number
Acceptable differences
Acceptable differences

Understanding sperm morphology

The following criteria should be applied when assessing the morphological normality of spermatozoa.

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Spermatozoa consists of a head, neck, middle piece( mid piece), principal piece and end piece.As the end piece is difficult to see, the cell can be considered to consist of a head( and neck) and tail( mid piece and principal piece).In order for a spermatozoa to be considered normal, both its head and tail must be normal.All borderline forms should be regarded as abnormal.

  • The head should be smooth, regularly contoured and usually oval. There should be a well- defined acrosomal region with 40- 70 percent of the head area.The acrosomal region should not contain large vacuoles and not more than two small vacuoles, which should not occupy more than 20 percent of the sperm head. The post- acrosomal region should not contain vacuoles
  • The middle part should be slender, regular, and about the same length as the sperm head. The principal axis of the midpiece should be aligned with the principal axis of the sperm head. Residual cytoplasm is considered an anomaly only when it is excessive, i.e. when it exceeds one third of the sperm head size
  • The principal part or piece should have a uniform caliber along its length, be thinner than the middle part and approximately 45 μm long( about 10 times the length of the head). It may be looped back on itself, provided there is no sharp angle indicative of flagellar break.
Sperm cell
Sperm cell

Procedure for examining and reporting spermatozoa morphology

  1. Carefully examine appropriate spermatozoa( according to their photomicrograph numbers on plate no. 7, 8 or 9 or 10)
  2. write down the classification: normal/abnormal
  3. Describe the defect when necessary: e.g. head without acrosme, amorphous head, thick midpiece, coiled tail, etc.

Reference values for semen variable

The following reference values describe a semen sample that has been analyzed properly

ParametersReference values
Volume1.5 ml or more
Sperm concentration15 x l0 6 spermatozoa/ml or more
pH7.2 or more
Total sperm number39 x l0 6 spermatozoa per ejaculate or more
Total motility (PR+NP) 40%
Progressive motility (PR)32%
Morphology*4% or more normal forms
Vitality58% or more live, i.e., excluding dye
“Round cells”fewer than 5 x 10 6 /ml
Leukocytesless than 1 x 106 /ml

Nomenclature for certain semen variables

Since it is often useful to describe deviations from reference semen variables with words instead of numbers, a nomenclature has been introduced( Eliasson et al., 1970).It is important to recognize that this nomenclature only describes some semen variables and does not imply any causal relationship.With this provision, the nomenclature should be used as follows:

NormozoospermiaNormal ejaculate as defined by the reference values
OligozoospermiaSperm concentration less than the reference value
AsthenozoospermiaLess than the reference value for motility
TeratozoospermiaLess than the reference value for morphology
OligoasthenoteratozoospermiaSignifies disturbance of all three variables (combinations of only two prefixes may also be used)
AzoospermiaNo spermatozoa in the ejaculate
AspermiaNo ejaculate
Semen Analysis: Sperm Counting Test Procedure and Results
Sperm count

Reference

  • WHO laboratory manual for the examination and processing of human semen. Fifth Edition. WHO Press, 2010,

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About the Author: Arthur Westmann

DEFFE ARTHUR (AMOEBAMANN) is the founder and author of MLTGEEKS and MLTEXPO.He’s from Cameroon and is currently a Final year State Medical Laboratory Technician (MLT MA). Beyond lab works, he’s a passionate internet user with a keen interest in web design and blogging. Furthermore He likes traveling, hanging around with friends and social networking to do in his spare time.

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