A semen analysis measures the volume, pH, sperm count, motility, morphology, and other parameters of a semen sample. It is performed to evaluate male fertility and determine if issues such as infertility, a failed vasectomy, or failed vasectomy reversal are present. Proper collection involves abstaining from ejaculation for 2-5 days prior and collecting the sample by masturbation within 1 hour of the analysis. The semen is then examined under a microscope to evaluate the various sperm characteristics and identify any abnormalities.

1. Semen analysis
 Semen consists of spermatozoa suspended in seminal
plasma
 A semen analysis measures the amount of semen a
man produces and determines the number and quality of
sperm in the semen sample.
 Its done to determine whether:
A man has a reproductive problem that is causing
infertility
A vasectomy has been successful
The reversal of a vasectomy has been successful.

2. Semen analysis: Preparation
 The patient may be asked to do the following
To avoid any sexual activity that results in ejaculation
for 2 to 5 days before a semen analysis
If possible, the patient should not avoid sexual activity
for more than 1 to 2 weeks before this test, because a
long period of sexual inactivity can result in less
active sperm.
To avoid drinking alcohol for a few days before the
test.
About any medications or herbal supplements he may
be taking.

3. Semen analysis: Preparation
 How to collect it
The most common way to collect semen is by
masturbation, directing the semen into a clean sample
container.
The patient can collect a semen sample during sex by
withdrawing his penis from his partner just before
ejaculating (coitus interruptus).This method is not
done to access infertility,
The patient can also collect a semen sample during sex
by using a condom

4. Semen analysis: Preparation
 After collecting
The sample must be received at the laboratory within 1
hour.
Keep the sample out of direct sunlight
Do not allow it to get cold or hot. If it is a cold day, carry
the semen sample container against his body to keep it as
close to body temperature as possible. E.g. In the armpit.
Do not refrigerate the semen sample.

5. Semen analysis
 Tests that may be done during a semen
analysis include
Volume: This is a measure of how much semen
is present in one ejaculation.
o Normal: 1.0–6.5 milliliters (mL) per ejaculation
o Abnormal: An abnormally low or high semen
volume is present, which may sometimes cause
fertility problems

6. Semen analysis
Semen pH: This is a measure of the acidity
(low pH) or alkalinity (high pH) of the semen.
oNormal: Semen pH of 7.1–8.0
oAbnormal: high or low semen pH can kill
sperm or affect their ability to move or to
penetrate an egg

7. Semen analysis
Sperm motility: This is a measure of the percentage of
sperm that can move forward normally.
o Normal: At least 60% of the sperm show normal forward
movement. At least 8 million sperm per milliliter (mL)
show normal forward movement
o Abnormal: Sperm must be able to move forward (or
“swim”) through cervical mucus to reach an egg. A high
percentage of sperm that cannot swim properly may
impair a man’s ability to father a child

8. White blood cell count: White blood cells are not
normally present in semen.
o Normal: No white blood cells or bacteria are detected
o Abnormal: Bacteria or a large number of white blood cells
are present, which may indicate an infection
Fructose level: This is a measure of the amount of a
sugar called fructose in the semen. The fructose provides
energy for the sperm.
o Normal: 300 milligrams (mg) of fructose per 100 milliliters
(mL) of ejaculation

9. Sperm count: This is a count of the number of
sperm present per milliliter (mL) of semen in one
ejaculation.
oNormal: 20–150 million sperm per milliliter (mL)
0 sperm per milliliter if the man has had a vasectomy
oAbnormal: A very low sperm count is present, which
may indicate infertility. However, a low sperm count
does not always mean that a man cannot father a
child.

10. Sperm shape (morphology): This is a measure of
the percentage of sperm that have a normal shape.
oNormal: At least 70% of the sperm have normal
shape and structure
oAbnormal: Sperm can be abnormal in several
ways, such as having two heads or two tails, a
short tail, a tiny head (pinhead), or a round (rather
than oval) head.

11. Liquefaction time: Semen is a thick gel at the time
of ejaculation and normally becomes liquid within
30 minutes after ejaculation. Liquefaction time is a
measure of the time it takes for the semen to
liquefy.
oNormal: Less than 60 minutes, ideally < 30
minutes
oAbnormal: An abnormally long liquefaction time is
present, which may indicate an infection

12. Gross Examination
 Macroscopic examination
Color: fresh semen is opaque, white or gray white
coagulum
pH: normal semen is mildly alkaline, with a pH of about
7.7.
Viscosity: Can be assessed by pouring semen, if it falls
drop by drop, its viscosity is normal. Increased viscosity is
important if it compromises the sperm motility.
Liquefaction:
Volume: The normal semen volume averages 3.5 mL, with

13. Gross Examination
 Microscopic Examination
Sperm Counts
Calculating sperm count on a Neubauer
hemacytometer.
The formula for calculating the sperm count when 5
small squares within the large center square are
counted is: (Number of sperm counted in 25 squares on
each of 2
sides × dilution factor ) /(volume × 1000) = sperm/mL.

14. Microscopic Examination: Sperm Counts
Example: 100 sperm are counted in the five
small squares of one side of the hemacytometer,
110 sperm are counted in 5 small squares of the
other. The dilution is 1:20.
Number of sperm in 25 squares on 2 sides = (110
+100) × 5 =1050
Sperm/mL = 1050 × 20 (dilution factor) divided by (0.2
mm3 × 1000) = 105 million sperm/ml.

15. Microscopic Examination: Sperm Counts
 Diluting a specimen for counting on a
hemacytometer.
Following liquefaction (20–30 minutes), mix the sample
manually
The diluent that may be used for sperm counts on a
hemacytometer can be as follows: 5 g of sodium
bicarbonate in 100 mL of distilled water, plus 1 mL of
formalin (neutral).
The dilution often used for routine sperm counts is 1:20
but the actual dilution factor will vary depending on the
total sperm count.

16. Sperm Morphology
• This is assessed by performing differential
count of morphologically normal and
abnormal spermatozoa types on stained
smears
• Smears are made on slides. The most
suitable stain is Papanicolaou.

17. Papanicolaus stain
• Place the smear immediately in a fixative
95% ethanol or 50% ethanol ether before
drying has occurred.
• 80％ ethyl alcohol
• 70％ ethyl alcohol
• Rinse in tap water
• Harris hematoxylin 5-10 minutes

18. Papanicolaus stain
• Rinse in running tap water
• Differentiate in 1％ acid in 70％ ethyl alcohol
• Rinse in running tap water
• Blueing in running tap water for 10 minutes
• 70％ ethyl alcohol 10 dips
• 80％ ethyl alcohol 10 dips
• 95％ ethyl alcohol 10 dips
• 95％ ethyl alcohol 10 dips

19. Papanicolaus stain
• Orange G for 3 minutes
• 95％ ethyl alcohol 10 dips
• 95％ ethyl alcohol 10 dips
• Eosin for 3 munites
• 95％ ethyl alcohol 10 dips
• 95％ ethyl alcohol 10 dips
• Absolute ethyl alcohol 10 dips
• Absolute ethyl alcohol 10 dips

20. Papanicolaus stain
• Xylene 4 dips
• Xylene 4 dips
• Mounting the slide with DPX and coverslip

21. Histopathology Fixatives
• 10% Formalin
• Buffered neutral formalin
• Zenker’s fluid
• Lugol’s solution
(Weigert’s Modification)
• Bouin’s fluid
• Carnoy’s fluid
• Absolute alcohol and acetone

22. Decalcification
5% aqueous solution of nitric acid
• only for small pieces of bone
• Decalcify for 1 to 4 days
• Sections of bone may be tested by bending,
piercing with a sharp needle or X-ray

23. Decalcification
Formic Acid Sodium Citrate Method
• Decalcify for 5–14 days in formic acid-sodium
citrate solution.

24. Processing of Tissues
I. Alcohol 80% 1–2 hours
II. Alcohol 95%—2 changes 1–2 hours each
III.Alcohol absolute—3 changes 1–2 hours each
IV.Xylene—2 changes 1–2 hours each
V. Melted paraffin—3 changes 1–2 hours each
VI.Embed in paraffin and cool quickly.

25. H-E Staining Procedure
• Xylene, absolute alcohol, 95% alcohol
• Rinse in tap water.
• Harris’s hematoxylin for 15 minutes.
• Rinse in tap water.
• Differentiate in acid alcohol—10 quick dips.
• Wash in tap water very briefly
• Blueing in running tap water for 10 minutes

26. H-E Staining Procedure
• Stain with eosin 2 minutes
• 95% alcohol.
• Absolute alcohol—at least two changes.
• Xylene—two changes.
• Mount in DPX mountant.
.

27. Which of the following is a correct order of
histological techniques
A. Fixation,cleaning,dehydration,embedding, cutting and
staining
B. Fixation,dehydration,cleaning,embedding, cutting and
staining
C. Cutting,Fixation,cleaning,dehydration,embedding and
staining
D.Cutting,Fixation,embedding,cleaning,dehydration, and
staining

28. The following are tissue fixatives except
A. Buffered formalin
B. Bowen’s fluid
C. Zenker’s formal saline
D. Cold acetone
E. Buffered gluteraldehyde

29. Concerning regressive hematoxylin
stain, choose incorrect statement
A. Differentiating step is involved
B. The hematoxylin contains an excess of
aluminum salts
C. Is accomplished by overstaining in
hematoxylin
D. Is one of the methods of hematoxylin
staining

30. Commonly used mordant in hematoxylin
stain
A. Lugol’s solution
B. Aluminum
C. Iron and chromium
D. copper or tungsten salts
E. Eosin and xylene

31. What is a cytological function of Mayer’s egg
albumin to be added to the glass slide before
fluid
A. It adhere fluid to the slide
B. It fix fluid to the slide
C. It prevent fluid cells to dry
D. To prevent degeneration of cells
E. None of the above is true