The document details the components and analysis procedures of semen, highlighting the importance of various parameters for evaluation, such as sperm count, motility, morphology, and seminal fluid characteristics. It outlines the collection process, macroscopic evaluations, viscosity, pH measurements, and specialized tests like fructose tests and immunological assessments for fertility issues. Terminology for abnormal findings in semen analysis, such as aspermia and oligospermia, is also defined.

1. Semen
• Normal semen is an admixture of
spermatozoa suspended in secretions
(SEMINAL PLASMA ) from the glandular tissue
of the male genital system.
• Semen analysis is an evaluation of
spermatogenesis and spermiogenesis

2. INDICATIONS FOR SEMEN ANALYSIS
• Infertility
• Hypogonadism
• Follow up after vasectomy
• storage of semen before radiotherapy

3. Sample Collection
• The container has to be sterile and known NOT to be
spermotoxic (i.e. provided by the lab)
• The man must have had 3 – 5 days of abstinence
• The man must have washed his hands before collection
(particularly if microbiological analysis is requested)
• The sample must be kept at 37°C until analysis, which
begins ideally within 30 min, but absolutely within 60 min,
of ejaculation

4. Macroscopic Evaluation-Appearance
• Colour (normal = white to grayish-yellow) – if
there is blood present, it may range from pink
to brown
• Opacity / translucence (normal = tends to
opaque)
• Whether mucus streaks or cell clumps are
present

5. Liquefaction
• Liquefaction is the breakdown of the gel
portion of the seminal plasma – the enzymes
for this are in the prostatic fluid
• At room temperature ,normal semen gets
liquefied within 30 minutes after collection.
• Presence of mucous streaks indicate
incomplete liquefaction

6. Viscosity
• After liquefaction aspirate sample by wide-
bore plastic pipette
• Normal Viscosity: Small discrete drops
• Abnormal Viscosity: Drop form thread > 2 cm

7. Volume
• The volume of sample should be measured in a graduated tube or a
small cylinder to the nearest 0.1 ml.
• Normal volume of ejaculate semen is 1.5 to 4.5 ml.
• If the volume of semen is below 1.5 ml , it is LOW VOLUME.
• Conditions which can lead to hypospermia or decreased volume are
• 1)DISORDERS OF PROSTATE GLAND & SEMINAL VESICLES.
2)RETROGRADE EJACULATION
• 3)CONGENITAL ABSENCE OF PROSTATE OR SEMINAL VESICLES.
4)OCCLUSION OF THE EJACULATORY DUCT
• 5)REPEATED SAMPLE COLLECTION.

8. PH
• The pH of the semen can be measured with pH paper ,
indicator dyes or pH mater. It depends upon sperms
conc.
• The pH should be measured within an hour of
ejaculation & should be in range of 7.2 – 8.0.
• If pH is less than 7.0 , dysgenesis of vas deferens,
seminal vesicles or epididymis may be present.
• Bacterial contaminated semen & semen with dead
sperms may produce ammonia which increases the pH
to make it alkaline.

9. SPERM COUNTING
• Visual assessment:
Examine semen slide under the high power objective
of a microscope to determine the need for any
dilution.
• The diluent used is 3.5% buffered formal saline.
Gentian violet can be added to this fluid to stain
the sperms.
• Improved Neubauer chamber (Haemocytometer)is
used for counting.

10. • At least 200 spermatozoa must be counted.
• Sperm count (million/ml) = CxDx1000/V
• Sperm count 60-150 million/ml

11. ASSESSMENT OF SPERM MOTILITY
• performed soon after production of sample, 3
and 6 hours later.
• Both motile and immotile sperms are counted
at least in 5 fields with a minimum count of
200.
• Only forward movement of the sperms is
taken as positive.

12. Motile sperm count=Sperm count/ml×%motility×semen vol/100
• Motility >70% at 1 hour and 50% at 3 hours after ejaculation
• Observe with a x40 objective and estimate the percentage of
spermatozoa moving at following speeds:
Grade 0: No movement at all
Grade 1: Moving with no forward progression
Grade 2: Moving with slow and wandering movement
Grade 3: Moving rapidly in almost straight line
Grade 4: Moving with high speed in straight line

13. • Grade x Percentage =
• 0 x 30 0
• 1 x 10 10
• 2 x 15 30
• 3 x 30 90
• 4 x 15 60
• Total score 190
Normal motility score for spermatozoa is ≥150.

14. ASSESSMENT OF SPERM
MORPHOLOGY
• A normal sperm consists of a head and a tail
joined together by a short neck.
• Assessment can be made in a wet preparation
or in a stained smear of semen.
• Stained by haematoxylin and eosin or
Papanicolaou or Giemsa.

16. • Abnormalities of the head including small,
large, tapering, pyriform, amorphous and
double heads.
• Abnormalities of the tail including double,
coiled or short tails
• >70% should be morphologically normal

18. • Also make a note of the presence of white
blood cells, epithelial cells, red blood cells,
lymphocytes, extraneous particles, protozoa
and bacteria

19. AGGLUTINATION
• Agglutination means motile spermatozoa stick to one another.
• Adherence of immotile sperms to one another or motile sperms to
mucous threads should be considered NON SPECIFIC
AGGREGATION.
• The presence of AGGLUTINATION is suggestive of immunological
cause of infertility.
• Agglutination should be assessed in 20 random fields & avg. %age
of sperms clumped together is estimated .
• Type of agglutination is also noted i.e. HEAD to HEAD, TAIL to TAIL
,HEAD to TAIL & MIDPIECE to TAIL.
• All types of agglutinations could be due to ANTI SPERM ANTIBODIES
& should be further subjected to IMMUNOLOGICAL TESTS

20. IMMUNOLOGICAL TESTS
• ANTI SPERM ANTIBODY ( ELISA ) :
• PRINCIPLE : The ELISA antisperm antibody kit uses sperm antigen
which are extracted from pool of treated sperms attached to wells
of microlitre plates.
• Serial dilutions of sera are attached to the wells & after incubation ,
the amt. of bound antibodies is measured by appropriate antisperm
ab’s . This test require serum.
• INTERPRETATION : a positive test is indicated by o.d of at least 0.3
at 405 nm.
• The dilution of 1/32 or more as antisperm ab’s is positive , while
dilution of 1/16 is considered borderline

21. FRUCTOSE TEST
• FRUCTOLYSIS :
• Fructose is main sugar present in seminal plasma & is imp. nutrient
for the sperms.
• The quality of semen can be assessed by measuring the rate of
utilization of fructose.
• FRUCTOLYTIC INDEX is the amount of fructose used or lactic acid
formed by spermatozoa per hour at 37 deg.
• The semen sample should be well buffered otherwise the
fructolysis will stop at certain stage & result will be erroneous.

22. FRUCTOSE TEST
• Fructose is absent from the semen of patients
with bilateral aplasia of the vasa differentia and
seminal vesicles. It is also absent in bilateral
obstruction of the ejaculatory ducts.
• Place 0.1 ml of semen in a test tube. Add to it 1ml
of resorcinol reagent. Boil for 5-10 min. The
solution turns reddish brown in the presence of
fructose. No change in colour indicates absence
of fructose from the semen

23. REPORTING
Some of the special terms used for reporting the results of
semen analysis are:
Aspermia: No ejaculate.
Oligospermia/Hypospermia: Reduction in volume of ejaculate.
Hyperspermia: Increase in volume of ejaculate.
Oligozoospermia: Low sperm count (<30 million/ml).
Polyzoospermia: High sperm count (>300 million/ml)
Asthenozoospermia: Absence or marked reduction in sperm
motility (Motility score<150)
Oligoasthenozoospermia: Low count with low motility.
Necrospermia: Dead sperm