1. Seminal Fluid
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Semen is body fluid that is ejaculated at the time
of orgasm, contain sperm & secretion of
seminal vesicle, prostate, Cowper's gland &
urethral gland.
2. Source Volume Characteristics
Urethral and
bulbourethral glands
0.1-0.2cc Viscous, clear
Testes,
epididymides, vas
deferentia
0.1-0.2cc Sperm present
Prostate 0.5-1.0cc Acidic,watery
Seminal vesicles 1.0-3.0cc Gelatinous, fructose
positive
Complete ejaculate 1.5-5.0cc Liquefies in 20-25min
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Semen
3. Semenis viscous,neutralor slightlyalkaline&
whitish opaque.
60 % semen volume is derived from seminal vesicle
which is also a major source of high FRUCTOSE
content of semen.
Seminal vesicle secretion also
provide the substrate for the
coagulation of the semen following ejaculation.
About 20% of the volume of semen is
contributed by the prostate gland.
It is milky in appearance & also rich in proteolytic enzymes
are responsible
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4. About 10 -15 % of semen volume is also
contributed by epididymis, vasdeferens, cowper’s
gland & uretheral gland.
Less than 5 % of semen volume is contributed by
Spermatozoa.
The process of ejaculation result in the mixing of
these distinct fraction of semen.
These enter the urethra individually in the rapid
succession.
The third & final fraction consist of mucoid secretion
resulting from emptying of seminal vesicle.
The semen specimen collected for routine
examination should contain all above mention
fraction.
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6. Steps:Semen Analysis
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In the first 5 minutes:
Placingthe specimen container on the
bench or in an
incubator (37 °C) for liquefaction.
Between 30 and 60 minutes:
Assessing liquefaction and appearance of the
semen.
Measuring semen volume.
Measuring semen pH (if required).
Preparinga wet preparation for
assessing microscopic
appearance, sperm motility
and the dilutionrequired
for
assessing sperm
number.
7. StepsOfSemenAnalysis
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Between 30 and 60 minutes:
Assessing sperm vitality (if the
percentage of motile cells is
low).
Making semen smears for assessing sperm
morphology.
Making semen dilutions for assessing sperm
concentration.
Assessing sperm number.
Performing the mixed antiglobulin
reaction (MAR) test (if required).
8. Steps
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Between 30 and 60 minutes:
Assessing peroxidase-positive cells (if round cells
are present).
Preparing spermatozoa for the immunobead test (if
required).
Centrifuging semen (if biochemical markers are to
be assayed).
Within 3 hours:
Sending samples to the microbiology laboratory (if
required).
9. Steps
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After 4 hours:
Fixing, staining and assessing
smears for sperm morphology.
Lateron the sameday (or on a subsequent
day if samples are frozen):
Assaying accessory gland markers (if
required).
Performing the indirect immunobead
test (if required).
10. SampleCollection:
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Preparation:
The sample should be collected in a private
room near the laboratory, in order to limit the
exposure of the semen to fluctuations in
temperature and to control the time between
collection and analysis.
The sample should be collected after a
minimum of 2 days and a maximum of 7 days
of sexual abstinence. If additional samples
are required, the number of days of sexual
abstinence should be as constant as possible
at each visit.
11. SampleCollection:
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Preparation:
The man should be given clear written and spoken
instructions concerning the collection of the semen
sample. These should emphasize that the semen
sample needs to be complete and that the man
should report any loss of any fraction of the sample.
The following information should be recorded on the
report form: the man’s name, birth date and personal
code number, the period of abstinence, the date and
time of collection, the completeness of the sample,
any difficulties in producing the sample, and the
interval between collection and the start of the
semen analysis.
12. SampleCollectionforDiagnostic/Research purpose:
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The sample should be obtained by masturbation and
ejaculated into a clean, wide-mouthed container made
of glass or plastic, from a batch that has been
confirmed to be non-toxic for spermatozoa.
The specimen container should be kept at ambient
temperature, between 20 °C and 37 °C, to avoid large
changes in temperature that may affect the
spermatozoa after they are ejaculated into it. It must be
labelled with the man’snameand identification number,
and the date and time of collection.
The specimen container is placed on the bench or in
an incubator (37 °C) while the semen liquefies.
Note in the report if the sample is incomplete, especially
if the first, sperm-rich fraction may be missing. If the
sample is incomplete, a second sample should be
collected, again after an abstinence period of 2–7 days.
14. Collectionofsemenat home:
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A sample may be collected at home in exceptional
circumstances, such as a demonstrated inability to produce a
sample by masturbation in the clinic or the lack of adequate
facilities near the laboratory.
The man should be given clear written and spoken instructions
concerning the collection and transport of the semen sample.
These should emphasize that the semen sample needs to be
complete, i.e. all the ejaculate is collected, including the first,
sperm-rich portion, and that the man should report any loss of any
fraction of the sample. It should be noted in the report if the sample
is incomplete.
The man should be given a pre-weighed container, labelled with his
name and
identification number.
The man should record the time of semen production and deliver
the sample to the laboratory within 1 hour of collection.
During transport to the lab., the sample should be kept between 20
15. Safehandlingofspecimens:
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Semen samples may contain dangerous
infectious agents (e.g. human immunodeficiency
virus (HIV), hepatitis viruses or herpes simplex
virus) and should therefore be handled as a
biohazard. If the sample is to be processed for
bioassay, intra-uterine insemination (IUI), in-vitro
fertilization (IVF) or intracytoplasmic sperm
injection (ICSI), or if semen culture is to be
performed, sterile materials and techniques
must be used. Safety guidelines as outlined in
Appendix 2 should be strictly followed; good
laboratory practice is fundamental to laboratory
16. Precautions
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Specimen should not be collected in ordinary
condoms since the powder or lubricant
applied to the condom may be spermicidal.
The container in which the semen sample is
collected should be free from detergent.
18. History To Be Noted:
Name
Date and time of collection
Length of abstinence
Interval between collection and analysis
History of fever
Drug intake
Alcohol abuse
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19. Assessment Of Semen:(according To WHO)
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Standardtests:
1. Volume
2. pH
3. Spermconcentration
4. Totalspermcount
5. Motility
6. Morphology
7. Vitality
8. Whitebloodcells
9. Immunobeadtest
10.MARtest
20. Optionaltests:
Alpha galactosidase (neutral): 20mU
or more.
Zinc (total): 2.4 micromol or more
Citric acid(total): 52 micromol or more
Acid phosphatase: 200U or more
Fructose: 13 micromol or more
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23. Viscosity:
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substrate
coagulum
produced by seminal vesicles.
liquefies spontaneously to form
Freshly ejaculated semen is highly viscous due to
The
a
translucent, viscous fluid in a three stage process.
Action of a prostatic clotting enzyme.
Liquefaction is initiated by enzymes of prostatic
origin.
Protein fragments are further degraded into free
amino acids and ammonia.
Failure to liquefy indicates inadequate prostatic
secretion. To liquefy, add bromeline, plasmin or
chymotrypsin.
24. Normal liquefaction time: 20-60min
Viscosity of liquefied semen can be
estimated by: Gentle aspiration into a 5ml pipette, then
allowing the semen to drop by gravity. Observe the length of the
thread.
Normal semen leaves as small discrete drops.
Increased viscosity is associated with poor
invasion of cervical mucus in post-coital studies as well as
decreased ability to fertilize ovum.
Absence of viscosity points to reduced cell content.
The semen from males with b/l congenital absence of vas
deferentia & seminal vesicles fails to coagulate due to absence of
substrate.
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25. pH:
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enzymes,
MeasuredbyusingapHmeterorpHpaper.
Normal:7.2-8
Semenisthestrongestbufferinthe body.
Seminalvesicle&vasdeferencesecretions alkaline
Prostaticsecretionacidic(duetocitricacid,proteolytic
acidphosphatase)
Motilityisreducedinacidic medium.
pH<7isassociatedwithlargelyprostaticsecretionsdueto congenital
aplasiaofvas&seminalvesiclesandwhencontaminated withurine.
pH>8 is associated with acute infection of prostate, seminal vesicles
orepididymis.
