2. Constituents of Semen
β Normal semen is an admixture of spermatozoa
suspended in secretions (seminal plasma) from
glandular tissues of male genital system.
3. β Testes produces spermatozoa and constitutes
5% of the semen volume.
β Vas deferens produces ergothionine
β Epididymis ( maturation/ storage of sperm)
produces:
β Choline - energy source of sperms.
β Alpha glucosidase
β Carnitine
4. β Seminal vesicle β nutritive fluid containing
fructose, and is secreted during ejaculation.
(50% of semen volume)
β Prostate produces (40% of semen volume)
β Citric acid
β Acid phosphatase
β Proteolytic enzyme
β Zinc
β Bulbourethral glands of Cowper produces
mucous. ( constitutes 5% of semen volume)
5. β Indications of Semen Analysis
β Investigation of infertility
β Check effectiveness of vasectomy
β Paternity testing
β Rape cases
β Selection of donors for artificial insemination/
assisted reproductive technology.
6. β Sample collection
β Sample should be collected after 48 hrs of
abstinence. Higher abstinence β decreased
motility. Lesser abstinence β decreased count.
β Collection is done by masturbation.
Not recommended: condom collection, coitus
interruptus. (loss of initial portion of the ejaculate)
β Collection should be done in a clean, wide mouth,
leak proof container.
7. β Transport
β Should be done within one hour to the laboratory.
β Temperature should be maintained as close to the
body temperature as possible (inside pocket)
β Two specimens should be examined at least 2
to 3 weeks apart.
8. Examination of Seminal fluid in
Infertility
β Physical examination
β Visual appearance : opaque to grey β white, slightly
yellow after abstinence.
β Inflammation of male accessory organs β yellow color of
semen β pyospermia
β White clear semen β azoospermia
β Brown or red color β hemospermia
9. β Viscosity
β Assessed by filling a pipette with semen and allowing it to
flow back to the container
β Normal semen fall drop by drop
β If droplet form threads > 2 cm long β increased viscosity
β Normal semen liquefies in 30 min. If liquefaction does not
occur in 60 min β abnormal increase in viscosity. This
decreases sperm motility.
β If sample does not liquefy β treat with plasmin or
chymotrypsin.
10. β Volume : more than 1.5 ml
β If the sample volume is less than 1 ml spillage or
incomplete collection must be ruled out
β Conditions leading to low semen volume
(hypospermia)
β Disorders of seminal vesicles or prostate
β Retrograde ejaculation
β Congenital absence of prostate or seminal vesicle
11. β PH : normal >= 7.2
β Seminal vesicle secretion is basic
β Prostatic secretion is acidic
β If pH = 7 with absence of sperm β indicates either
obstruction of ejaculatory duct or absence of vas
deferens.
12. Microscopic examination
β Motility
β Ability of the sperm to move
β 3 types of motility
β Rapidly progressive β moving fast and forward in a
straight line
β Slowly progressive β crooked, curved, slow forward
movement
β Non progressive β movement of tail only
13. β Only those sperms with rapid progressive
movement are capable of fertilizing an ovum.
β Method
β A drop of semen is placed on a slide, covered with
coverslip and sealed with petroleum jelly.
β Examination is done under 40x
β Count at least 200 spermatozoa
β Find the percentage of rapidly progressive, slowly
progressive, non progressive and non motile sperm.
β Normal values
β > 32 % progressive motility
β > 40 % progressive + non progressive motility
14. β Vitality
β Number of live sperms are called viable
β A viable sperm will have intact cell membrane and
will not take up eosin Y
β Method
β 1 drop of semen + 1 drop of eosin β nigrosin
β Wait for 30 sec
β Put a drop on a slide
β Air dry
β Examine under oil immersion and count 200 sperms
β Red sperms not viable; white sperm viable
β Normal viable count > 58%
15. β Count
β Wait for liquefaction
β Mix 1ml semen with 20 ml diluting fluid(sodium
bicarbonate β formalin)
β Charge Neubauerβs chamber with pateurβs pipette
β Place chamber in humid conditions for 10 β 15 min
β Count in 4 large chambers
16. β Calculation
count = sperm counted x correction for dil. Fluid x1000
β--------------------------------------------------
No. of squares counted x vol of 1 square
= N x 20 x1000
-------------
4 x 0.1
= N x 50,000
β Normal count > 15 million/ ml
17. β Morphology
β Drop of seminal fluid on the slide
β Stain with pap/eosin-nigrosin/rose bengal-toludine
blue
β Examine the morphology of at least 200 sperms
β Normal > 4 % of sperm should have normal
morphology.
18. β Normal morphology of spermatozoa
β Head : consists of nucleus with condensed chromatin
and some nuclear vacuoles.
β Acrosome: anterior 2/3rd of the head shows an
acrosom cap, secrets enzymes that dissolve the cells
of corona radiata and zona pellucida of the ovum
during fertilization.
β Middle piece contains mitochondria β provides
energy.
β The tail used for motility.
19.
20.
21. Immunological analysis (antisperm antibody
determination )
β Sperm Mar Test
β Direct SMT
β For detection of sperms coated with IgG/IgA
β Indirect SMT
β For detections of antisperm IgG/A antibodies in serum.
22. β Immunobead test
β Similar to sperm mar test but uses plastic beads
instead of latex particles to detect
antigen/antibodies
β Normal
β <50 % motile spermatozoa with bound particles.
23. Biochemical Analysis
β Seminal vesicle marker (Fructose)
β 50 mg of resorcinol in 33ml of conc. Hcl then diluted
with 100 ml of Distilled water.
β 0.5 ml of seminal fluid is added
β The mixture is heated β produces red precipitate in
30 seconds
β Presence of red precipitate indicates presence of
Fructose
β Absence of fructose β no seminal vesicle present
β D/t obstructed vas deferens or absent seminal vesicle.
24. Sperm function test
β Post coital (Sims-Huhner) test
β Principle
β Examination of the quality of cervical mucus post coitus can
give an idea about the quality of cervical mucus and the ability
of the sperm to penetrate it.
β Normally in proliferative phase (estrogen phase), mucus is
watery and sperm can penetrate easily.
β During secretory phase (progesterone phase), mucus is viscus.
β Hence testing mucus is scheduled just before ovulation.
25. β Method
β Cervical mucus is aspirated 2 β 12 hrs after intercourse.
β Gross examination
β Normal β mucus stretches at least 2 inches, dries in fern
like pattern.
β Abnormal β can not stretch 2 in. No fern like pattern on
drying.
β Microscopic examination
β Normal β more than or equal to 10 motile sperms
β Abnormal β less than 10 motile sperms
β Causes β antisperm antibodies, cervicitis, wrong judgement of
date.
26. β Cervical mucus penetration test
β Evaluation of the distance traveled by sperm in bovine mucus.
β Fertile sperm travel > 30mm
β Infertile sperm travel <20 mm
β Hamster egg penetration assay
β Hamster egg β enzymatically treated β removal of outer coat
β Incubate with sperm
β Look for number of eggs penetrated ( <15% indicate low fertility ) and
number of sperm penetrating the egg (normal > = 5%)
27. β Hypoosmotic swelling of flagella
β If sperm is exposed to hypoosmotic solution, sperm
curls up if plasma membrane is abnormal.
β Assessment of functional integrity of plasma
membrane.
β Computer assisted semen analysis
β All above parameters are measured by automated
machines.