EXAMINATION OF

PHYSICAL CHARACTERS,
COUNT, MOTILITY,
VIABILITY AND
SEMEN MORPHOLOGY
 CONSTITUENTS OF SEMEN

Normal semen is an mixture of spermatozoa

suspended in seminal plasma from glandular
tissues of male genital system
 FRACTION OF SEMEN
CONTRIBUTED BY VARIOUS
GLANDS
1. Urethral glands (2-5%) - small mucus secreting
glands.
2. Prostate: 20-30% of the volume, acidic fluid
produced by the prostate gland, the secretion
contains citrate, zinc, acid phosphatase and
proteolytic enzymes liquefaction of the semen.
3. Seminal vesicles: 46-80 % of the semen volume,
alkaline viscous, yellowish secretion is rich in
fructose, vitamin C, prostaglandin, protein kinase,
and other substances, which nourish and activate
the sperm.
 WHAT IS THE PURPOSE OF
THE TEST?
 Investigation of infertility ( Primary or Secondary)
Identify treatment options
Surgical treatment.
Medical treatment.
Assisted conception treatment.
Determine the suitability of semen for ICSI/IVF
 Pre and Post vasectomy – Confirmation.
 Following vasectomy reversal
• Human sperm cell is about 70 µm
long.
The head size: 4-5µm
Nucleus - contains the 23
chromosomes.
Acrosome
Mid-piece: 4-5µm
The energy for motility is
generated.
Tail: 55µm
Motility -Propagated along
the tail.
 STANDARD GUIDELINES FOR
THE COLLECTION OF SEMEN
There should be 2 to 7 days of sexual abstinence before
collection.
Two separate samples at least 7 days apart should be
analyzed.
The duration of abstinence should be constant
Collection - Private room in the same centre where the
semen will be analyzed.
Pre warmed (21oC), sterile, non-toxic, wide-mouth
container.
 LABELLING OF SAMPLE
Patient name
Age
Clinic or Doctor name

Laboratory analysis form:

The period of abstinence (in days).
Date &Time of collection.
Mode of collection.
Complete or incomplete.
The time interval from collection to analysis.
 TIMING OF ANALYSIS
• Semen is placed in a 37° C gently shaking
incubator for 30 minutes.

• The semen sample should be examined, Ideally

within 30 mins Absolutely within 1 hour of
collection.

• Motility decreases significantly after 2 hours

 WHO 2010
Parameter 1992 Lower Reference Limit
2010
Semen volume 2 ml 1.5 ml
Sperm concentration 20 M 15 x 106 /ml
Total sperm number 39 x106 /ejaculate
Progressive motility >50 % 32% A
Total motility 40% A+B
Vitality (live sperms) 58%
Sperm morphology >15 % 4%
pH >/=7.2 >/=7.2
Leucocyte <1M <1x 106 /ml
 EXAMINATION OF SEMINAL
FLUID IN INFERTILITY
 Physical examination
 Visual appearance : opaque to grey – white, slightly
yellow after abstinence.
 Inflammation of male accessory organs → yellow color
of semen → pyospermia
 White clear semen → azoospermia
 Brown or red color → hemospermia
 VISCOSITY
 Assessed by filling a pipette with semen and allowing it to flow
back to the container
 Normal semen fall drop by drop
 If droplet form threads > 2 cm long → increased viscosity
 Normal semen liquefies in 30 min. If liquefaction does not occur
in 60 min → abnormal increase in viscosity. This decreases sperm
motility.
 If sample does not liquefy → treat with plasmin or chymotrypsin.
 Volume : more than 1.5 ml
 If the sample volume is less than 1 ml incomplete
collection must be ruled out
 Conditions leading to low semen volume
(hypospermia)
Disorders of seminal vesicles or prostate
Retrograde ejaculation
Congenital absence of prostate or seminal vesicle
 pH : normal >= 7.2
 Seminal vesicle secretion is basic
 Prostatic secretion is acidic
 If pH = 7 with absence of sperm → indicates
either obstruction of ejaculatory duct or
absence of vas deferens
 MICROSCOPIC EXAMINATION
 Motility – Ability of the sperm to move – 3 types
of motility
 Rapidly progressive – moving fast and forward
in a straight line
 Slowly progressive – crooked, curved, slow
forward movement
 Non progressive – movement of tail only
Method :
• A drop of semen is placed on a slide, covered with coverslip and
sealed with petroleum jelly.
• Examination is done under 40x
• Count at least 200 spermatozoa
• Find the percentage of rapidly progressive, slowly progressive, non
progressive and non motile sperm.
• Normal values – > 32 % progressive motility – > 40 % progressive
+ non progressive motility
 Vitality
 Number of live sperms are called viable
 A viable sperm will have intact cell membrane and will not take up
eosin Y
 Method
● 1 drop of sample + 1 drop of eosin – nigrosin
● Wait for 30 sec
● Put a drop on a slide
● Air dry
● Examine under oil immersion and count 200 sperms
● Red sperms not viable; white sperm viable
● Normal viable count > 58%
 WET SMEAR PREPARATION
Count
 Wait for liquefaction
Mix 1ml semen with 20 ml diluting
fluid(sodium bicarbonate – formalin)
Charge Neubauer’s chamber with pateur’s
pipette
Place chamber in humid conditions for 10 –
15 min
Count in 4 large chambers
 CALCULATION

(Number of sperm counted x dilution factor x 1000)/

volume = sperm/ml.

Example:N sperm are counted in the four small squares.

The dilution is 1:20. One small square of Neubaur’s
chamber volume = (1x1x0.1) mm3

Sperm/ml = (N x 20 x 1000 )/ (0.1 x 4 )mm3/ml = N x

50,000 sperm/ml.
 MORPHOLOGY
 Drop of seminal fluid on the slide
 Stain with pap/eosin-nigrosin/rose bengal-toludine blue
 Examine the morphology of at least 200 sperms
 Normal > 4 % of sperm should have normal
morphology
 NORMAL MORPHOLOGY OF
SPERMATOZOA
Head : consists of nucleus with
condensed chromatin and some nuclear
vacuoles.
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.
Middle piece contains mitochondria →
provides energy.
The tail used for motility