SEMEN EXAMINATION

Submitted By :
Vasant R. Parmar.
M.V.Sc ( Gynaecology)
Reg. No.: 04-00317-07
E mail; vasantvet@gmail.com
 Introduction
• Semen is collected for examination of breeding
soundness, infertility, Artificial insemination and
Parasitic diseases.
• Semen quality of the first ejaculate after a long period
of sexual rest may have a lowered motility and an
increased no of dead spermatozoa.
• Semen quality in many rams and a few bulls may be
decreased during the hot summer months.
• Season of the year had a significant effect on semen
quality in the stallions with the best quality of semen
produced late in spring or early summer and the
poorest quality semen in the winter months
 Important features of semen evaluation:

• While handling semen samples avoid exposure to water,

changes in pH and temperature.
• All laboratory equipments (microscope slides, cover slips,
pipettes) stains and diluents should be warmed at 37°C.
• Equipment should be clean and dry. Room should be
warm and dust free.
• Examine the semen ejaculate for volume, gross
appearance, wave motion, microscopic motility,
spermatozoa morphology and concentration, ratio of live
to dead and presence of other materials.
• For record note age, history, breed, other abnormalities if
any and methods of semen collection (massage, artificial
vagina or electroejaculation)
 Semen is subjected to the evaluation of the
following aspects:
(A) Physical characters (Macroscopic
Characters)

(C) Physiological and Biochemical studies

(E) Microscopic Evaluation

(G) Examination for genital infections

 (A) Physical characters (Macroscopic Characters)
1. Colour :
• Creamy
• Milky
• Watery
• Cloudy
Bull & Ram Concentrated Milky or creamy white & opaque

Stallion, Boar Less

& Dog Concentrated Pearl white to grey and translucent

Brownish color Blood Pigment Orchitis

Light yelllow color Riboflavin secreted by accessory glands

Yellowish – Green Presence of Pseudomonas aeruginosa

Clots and Flakes Presence of Pus

Semen samples from a bull (left)
and dog (right), showing differences
in opacity and concentration

Blood in a canine ejaculate

(hemospermia)
 Sperm cell Concentration and Semen Color
in Bulls and Rams

Color of Semen Bulls & Rams (per cmm)

Thick Creamy 25,00,000
Creamy 20,00,000
Light Creamy 10,00,000
Milky 5,00,000
Cloudy, watery, translucent 1,00,000
Almost clear, transparent, Less than 50,000
watery
(Gunn et al., 1942)
2. Volume :

Normal volume of semen in different animals

 Bull - 4 (1-15) ml
Stallion -70 (30-250) ml
 Ram/Buck - 1 (0.7-3.0) ml
 Boar - 250 (125-500) ml
 Dog - 10 (1-25)
 Cat - 0.04 (0.01-0.12) ml

(Roberts, 1971)
 Ejaculate consist of 3 portions :
1. Sperm free watery secretion
2. Sperm rich secretion
3. Sperm poor fraction

In stallion, Dog & Boar third secretion is voluminous than

first two fractions may be collected.

In Tom, Bull, Buck and ram – all three fractions are collected
Physiological and Biochemical
studies
1. Mass motility
• Immediately after collection take a small drop of
semen and examine under low power (10x)
Scales Criteria for semen
5 (100%) Excellent motility, extremely rapid swirls
and eddies
Very good motility, waves and eddies
4 (90%)
observed
Good motility, slow formation of waves and
3 (75-85%)
eddies
Fair motility, vigorous movement but no
2 (50-75%)
waves or eddies
Poor motility, weak and oscillatory
1 (<50%)
movement of spermatozoa
0 No motility
(Herman and Madden,
Individual motility:
Take a small drop of semen on warmed glass slide
put a cover slip and estimation under 40x
Scales Criteria for semen
5 Straight and rapid displacement of spermatozoa

4 Rapid displacement, some sperm cells with straight

trajectory, others with circular trajectory
3 Spermatozoa follow curvilinear displacement with no
trembling movement
2 Slow displacement, trembling, disorganized movement,
some spermatozoa move more rapidly
1 Very slow (or no) displacement, trembling of
spermatozoa, tail oscillation
0 No displacement of sperm cells
A progressively motile sperm swims forward
in an essentially straight line,
whereas a non-progressively motile sperm swims,
but with an abnormal path, such as in tight circles.
Cold shock resistance test:
• Take one ml of semen in test tube
• Put in a beaker containing ice (0°C) for 10 min
• Determine motility and live sperm percentage
• Compare the values before and after cold shock
• Estimate storage ability and freezability of
spermatozoa
Hydrogen ion concentration:
In bulls, rams and dogs About 6.7
In Stallions, Boars and cats About 7.4
In Bulls pH of 7.0 or higher seen in incomplete
ejaculation and in pathological or inflammatory condition
 Methylene blue reduction time test:
Principal :
Hydrogen ions are released during sperm metabolism.
These ions reduce the methylene blue to leucomethylene.
The liberation of hydrogen ions is due to dehydrogenase enzyme
present in active sperm.

Method:
1. Prepare methylene blue solution by dissolving 50
mg of methylene blue in 100 ml of citrate buffer
2. Dilute 0.2 ml of semen with 0.8 ml of egg yolk
citrate dilutor in a 10 ml vial and mix.
3. Add 0.1 ml of methylene blue solution and mix.
 4. Seal the mixture by layering with liquid
paraffin, 1.0 centimeter thick.
5. Observe the time required for the sample to
lose its blue color.
The semen which bleaches the blue color within 9
minutes is considered to be of good quality.

Resazurin reduction:

Resazurin, a hydrogen acceptor which changes color

upon reduction replaces methylene blue in the above
test
 Microscopic Evaluation
Sperm concentration:
Five Methods:
1. Direct sperm count of semen
by hemocytometer
2. Use of Spectrophotometer
3. Macroscopically by consistency
or density of semen
4. Opacity tube method by
comparing with standard
opacity solution
5. Compare PCV after centrifugation
vs Hemocytometer
Procedure:
1. Thoroughly mix specimen and dilute 1:20 with diluent.
(To obtain this dilution, dilute 50 uL of liquefied semen
with 950 uL of diluent)
2. Thoroughly mix diluted specimen and allow a drop to
into each side of the hemocytometer covered with a
coverglass.
3. Allow chamber to stand for about 5 minutes in a humid
container to prevent drying. During this period, the cells
settle and can be more easily counted.
4. After cells have settled, place chamber under phase
contrast microscope (preferably), using 40x
 Calculation:
1 primary square – 1mm length & 1mm width = 1 sq.
mm area
Total 8o squares are counted So,
Surface area of 80
80/400 × 1 sq. mm = 1/5 sq. mm
tertiary squares
Height betn counting chamber & coverslip – 0.1 mm
So, cubic volume
1/5×1/10 = 1/50 cmm
of 80 squares
No. of sperms (N) present 1/50 cmm of diluted semen

So, No. of sperm in 1 cmm diluted semen = N×50

Semen is diluted 1:200 times and 1ml = 1000 cmm
No. of sperms in 1 ml = N×50×200×1000
of undiluted semen
= N × 107
 Electronic Counting

• Photometers such as the Photometer SDM5are in common

use.
• A sample of raw semen is diluted in a cuvette with a
predetermined volume of diluent and analyzed with the
photometer.
• This analysis takes about 30 seconds.
• The volume, progressive motility, number of sperm per
dose and the volume per dose is entered.
• The number of doses to be frozen and the final extender
volume will be calculated.
 CASA System

• Systems such as Sperm Vision use computer programmed

digital analysis of microscope fields of moving sperm.
• Several characteristics of motility are quantified, and sperm
concentration is also determined.
• These systems require appropriate dilution of semen
samples before filling of one or more commercially
supplied disposable counting chambers with a depth of 20
microns.
• A counting chamber is placed under a microscope and up
to 7 microscope fields are analyzed in just seconds per
field.
• Data can be stored and provided on customized printed
forms.
Sperm Morphology Evaluation

• Sperm cells are translucent when observed with

bright field microscopy; therefore, phase contrast
microscopy or the use of sperm stains are needed for
analysis of sperm morphology.

• Eosin-nigrosin stain is commonly used as a

"live/dead" stain because in addition to providing
background-contrast for sperm cells with the
nigrosin component.

• Sperm membrane penetration by eosin, or lack

thereof, is an indicator of sperm membrane integrity
and thus of sperm viability.
 Technique:
• Put a glass slide on a warming plate (37°C) for 30 - 60 seconds.
• Put a 5 - 6 mm droplet of eosin-nigrosin stain at one end of the
glass slide.
• Put a droplet of semen beside the droplet of stain
• The droplet’s size depends on the density of the semen

• Mix the stain and the semen on the slide.

• Spread the mixture slowly on the
slide from one end to the other
using the edge of another glass
slide.
• Dry the smear quickly by blowing
air over it.
• Perform the sperm morphology
evaluation at 1000 x magnification
using immersion oil, counting at
least 100 sperm per sample.
• If a high number of abnormalities
are observed, a count of 300 or
more sperm will give a more
accurate differential count.
Other staining techniques

1. Toluidine blue stain

2. Eosin-opal blue stain
3. Eosin fast green stain
4. Eosin-Aniline blue stain
5. Rose bengal stain
Double headed sperm Misshapen head along with 4
normal sperm

Pyriform (pear-shaped) head

Elongated head and bent, abnormal midpiece
Proximal droplet Distal droplet

Detached head Coiled tail

 Examination of semen for Genital Infections
Microorganism like Mycobacterium tuberculosis,
Brucella abortus, Corynebacterium pyogenes,
Staphylococci and Streptococci can be demonstrated
by culture methods.

Campylobacter fetus :
Primary cultures must be incubated under 10% CO2
or in a gas mixture containing not more than 5%
oxygen.
The commonly employed method for diagnosis of
vibriosis is to test presence of antibodies for C. fetus
in the vagina mucus
 Brucellosis:
Agglutination test on seminal plasma can be conducted
for detection of antibodies against Brucella abortus by
the conventional tube method
Viral infectious agents:
Estimated by tissue culture technique

Trichomonas fetus:
Detected by microscopic observation of moving protozoa.
Also cultures from preputial washings can be obtained.