Prepared by:

Dr. Jun Carino Corpuz

RMT.,RN.,MACT.,US-RN.,PH.D.
DOH Certified Drug Analyst
 MEDICOLEGAL
SIGNIFICANCE OF
DETECTION OF SPERM &
SEMEN
a. Rape

b. Sodomy (Anal intercourse),

c. Bestiality (Sexual intercourse

by a human being with a lower
animals like dogs, calves,
sheep etc.)
d. In case of false Accusation by
a
women
e. Incest (Sexual intercourse in
blood
relation)
f. Sexual Murders.
Definition:
“ It is a penile ejaculate
consisting
of a thick, opaque, yellowish-
white, viscid fluid containing
spermatozoa”
- A mixture produced by
secretions of the:
a. Testes & Epididyis
b. Seminal vesicles
c. Prostate
d. Bulbourethral glands.”
3 Sets of Glands
a. Prostate Gland
- a lobular gland that surrounds
the male urethra, located in front
of the urinary bladder.
b. Bulbourethral/Cowper’s Gland
c. Seminal Vesicles
- Secrete the seminal fluid
Testes/Testicles
- Male gonads or Sex organs
a. Semineferous tubules
- Procudes spermatozoa
b. Interstitial Cells
- Produces male hormone
Epididymis
- Tiny ducts, coiled tube
- Stores sperm cells
- Released in Ejaculation
Vas Deferens
- Long sperm ducts
- Passes through inguial canal
- Arises from the bladder
Urethra
- An excretory and
reproductiveorgan
- Urine passes during excretion
- Semen passes during
ejaculation
Penis
- Muscular organ
- Transfers semen in the vagina
a. Human body fluid present in
human males.
b. It is a viscid mucilaginous
fluid
c. Faint yellow colour/Flavin
d. Characteristic odour called
seminal odour.
e.Volume about 3 ml per
ejaculate.
f. No. of Spermatozoa
- 50,000,000 -350,000,000 per
ml.
- 10% of Seminal Plasma
- Seminal Plasma
- A suspending fluid for
Semen consist of the following:
 1. Spermatozoa (10%)

 2. Seminal Plasma (90%)

 3. Epithelial Cell (< 1%)

Spermatogenesis:
- Production and development
of spermatozoa in the testis
- A mature male sex germ cell
with the capacity to fertilize a
mature ovum.
a. Total length: About 50 Microns.
b. Consists of:
- Head
- Flat, oval shaped
- Nucleus occupies major
portion
- Contains genes
 - Tail
- For the movement of sperm.
- Mitochondria supplies
energy
for movement
c. Fructose
- Energy source of
spermatozoa
d.Spermatozoa contains :
1. Lipid
2. Proteins
- Protamine & histone etc
- Enzymes
- Dehydrogenises &
Transaminases.
Spermatid: an immature sperm cell.
Azoospermia
- Absence of spermatozoa in
semen
Oligospermia
- Fewer than 20 million/mL.
sperm cells
Necrospermia
- Dead sperm cells
- Mixture of secretion
- Derived from the male accessory
reproductive organs
Epididymis
Seminal vesicles
Prostate
Vasa-deferantia,
Bulbourethral
Urethral glands
 Citric Acid  Enzymes :
 Ascorbic Acid, Fibrinogenase,
Lactic Acid, Diastase, Acid &
Fructose, Potassium Alkaline
Choline Phosphate, Phosphatase,
Proteases, free Glysidases,
Amino Acids, Mannosidases
Ergothioniene, Zinc, Glucosidases,
Calcium, Spermine, Givcouridases
Lipids, Blood Group Factors
1. Clothes : Underwear, Bed sheet,
Carpet, Towel, Pillow cover.
2. Body :Perineum, thigh, Vagina &
pubic hair.
3. Seen of crime : On the floor or
grass, vehicle
Accused:
a. Flies and handkerchief
b. Front side of trouser,
pockets
c. Undergarments
d. Front and back flap of t-shirt
e. Penis & Pubic Hair
f. Skin close to penis
Bestiality
a. Vaginal and Rectal Swabs
b. Animal Secretions or Feces
- Garments
- Body parts
White cloth appear slightly
yellowish
On colored cloth , almost invisible
 Handling of articles bearing
stains should be done very
carefully to avoid damage to
spermatozoa
 Do not apply heat or expose to
direct sunlight
 Don’t fold cloth in the region
of the stain
 Vaginal / anal / penile swabs
should be sent along with their
smears on slides.
 Swabs should be taken on
sterile gauze / cloth and their
smears prepared on sterile
slides
 Swabs dispatched in sterile test
tube and slides in clean
wrappers.
 Dried in air at room
temperature (37 degrees
celcius)
a. Individualization
- Determined using DNA Typing
b. Time
- 2 to 3 wks (pale yellow to
brown)
- Fresh semen has complete parts
- Old semen disintegrate
c. Viability
- Survive for few hours in normal
conditions
- Freezing stores and preserves
viability of sperm cells
- Sperm cells die sooner in humid
and warm climate
-
- Sperm cells are identified even
after few months in dry and
cool climate
- Sperm cells are detected 5-10
days in vaginal swabs,
decreased number
Dead Persons
- Detection of sperm cells
depends
on condition the dead body
remained
Methods Applied for Detection
of Seminal Stains

1. Physical Examination
2. Chemical Examination
3. Microscopic Examination
4. Biological Tests
A. Physical Examination
- Visual Examination of Seminal
Stains
a. Translucent or opaque spots
b. Yellowish tint and darker
border
c. Stiffness due to dried semen.
d. Fluorescent under ultraviolet
light.
 B. Chemical Method
a. pH = 7.4 Alkaline

b. Chemical tests used to detect

Seminal Stains
- Based on the chemical
composition of the semen
1. Florence Test – Choline as dark 6. Creatinine in Phosphokinase
brown crystals, needle shaped

2. Barberio Test – Spermine as 7. Ammonium Molybdate Test

needle shaped rhombic crystals (Phosphorus)

3. Acid Phosphatase Test 8. Semen Specific Glycoprotein

(P30 ) Test

4. LDM Isoenzyme Method 9. Enzyme-linked immunosorbent

assay (ELISA), assay, for a seminal
vesicle-specific antigen (SVSA)

5. Acid Phosphatase Isoenzyme 10. Puramen Test – Spermine as

Test cross like crystals
Florence Test
Basis: Choline is detected
Result:
- Dark brown crystals of choline
periodide, generally needle
shaped,
formed with a few minutes.
Comments: Non-specific & false
negative results are common.
 A few drops of watery solution
of the stain is extracted and taken
on a slide and a drop of Florence
reagent (8%) W/V solution of
Iodine in water containing 5%
W/V of Potassium Iodide) is
poured & allowed to mix slowly
under a cover slip.
Barberio’s Test:
Basis: Detection of Spermine
Result:
- Crystals of sperm in picrate
(Needle shaped, rhombic & of
yellow colour).
Comments: Non-specificity and
lack of reproducibility
 Soak a small piece of cloth
believed to be stained with
semen in 1 ml of 12.5% TCA
using a centrifuge tube for an
hour or longer.
 Centrifuge the preparation and
decant the clear liquid.
 Add equal volume of a saturated
picric acid.
Note:
 Florence and Berberio’s tests
have
not been accepted universally.
Acid Phosphatase Spot Test:
Basis: Acid Phosphatase
Enzyme
Modified Fishman and
Lerner’s Method.
Result: Blue Colour Developed
Comments: Semen produces a
very high acid phosphatase
activity as compared with
LDH Isoenzyme Method
Basis: Detection of Spermatozoa
Result:
- Electrophoresis is carried out in
refrigerators for 150 minutes.
- Isoenzyme bands are revealed
by
staining.
Comments:
- Specific biochemical detection
of
spermatozoa in semen in the
presence of Vaginal Fluid,
Blood,
Nasal Secretion, Saliva &
Urine.
- LDH isoenzyme is stable in
Procedure:
Seminal stains are extracted
with 1 ml of water. 0.25 ml of
clear extract is mixed with 0.25
ml of 40% W/V of sucrose. 0.1
ml of this mixture is subjected to
vertical polyacryl amide gel
electrophoresis.
Acid Phosphatase Isoenzyme
Method
Basis: Detection of Spermatozoa
Result:
Phosphate reagent enable the
seminal acid phosphatase to be
distinguished from that of
other substance like vaginal
secretions.
Procedure:
Seminal stains are extracted
with water and is used in
polyacrylmile gel
Electrphoretic method
followed by staining with
methyl belliferyl.
Advantages:
 Isoenzyme pattern of human is
different from that of animal.
 Positive results are obtain in
large number of cases.
 Can differentiate from vaginal
secretions on pattern of bands.
Creatinine Phosphokinase
Bases: Detection of creatinine
phosphokinase.
Normal Seminal Fluid Content:
385 - 14000 U of CPK.
Diagnosis:
>400V of CPK / ml.
Advantage:
- Enzyme is stable & can be
demonstrated in old status of
six
months.
Choline & Spermine Test
Bases:
- Combination of choline &
spermine is present only in
semen.
Procedure:
 Liquid semen & dried seminal
stains
 Thin Layer Chromatographic
Puramen Test
Basis:
- Presence of Spermine in the
prostate fluid
Result: Small cross-like crystals
microscopically
- Yellow precipitate of Spermine
Flavinate
 The Micro Scopic detection of the
Seminal stains is based in
morphology of spermatozoa.
 Cloth pieces from different
stains are taken in 0.5 ml of 0.01
N HCL in small test tubes
placed in a beaker containing
water.
 After sonication for 5 minutes
the extracts and the cloth
pieces are transferred to
separate micro scope slides
and cloth pieces delicately
teased with a needle.
 Threads are removed and the
residual liquid is gently
evaporated to dryness.
 Residue obtained is stained
Haematoxylin and Eosin.
 Check the slide for the
presence of spermatozoa
Fluorescence MicroScopy:
Basis:
- Detection of spermatozoa.
- It is based on the principle
that Y
Chromosome is fluorescent to
Quinacrine.
Comments:
 Possible to detect both intact
spermatozoa as well as the
disconnected heads.
Precipitin Reaction
Basis: Determines if semen came
from humans
- Uses Human Anti sera
Result: Precipitation Reaction
 Positive Florence test is obtained
from other body tissue
containing choline.
 Negative Florence when choline
content is low or in cases where
the stains are decomposed.
 False positive test are not
obtained in cases of vaginal
secretion.
 Acid phosphatase of prostatic
origin is also utilized for the
bio-chemical detection of
semen.
 Vaginal Acid phosphatase
could also be inhibited by 1-
 Other Tests:
a. Radio-Immuno Assay (RIA)
b. Enzyme-Linked
Immunosorbent Assay
(ELISA)
c. Seminal Vesicle-Specific
Antigen (SVSA) provides
highly
sensitive detection of semen
 But these tests are costly
enough restricting their use for
research purpose only.