FORENSIC CHEMISTRY & TOXICOLOGY

SEMEN AND SEMINAL FLUID

MEDICO-LEGAL SIGNIFICANCE OF DETECTION OF SPERM
• Rape - Sexual assault
• Sodomy (Anal Intercourse)
• Bestiality (Sexual intercourse by a human being with lower animals like dogs, cow, sheep, etc.)
• In case of false accusation by a woman - need to proof if there is a presence of semen or sperm.
• Incest (Sexual intercourse within blood relations) -
• Sexual Murders - seriaal rape.

SEMEN: A penile ejaculate consisting of a thick, opaque, yellowish-white, viscid fluid containing spermatozoa.
(Vuscid means viscous)
WHITE- sexually active.

Semen is a mixture produced by the secretions of the:

a. Testes & Epididymis
b. Seminal Vesicles
c. Prostate
d. Bulbourethral Glands

Three 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 of male

a. Seminiferous tubules – produces
spermatozoa.
b. Interstitial Cells – produces male
hormones. (testosterone)

Epididymis: Tiny ducts, coiled tube

Stores sperm cells released in ejaculation.

Vas Deferens: Long sperm ducts

Passes through the inguinal canal
Arises from the bladder

Urethra: An excretory and reproductive organ (The urethra serves as passage)

Urine passes during excretion
Semen passes during ejaculation

Penis: Muscular organ

Transfers semen in the vagina
NATURE OF SEMEN
• Human body fluid present in human males
• It is viscid mucilaginous fluid
• Faint yellow color (caused by Flavin, Flavin is protein t hat causes that is present in production of semen.)
• Characteristic odor called Seminal Odor (smells like bleach).
• Volume: about 3mL per ejaculate
• Number of Spermatozoa: 50,000,000 – 350,000,000 Per mL

COMPONENTS OF SEMEN
Semen consists of the following:
a. Spermatozoa (10%)
b. Seminal Plasma (90%) - WHERE THE SPERM COULD SWIM
c. Epithelial Cell (<1%) – skin cells

SPERMATOZOA – 10%

Spermatogenesis (“genesis” – creation/formation of sperm)

- Production and development of spermatozoa in the testEs.
- A mature male sex germ cell with the capacity to fertilize a mature ovum.

Morphology of Spermatozoa
a. Total length: About 50 microns
b. Consists of:
Head: Flat, oval shaped Tail: For the movement of the sperm
Contains genes Mitochondria supplies energy for
movement
Fructose: Energy source of spermatozoa
c. Spermatozoa contains:
Lipids
Proteins: Protamine & histones etc.
Enzymes – Dehydrogenase & Transaminases

***Spermatid: an immature sperm cell

Terminologies to describe Spermatozoa
Azoospermia – Absence of spermatozoa in semen.
Oligospermia – Fewer than 20 million/mL of sperm cells
Necrospermia – Dead sperm cells

SEMINAL PLASMA – (90%)

Mixture of secretion
Derived from the MALE ACCESSORY REPRODUCTIVE ORGANS:
• Epididymis
• Seminal Vesicles
• Prostate
• Vasa-differentia (Vas Deferens)
• Bulbourethral Gland
• Urethral Gland

Composition of Seminal Plasma

• CITRIC ACID ENZYMES:
• ASCORBIC ACID FIBRINOGENASE
• LACTIC ACID DIASTASE
• FRUCTOSE PHOSPHATASE
• POTASSIUM GLYSIDASES
• CHOLINE PHOSPHATE MANNOSIDASES
• PROTEASES GLUCOSIDASES
• FREE AMINO ACIDS GIVCOURIDASES
• ERGOTHIONENE BLOOD GROUP FACTORS
• ZINC
• CALCIUM
• SPERMINE
• LIPIDS

WHERE TO LOOK FOR SEMINAL STAINS?

1. Clothes: Underwear, Bed Sheet, Carpet, Towel, Pillow Cover
2. Body: Perineum, Thigh, Vagina, Pubic Hair
3. Scene of the Crime: On the floor, Grass, Vehicle
4. From the Accused: Flies and handkerchief

5. In Cases of Bestiality: Vaginal and rectal swab

Animal secretions or Feces from Garments or Body Parts
** Semen on white cloth appears slightly yellowish. It is almost invisible on colored cloth.

METHODS OF COLLECTION
• Handling of articles bearing stains should be done very carefully to avoid damage to spermatozoa.
• Do not apply heat or expose to direct sunlight.
• Do not 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 °C)
PROBLEMS IN HANDLING SEMEN
• Individualization: Determined using DNA Typing
• Time: 2 – 3 weeks (pale yellow to brown) Fresh
semen has complete parts Old semen
disintegrate
• 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 a few months in dry and cool climate
Sperm cells are detected 5 – 10 days in vaginal swabs but there can be a decrease in number

METHODS APPLIED FOR THE DETECTION OF SEMINAL STAINS

a. Physical Examination
b. Chemical Examination
c. Microscopic Examination
d. Biological Tests

A. PHYSICAL EXAMINATION
• Physical Identification: Visual Examination of Seminal Stain (Using UV light)
• Translucent or opaque spots
• Yellowish tint and darker border
• Stiffness due to dried semen
• Fluorescent under ultraviolet (UV) light
B. CHEMICAL METHOD
• pH = 7.45 (Slightly Alkaline)
• Chemical tests used to detect Seminal Stains:
Based on the chemical composition of the semen

1. Florence Test
Basis: Choline is detected (It is the basis of florence because choline have higher level in seminal
fluid)
Result: Dark brown crystals of choline periodide, generally needle shaped, formed within a few
minutes

Florence Reagent = 8% sol’n of Iodine in H2O containing 5%

Potassium iodide

*Non-specific & false negative results are common

2. Barberio’s Test
Basis: Spermine is detected (Spermin has
Levels in seminal fluid.
Result: Crystals of sperm in picrate Needle
shaped, rhombic and of yellow color

Alcoholic solutiion of Picric Acid – Needle

shape crystal

*Non-specific & lack of reproducibility

(meaning when first tested a specimen and
positive and when you repeat it, the
re is a possibility that they have different
result.)

Note:
FLORENCE AND BARBERIO’S TESTS HAVE NOT BEEN
ACCEPTED UNIVERSALLY
 3. Acid Phosphatase (Spot)
Test Sensitive (Acid
phospatase enzyme is only
found in the seminal fluid.)
-This is more suggested test.
Can detect invisible stains
Disadvantage: Water soluble (washing removes AP activities)
Basis: Acid Phosphatase Enzyme which has high levels in seminal fluid

Result: Purple Color (color change within 2 minutes)

**False Positives of AP Test: (AP test is just

preliminary test) Vegetable extracts
Vaginal Fluid: - pink/brown (cauliflower, sprouts)
- pink
- bacteria (blue/gray)

Feces and urine Tea

- purple -strong purple

Perfumes
- strong purple

4. Lactate Dehydrogenase (LDH) Isoenzyme Method

Specific biochemical detection of spermatozoa in Semen in the presence of Vaginal Fluid, Blood,
Nasal Secretion, Saliva and Urine (Can detect semen even if these fluidas are present)
Basis: Detection of Spermatozoa
Result: Electrophoresis is carried out Isoenzyme
bands are revealed by staining
5. Acid Phosphatase Isoenzyme Method (confirmatory test
Specific biochemical detection of spermatozoa in Semen in the presence of Vaginal Fluid, Blood,
Nasal Secretion, Saliva and Urine
Basis: Detection of Spermatozoa
Result: Phosphate reagent enables the seminal acid phosphates to be distinguished from that of other
substances (e.g. vaginal secretions)
Advantages:
- Isoenzyme pattern of human is different from that of animal
- Positive results are obtained in large number of cases.
- Can differentiate from vaginal secretions on pattern of bands

6. Creatinine Phosphokinase (presence of enzymes)

Creatinine Phosphokinase enzyme is doubled in sperm compared to other body parts. Basis: Detection
of Creatinine Phosphokinase

- Enzyme is stable and can be demonstrated in old status of six months.

Because the creatine phosphokinase enzyme is stable and does not
disentegrate easily.

7. Choline and Spermine Test

Basis: Combination of choline and spermine is present only in semen
Why is it acceptable? - Because, other test separately detects the choline and spermine, And It is only
present in semen.) If both are detected, meaning it is positive.
High levels of choline and spermine in seminal fluid
Procedure: Liquid semen & Dried Semen
By the use of machine called Thin Layer Chromatography

8. Puramen Test:
Basis: Presence of Spermine in the prostate fluid
**Spermine causes the odor of Semen
Result: Small cross-like crystals microscopically Yellow
precipitate of Spermine Flavinate

9. p30 Test (Kit test)

• The p30 test is a presumptive test for the presence of semen &/or seminal fluid.
• Presumptive test for the presence of human prostate-specific p30 protein found in seminal fluid.
• Works through an antigen-antibody reaction.
• Antibodies are present on the p30 card and if the antigen is present (p30 protein) in a sample, then a
reaction occurs on the test site (T) of the card.
• The antigens and antibodies bind to each other and form a lattice (chain) which causes a pink
dye band to be visible.
• The control region of the test strip should always have a reaction, and the pink dye band should be
visible on both positive and negative results.

- C- means control. Meaning the test is negative. If negative only one band, and it is only
present in “Control band” and because there is no band in the “Test” part.

-Positive if there is an antigen present and it will reacts with the test kit.
C. MICROSCOPIC EXAMINATION - more on confirmatory testing
• Based on the MORPHOLOGY of Spermatozoa
(Looking the absence or presence of the spermatozoa)
1. Microscopic Examination Of Spermatozoa
• Cloth pieces from different stains are taken in 0.5 mL of 0.01 N HCL in a small test tubes placed in a
beaker containing water.
• After sonification for 5 minutes, the extracts and the cloth pieces are transferred to separate
microscope slides and cloth pieces delicately teased with a needle.
• Threads are removed and residual liquid is gently evaporated to dryness.
• Stain with Haematoxylin and Eosin Stain
• Check slide for presence of spermatozoa.
2. Fluorescence MicrosCopy -(PrESENCE OF FLOURESCE - GLOW)
Based on the principle that Y chromosome is fluorescent to Quinacrine. Basis:
Detection of spermatozoa
Possible to detect both intact spermatozoa as well as disconnected heads.
3. Sperm Hyliter
• Microscopic confirmatory test for semen identification.
• A slide staining technique used to microscopically identify sperm.
• Also works through an antigen-antibody reaction.
• During the staining process, the sperm heads react with an antibody found in the stain. This reaction
causes the sperm heads to fluoresce a bright green color.
• A second stain is added that stains all of the nuclei in a sample. Therefore, when sperm are present in a
sample they will fluoresce green or blue when viewed under two different contrasts on the microscope.
• This fluorescence makes the sperm heads easy to distinguish from the surrounding cells/debris.
This reaction is specific to human sperm heads.
• When identifying sperm heads one looks for the correct fluorescence along with the right size, shape,
and the presence of an acrosome; which is a small unstained part of the sperm head.
 What is viewed under the Sperm Hyliter

4. Christmas Tree Stain

• Another slide staining technique used to microscopically identify sperm.
• Much quicker than Sperm Hyliter.
• The sperm heads stain red and the sperm tails and other cells stain green. When viewed under a
microscope, the sperm heads are fairly easy to spot and differentiate from the surrounding cells.
• Similar to SpermHyliter™, when identifying sperm heads one looks for the right color, size, shape, and
the presence of an acrosome; which is a small unstained part of the sperm head.

D. BIOLOGICAL TEST
1. Precipitin Reaction
Basis: Determines if semen came from humans Uses
human Anti-Sera
Result: Precipitation Reaction
Can you identify the blood type of a person using sperm or semen?
-Yes, we can use the sperm or semen for blood typing. Because in the sperm cells, there is also a
presence of antigens, however the antigens present is not the same with the antigens present in blood
typing. The antigens present is Not Antigens A and B. emaning there is different antigen present in the
sperm.
How? Blood typing detects antigens A and B present in the red blood cells.

**Possible Blood Typing using Sperm: Antigens are present on the membrane of human spermatozoa.

**OTHER TESTS:
(Machine that have high price! Mahal sizz..HAHAHAHHA)
1. Radio – Immunoassay (RIA)
2. Enzyme – Linked Immunoabsorbent Assay (ELISA)
3. Seminal Vesicle – Specific Antigen (SVSA): provides highly sensitive detection of semen.

Note: These tests are costly enough restricting their use for research purposes only.