Semen As A Forensic Evidence

Semen is a thick, yellowish white, glairy, opalescent, secretion having a characteristic odour known as seminal odor.It is also called seminal fluid, fluid that is ejaculated from the male reproductive tract and that contains sperm cells, which are capable of fertilizing the female eggs.

Semen also contains other liquids, known as seminal plasma, which help to keep the sperm cells viable. The total volume of semen for each ejaculation of a human male averages between 2 and 5 ml. Each ejaculation contains normally 200 to 300 million sperm. The pH of semen(also referred to as seminal fluid) is slightly alkaline, ranging between pH 7.2 and 7.4.

COMPOSITION OF SEMEN

Several components including sperm cells, enzymes, sugars, minerals, organic chemicals and vitamins composes semen.

Four glands contribute their secretions to the seminal fluid, viz., Testes, Seminal Vesicle, Prostate and Bulbourethral gland.

—The seminal vesicles produce a yellowish viscous fluid rich in fructose and other substances that makes up about 70% of human semen.

—The prostatic secretion, influenced by dihydrotestosterone, is a whitish (sometimes clear), thin fluid containing proteolytic enzymes, citric acid, acid phosphatase and lipids.

— The bulbourethral glands secrete a clear secretion into the lumen of the urethra to lubricate it.

The three important components of Semen are produced by two organs.

1.Testes produce spermatozoa that can be considered as confirmatory evidence for the presence of semen.

2.Prostate, on the contrary, produces two very important proteins, namely, Acid phosphatase enzyme and prostate specific antigen.

SPERMATOZOA

A normal spermatozoa consists of three main parts, namely, Head, middle part and tail. The tip of the sperm cell is known as acrosomal cap or acrosomal vesicle. Acrosomal vesicle containing hydrolytic enzyme facilitate the penetration of sperm into the egg.

The head also contains DNA inside the nucleus.
The mid-piece of sperm contains several mitochondria.
The last part is the tail called flagellum which is responsible for its motility.

As with other forensic exhibits, the investigation of semen is also carried out in a specific way using various tests.

First, the screening tests are performed in order to identify whether the questioned stain is semen or not. Once the preliminary screening tests are positive, a more detailed confirmatory analysis is carried out to establish that the stain is indeed semen.

Further exhaustive analysis of seminal stains is performed in order to individualize the stain to a particular individual.

The identification of one or more sperms is a conclusive proof of the presence of semen.However, there are difficulties in the identification of sperms due to several reasons.

1. If a criminal is oligospermic, then his ejaculate may contain only a few sperms.

2. Another reason could be the condition of Aspermia where no sperms are produced by the seminiferous tubules of the testes.

3. If the criminal has undergone Vasectomy which is the surgical procedure for male sterilization to prevent sperm from entering into the seminal stream.

4. Use of a contraceptives e.g. Condoms

5. In addition to these, there may be other reasons such as sperms trapped in the clothing not extracted into the testing extracts.

6. Disintegration of sperms can also take place during handling of the material.

Finding sperm cells in body cavities of a victim of sexual crime is the utmost priority for obvious reasons. Several research findings indicate that motile and non-motile sperms can be obtained at different duration in different body cavities.

The table below shows the approximate duration for which motile and non-motile sperms can be found in the body cavities of a victim of sexual crime.

COLLECTION & PRESERVATION OF SEMEN

Collection of semen stain has much precaution. Semen converts in to brittle after drying. So if cloths or other articles are not handled properly or folded it then spermatozoa are breakdown in to pieces.

Preservation of semen stain always performed after complete dryness of stain. If stain is wet then bacterial growth is started and putrefaction may occur due to presence of protein in semen.
Any infection in testis due to presence of some bacteria also secretes liquid like seminal stain from testis which creates doubt about semen.
It is always preserved in air bag not in airtight bag or plastic bag.
If stain found on immovable or small article, then preserved whole article or cloth and sent sample to FSL.
If stain present on immovable article then sharp knife or scalpel is used to scratch the stain and packed in clean glass bottle.
Seminal stain may be present on pubic hair so it is also preserved in bag or clean plastic tube.
If seminal stain present on any body part then take cotton swab is taken, dipped in saline water. Then cotton applies on body and prepare cotton swab. After that drying the cotton swab and preserved in bag.
Never touch sample with bare hand because it can contaminate and spoil the specimen.
In fresh seminal stain, live spermatozoa are present so keep safe the cloth and send properly to FSL.

LOCATION

• Stains will usually be found on the clothing of the victim or the perpetrator , particularly the undergarments. stains may also be found on bedding, mattresses, car seats etc.

• Clothing and articles suspected to contain seminal stains should be seized quickly as possible because an attempt may be made to remove the traces by washing.

• Often, seminal stains are readily visible on a fabric because they exhibit a stiff, crusty appearance. Fresh semen has a characteristic odour.

• The female victim of an assault may have a considerable quantity of semen in or around the vagina. In such cases vaginal swabs should be obtained through the medical examiner.

• Controlled samples should be collected from both the suspect and the victim.

FORENSIC EXAMINATION OF SEMEN

• Visual & Physical examination

Color: Thick, yellowish white, glairy, opalescent, secretion having a characteristic odor known as seminal odor.

Texture: On touch, seminal stains are starchy.

Appearance: Garments sent for forensic examination are usually dirty having variety of stains, in natural light some stains are reddish coloured, while others are brown, yellow or faint grey in colour. These are often mixed with stains of blood vaginal discharge, urine and semen, so as to restrict the investigation to seminal stains only, preliminary examination is done under filtered UV light.

• Microscopic examination

Semen can be conclusively identified by the presence of spermatozoa in the stain. When stain is subjected to a microscopic examination, spermatozoa can be identified as having been derived from semen. Spermatozoa contain gram positive protein, the presence of spermatozoa will be confirmed by microscopic appearance of purple bodies.

Procedure :

Stain should be washed in acidulated water in a test tube.
The test tube should be subjected to ultrasonic oscillation for the separation of spermatozoa from dried stain.
Then,it should be fixed with dil. sulphuric acid.
Appearance of purple bodies under the microscope can be observed.

PRESUMPTIVE TEST OF SEMEN

• Acid Phosphatase Test

Acid phosphatase is an enzyme present in seminal fluid in concentration far more than that found in any other body fluid. An extract of the suspected stain is treated with an acidic solution of alpha naphthyl phosphaye followed by bentamine Fast Blue B dye. Appearance of intense purple color will determine the presence of acid phosphatase enzyme in the semen stain.

PROCEDURE

Reagent Preparations:

Buffer

Glacial Acetic acid- 1ml
Sodium acetate anhydrous-2gm
Distilled water -100ml

Step 1 Reagent

Buffer – 50ml

Sodium alpha-naphthyl Phosphate, 0.25% (w/v) – 126gm

Step 2 Reagent

Buffer- 50ml

Naphthanil diazo blue B, 0.5% (w/v) – 250gm
Step 1 Reagent and Step 2 Reagent can be made up in bulk and aliquoted into test tubes and frozen.When needed, one tube of each reagent can be thawed under warm running water for use.

Procedure:

Place a small piece (2 x 2 mm) of suspected seminal stain

1. Stain material on Whattman filter paper or other suitable test paper. Use proper standards and controls including positive, negative and unstained controls.

2. Add 1-2 drops of Step 1 Reagent and allow to react for 30 seconds. (No colour should develop at this stage)

3. Add 1drop of Step 2 Reagent. Record the result after 10 seconds.

4. A positive reaction is recorded upon rapid development of a purple colour, which is indicative of semen.

This is not a confirmatory test for semen.

NOTE: Some vegetable juices , contraceptive creams, vaginal secretions do give a positive response to this test but none of these react with speed of seminal fluid. The test is of immense value when an individual has undergone vasectomy operation or shows no sperms at all in his seminal fluid. Acid phosphatase present in semen and vaginal fluid can be discriminated by the electrophoresis method.

• THIN LAYER CHROMATOGRAPHY

This method is used for the detection of choline and spermine. In TLC, it is possible to detect choline and spermine simultaneously in seminal stain. It has been possible to detect choline and spermine in old seminal stains events after the lapse of 5 years by this method. There are 2 types of reagents used in TLC method they are

—Dragendroff’s reagent

—Potassium iodoplatinato reagent

• Examination through UV light

The alternative light source is used to detect semen if not visible . Under UV light, semen fluoresces making it visible to investigators to collect samples from a crime scene. Seminal stains give bluish white fluroscence.

The fabric on which stain is found was classified into three groups:

—Bright (white, bright blue and pink),

—Medium (blue, green, yellow, orange, red) and

—Dark (black, brown).

CONFIRMATORY TEST FOR SEMEN

Christmas Tree Test

The sample is extracted with sterile water in order to make a wet mount on a microscope slide.
The sample is then heat-fixed to the slide and stained with Nuclear Fast Red for 15 minutes, then rinsed with deionized water.
Next, a green stain is applied for 10 seconds, then rinsed with ethanol.
The slide is placed under a compound light microscope for sperm observation.
If sperm are present, following observations will appear :
tip of the head – pink
bottom of the head – dark red
middle piece – blue
tail – yellowish green
stem cells – bluish green

Cross Over Electrophoresis

Seminal Material can be identified by demonstrating the presence of p30, a semen specific protein. One method of doing this is Cross-Over Electrophoresis. An extract of the suspected stain is placed in the cathode well of a gel plate and anti p30 is placed in the anode well. Electrophoresis is commenced for 20 minutes at 200 volts, forcing two components together. When the antigen (p30) meets the antibody, a precipitin band is formed. The presence of a precipitin band within an extract of an unknown stain proves that the stain contains seminal material,anti-p30.

PROCEDURE

Plate preparation –

Clean 1” x 3” or 2” x 3” microscope slides with detergent. Rinse and polish dry. Slides should be consecutively numbered.

Pre-coating Plates:

Add 1 gm of 0.2% stock agarose to 10 ml distilled water and boil until dissolved.Paint a thin coating of this solution onto the glass slides with a soft brush. Allow to air dry.

Coating Plate with Gel:

Pour the liquefied 1% agarose onto the slide. Surface tension and pre-coating of slide will prevent the liquid from overflowing the edges. A smooth level surface is formed. Allow it to cool and harden.

CRYSTAL TESTS FOR SEMEN

Florence test :

This test was discovered by Dr.Florence in the year 1886. When Florence reagent (PotassiumIodide+Iodine+Water) is applied to the slide it produces rhomboidal shape dark crystals of choline periodide. Similarly, any tissue or biological material containing sufficient high choline concentration would give positive Florence Test.

Barberio’s test

Barberio’s test was invented by Barberio in the year 1905. When the questioned stain is allowed to react with picric acid it leads to the formation of yellow needle shaped spermine picrate crystals, including the presence of seminal stain.

Identification of blood group in semen

semen and other body fluids can be used to determine the ABO blood group of the individual from whom they originated. About 80% of the population is secretors who secretes blood group substances in their body fluids. Both saliva and semen are rich sources of secreted blood group substances.Polymorphic enzymes have also been detected in semen and other body fluids.

FORENSIC IMPORTANCE OF SEMEN AS AN EVIDENCE

1-Semen analysis is an important laboratory test and should be thought of in the same way as any other diagnostic assay is used in determining treatment plans for infertility.

2-Semen analysis is very important in assault cases as it is very useful for detection of semen in cloths.

3-Isolation and identification of seminal stain found on various suspected area such as cloths, bed sheet, pillow, blanket, etc.

4-Analysis of various protein present in semen or seminal stain.

5-Seminal stains are examined generally in rape cases, Sodomy, Bestiality, Buccal coitus and in cases of Sexual perversions.

6-Detection of sperm cell in Vagina and other body parts.

7 -It is useful because of the relative quantity of spermatozoa and epithelial cells can be assessed. This determination becomes important during subsequent DNA analysis because spermatozoa contain male DNA while most epithelial cells in a male–female sexual assault will contain female DNA from the complainant.