Seminal fluid Analysis in forensics

Seminal fluid is a complex mixture of secretions from at least four male urogenital glands.  The seminal vesicle gland contributes approximately 60% to this mixture, the prostate gland contributes approximately 30%, and the combined contribution of the epididymis and bulbourethral glands account for the remaining 10%.

Number of Spermatozoa – 50,000 to 3,50,000.

Color – Greyish Yellow / Greyish White.

Odour – seminal odour.

pH – 7.3-7.5.

Appearance – Thick and Semitransparent,

Density – 1.028.

Semen consists of the following

1. Spermatozoa (10%)

2. Seminal Plasma (90%)

3. Epithelial Cell (<1%)

An average male ejaculate measures around 3.5 milliliters.  Each milliliter can contain between 10 and 50 million sperm cells.  This number can be affected by age of the male, medical conditions, genetic background, diet, and other habits such as smoking and illicit drug use, etc.


Some males in the population have a condition known as Oligospermia, which defines an abnormally low sperm count.


Aspermia refers to another condition where the affected male produces no sperm.


Vasectomy, which is a surgical sterilization option, renders the male incapable of producing sperm somewhere between two and four months following that procedure.

Vasectomized, Oligospermic, and Aspermiac males can still produce normal amounts of seminal fluid containing both prostate gland and seminal vesicle secretions.

Semen analysis is very important in sexual assault case.


The Spermatozoa is produced in the testis by the process of spermatogenesis. Spermatozoa contain Lipid, Proteins like Protamine and Histone etc. and Enzymes like Dehydrogenases and Transaminases. The head is flat, oval shaped: 4.6 × 2.6 × 1.6 μ in Length Width Thickness. The nucleus occupies major portion of the head. The tail portion is responsible for the movement of sperm.

Where to look for Seminal Stains

Seen Of Crime:
  • On The Floor
  •  Grass Etc.

Collecting DNA Evidence with Semen

1. Absorb suspected liquid semen on a clean cotton swab. Air-dry the swab and pack in a clean envelope with sealed corners.

2. Submit small and dry suspected semen-stained objects to the laboratory. Pack in clean envelope with sealed corners to prevent stain removal.

3. Documenting the scene by video taping, written notes and Photography.

4. Processing evidence at the scene

5. Completing and recording the scene investigation.

Physical/Visual Test

First step of every examination is visual examination. If the area to be examined and analyzed for semen is larger than an individual swab, investigator or forensic scientist should process visual identification first.

Clothing, undergarments, and bedding can be quickly surveyed for potential semen stains using the naked eye.

Microscopic Examination

Microscopically, detection of spermatozoa is also considered as confirmatory evidence for the presence of semen in a suspected stain. For microscopic examination stained part of fabric should dissolved in acidulated water in a test tube.

Presumptive Tests

Alternate Light Tests

Semen can also be visualized using blue light, ultraviolet light (also known as Wood’s Lamp), or a modern light source such as Crime Scope that is properly configured with optimum wavelength filters.

Under those specialized lights, semen will fluorescence due to the presence of molecules such as Flavin and Choline-conjugated proteins.  The color of this fluorescence will vary from blue to yellow, depending on the light equipment used.

The semen responds to illumination by longer wavelength frequencies of UV light (~350 nm) which is invisible to the human eye.

Factors which affect Fluorescence

There are many molecules (natural and artificial) that will fluorescence in a similar way as semen, and therefore, this detection technique is highly presumptive.

Exposure of the sample to factors such as heat, humidity, oxidizing agents, and microorganisms such as bacteria can affect this fluorescent activity.

Acid Phosphatase Test

The male prostate gland produces a high amount of the enzyme acid phosphatase (AP) in semen.

In the presence of Alpha-Naphthyl acid phosphate and Brentamine Fast Blue, AP will produce a dark purple color in less than a minute. The color will depend on the activity of the enzyme, which can be negatively impacted by the age of the stain and the storage conditions.

The test for AP also known as Brentamine spot test remains highly presumptive due to the fact that other bodily fluids contain detectable levels of this enzyme.

Prostate Specific Antigen

Another presumptive test for semen is the detection of prostate specific antigen (PSA) or the P30 molecule.  Forensic labs utilize a test known as ABA card or P30 test to screen for PSA.

PSA is produced in high amounts by the male prostate gland. However, this antigen can also be found in very small amounts in fecal material and sweat.

Studies have shown PSA can also exist in female urine and breast milk. 

While the PSA test remains a strong test for the presence of male semen, caution is always urged when interpreting positive PSA results which are not confirmed by the actual presence of sperm.

MUP (4-MethylUmbelliferyl Phosphate) Test

•MUP test is also used for detection of seminal stain as preliminary stage.

•This test is more sensitive than acid phosphatase test.

•Acid phosphatase catalyses the removal of phosphate residue on the 4-MethylUmbelliferyl Phosphate which generate fluorescence effect on under UV light.

Confirmatory Tests

The Christmas Tree Stain

The most reliable confirmation for the presence of semen is the positive visual identification of sperm cells (or spermatozoa) using the Christmas tree stain.

Two main reagents are used consecutively to produce this distinctive stain:

1.Picroindigocarmine – Neck and tail portions of the sperm in green and blue.

2.Nuclear Fast Red (also known as Kernechtrot)- Sperm heads in red color and the tips of the heads (acrosomal cap), a pink color.

Although this color pattern seems quite unique and may render sperm cells easily distinguishable under a microscope, sperm cells tend to deteriorate quickly after ejaculation.

RSID (Rapid Stain Identification Series) (Semen Strip Test)

The RSID-Semen test provides sensitivity as well as specificity to human semen.

The RSID-semen test identifies the presence of the seminal vesicle-specific antigen, or semenogelin. 

This test can also identify semen even if the stain was stored under less favorable conditions which have been shown to affect other tests such as the Acid Phosphatase test.

Berberio’s Test

Berberio’s test was invented by Barberio in 1905.

This test is based on Detection of Spermin.

A few drops of picric acid on suspected stain produces crystal of spermin picrate yellow needle shape.

Puramin Test

This test is based on Spermin protein.

Suspected stain treated with Nephtal yellow S which produces Yellow lenticular crystal of Spermin Flavinate.

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  15. American Urological Association: “Vasectomy Guideline (2015).”
  16. University Hospitals: “Semen Analysis.”

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