Semen contains spermatozoa and seminal plasma secreted from glands. Semen stains may be examined in criminal cases like rape. Samples are collected without heat and packaged carefully. Screening tests examine color, fluorescence under UV light, and chemical tests for choline or spermine. Confirmatory tests identify intact spermatozoa under microscopy or seminal markers like acid phosphatase. Motility can determine timing of deposition. Species origin is confirmed by precipitin tests, LDH patterns, or presence of human-specific Y bodies in sperm.

2. Semen serology
Dr Hasnat

3. Seminal Stains
Introduction
• Semen is the fluid discharged from the penis during
ejaculation, usually at the time of orgasm. Like blood, semen consists of two compartments:
1, the cellular compartment (spermatozoa)
2, noncellular compartment (seminal plasma) which is secreted from the
prostate gland, seminal vesicles, Cowper’s glands and
the glands of Littre.

4. • The normal quantity of seminal fluid in a single emission is 2-5 ml and
contains about 60-150 million sperms/ml of which 90% are motile at time
of ejaculation.
• Spermatozoa constitute about 10% of the volume of the semen which
contains water and small amounts of salt, protein and fructose.
• Prostatic secretions in humans contain high levels of citric acid, acid
phosphatase and zinc. The secretion is alkaline with a pH of 7.4.

5. Purpose of Seminal Identification
• Seminal stains may have to be detected/examined in
criminal and civil cases.
Criminal cases Civil cases
• Rape/attempted rape • Disputed paternity
• Sodomy • Legitimacy
• Bestiality • Artificial insemination
• Sexual murder of the • Divorce
female • Compensation on grounds of
acquired sterility/failure of vasectomy cases

6. Collection of Material
• The stains are usually found on the clothing, but may be found on the
person of either the victim or the accused. They may also be found on
bedclothes, furniture, vehicles, carpet, floor or grass, where the
offence was committed or any item the victim may have used to clean up after
the assault (tissue or washcloth). Seminal stains have to be differentiated from
those due to starch, pus, leucorrhoeal discharge
and egg albumen.

7. Before proceeding with the examination, stains may have to be collected and preserved
from different sources:
• i. Clothing: Portion of cloth with the stain is cut, dried in shade (not heated) and
preserved.
• ii. Vaginal fluid: Fluid from the vagina is collected with a pipette or vaginal washing
is done which is concentrated by centrifugation. Swabs are taken with sterile gauze
and smears are prepared on sterile slides.

8. • iii. Dried stains on other parts of body: Dried seminal
fluid on the perineum or thighs is collected with a
wet swab.
• iv. Matted pubic hair: It is plucked/cut and placed
in a small container.
• v. Stains on smooth surface: These are gently scraped
off into a glass container and preserved.

9. • Samples must be packed in containers that allow air circulation; never in plastic bags
or sealed nonporous tubes, jars or boxes. Chain of custody must be documented
and adhered to the prevailing polices.
Semen samples may contain dangerous infectious agents and should be handles as bio - hazard
– human immunodeficiency virus (HIV),
– hepatitis viruses
– herpes simplex virus

10. Examination of Seminal Stains
• Evidence material for analysis of possible semen consists
of swabs taken from various locations on or in the victim
and objects that may contain semen.

11. Screening Tests
• Physical Examination
When fresh, semen is a whitish or yellowish-white in color, slightly viscous, jelly-like, sticky
and has a characteristic odor. On standing, viscosity is lost due to prostatic fibrolysin and it
becomes thin.
Dried seminal stains on clothes are grayish-white or yellowish-gray color, show an irregular
outline and starchy hard in feeling. When examined under filtered UV light, they fluoresce with
a bluish-white color (due to choline in semen) which is not specific, as other albuminous
materials, such as nasal or leucorrhoeal discharges and detergents also fluoresce.
A fresh stain on a non-absorbent material appears translucent. After a month, it becomes
yellow to brown.

12. Presumptive Chemical Examination
• Presumptive Chemical Examination
Presumptive tests for semen are based on colorimetry and are qualitative in nature.
Positive presumptive tests must be followed by a confirmatory test, such as
microscopic examination, quantitative acid phosphatase test or detection of p30.
• i. Florence test: The stain is extracted, dried on a glass slide and covered with a
coverslip and a drop of Florence solution (8% w/v of iodine in water containing
5% w/v of potassium iodide) is allowed to run under the coverslip.

13. • If semen is present, dark brown rhombic crystals resembling hemin (but larger)
arranged in clusters or rosettes of choline periodide appear immediately (Fig.
31.1).2 Choline originates from the seminal vesicles. A positive test is not proof of
seminal fluid, but confirms the presence of some vegetable or animal substance. A
negative reaction proves that the stain is not semen, but may occur if choline
content is low or the stain is decomposed.
• ii. Barberio's test: A saturated aqueous or alcoholic solution of picric acid when
added to dried stain extract on a glass slide covered with a coverslip, produces yellow
needle-shaped crystals of spermine picrate (Fig. 31.2). The reaction depends on the
presence of spermine from prostatic secretions.3,4 This test is positive without the
presence of spermatozoa.

14. • iii. Brentamine fast blue test
It is the most common presumptive test for seminal acid phosphatase. Acid phosphatase activity is 500-
1000 times greater in human semen than in any other bodily fluid. An enzyme substrate, sodium -
naphthyl
phosphate is converted to sodium phosphate and naphthol by the acid phosphatase enzyme in the
semen and a coupled reaction with bentamine fast blue dye takes place, forming a purple color.
• It can produce false positives because similar enzyme activity is found in other body fluids (e.g.
vaginal secretions and fecal stains), human red cells, semen of higher apes as well as in presence of
fungi, bacteria and even plants (juice of cauliflower). Moreover, pregnancy, menstruation, bacterial
vaginosis may also elevate its level.
• Dried and old seminal stains which have not undergone putrefaction give positive reaction.

15. • Barberio’s test (spermine picrate crystals)

16. Confirmatory Tests
• Confirmatory testing involves solubilization of sample followed by centrifugation which
yields a supernatant and a cell pellet. The cell pellet is used to detect spermatozoa and for
DNA analysis, whereas the supernatant is useful to detect noncellular markers when sperms are
not detected and for grouping or genetic profiling.
• Sometimes, the sample is contaminated by other bodily fluids (saliva, vaginal secretions),
epithelial cells, cellular debris wherein selective degradation may be done by treating the cell
extract with a mixture of proteinase K and sodium dodecyl sulfate before staining and
microscopic examination.
• Most commonly used confirmatory test for semen is visualization of one or more intact
spermatozoa after staining with dyes such as hematoxylin and eosin and ‘Christmas tree’
stain.

17. Microscopic Examination

18. morphology of spermatozoa

19. Motility of Sperms
• At room temperature, motility depends on time elapsed since ejaculation.
• At body temperature (in living victims), sperms retains full motility in vagina
between 6-12 h. The sperms remain motile in the uterine cavity for 3-7 days.
Later, the sperms disintegrate into head and tails which may be recovered
from the vagina upto 7-10 days and 12-14 days in the cervix and uterus.
• Complete motile sperms may be seen upto 28 h in vagina after ejaculation
(non-motile sperms may be found upto 10 days).

20. Non-cellular Semen Markers
• Markers are specific and unique to seminal plasma but independent of
spermatozoa. The two most commonly employed constituents are acid
phosphatase and the prostate-specific glycoprotein p30 (PSA).These tests are
conclusive even in the absence of demonstrable sperms, azoospermia or
vasectomized individuals.

21. Identification of Species Origin
• Confirmation of species is done by precipitin test. Specific anti-human-semen
serum may be used in place of anti-human serum which is commonly used.
• LDH isoenzyme pattern may be used for detection of human origin of
semen as it is different in animals.
• Detection of Y bodies in spermatozoa heads using fluorescent microscope
which is not seen in animals.