Analysis of Semen

Analysis of Semen

  • Course Code: FRSC370 F24

  • Associated Readings: Li, Chapter 14

Relevance of Semen in Forensics

  • Most often connected to sexual assaults.

  • Also relevant in cases of homicide, harassment, and stalking.

  • Types of evidence that can be collected include:

    • Body or orifice swabs

    • Condoms

    • Clothing

    • Bedding

Composition of Semen

  • Average ejaculate volume: 2-5 mL.

  • Sperm count: 10^7 - 10^8 spermatozoa per ejaculate.

  • Neutral pH level: 7-7.5.

  • Fluid composition sources:

    • 60% from seminal vesicles

    • 30% from prostate

    • 5% from epididymis

    • 5% from bulbourethral (Cowper’s gland)

Anatomy of the Male Reproductive System

  • Key structures involved in semen production:

    • Bladder

    • Ejaculatory duct

    • Prostatic gland

    • Seminal vesicle gland

    • Bulbourethral gland

    • Testicular Structures: Epididymis, seminiferous tubules, and external urethral orifice.

Prostatic Secretions

  • Key components include:

    • Acid Phosphatase (AP)

    • P30 (Prostate Specific Antigen)

    • Citric acid, enzymes for protein digestion (e.g., fibrolysin).

    • Ions: Ca2+, Na+, Zn2+, K+.

Seminal Vesicle Secretions

  • Important chemicals:

    • Fructose (energy source for sperm)

    • Ions, citric acid, amino acids, and prostaglandins (hormones).

    • Semenogelin/Seminal Vesicle-Specific Antigen (SVSA).

Bulbourethral Secretions

  • Primary function: lubrication.

  • Also helps remove urine from the tract and sets a suitable pH for spermatozoa.

Epididymis Secretions

  • Spermatogenesis begins in the testes:

    • Process: spermatogonia → spermatids → spermatozoa.

    • Mature spermatozoa are stored in the epididymis for about 3 months.

Terminology Related to Spermatozoa

  • Singular: spermatozoan.

  • Plural: spermatozoa.

  • Conditions:

    • Oligospermia: low sperm count.

    • Azoospermia: no sperm production.

    • Vasectomy: surgical removal of part of the ductus deferens, affecting sperm inclusion in ejaculate.

Important Concepts Around Spermatozoa

  • Spermatozoa are present only in semen.

  • They lack many organelles found in other cells.

  • Distinct morphology: Over 60% should have normal morphology in a healthy male.

Morphological Characteristics of Spermatozoa

  • Head: densely packed nucleus with acrosomal cap and enzymes necessary for fertilization.

  • Midpiece: contains mitochondria for energy.

  • Tail: responsible for motility.

Forensic Analysis of Semen

  • Methods for identification include:

    • Locating stains on evidence using alternate light sources (450-495 nm excitation wavelength).

    • Acid Phosphatase assays as preliminary indicators.

Acid Phosphatase (AP)

  • Group of enzymes that hydrolyze phosphate groups:

    • Optimal activity at acidic pH (seminal pH 7-7.5).

  • Prostatic acid phosphatase is a dominant source in semen.

Importance of Analyzing Acid Phosphatase

  • Levels unaffected by vasectomy, oligospermia, or azoospermia.

  • Activity detectable for up to 50 years with proper storage.

Conducting Acid Phosphatase Assays

  • Methods include colorimetric and fluorometric tests:

    • Indicate the presence of semen but are not specific.

Increasing Specificity in AP Assays

  1. Use faster hydrolyzing substrates for prostatic AP.

  2. Remove some sample with water for testing.

  3. Employ inhibitors during testing.

  4. Use gel electrophoresis for distinction between vaginal and seminal AP.

Practical Application: Acid Phosphatase Assays

  • Utilizing substrates such as ⍺-naphthylphosphate for detection.

  • Hydrolysis produces colorimetric results indicating presence.

Semenogelin

  • Two major types: Semenogelin I (Sg I) and Semenogelin II (Sg II).

  • Functions to form a coagulum that protects sperm cells.

  • Coagulum is liquefied after a few minutes, allowing for sperm motility.

  • Present in several tissues but not urine or other body fluids, which impacts its use in identification.

P30 and Its Implications

  • A protein hydrolyzing Semenogelin, detectable for long periods post-deposit.

  • Not exclusive to semen; found in other fluids, thus a positive test indicates, but does not confirm, semen.

Spermatozoa Identification

  • Spermatozoa must be correctly identified through microscopic examination.

  • Key morphological criteria include:

    1. Head size and shape.

    2. Presence of acrosomal cap.

    3. Midpiece and tail structure.

  • Lack of tails (common in casework) is noted; identification relies on head morphology.

Advances in Spermatozoa Analysis

  • Alternative methods include:

    • SPERM HY-LITER: fluorescence labeling for sperm identification.

    • Laser Capture Microdissection: isolates spermatozoa from other cell types.

    • RNA-based assays: target mRNA specific to sperm cells.

Recap of Forensic Laboratory Techniques

  1. Use ALS to locate stains when necessary.

  2. Apply Acid Phosphatase tests (quick, easy).

  3. Use P30 for less demanding tests.

  4. Conduct KPIC microscopic tests for definitive identification of sperm.