CHAPTER 10: SEMEN

PHYSIOLOGY

Ejaculatory Ducts

Semen

AUBF

Composed of four fractions contributed by:

o Testes
o Epididymis
o Seminal Vesicles
o Prostate Gland
o Bulbourethral Glands
Composition
o Spermatozoa: 5%
o Seminal Fluid: 60 70%
o Prostate Fluid: 20 30%
o Bulbourethral Glands: 5%

Seminal Fluid

TESTES

Paired glands in the scrotum

Contains SEMINEFEROUS TUBULES: secretion of
sperm
Lower scrotum temperature: optimal for SPERM
DEVELOPMENT

Germ Cells

For the production of spermatozoa

Located: epithelial cells of the seminiferous
tubules

Provide support and nutrients for the germ cells

as they undergo mitosis and meiosis
(spermatogenesis)

EPIDIDYMIS

Where immature (nonmotile) sperm enters

after complete spermatogenesis
Where SPERM MATURES & DEVELOP FLAGELLA
Maturation: 90 days
Storage of sperm cells until ejaculation
o epididymis -> DUCTUS DEFERENS ->
ejaculatory ducts

transport medium for the sperm

contributes 60 70% of fluid
contains FRUCTOSE and FLAVIN
o FRUCTOSE:
metabolized
by
spermatozoa for energy to propel
through female reproductive system
o FLAVIN:
responsible
for
grayappearance of semen
o VARIOUS PROTEINS: secreted by
seminal vesicles; for coagulation of the
ejaculate

PROSTATE GLAND

Specialized Sertoli Cells

receive sperm from epididymis through ductus

deferens
receive seminal fluid from seminal vesicles

Located: below the bladder, surrounds upper

urethra
Aids in propelling the sperm through the
URETHRA by contractions
Semen volume: 20-30% acidic prostate fluid
o Contains:
Acid phosphatase
citric acid
zinc
proteolytic
enzymes:
for
coagulation and LIQUEFACTION

BULBOURETHRAL GLANDS/COWPERS GLAND

located: below prostate

contributes thick, alkaline mucus to neutralize
acidity from prostate secretions and vagina
no neutralization: diminished sperm motility

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STRUCTURE
Seminiferous Tubules
Epididymis
Ductus Deferens
Seminal Vesicles
Prostate Glands

Bulbourethral Glands

FUNCTION
Spermatogenesis
Sperm maturation
Propel
sperm
to
ejaculatory ducts
Provide nutrients for
sperm and fluid
Provide enzymes and
proteins for coagulation
and liquefaction
Add alkaline mucus to
neutralize prostatic acid
and vaginal acidity

Specimen Handling

All semen = potential reservoirs for HIV and

Hepatitis viruses
Discarded as biohazardous waste
Sterile materials and techniques used in:
o Semen Culture
o Bioassay
o Intra-uterine Insemination (IUI)
o In-vitro Fertilization (IVF)

SEMEN ANALYSIS
SPECIMEN COLLECTION

Most of the sperm are contained in the first

portion of ejaculate
When a part of the FIRST PORTION of the
ejaculate is missing:
o Sperm count
o Falsely pH
o Specimen will not liquefy
When a part of the LAST PORTION of the
ejaculate is missing:
o Semen volume
o Falsely sperm count
o Falsely pH
o Specimen will not clot
PERIOD OF SEXUAL ABSTINENCE: at least 2 days
BUT not more than 7 days
Prolonged abstinence: volume, motility
WHO: 2 or 3 samples collected not less than 7
days or more than 3 weeks apart; 2 abnormal
samples are significant
Warm sterile glass or plastic containers
Should be kept at RT
Delivered within ONE HOUR of collection
Specimens collected by masturbation
If not possible, only nonlubricant-containing
rubber or polyurethane condoms must be used
Ordinary condoms contains SPERMICIDES

Consists of macroscopic and microscopic

examinations.
Parameters reported:
o Appearance
o Volume
o Viscosity
o pH
o sperm concentration and count
o motility
o morphology

APPEARANCE

normal semen: gray-white color, translucent,

musty odor
very sperm concentration = almost clear
white turbidity = presence of WBCs and
infection within reproductive tract
Specimen culturing is done prior to semen
analysis
Microscopic examination: WBCs differentiated
from immature sperm (spermatids)
Leukocyte esterase reagent strip test
o Screen test for WBCs
Red coloration = abnormal presence of RBCs
Yellow coloration = urine contamination
Urine = toxic to sperm, affects motility

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LIQUEFACTION

Fresh semen specimen:

o Clotted
o Should liquefy within 30-60 minutes
Failure to liquefy within 60 minutes:
o Deficiency in prostatic enzymes
o Should not be reported
Analysis of specimen cannot begin until
liquefaction
After 2 hours still not liquefied:
o Add equal amount of:
Physiologic
Dulbeccos
Phosphate-buffered Saline
Proteolytic enzymes (Alphachymotrysin or Bromelain)
o However may affect biochemical tests
o Dilution of sperm with bromelain must
be reported in calculating sperm
concentration
o Liquefied semen contains jelly-like
granules = no clinical significance
o Mucus strands interferes with semen
analysis

VOLUME

Normal semen volume = 2-5 mL

Measured through graduated cylinder with 0.1
mL increments
volume = extended abstinence
volume = infertility, improper functioning of
seminal
vesicles,
incomplete
specimen
collection

pH

Refers to the consistency of the fluid

Related to specimen liquefaction
Incompletely liquefied specimens = clumped
and highly viscous
Normal specimen should be easily drawn into a
pipette and form small discrete droplets that do
not appear clumped

Should be measured within 1 hour

Normal pH = alkaline (7.2 - 8.0)
pH = infection within reproductive tract
pH = increased prostatic fluid, ejaculatory
duct obstruction, poorly developed seminal
vesicles

SPERM CONCENTRATION AND SPERM COUNT

VISCOSITY

Droplets with threads >2 cm = viscous,

abnormal
Reportings: 0 (watery) to 4 (gel-like) or low,
normal, high

Factors that affect sperm concentration:

o Days of sexual abstinence before
collection
o Infection
o Stress
Reference range:
o 20 250 million sperm/mL
Borderline:
o 10 20 million sperm/mL
TOTAL SPERM COUNT
= (sperm concentration)(specimen volume)
Considered normal
o 40 million sperm/ejaculate
o (20 million sperm/mL)(2 mL)
Neubauer Counting Chamber measures sperm
concentration
Most commonly used dilution is 1:20
Dilution immobilizes sperm before counting
Traditional Diluting Fluid contains Sodium
bicarbonate and formalin
Saline and Distilled Water can also be used
Neubauer Hemocytometer:
o Four corner and center squares of the
large center square
o Both sides are loaded
o Settle for 3 5 minutes
o Counts should agree within 10%
o Phase or Bright-field microscopy
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Addition of CRYSTAL VIOLET STAIN aids

in visualization when using Bright-field
microscopy
ONLY FULLY DEVELOPED SPERM SHOULD BE
COUNTED
Immature sperm and WBCs, round cells must
not be included
> 1 million leukocytes/mL = inflammation or
infection of the reproductive organs that can
lead to infertility
> 1 million spermatids/mL = disruption of
spermatogenesis

When using 1:20 dilution:

o SPERM CONCENTRATION/mL
= (no. of sperm)(1 000 000)
o TOTAL SPERM COUNT
= (sperm conc./mL)(specimen vol.)

SPERM MOTILITY

Forward, progressive movement

Cervix -> cervical mucosa -> uterus -> fallopian
tubes -> ovum
Must be assessed using well-mixed, liquefied
semen specimen within 1 hour
Allow to settle for 1 minute
Minimum motility of 50% with a rating of 2.0
after 1 hour = normal
WHO: within 1 hour, >50% should be motile in
category a, b, c, d or 25% should show
progressive motility (a and b)
Computer-Assisted Semen Analysis (CASA)
o Provides objective determination of
sperm velocity and trajectory (direction
of motion)

SPERM MOTILITY GRADING

GRADE
WHO Criteria
Motility Action
Rapid, straight4.0
a
line motility
Slower speed,
3.0
b
some lateral
movement

1.0

Slow forward
progression,
noticeable
lateral
movement
No forward
progression
No movement

SPERM MORPHOLOGY

Calculating Sperm Concentration and Sperm Count

2.0

Evaluated with respect to the structure of:

o Head
o Neckpiece
o Midpiece
o Tail
Abnormal head morphology = poor ovum
penetratrion
Abnormal neckpiece, midpiece, tail = poor
motility
Normal sperm = 5 m long and 3 m wide, 45
m long flagellar tail
Acrosomal cap
o critical to ovum penetration
o located at tip of head
o should cover 2/3 of the sperm nucleus
Neckpiece
o Attaches head to the tail and the
midpiece
Midpiece
o 7.0 m long, thickest part of the tail
o Surrounded by mitochondrial sheath for
energy
Evaluated from thinly smeared, stained slide
under oil immersion
Stains: Wrights, Giemsa, Shorr, Papanicolaou
Air-dried samples are stable for 24 hours
ABNORMAL HEAD STRUCTURES:
o Double heads
o Giant heads
o Amorphous heads
o Pinheads
o Tapered heads
o Constricted heads
ABNORMAL SPERM TAILS:
o Doubled
o Coiled
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Bent (abnormal long neckpiece)

Calculating Round Cells

N = no. of spermatids or neutrophils/ 100

mature sperm
S = sperm concentration in millions/mL
C = (N)(S)
100

REFERENCE VALUES
Volume
2 to 5 mL
Viscosity
Pours in droplets
pH
7.2 to 8.0
Sperm concentration >20 million/mL
Sperm count
>40 million/ejaculate
Motility
>50% within 1 hour
Quality
>2.0 or a, b, c
Morphology
>14% normal forms (strict)
>30% normal forms (routine)
Round cells
<1.0 million/mL

ADDITIONAL TESTING

Most common tests:

o Sperm vitality
o Seminal fluid fructose level
o Sperm agglutins
o Microbial infection

SPERM VITALITY

sperm vitality = normal sperm concentration,

decreased motility
Should be assessed within 1 hour
Mix specimen with eosin-nigrosin stain
Counting number of dead cells in 100 sperm
Bright-field or Phase-contrast microscopy
Living cells = remain bluish white
Dead cells = stain red against purple
background
Vital but immobile cells = defective flagellum
Nonviable and immotile cells = epididymal
pathology

SEMINAL FLUID FRUCTOSE

Low sperm concentration = lack of support

medium by seminal vesicles (low fructose
levels)
Resorcinol Test: screening for fructose
o Orange color when fructose is present
Normal fructose level: >13 mol/ejaculate
o Determined by SPECTROPHOTOMETRIC
METHODS
Tested within 2 hours or frozen to prevent
fructolysis

ANTISPERM ANTIBODIES

May be detected in:

o Semen
o Cervical mucosa
o Serum
Possible cause of infertility
Presence of antibodies in males = clumps of
sperm during routine semen analysis
Two Tests:
o MAR Test detechs IgG antibodies
Incubate sperm with IgG
antihuman globulin (AHG) and
a suspension of latex particles
or treated RBCs coated with
IgG
<10% of the motile sperm
attached to the particles =
normal
o Immunobead Test detechs IgG, IgM,
IgA antibodies and what area is
affected
Sperm
mixed
with
polyacrylamide beads coated
with IgG, IgM, IgA
<50% presence of beads =
normal

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MICROBIAL AND CHEMICAL TESTING

>1 million leukocytes = infection in reproductive

system
Tests for:
o Chlamydia trachomatis
o Mycoplasma hominis
o Ureaplasma urealyticum
Motile sperm detected 24 hours after
intercourse
Nonmotile sperm can persist until 3 days
As sperm dies, head remains for 7 days
Detection of Acid phosphatase = positive
presence of semen

ADDITIONAL TESTING
Abnormal Result Possible
Test
Abnormality
Decreased
vitality
Eosin-nigrosin
motility, normal
stain
count
Decreased count Lack of seminal Fructose level
vesicle support
medium
Decreased
Male antisperm MAR Test and
motility
with antibodies
immunobead
clumping
assay
Normal analysis Female
Sperm
with continued antisperm
agglutination
infertility
antibodies
with
female
serum

POSTVASECTOMY SEMEN ANALYSIS

Detects the presence of spermatozoa after

vasectomy
Specimens routinely tested at monthly
intervals, 2 months postvasectomy until 2
months show no spermatozoa

REFERENCE SEMEN CHEMICAL VALUES

Neutral-glucosidase
20 mU/ejaculate
Zinc
2.4 mol/ejaculate
Citric acid
52 mol/ejaculate
Acid phosphatase
200 units/ejaculate

SPERM FUNCTION TESTS

Hamster Egg Penetration

o Sperm are incubated with speciesnonspecific
hamster
eggs
and
penetration is observed microscopically
Cervical Mucus Penetration
o Observation of sperms ability to
penetrate partners midcycle cervical
mucus
Hypo-osmotic Swelling
o Sperm
exposed
to
low-sodium
concentrations are evaluated for
membrane integrity and sperm viability
In-vitro Acrosome Reaction
o Evaluation of the acrosome to produce
enzymes essential for ovum penetration

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