in the context of higher organisms suchb. as mammals.

semen is defined as the viscous

biological fluid produced from the reproductive organ of reproductively mature male members of
a species during the act of ejaculation which usually occurs at the culmination of sexual
intercourse or masturbation semen consists of two main parts the spermatozoa or sperm cells
which comprises of about five percent of semen and the seminal plasma or seminal fluid the
spinal plasma consists of secretions from the saminal vesicles the prostate and the bulbo urethral
glands among the many sugars amino acids enzymes and minerals present in the saminal fluid
the most noteworthy and major energy source for sperm cells is fructose secretions of the spinal
vesicles contain fructose along with substances that suppresses any immune response against the
foreign sperms inside the female reproductive tract secretion of the prostate gland contains
substances that help to stabilize and protect the genetic material and thereby the fertility of the
sperm cells secretion of the bulbo urethral glands also known as cooper's glands contain
substances that provide viscosity and slimy textures to the semen thereby increasing sperm
motility within the female reproductive tract it is also the source of the pre-ejaculate semen
analysis or seminogram or spermiogram is a comprehensive series of clinical tests that evaluates
the quality and quantity of

a siemens sample the most important and

commonly analyzed parameters included

under semen analysis are liquefaction

time

amount color ph odor

viscosity sperm motility sperm viability

sperm morphology sperm count and

fructose content

in this video we'll be performing semen

analysis of human semen based on the

previously mentioned parameters

for this experiment you will need the

following freshly collected

human semen sample 10 ml graduated

cylinder or syringe
ph paper preferably narrow range

a narrow glass tube or pipette droppers

glass lights and coverslips eosin and

nigration stain solutions

harris hemorrhox island stain eosin

stain

one percent acid alcohol solution

alkaline water or 0.2 ammonia water

95 ethanol absolute ethanol

xylene dpx or canada balsam

coupling jars thumb forceps and pins

hemocytometer counting chamber with wbc

pipet

semen diluting fluid compound light

microscope

immersion oil cell one of the reagent

test tube and holder spirit lamp

5 ml and 1 ml measuring pipettes

the first and foremost parameter in

semen analysis is the liquefaction time

liquefaction of semen is the process

that results in the conversion of

freshly ejaculated semen

from a thick whitish opaque viscous

and gelatinous consistency to a watery

less viscous translucent fluid with the

help of proteolytic enzymes present in

the saminal fluid

liquefaction time is the time taken for

this conversion process to occur

to perform this test note down the time

when the semen sample is collected

observe the semen sample and note down

the time when the liquefaction is

complete

the normal liquefaction time for human

semen is 10 to 30 minutes

any period of time longer than 30

minutes is indicative of infection

before estimating semen volume the semen

sample needs to go through the

liquefaction period

allow 10 to 30 minutes after collection

for the sample to liquefy before

measuring the volume

to determine the volume of the semen

sample tilt the sample container for a

couple of minutes and then carefully

pour

the liquefied semen sample into a 10 ml

graduated cylinder completely till the

last drop

wait a couple of minutes for the sample

along the inner walls of the cylinder to

settle down

and note down the volume add a volume of

0.5 ml to the observed volume in order

to compensate for the semen sample stuck

to the walls of the collection container

a alternative method of measuring semen

volume is to directly aspirate the

liquefied semen sample

into a 10 cc needless syringe after

positioning the container in a slanting

position for a couple of minutes prior

to aspiration

make sure to aspirate completely till

the last drop

while at the same time taking extreme

care not to aspirate any air bubbles

note down the volume as before add a

volume of 0.5 ml to the observed volume

in order to compensate for the semen

sample stuck to the walls of the

collection container
a third method which is also considered

the most accurate method

according to some published papers is to

directly weigh the sample

in an analytical weighing scale one gram

is generally agreed to correspond to one

ml of the sample

this method eliminates the loss in

volume when transferring the sample from

the collection container

into the measuring receptacle one

important point to keep in mind when

applying this method is to note down the

weight of the empty container

prior to sample collection approximately

two to five ml

is considered as the normal volume of

semen per ejaculation

low semen volume also known as

hypospermia could be an indication of

partial or complete blockage in the

reproductive tract

especially the seminal vesicles it could

also be the result of certain

infections such as stds high

semen volume also called hyperspermia

can be caused by extended periods of

sexual arousal and stimulation

abstinence from sex and masturbation

excess intake of fluids etc

the normal color of human semen is white

to grayish white

semen color is influenced by diet age

medication and certain underlying

medical conditions

red to brownish semen color indicates

the presence of blood

a condition called hematospermia this

could be a result of infection in the

reproductive tract

take a strip of either broad range or

narrow range ph paper and touch

one surface onto the semen sample

observe the ph

paper for color change a more accurate

laboratory ph meter can also be used to

determine the ph

normal semen has a slightly alkaline ph

ranging from

approximately 7.2 to 8. acidic semen

could be indicative of a blockage in one

or both seminal vesicles

high alkaline ph could be a result of

infection

normal semen has a characteristic bleach

or musky smell

due to the presence of tiny amounts of

basic amines

like cadaverine putrescine spermine and

spermidine

slight variations to this smell could

result from a person's diet including

certain medications

the viscosity of semen is determined on

a sample that had already been through

liquefaction

when normal semen is drawn in a narrow

tube or dropper and allowed to dispense

from the tube by the force of gravity

it flows out of the tube in the form of

medium-sized discrete drops without any

trailing strands

abnormally viscous semen however flows

out of the tube in the form of long and

thick viscous
strands that are more than two

centimeters this condition is called

semen hyper viscosity or shv

and is an indication of inadequate

functioning of the prostate

or seminal vesicles and also infection

hyper viscous semen can severely impede

sperm motility and can lead to male

infertility

hypoviscos semen or watery semen on the

other hand could be due to excessive

masturbation

ejaculation after an extended period of

stimulation

and arousal leading to a low sperm count

and also due to a certain underlying

infections

the sperm motility test is done within

60 minutes of sample collection

sperm motility is the ability of a sperm

cell to move in the surrounding fluid

medium

a drop of liquefied semen sample is

taken in a glass light and gently

covered with a coverslip

the slide is immediately observed under

40x objective of the microscope

100 or 200 random sperm cells are

briefly observed one by one to check for

motility or the lack thereof

to minimize the chances of repeated

assessment of the same sperm cell

formatility

the microscopic field of view is changed

frequently in a specific order of

movement

such as from the left corner of the

slide to the right corner etc

until 100 or 200 cells have been

assessed

based on the extent of motility human

sperms are divided into

four grades also known as motility

grades the various grades of motility

are calculated in terms of percentage by

counting hundred or two hundred random

sperm cells

grade a or motility four sperm cells

have rapid progressive motility

these are the strongest and swim fast in

a straight line

grade b or motility 3. these sperm cells

have a slow

or sluggish or non-linear progressive

motility

these also move forward but tend to

travel in a curved or crooked motion

grade c or motility 2 or non-progressive

motility

they move their tails but do not move

forward

grade d or motility 1 or immotility

these are immotile and fail to move at

all

the immotile sperms may either be dead

or alive

which will be ascertained in the next

test which is known as sperm viability

test

in this video taken through the

microscope you can see sperm cells

exhibiting different motility grades

observe the individual sperms followed

by the cursor

normal semen contains at least 50 grade

a

plus b sperm cells combined low

spermatility of below 50 percent

also known as asthenospermia can be a

symptom of disorders related to

testicular function and male accessory

sex glands

it could also be a sign of infection or

injury in the male reproductive tract

in cases of reduced sperm motility the

semen sample could also be checked for

sperm viability

in the sperm motility test if non-motile

sperms are found

one has to assess what fraction of these

non-motile sperms are still alive and

what fraction are dead

sperm viability test is therefore used

to determine if non-motile sperms are

alive or dead

this particular test is applicable in

cases where the motility

of the sperms are drastically low that

is less than 20 percent

viability testing is based on the

principle of differential staining

property of life and dead sperms also

known as dye exclusion assays

dye exclusion assays rely on the ability

of life sperms to resist absorption of

certain dyes

whereas these dyes penetrate and stain

non-viable or dead sperms

a combination of eosin and nigrosin

stains is commonly used in sperm

viability testing

make sure to watch my video on how to

prepare eosin and nigrosin stain for

sperm viability testing

by clicking on the link given on the top

right corner of the screen right now

or the link given in the video

description below

just as in the sperm motility test sperm

viability test is also assessed within

60 minutes of collection of the sample

a drop of liquefied semen sample is

taken on a glass slide

five drops of eosin nigrosin stain or

two drops of one percent eosin and three

drops of ten percent nigrosin stain

is added to the sample

the stain and the sample are gently

mixed on the slide and allow to stand

for 10 to 20 seconds

take a second slide touch one edge of

the slide

on the sample and prepare a smear of the

sample onto a third slide

allow the smear to air dry

observe the slide under high power 40x

or 100x of the microscope

dead sperms that is the sperm cells that

were already dead

in the fresh semen sample are stained

pink in their head region

while life sperms that is the sperm

cells that were motile or immotile

but alive in the fresh semen sample will

remain unstained

observe 100 or 200 random sperm cells

and calculate the fraction of live and

dead sperms in percentage

normal semen contains more than 60

immotile but viable sperms

viable sperms below 60 can be caused by

excessive heat exposure of the testes

exposure to certain substances that are

toxic to sperms

certain infections etc necrospermia is a

condition where there are no

viable or living sperm cells in semen

sperm morphology assessment looks at the

proportion of sperm cells with abnormal

shape or morphology against normal cells

defects in the morphology of a sperm

cell also known as

teratospermia can be classified as

head defects neck and midpiece defects

tail defects and cytoplasmic droplets

the assessment of sperm morphology is

done by microscopic

observation of a stained smear of the

semen sample different staining

techniques can be employed

in this video we'll be using the

hematoxylin eosin or hne staining

technique which is simple

quick and reliable for this experiment

you will need the following

freshly collected human semen sample

droppers

glass lights and coverslips harris

hemorrhox island stain

eosin stain one percent acid alcohol

solution

alkaline water or 0.2 ammonia water

95 ethanol absolute ethanol

xylene dpx or canada balsam

coupling jars thumb forceps and pins

compound light microscope immersion oil

h and e staining is performed as follows

take a small drop of the liquefied semen

sample on a glass light and make a smear

using a second glass light as shown here

allow the smear to air dry at room

temperature immerse the slide in harry's

hemorrhage island stain for about 20

minutes

remove the slide and give it two to

three quick dips in one percent

acid alcohol solution rinse the slide

with slightly alkaline water or with 0.2

ammonia water the stain smear now

becomes bluish
transfer the slide into eocene stain

solution and leave it for two to three

minutes

give the slide three to five dips in 95

ethanol

followed by another three to five dips

in absolute ethanol with two changes

which is then followed by three to five

dips in xylene with two changes

add a drop of mounting medium such as

dpx or canada balsam to the dehydrated

and cleared smear

cover it with coverslip allow the slide

to air dry overnight

and observe under oil immersion

objective of the microscope

study the morphology of 100 to 200

individual sperm cells and assess the

morphological defects of the various

parts of the sperm cells

calculate the fraction of abnormal sperm

cells in terms of percentage

a semen sample that contains more than

30 percent of morphologically normal

sperm cells is considered normal semen

abnormal sperm morphology can be caused

by a number of factors

sperm count is the number of sperm cells

found in one ml of semen

total sperm count is the total number of

sperm cells found in the total volume of

semen

obtained in a single ejaculation event

that is sperm count multiplied by semen

volume

in this video we'll be using the

hemocytometer with wbc pipette to

perform the sperm count

for this experiment you will need the

following freshly collected human semen

sample

thumb forceps and pins hemocytometer

counting chamber with wbc pipet

semen diluting fluid compound light

microscope

first prepare the counting chamber by

placing the coverslip in position

keep this aside

now draw liquefied siemens sample till

the 0.5 mark of the wbc pipette

then draw the semen diluting solution

until the 11 mark on the pipette

gently wipe off excess fluid from the

sides of the pipette tip

now place the loaded pipette in a

horizontal position and gently mix the

contents for a minute or so

by rotating the pipet between the palms

of the hands

discard the first two or three drops

from the pipet

load the counting chamber with the

diluted semen sample

place the loaded chamber under the

microscope and let it rest for a minute

or so

under 40x objective begin counting the

sperm cells within the four corners at

the central small squares of the central

large

square remember to apply the thumb rule

of cell counting by excluding cells

found along the bottom and left

borders of the squares you can also

check out my video on the hemocytometer

click on the link in the video screen

and it will take you there

this is how sperm cells will appear

within the small squares of the counting

chamber

repeat the counting process in the

second counting grid of the counting

chamber as well

take at least two readings for both

counting grids and take the average of

the readings

calculate the sperm count per ml using

the formula

sperm cells per ml equals number of

sperm cells into 20 into 1000

by 0.02 or in short

sperm cells per ml equal sperm counted

multiplied by 1 million

total sperm count equals sperm count per

ml

multiplied by ml of semen sample or

ejaculate

this formula is used when counting is

done in the five small squares of the

central large
square wbc pipette having one is to 20

dilution mark is used sperm count above

15 million cells per ml

of semen is considered normal sperm

count below 15 million cells per ml

also known as oligospermia is one of the

leading causes of male infertility

oligospermi can have a variety of causes

such as

infection and other medical conditions

in the reproductive tract or in the

testes

chemo and radiotherapy varicocele

stds vasectomy frequent overheating of

testicles

occupational exposure to chemicals and

radiation

drugs alcohol or tobacco use anxiety

depression

and other mental issues

fructose is produced in the seminal

vesicles and constitutes about 99

of the reducing sugars present in semen

it is the main source of energy for the

sperm cells
semen samples normally contain fructose

and can be detected by qualitative

analysis using cellular one of the

reagent

quantitative calorimetric estimation can

also be performed

to find out the exact amount of fructose

present in the sample

fructose test is an optional parameter

in routine semen analysis

and is performed especially when the

semen sample contains no sperm cells a

condition known as

azuspermia or when ejaculate volume is

below normal

qualitative fructose test is performed

using salon of the reagent

the test is based on the principle that

when fructose is heated with resorcinol

in an acidic medium

fructose is converted to hydroxy methyl

furfural

which then condenses with resource in

all to form a red precipitate

to perform qualitative tests for

fructose in a semen sample

take 5 ml of salivano's reagent in a

test tube

add 0.5 ml of the semen sample to the

tube and boil the mixture for a minute

or so

the formation of a red color in the tube

is considered as positive test for

fructose

no change in color indicates an absence

of fructose in the sample

to know the exact amount of fructose

present in the sample

quantitative fructose test may be

further performed in a calorimeter using

standard fructose and resources

reagent semen fructose content of 200 to

400 milligram per deciliter is

considered normal

low or no fructose levels in semen is

indicative of conditions such as

testosterone deficiency obstruction

complete absence or developmental

defects of seminal vesicles

and or vast difference so this is all

about the siemens analysis as performed

routinely in a clinical lab

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Inggris (dibuat secara otomatis)