SEMEN EVALUATIONSEMEN EVALUATION

EQUIPMENTEQUIPMENT

Microscope RequirementsMicroscope Requirements

�� Good quality lensesGood quality lenses

�� PhasePhase--contrast preferred for % contrast preferred for % progressive motility evaluationsprogressive motility evaluations

�� Objectives Objectives –– 10X, 20X*, 40X*, 100X, 10X, 20X*, 40X*, 100X, minimumminimum

�� Heated stage preferredHeated stage preferred

*Preferably phase*Preferably phase--contrastcontrast

EVALUATION OF MOTILITYEVALUATION OF MOTILITY

�� Semen must be kept warm (33Semen must be kept warm (33--3737ooC)C)

�� Evaluate immediately after collectionEvaluate immediately after collection

�� Motility declines rapidly on an unheated Motility declines rapidly on an unheated stagestage

�� On an unheated stage, the first slide is On an unheated stage, the first slide is usually for focusing so subsequent usually for focusing so subsequent slides can be evaluated immediatelyslides can be evaluated immediately

�� If no phaseIf no phase--contrast, use low lightcontrast, use low light

Evaluation of Wave MotionEvaluation of Wave Motion

(Gross Motility)(Gross Motility)

�� Only useful for Only useful for

concentrated samples concentrated samples

(usually ruminant (usually ruminant

semen)semen)

�� Place a drop of semen Place a drop of semen

on a warm slide (no on a warm slide (no

cover slip) and cover slip) and

evaluate under evaluate under <<125X 125X

magnificationmagnification

Wave MotionWave Motion

�� Depends on:Depends on:

�� High concentrationHigh concentration

�� Percentage of progressively motile Percentage of progressively motile

cellscells

�� Speed of progressionSpeed of progression

Gross MotilityGross Motility

�� Rating:Rating:�� VG VG –– rapid dark swirlsrapid dark swirls

�� G G –– slower swirls and eddiesslower swirls and eddies

�� F F –– no swirls, but prominent individual cell no swirls, but prominent individual cell motionmotion

�� P P –– little or no individual cell motionlittle or no individual cell motion

�� If gross motility is If gross motility is >>good, further motility good, further motility evaluation is usually unnecessaryevaluation is usually unnecessary

Evaluation of Percentage MotilityEvaluation of Percentage Motility

�� 5 5 µL semen on a semen on a

warm slidewarm slide

�� Gently press Gently press

down warm down warm

cover slipcover slip

Evaluation of Percentage MotilityEvaluation of Percentage Motility

�� Evaluate at 200Evaluate at 200--

500X500X

�� Avoid examining Avoid examining

near cover slip near cover slip

edge and near air edge and near air

bubblesbubbles

�� Evaluate several Evaluate several

fields and fields and >>2 slides

Standards Standards –– Progressive MotilityProgressive Motility

�� Minimum Minimum >>60%60%

��VG VG –– 8080--100% 100%

��G G –– 6060--79%79%

��F F –– 4040--59%59%

��P P -- <40%<40%

SPERM CELL CONCENTRATIONSPERM CELL CONCENTRATION

�� Only estimated with routine evaluation on Only estimated with routine evaluation on

ruminants:ruminants:

�� VG VG –– grainy appearance with 0.75grainy appearance with 0.75--1+ billion 1+ billion

sperm/sperm/mLmL

�� G G –– opaque milkopaque milk--like with 0.4like with 0.4--0.75 billion sperm/0.75 billion sperm/mLmL

�� F F –– skim milkskim milk--like with 0.25like with 0.25--0.4 billion sperm/0.4 billion sperm/mLmL

�� P P –– translucent or watery with <0.25 billion sperm/translucent or watery with <0.25 billion sperm/mLmL

�� Semen obtained by Semen obtained by electroejaculationelectroejaculation may not may not

reflect true volume and concentration reflect true volume and concentration

VOLUME & CONCENTRATIONVOLUME & CONCENTRATION

�� Precise estimates required whenPrecise estimates required when

�� Evaluating semen when the entire ejaculate Evaluating semen when the entire ejaculate

is obtained: concentration x volume = total is obtained: concentration x volume = total

sperm in ejaculatesperm in ejaculate

�� Calculating dose volume: concentration x Calculating dose volume: concentration x

%motile (+/%motile (+/-- x %normal) = %(normal) x %normal) = %(normal)

motile/motile/mLmL..

�� E.g., If this is 50x10E.g., If this is 50x1066 and 500x10and 500x106 6 are required, are required,

the dose is 10 the dose is 10 mLmL..

SEMEN VOLUMESEMEN VOLUME

SPERM CELL CONCENTRATIONSPERM CELL CONCENTRATION

�� Focus on the grid Focus on the grid

in in hemacytometerhemacytometer

chamber firstchamber first

�� This avoids broken This avoids broken

cover slipscover slips

SPERM CELL CONCENTRATIONSPERM CELL CONCENTRATION

�� Usually dilute to 1:100 Usually dilute to 1:100

with water or with the with water or with the

UnopetteUnopette systemsystem

�� Platelet/leukocyte Platelet/leukocyte

system dilutes 1:100system dilutes 1:100

�� Sometimes get Sometimes get

clumping of cells with clumping of cells with

straight leukocyte straight leukocyte

systemsystem

The The UnopetteUnopette SystemSystem

Loading the ChamberLoading the Chamber

�� Flush capillary tubeFlush capillary tube

�� Mix solution wellMix solution well

�� Fill both chambersFill both chambers

�� Let sperm settle a few Let sperm settle a few

minutes before minutes before

countingcounting

Pipette MethodPipette Method

�� Dilute 1:100 Dilute 1:100

�� E.g., 20 E.g., 20 µL semen into into 1.98 mL water

Pipette MethodPipette Method

�� Mix well before Mix well before

withdrawing diluted withdrawing diluted

samplesample

�� PipetterPipetter used to fill used to fill

both chambersboth chambers

�� Let sperm settle Let sperm settle

before countingbefore counting

Assuming a 1:100 DilutionAssuming a 1:100 Dilution

�� Count at ~200X, phaseCount at ~200X, phase--

contrast or low lightcontrast or low light

�� Count all cells in the 4 Count all cells in the 4

corner squares (each corner squares (each

contains 16 smaller contains 16 smaller

squares) squares)

�� Divide by 4 and multiply by Divide by 4 and multiply by

101066 to get concentration of to get concentration of

cells/cells/mLmL

Automated Methods to Determine Automated Methods to Determine

Sperm Cell ConcentrationSperm Cell Concentration

SPERMATOGENESISSPERMATOGENESIS

�� Sperm develop in the Sperm develop in the seminiferousseminiferous tubulestubules

�� This takes ~40This takes ~40--70 days, depending on the 70 days, depending on the

speciesspecies

SPERMATOGENESISSPERMATOGENESIS

�� Sperm pass from the Sperm pass from the

seminiferousseminiferous tubules tubules

(ST) into the (ST) into the reterete

testes (RT) in the testes (RT) in the

mediastinummediastinum

�� Efferent ducts (ED) Efferent ducts (ED)

then transport sperm then transport sperm

to the to the epididymisepididymis

EPIDIDYMAL TRANSPORTEPIDIDYMAL TRANSPORT

�� This requires 7This requires 7--16 16

days, depending on days, depending on

the species the species

�� The The cytoplasmiccytoplasmic

droplet passes down droplet passes down

the tail and is usually the tail and is usually

shed in the ejaculateshed in the ejaculate

EVALUATION OF MORPHOLOGYEVALUATION OF MORPHOLOGY

�� Sperm in the Sperm in the

ejaculate reflect ejaculate reflect

events that occurred events that occurred

<<2 months ago2 months ago

�� Abnormal sperm were Abnormal sperm were

formed weeks before formed weeks before

appearing in the appearing in the

ejaculate ejaculate

Sperm Cell MorphologySperm Cell Morphology

�� Use eosinUse eosin--nigrosinnigrosin

stainstain

�� NigrosinNigrosin provides a provides a

dark background dark background

�� Sperm cells are white Sperm cells are white

unless eosin unless eosin

penetrates a damaged penetrates a damaged

membranemembrane

SamplingSampling

�� Use a nonUse a non--heparinizedheparinized

capillary tube or capillary tube or

wooden applicator wooden applicator

stick stick

�� The higher the The higher the

concentration, the concentration, the

smaller the amount to smaller the amount to

get correct get correct

concentration on slideconcentration on slide

Preparing the Slide 1Preparing the Slide 1

Preparing the Slide 2Preparing the Slide 2

Examination of the SlideExamination of the Slide

�� Dry slide immediatelyDry slide immediately

�� Check slide at 200XCheck slide at 200X

�� Evaluate morphology Evaluate morphology

under oil immersion under oil immersion

at at >>1000X1000X

�� Evaluate at least 100 Evaluate at least 100

cells cells

Normal MorphologyNormal Morphology

From Garner DL, Hafez ESE. Spermatozoa and seminal plasma. In:From Garner DL, Hafez ESE. Spermatozoa and seminal plasma. In: Hafez B, Hafez Hafez B, Hafez

ESE, eds. Reproduction in Farm Animals. 7th ed. Philadelphia: LESE, eds. Reproduction in Farm Animals. 7th ed. Philadelphia: Lippincott Williams ippincott Williams

& Wilkins, 2000:98.& Wilkins, 2000:98.

Comparative MorphologyComparative Morphology

From Garner DL, Hafez ESE. Spermatozoa and seminal plasma. In:From Garner DL, Hafez ESE. Spermatozoa and seminal plasma. In: Hafez B, Hafez B,

Hafez ESE, eds. Reproduction in Farm Animals. 7th ed. PhiladelpHafez ESE, eds. Reproduction in Farm Animals. 7th ed. Philadelphia: Lippincott hia: Lippincott

Williams & Wilkins, 2000:98.Williams & Wilkins, 2000:98.

Head DefectsHead Defects

�� Pinched base or Pinched base or

moderately moderately pyriformpyriform

heads probably do heads probably do

not cause infertility if not cause infertility if

nearly all cells have nearly all cells have

these shapes these shapes (photos courtesy of AD Barth)(photos courtesy of AD Barth)

Head Defects That Affect FertilityHead Defects That Affect Fertility

�� Severe Severe pyriformpyriform

shapeshape

�� Equatorial vacuolesEquatorial vacuoles

�� Large vacuolesLarge vacuoles

�� The significance of The significance of

small, single apical small, single apical

vacuoles is unknownvacuoles is unknown

(photos courtesy of AD Barth)(photos courtesy of AD Barth)

Examples of Other Head Defects Examples of Other Head Defects

That Affect FertilityThat Affect Fertility

�� Large or small headsLarge or small heads

�� Usually not seen in high numbersUsually not seen in high numbers

�� Abnormal DNA condensationAbnormal DNA condensation

�� Not detected with ordinary stains; requires Not detected with ordinary stains; requires

FeulgenFeulgen stainstain

Knobbed Knobbed AcrosomesAcrosomesUnable to fertilizeUnable to fertilize

((Photos courtesy of AD Barth)Photos courtesy of AD Barth)

AcrosomeAcrosome Defects Defects –– usually only usually only

seen on frozenseen on frozen--thawed semen thawed semen

Defects That Apparently Do Not Defects That Apparently Do Not

Affect FertilityAffect Fertility

�� AbaxialAbaxial tail tail attachment, if no attachment, if no accessory or double accessory or double tail is presenttail is present

(photo courtesy of AD Barth)(photo courtesy of AD Barth)

�� Distal Distal cytoplasmiccytoplasmicdropletsdroplets

Defects That Affect Fertility Defects That Affect Fertility

(photo courtesy of AD Barth)(photo courtesy of AD Barth)

Possible ArtifactsPossible ArtifactsVerify by Checking Wet MountVerify by Checking Wet Mount

�� Distal Distal midpiecemidpiece

reflexes and bent reflexes and bent

principal pieces with principal pieces with

no droplet enclosedno droplet enclosed

�� Bowed Bowed midpiecesmidpieces

�� Loose normal headsLoose normal heads

(photo courtesy of AD Barth)(photo courtesy of AD Barth)

Minimum Standards Minimum Standards -- MorphologyMorphology

�� A minimum of 70% normal cellsA minimum of 70% normal cells

�� ‘‘CompensableCompensable’’ defects such as defects such as midpiecemidpiece

defects may be of less concern as long as defects may be of less concern as long as

there are sufficient normal, motile cellsthere are sufficient normal, motile cells

�� Certain head defects are Certain head defects are ‘‘nonnon--compensablecompensable’’

and therefore of more concernand therefore of more concern

CELLS OTHER THAN SPERM CELLS OTHER THAN SPERM

(COTS)(COTS)

CELLS OTHER THAN SPERMCELLS OTHER THAN SPERM�� White blood cells (and other COTS) are best White blood cells (and other COTS) are best

detected by staining a dried semen smear with detected by staining a dried semen smear with

WrightWright--GiemsaGiemsa or Diff or Diff QuikQuik stainsstains

�� WBCsWBCs indicate infection in the tractindicate infection in the tract

CELLS OTHER THAN SPERMCELLS OTHER THAN SPERM�� Cells shed from the Cells shed from the seminiferousseminiferous

epithelium indicate testicular degenerationepithelium indicate testicular degeneration�� This may condition may improve or worsen This may condition may improve or worsen

with timewith time

ASSIGNMENTASSIGNMENT

�� Classify Classify >>100 cells100 cells

�� Work alone or in small groupsWork alone or in small groups

�� Hand in resultsHand in results

�� Include species evaluatedInclude species evaluated

�� List List name(sname(s) of ) of evaluator(sevaluator(s) )

Categorize Cells By PercentageCategorize Cells By Percentage

�� Head defectsHead defects

�� MidpieceMidpiece defectsdefects

�� Principal piece defectsPrincipal piece defects

�� Normal detached headsNormal detached heads

�� Proximal dropletsProximal droplets

�� AcrosomeAcrosome defectsdefects

�� Minor defects (e.g., distal droplet, simple bent Minor defects (e.g., distal droplet, simple bent principal piece)principal piece)

�� Normal Normal