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1. Artificial insemination
Prof. Dr. Abdel Razik Khalifa
Professor of Therigenology

2. ٢
Sperm Metabolism
(Fructose Utilization)
Anaerobically: Fructose
Ø2
2lactic acid + H+2ATP (net yield)
•Aerobically : Fructose
O2
CO2+ H2O +38ATP (net yield)
ATP + H2O ADP + H3PO4+7,000calories /mole
•Measurement of metabolism rate
a.Under aerobic condition:1). O2consumption2). CO2
liberation b. Under anareobic condition:1) Increase in
lactic acid2) Decrease in fructose3) Methylene blue
reduction tiume

3. Relationship of fertile life to metabolic rate
of sperm:
a. Reduced metabolic rate : extend the
storage life of semen;
b. Reduced metabolic rate in epididymis:
extend life of epididymal sperm
c. Sperm in fresh ejaculate of semen: fertile
for a few hours under high rate of
metabolism
٣

4. Factors affect the rate of metabolism:
1-Temperature :
a) Increase temperature leads to increase the rate of
metabolism and decrease sperm life span
*if temperature reach50C this leads to irreversible
loss of motility
*causes of death are due to exhaustion of substrate
and decrease pH.
b)Decrease temperature leads to slow metabolic rate
and extend fertile life if coldshock avoided
2-pH:optimum activity of sperm enzymes at pH7.0.
3-Osmotic pressure
٤

5. Standard Technique of Freezing Bull Semen
6-Freezing
A.Freezingprocedure:Asinglelayerofstrawsareplacedona
trayat5.5cmabovetheliquidnitrogenlevel;Strawswillreach
thetemperatureofliquidnitrogenvaporinabout2min.
B.B.Optimumfreezingrate::-50C/minto-7C/min.:satisfactory
results:influencedbythetypeofpackage,glycerollevel,
thawingrateanddilutercomposition
7-Storageofbullsemen:
Semen storage in liquid nitrogen container: Double wall stainless
steel or aluminum container with a vacuum between the walls:
Large storage unit and small field unit

10. Evaluation of frozen semen
ASSESSMENT OF QUALITY OF FROZEN SEMEN
METHODSUSEDTOEVALUATEFROZENSEMEN:
1-Motility:2-Numberofmotilespermatozoaper
inseminate3-AcrosomalIntegrity
1-Motility:
Itisadopted20%progressivelymotilespermatozoa
astheminimumacceptablemotilityforfrozen
semen.Thespermatozoamustshowareasonable
rateofforwardmovement

11. Evaluation of frozen semen
2-Number of motile spermatozoa per inseminate:
Thenumberofmotilespermatozoaperinseminatemustbe
determinedtoknowiftoofewortoomanyspermare
presentformaximumreproductiveefficiency.Final
conclusionthatformostbullseachinseminateshould
containatleast10millionmotilespermafterthawing.
3-Acrosomal Integrity :
Sincetheacrosomeisinvolvedinthefertilizationprocess,it
hasbeenpostulatedthatspermatozoawithdeterioratedor
damagedacrosomeareunlikelytobecapableof
fertilization.
Arelativelyhighpositivecorrelationhasbeenreportedfor
acrosomalintegrityandfertilityofHolsteinbulls.

12. Handling Frozen Bovine Semen in the Field
Mostoffertilityproblemsassociatedwiththeuseoffrozen
semenareduetoimproperhandlingordepositionofsemen
bythetechnician.Herewith,wewilldiscushowto
minimizethereductioninfertilityresultingfromimproper
handlingtechniquesforfrozensemenpackagedinstraws.
1-Maintainingtemperatureoffrozensemen:
Formaximumreproductiveefficiency,thetemperatureof
semenshouldbemaintainedat-130Corloweratalltimes
Ifthetemperaturehasrisenabove-80Cthesemenshould
notbeusedtobreedcowsbecausethefertilityisgreatly
reduced.

13. Handling Frozen Bovine Semen in the Field
2-Transfer of semen:
•Thefirstopportunityforimproperhandlingoffrozensemen
comesduringdeliveryorpickingup.Semencanbeexposed
toelevatedtemperatureswhenittransferredfromonetank
toanother.Seminaltemperaturerosemorequicklywithin
longstrawwithsmalldiameterthantheshortonewith
greaterdiameter.
•Inthesametime,semenexposetoriseintemperatureand
immediatelyplungedbackintoLN,produceareductionin
postthawmotilitywhichisareliablemeasurementof
fertilityandthatoccursmorerapidlyinsmalldiameter
straws.

14. •Finally,Weconcludedthat,strawsshouldbetransferredwhilein
gobletscontainingLN,andthetransfersshouldbecompletedas
quicklyaspossible,awayfromwindandsunlight.
Exposure of semen during removal:

15. •Semenmayalsobeexposedtodamagingtemperatureeachtimeit
israisedintotheneckoftanktoremoveanindividualstrawfor
insemination
•Thetemperaturewithintheneckoftankismuchhighertoward
thetopofthetank.Sincespermatozoawillbedamagedatthese
temperatures,thecanistercontainingthestrawsshouldberaised
nohigherthanabsolutelynecessary,andtheindividualstraw
shouldberemovedforthawingasquicklyaspossible.The
damagefortherestofstrawsfromexposuretoelevated
temperatureisadditive.
So,seminaltemperaturereachedduringexposuretotheneckofthe
tankdependsupontheheighttowhichsemenisraised,lengthof
exposure,levelofLNinthetankandtheintervalbetween
exposures.
Therefore,semenshouldberaisednohigherthannecessaryto
facilitateremovalofstrawforthawingandremovalshouldbe
quicklyaspossible.

17. Thawing of Frozen Straw
•Preparethethawingbathcontainingwaterat35C
•RemovethelidfromtheLNtank,identifythecanisterholding
thestrawsthataretobeused,liftthecanisternohigherthan
thefrostlineintheneckoftheLNtank
•Withthethumbandforefinger,quicklygraspthetipofthe
gobletholdingthestrawsandlowerthecanisterbackintothe
tank
•Withalongforcepsgraspanindividualstrawfromthegoblet
andlowerthegoblettothebottomofthecanisterwithinthe
tank.
•Immediatelytransferthestrawfromthestoragetanktothe
thawingbath.WhereFrenchstrawsshouldbeheldinthe
waterfor6seconds.

18. Thawing of Frozen Straw
•Afterremovingthestrawfromthethawingbath,carefullydrythe
strawwithtaolitpaperandinspectitforanycracksordefective
seals.
•Carefullycliponeendofthestrawwithespecialsharpcutteror
scissors.Thecliplinemustbestraight
•Placethestrawsintothefunneledendoftheplasticinsemination
sheath
•Completethepartsofinseminatingdevice
•Inseminatethecowaswillmentionedafter.
Thetemperatureofthesemenmustbepreventedfromreaching
excessivelevelsandthethawingtimemustbecontrolled.Inthe
sametimebecarefulandavoidconditionthatwillreducethe
temperatureofthethawedsemen.

19. Insemination of Cow
Insemination by Recto-vaginal techniques:
Althoughitissomewhatdifficultforthebiginnerbutitis
themethodofchoiceforitshighconceptionrate.The
techniqueisdoneasfollow:
1-introducethelefthandthroughtherectumandmake
packracking,afterthatmakedrycleaningofthe
vulvawithtaollitpaper.
2-Graspthecervixwithlifthand
3-Insertinseminatinginstrumentthroughvagina.
4-Holdcervixbyitsposteriorendwithindexandmiddle
fingerandthumb,leavingtheothertwofingersfreeto
helpguidetheinseminatinginstrument.

20. 5-Guidetheinstrumentintotheopeningofcervixand
manipulatethecervixinalldirectionstopassthe
instrumentthroughthecervix.
6-Movethefingersandthumbforwardsothatthe
manipulationistakingplacejustforwardtotheend
ofinstrument.
7-Stoptheinstrumentassoonasitreachesthe
anteriorendofthecervix,anddonotwithdraw
theinstrument,especiallywhenthecowurinates.

22. Some Problems during insemination:
•Inserttheinstrumentintovaginanottointersub-urethral
diverticulum'sorexternalurethralorifice
•Whenmuscularcontractionforcethereproductivetracttoward
anusandcausevaginatobecomefolded,graspthecervixwith
lefthandandpushitforwardtostraightenthevagina.
•Whenthecowattempttoexpellefthandfromrectumwith
peristalticmuscularcontractions,waituntiltherectumwillbe
relaxed.
•Whenthecervixcannotbegraspedormanipulatedthrough
contractionsofrectalmuscles,cupthefingersoverthehanded
rectalwallandpullthehandtowardtheanustocausethe
contractedrectalmuscletoberelaxedandsoftened.
•Whenthevaginafillswithair,dispeltheairbyfirmpressure
withthehandtowardthevulva.
•Whenthebladderisextremelyfull,manipulatetheclitoristo
causeurination.