Jorge M Sztein DVM PhD2016

Spallanzani 1776? Cold of winter snow……..

Prevost and Dumas 1840? Fertilization…

Mantegazza 1866? Human sperm to -17 ºC. Semen Bank concept

Schenk 1870? Frog, turtle, rabbit, dog exposed to -6 ºC

Pawlow 1927? First time human sperm cooled to -11 ºC

(cockroach?)

AntonyvanLeeuwenhoek(1632-1723)

Animalculesorparasites?

Philosophical Transactions of the Royal Society, 1679

Orshouldbe………….

• 1683- ThefirstdocumentedcryobiologicalstudybySirRobertBoylewhodocumententedtheeffectsoffreezingtemperaturesonlivinganimalsinhismonograph"NewExperimentsandObservationsTouchingCold"

NASA'sGlennResearchCenter

Boyle’sLaw

0.0 0.3 0.6 0.9 1.2 1.5Osmolality (Osm)

0

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100

150

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250

300

Rela

tive

Volu

me

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Mouse Zygote Volumes and Solution Osmolality

Source: S. Leibo

0 20 40 60 80 100Time (minutes)

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20

40

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elat

ive

Cel

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ume

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Calculated Volume of Embryo in Two CPA Concentrations

non-osmotic cell volume

0.0 0.3 0.6 0.9 1.2 1.5Osmolality (Osm)

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50

100

150

200

250

300

Rela

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Mouse Zygote Volumes and Solution Osmolality

Source: S. Leibo

Van’t Hoff plot

Jacobus Van’t Hoff 1901

(1883) Zygmunt Wróblewski Karol Olszewski

From Wikipedia, the free encyclopedia

Which one is correct?

TraditionalMilestones

• 1930WaltonandHammond:J.Exp.Biol.:Effectoftemperatureonrabbitsperm

• 1938Jahnel Spermcooledto-79ºC• 1938Luyet andHoddapp:Biodynamica :Revivalofspermatozoavitrifiedinliquidair

• 1940Shettles frozespermat-269ºC• 1945A.Parkes:BJM:Humanspermatlowtemperatures

Cryoprotectants Milestones

• 1949:Polge,SmithandParkes:Nature:RevivalofSpermatozoaafterVitrification andDehydrationatLowTemperatures

• 1949:Polge,SmithandParkes:discoveryofglycerolascryoprotectant (Eureka!)

• 1959:LovelockandBishop:discoverDMSO(Me2SO)

• 1965:PMazur:publishthestudyonintracellulardamage

Milestones• 1952PolgeFirstlivinganimalfromfrozensperm:Frosty

• 1953BungeandSherman:Nature:Fertilizingcapacityoffrozenhumanspermatozoa.

• 1972Witthingham,LeiboandMazur:Science.Mouseembryofreezing

• 1985RallandFahy:Nature:Ice-freecryopreservationofmouseembryosat-196degreesCelsiusbyvitrification.

With CCR Press authorization. In: Life in the Frozen State - CRC Press

Survivalofmouseembryosfrozenat-196and-269

CryopreservationPioneers

The original paper on Intracellular damage is from 1965

Leibo, SP. Preservation of ova and embryos by freezing. New Technologies in Animal Breeding. NY: Academic Press; 1981

W. Rall 1985

Leibo S., Theriogenology. 1984 May;21(5)

SUCCESFULCRYOPRESERVATIONOFMAMMALIANEMBRYOSYELDINGLIVEOFFSPRING

Species FirstReport

Mouse Whittingham etal.1972

Cattle Willmut &Rowson ’73(Poldge etall.Frosty,’52)

Rabbit Bank&Maurer1974

Sheep Willadsen etal.1974

Rat Whittingham 1975

Goat Bilton &Moore1976

Horse Yamamotoetal.1982

Eland Krameretal.1983

Human Trunson &Mohr 1983

Baboon Popeetal.1984

Marmoset Summersetal. 1986

MacaqueMonkey Balmaceda etal.1987

Cat Dresser etal.1988

MilestonesonMouseSpermcryo

• 1990 Yokoyamaetal.Raffinose andglycerol.Productionofnormalyoungfollowingtransferofmouseembryosobtainedbyin-vitrofertilizationusingcryopreserved spermatozoa.

• 1990Tadaetal.Cryopreservationofmousespermatozoainthepresenceofraffinose andglycerol.

Yokoyama M,Akiba H,Katsuki MandNomura (1990)

MilestonesonMouseSpermCryo

• 1990.Okuyamaetal.In-vitrofertilizationandartificialinseminationbycryopreservedspermatozoainmouse.

• 1991 Takeshima,NakagataandOgawa:Cryopreservationofmousespermatozoa.

• 1992 NakagataandTakeshima:Highfertilizingabilityofmousespermatozoadilutedslowlyaftercryopreservation

MilestonesonMouseSpermCryo

• 1992 Szteinetal.CryopreservationofmousesperminaGlycerol/Raffinosesolution.

• 1993 PenfoldandMoore.Newmethodforcryopreservationofmousespermatozoa.

• 1993Nakagata:Productionofnormalyoungfollowingtransferofmouseembryosobtainedbyinvitrofertilizationbetweencryopreservedgametes.

Moreofthesame…….

• 1995 Taoetal.Theeffectofcollectiontemperature,coolingrateonchillinginjuryandcryopreservationofmousespermatozoa.

• 1997 SongsasenandLeibo:Fertilizingabilityofcryopreservedmousespermatozoa.

• 1997 Szteinetal.Motilityofcryopreservedmousespermatozoaaffectedbytemperaturecollectionandrateofthawing.

Endofthestory……• ‘95-2000Nakagata tonspapers………..• 2000 Sztein etal.Invitrofertilizationwithcryopreserved inbredmousesperm

• 2001 Sztein etal.Comparisonofpermeatingandnonpermeating cryoprotectants formousespermcryopreservation.

• 2008 Ostermeier etal.Conserving,distributingandmanaginggeneticallymodifiedmouselinesbyspermcryopreservation

CARD• 2008 Takeo,Tetal.Methyl-beta-cyclodextrinimprovesfertilizingabilityofC57BL/6mousespermafterfreezingandthawingbyfacilitatingcholesteroleffluxfromthecells.

• 2010 TakeoandNakagata.Combinationmediumofcryoprotective agentscontainingL-glutamineandmethyl-{beta}-cyclodextrin inapreincubation mediumyieldsahighfertilizationrateforcryopreserved C57BL/6Jmousesperm.

Nailing…..• 2011.Takeo etal.Reduced glutathione enhancesfertility offrozen/thawed C57BL/6mousespermafter exposure to methyl-beta-cyclodextrin.

• 2014.Nakagata etal.Rescueinvitrofertilizationmethodforlegacystockoffrozenmousesperm.

• 2014.Takeoetal.Investigationsofmotilityandfertilizationpotentialinthawedcryopreservedmousespermfromcold-storedepididymides.

• 2015.TakeoandNakagata.Superovulation usingthecombinedadministrationofinhibin antiserumandequinechorionicgonadotropin increasesthenumberofovulatedoocytes inC57BL/6femalemice

SpermBike

www.copenhagenize.com

Thanks!!!