Manual of Sperm Cryopreservation and in vitro fertilization using ...

Sperm Cryopreservation2) Put the caudal portion into a 4-well dish.3) Wash off extra fat gently with storagesolution 3 times and put it into the 4th well.1) Remove the caudaepididymidis from male mice(12- to 70-week-old).* Vortex the FERTIUP TM MouseSperm Cryoprotectant (CPA)gently before use.wellFERTIUP TM Mouse SpermCryoprotectant (CPA)* Dispense Drop CPA solution(120 L /well) at the edge.Incisions6) Aspirate approximately 100 L ofHTF, and approximately 10 L ofsperm suspension into the straw withair pocket between them.7) Seal both ends of thestraw with a sealer.air4) Incise 5 or 6 points of thecauda epididymidis and leavethem for 3 minutes.airSperm suspensionHTFCotton plugSealer 5) Remove the caudaepididymidis from the Drop andshake the mixture gently.Straw Floater8) Suspend the straw in air for 10minutes using a floater, and thenimmerse it in liquid nitrogen. Straw caseNote1) Do not let the caudaepididymidis become dry.2) Removal of the caudaepididymidis and its exposure toair must be within 15 minutes atmost.

Thawing frozen spermsandin vitro fertilization1) Prepare a 90 L of HTF drop onthe day before IVF, and equilibrateit with the gas (for the oocytecumuluscomplex collection andIVF).HTF drop2) On the day of IVF, equilibrate120 L of FERTIUP TM MouseSperm Preincubation medium(PM) with the gas (0.5-3 hours).3) Isolate the oocyte-cumuluscomplexes in a 90 L drop of HTFmedium from hyperstimulation ovulate(one drop for 2-4 mice).The oocyte-cumuluscomplexesUse liquid paraffin for covering.PM dropUse liquid paraffin for covering.* Vortex the FERTIUP TM PM gently before use.4) Thawing frozen sperms ;Remove the straw from liquid nitrogen, leave themin air for 5 seconds, and put it in a thermostat bathat 37 C for 10 minutes.Thermostat bathLeave them in air for 5 secondsThe straw with frozen spermPut the straw in a thermostat bathat 37 C for 10 minutes5) Culture the thawed sperms inPI medium (30-45 minutes)PM drop* Do not exceed 1 hour!Frozen-thawedspermatozoa6) After the preincubation,collect 10 L of sperms from thetip of the drop with a pipette andinseminate them.NoteInseminate 10 L spermsPM dropIVF drop(HTF)1) Be sure to check that thehumidity in an incubator is95% RH in advance.2) Do pipette operationgently!

A list of chemical reagent / equipment partschemical reagent/ equipment partsmakerCatalog No.remarksFERTIUP Mouse SpermCryoprotectant : CPAKYUDO CO., LTD.(Tosu, Japan)FERTIUP-CRPFERTIUP Mouse Sperm PreincubationMedium : PMKYUDO CO., LTD.(Tosu, Japan)FERTIUP-TMmWMKYUDO CO., LTD.(Tosu, Japan)WMHTFKYUDO CO., LTD.(Tosu, Japan)HTFParaffin OilNACALAI TESQUE Inc.(Kyoto, Japan)SP 26137-85For Cell Culture4-well disposable multi-dishNunc A/S(Roskilde, Denmark)17674035mm Plastic DishNunc A/S(Roskilde, Denmark)153066For SpermPreincubation andIVF0.25-ml Plastic StrawIMV Technologies(L'Aigle, France)AAA201Triangular cassette, long version 280 mmMTGMedical TechnologyVertriebs-GmbH(Altdorf, Germany)16981/0140For Straw Case0.5-10 LPipette TipPorex Bio ProductsGroup(Petaluma, USA)#114For SpermCollectionCorrespondence;Animal Development Division,KYUDO CO., LTDOffice : 557 Tashirohoka-machi, Tosu, Saga841-0016, JapanFAX : +81-942-81-2168E-mail : fertiup@kyudo.co.jpURL : http://www.kyudo.co.jpDistributorTOYO 2CHOME, KOTO-KU, TOKYO, 135-0016, JAPANhttp://www.cosmobio.co.jp e-mail : export@cosmobio.co.jpPhone : +81-3-5632-9617 FAX : +81-3-5632-9618