FourhoursafterboostingwithhCG,thefemalesarereadytobeginlayingeggs.Thebesttestistoseeifthereareeggsintheholdingbuckets.Also,thecloacagetsredandswollenlikeinX.laevis. WerecommendprecoatingthedishesthatyouwillbeputtingeggsintowithalittlebitofL15/CS(seetestesisolationsectionfordescriptionofL15/CS).Thisfacilitatesthemanipulationandtransferringofeggsandembryos.Youcanuseaslittleas1/2mlandthentransferthatsolutionfromdishtodish.Coatyourtransferpipets(weuseplasticones)withtheL15/CSaswell.Weuseonedishperfemalesothatwecankeepeachfemale"seggsseparateincaseanindividualisproducingpoorqualityeggs.Asanalternative,youcancoatyourdisheswithathinlayerofagarose(E.Amaya). Therearemanywaystoholdthefemalesforharvestingtheeggs,butwerecommendthefollowing.Holdthefemalesothatthelegsarepulledbackwiththecloacaatthevertex(seephoto).Trytokeepthecloacaasthelowestpointsothattheeggsfalloutandlandinyourdish.Theeggsarequitestickyandwillrundownyourfingersanddownherlegs–anywherebutinthedishwhereyouwantthemunlessyoukeepthecloacaatthelowestpoint.GentlepressureonthelowerbackandaBDomenwillexpeltheeggs.Examinetheeggstomakesurethattheylookgoodwithanicepigmentedanimalpoleandpalevegetalpole.Thereisnopointinwastingspermonpoorqualityeggs.Ifthemalebeingsacrificedforthefertilizationisparticularlyvaluable,youshouldcheckthequalityoftheeggs(eitherbyaverysmallsqueezeorcheckingtheeggspresentinthebucket)priortosacrificingthemalefortestesremoval. Afteratleastanhourofrestafterthefirstsqueeze,thefemalescanbere-squeezedforasecondfertilizationifdesired.Agoodfemalecanexpelover1000eggsinasqueeze,andwithseveralsqueezes,wecanget2000-3000eggs.Inanaturalmating,thenumberoffertilizedembryoscanbewellover3000. ContributedbyTimGrammerandMustafaKhokhaSqueezethefemalesothathercloacaisclosesttothedish.Thispreventstheeggsfromrunningdownyourfingersandherlegs.