WhenwefirststartedusingX.tropicalis,invitrofertilizationhadanextremelypoorefficiency.However,withthecarefulselectionofamaturemaleandtheuseofL15/CS(L15Leibovitch"sMediasupplementedto10%calfserum),theinvitrofertilizationefficiencyhasbeenimproveddrastically.Directcomparisonofdifferentmedias(1xMR,L15alone,L15/CS),clearlyshowedthatL15/CSgivesthebestratesoffertilizationwithL15alonebeingbetterthan1xMR.OthershavereportedthatanalbuminsolutionworkswellalthoughwehavenottestedthisdirectlyagainstL15/CS.Onceweusedgoatseruminsteadofcalfserumwhichseemedtoworkwellalthoughnottestedinasidebysidecomparison.Othertypesofserum(fetalcalfserumforexample)havenotbeentried.(manythankstoJ.GerhartforsuggestingtheserumandL.ZimmermanfortheL15).

AnotherimprovementforIVFefficiencyistoboostthemaleswith100uhCGthenightbeforeyoudotheIVF.Weusuallyprimefemaleswith10uhCGaswellatthesametime(weusedtoprimewith20uhCGbutnoticedthatthefemalessometimeslaidafairnumberofeggsatthatdose).Wehaven"tscientificallytestedwhetherboostingthemalethenightbeforeactuallyimprovesfertilization,butthatisourgeneralfeeling.

Oncetheeggsaresqueezedandreadyforfertilization,wemaceratethetestisin500µlcold(onice)L15/CSina1.5mlEppendorftubeusinga1.5mlmicrotubepestle.Unlesshaploidsarebeinggenerated(see"makinghaploids"section),weimmediatelypipetthespermsUSPensionontotheeggsandgentlymixwelltoensureexposureofalltheeggstothesperm.Allowthespermtoattachtotheeggsforabouttwo-threeminutes,then"activate"thefertilizationbyfloodingthedishofembryoswith1/9xMR.Thelowsaltsolutionactivatesthesperm.Formicroinjection,floodwith3%Ficoll+1/9xMR.

UnlikeX.laevis,wefindthat"turning",ortheuprightrotationoftheanimalhemisphereinthevitellineenvelope,isnotalwaysagoodindicationofsuccessfulX.tropicaliseggfertilization.Manytimeswehavehadverygoodfertilizationrateswithlittleobviousturning.Weoftendoseecorticalcontractionoftheanimalhemisphereinfertilizedeggs,buttheabsenceofeithercontractionorturningisnottrulypredictiveofafailedfertilization.Usuallythefirstcelldivisionoccursaboutanhourandtenminutesafterfloodingtheeggs.Iftwohourshaveelapsedandnocelldivisionisseen,thenthefertilizationwasunsuccessful.

Ifoneneedstoobtainonecellstageembryosformanipulationandthereforecannotwaituntilfirstcleavagetodeterminefertilization,werecommenddejellyingtheeggsabout30-40minutesafterflooding.Oncedejellied,thefertilizedeggscanusuallybeidentifiedbasedontheirrigidity,presenceofspermentrypoint(seemicroinjectionprotocol),and(sometimes)corticalcontractionoftheanimalhemisphere.

ContributedbyTimGrammerandMustafaKhokha