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Survival of rabbit eggs shrunken to aid in sperm microinjection
THERIOGENOLOGY
SHWNKEN'ICAIDINSPERMMICROINJEClTON SURVIW&OFRABBITECGS X. Yang, J. &en, Y. Chen and R.H. Foote Department of Animal Science, Cornell University Ithaca, New York 14853 USA Microinjection of sperm into oocytes hasbeenconductedina rnmber of species, but the fertilizationrate is lclw. Gne reason for failuremaybethemechanical damageduringmanipulation. Experiments were conductedto minimize this damage. ~cfllcr0sei.n ~~~~fferedsaline(SPBS)wasemployedtodehydmteoocytesand uxreasetheperivitelllnespace. When 0.5 M or 1.0 M SPBS was used, cocytes and embryos shrank to about 40% or 37% of their initial size. This provided a large space for spennmicroinjection. In experirrant1, 126 pronuclear stage embryos 18 to 20 h postmtion were collected from fcur superovulated D_rtch-belted rabbits. They were then plaoed in (A) 0.5 M, or (B) 1.0 M sucroseSPBS, or (C) accidentallyin hypexsmo tic (5X normal) concentratedPBS for 30-40 min and then recoveredin normal PBS medium. In (D) occytes were added to 1.0 M sucrcse-PBS for lo-15 min and then placed in droplets of the sams medium and the zona pellucidawas w with abeveledmicropipet20-30 um in diameter. Embryos inmediumwithout Following culture of sucrose expxure (E) served as controls. treatmentsA to E at 39OC, 5% Co2 and 95% humidifiedair for 4-5 d in BSM II (Kane and Foote, P.S.E.B.M. 133:921. 1970), 88% (n=24), 83% (1~18),32% (r~25),82% (~44) and 93% (1x=15) of the pronuclearstage embryce developed to blastocysts,respectively. Neither hyperosmotic SPBS (0.5 M or 1.0 M sucrcse) nor miv influencedembryonic development (p,O-05), in contrast to reduced developnnent(RO.05) follcwingexposwx to the hyperosnoticPBS solution. Experiment 2 was conducted to investigate the viability of oocytes following hyperosmotic treatment (sucrose) and microfertilizationby injecting sperm. oocytes were collected 13 to 14 h after luteinizing hormone injection and treated with hyaluronidase to remove amnitus cells. They were then assigned randomlytoseveral~~. In (A) several spermatozoa were injected intotheperivitellinespaceofoocytespretreatedwith 0.5M sucrose-PBSusing spenncapacitatedwithdefinedmedium (CM) or (B) W plus 60 ug/mllysophosphatidylcholine.Cocytespretreated (C) with or (D) without sucrose and mixed with sperm capacitated in CMserved as IVF controls. In(E) V'=doocytes (pmcturedasin experiment 1) and (F) oocytes without sperm coincubation or other manipulation served as parthenogeneticcontrols. Cleavage to 2-4 cells, was 81% (n=29), 67% (r~12),74% (n=34), 100% (1~15), 0% (1~10) and 0% (IFll), for treatments A to F, respectively. Corresponding developmentrates (to morula) were 58%, 50%, 47%, 60%, 0% and 0%. The sucrose pretreatit of oocytes facilitatedmicroinjectionof spenn, with development in culture of sperm-exposed oocytes similar to controls (mO.05). Sham operation did not ixrease the chance of parthenogenesis(-0.05). Furtherresear&isinprqresstotestthe normalityof embryonicdevelopamant.
336
JANUARY
1988 VOL. 29 NO. 1
SHWNKEN'ICAIDINSPERMMICROINJEClTON SURVIW&OFRABBITECGS X. Yang, J. &en, Y. Chen and R.H. Foote Department of Animal Science, Cornell University Ithaca, New York 14853 USA Microinjection of sperm into oocytes hasbeenconductedina rnmber of species, but the fertilizationrate is lclw. Gne reason for failuremaybethemechanical damageduringmanipulation. Experiments were conductedto minimize this damage. ~cfllcr0sei.n ~~~~fferedsaline(SPBS)wasemployedtodehydmteoocytesand uxreasetheperivitelllnespace. When 0.5 M or 1.0 M SPBS was used, cocytes and embryos shrank to about 40% or 37% of their initial size. This provided a large space for spennmicroinjection. In experirrant1, 126 pronuclear stage embryos 18 to 20 h postmtion were collected from fcur superovulated D_rtch-belted rabbits. They were then plaoed in (A) 0.5 M, or (B) 1.0 M sucroseSPBS, or (C) accidentallyin hypexsmo tic (5X normal) concentratedPBS for 30-40 min and then recoveredin normal PBS medium. In (D) occytes were added to 1.0 M sucrcse-PBS for lo-15 min and then placed in droplets of the sams medium and the zona pellucidawas w with abeveledmicropipet20-30 um in diameter. Embryos inmediumwithout Following culture of sucrose expxure (E) served as controls. treatmentsA to E at 39OC, 5% Co2 and 95% humidifiedair for 4-5 d in BSM II (Kane and Foote, P.S.E.B.M. 133:921. 1970), 88% (n=24), 83% (1~18),32% (r~25),82% (~44) and 93% (1x=15) of the pronuclearstage embryce developed to blastocysts,respectively. Neither hyperosmotic SPBS (0.5 M or 1.0 M sucrcse) nor miv influencedembryonic development (p,O-05), in contrast to reduced developnnent(RO.05) follcwingexposwx to the hyperosnoticPBS solution. Experiment 2 was conducted to investigate the viability of oocytes following hyperosmotic treatment (sucrose) and microfertilizationby injecting sperm. oocytes were collected 13 to 14 h after luteinizing hormone injection and treated with hyaluronidase to remove amnitus cells. They were then assigned randomlytoseveral~~. In (A) several spermatozoa were injected intotheperivitellinespaceofoocytespretreatedwith 0.5M sucrose-PBSusing spenncapacitatedwithdefinedmedium (CM) or (B) W plus 60 ug/mllysophosphatidylcholine.Cocytespretreated (C) with or (D) without sucrose and mixed with sperm capacitated in CMserved as IVF controls. In(E) V'=doocytes (pmcturedasin experiment 1) and (F) oocytes without sperm coincubation or other manipulation served as parthenogeneticcontrols. Cleavage to 2-4 cells, was 81% (n=29), 67% (r~12),74% (n=34), 100% (1~15), 0% (1~10) and 0% (IFll), for treatments A to F, respectively. Corresponding developmentrates (to morula) were 58%, 50%, 47%, 60%, 0% and 0%. The sucrose pretreatit of oocytes facilitatedmicroinjectionof spenn, with development in culture of sperm-exposed oocytes similar to controls (mO.05). Sham operation did not ixrease the chance of parthenogenesis(-0.05). Furtherresear&isinprqresstotestthe normalityof embryonicdevelopamant.
336
JANUARY
1988 VOL. 29 NO. 1