A Technique for Bisection of Embryos to Produce Identical Twins
A successful technique for bisection of postcompaction-stage embryos has been devised that combines methods of micromanipulation developed by other workers. Several papers describe generally and in detail various equipment, microtools, and methods of negotiating the bisection of bovine and ovine embryos (6). Willadsen (4,5) used a fine glass needle to pene trate the zona pellucida and to sever the embryo against a holding pipette using a Wild M5 Stereozoom microscope. Ozil (3), using 6 micromanipulators, opened the zona pellucida with needles, ejected the embryo with medium from a suction pipette, then cut the embryo with a specially machined microscalpel. Needles were also used by Lambeth et al. (2) and Gatica et al. (1) to access the embryo and cut it against the bottom of a petri dish. Williams and Seidel (7) used a fine microsurgical blade for opening the zona pellucida and bisecting the embryo in a single step. These methods were attempted with various difficulties in our laboratory. Spatial visualization was difficult when attempting needle cutting against the holding pipette with a Leitz Diavert inverted microscope. The alternative microsurgical blade made a cut in the zona pellucida suitable for introducing a suction pipette, but the embryo tended to stick when the blade was made to cut completely through it. The blade approach was generally more favorable but only for the initial stage of cutting. A method was needed to finish cutting the embryo without the problem of sticking. The fine glass needle seemed suitable for this task, which could be accomplished against the floor of the petri dish after the embryo was gently removed from the zona by blowing in medium from the suction pipette. The resultant technique utilizes a holding pipette, a microsurgical blade for splitting the zona and embryo, and a suction pipette for withdrawing half a severed embryo and inserting it into an empty zona pellucida or for ejecting the semidivided embryo to the floor of the petri dish for completion of the separation with a fine glass needle. Since the blade and needle tools do not require a pressure line to a microinjection syringe, they are easily interchangeable, and only one holder is required for them. Only 2 micromanipulators are required with one single and one double holder for all 4 microtools.
KeywordsZona Pellucida Silastic Tubing Eyepiece Reticle Knife Blade Pipette Puller
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