A new method of splitting embryos without the use of a commercial micromanipulator unit

Abstract

The objective of this study was to develop a simplified method for bisecting bovine embryos without the use of a commercial micromanipulator (NICM) unit. Two simplified MICM unit systems were constructed out of glass microscope slides. The first of these unit systems (Method I) was patterned after the glass-slide apparatus used to remove the zona pellucida (ZP) from rat and cattle embryos (after Hoppe & Bavister, Theriogenol. 19(3):391,1983). Method I used a fine glass surgical needle to bisecfintact and ZP-free embryos. During the micromanipulation procedure, embryos (submerged in medium) were viewed (75X) under a stereonlicroscope (Wild M-8). A fine glass suction pipette attached (via polyvinyl tubing) to a 1 ml syringe was used to hold intact embryos for mechanical ZP removal. ZP-free embryos were uroduced by submerqinq intact embryos in 2.5% oronase (Streptomyces griseus protease:S;gma Chemical; St. Louis, 'MO) in Dulbecco's ohosohate-buffered saline (PBS: GIBCO, Grand Island, NY) for a 5 to io minute interval. Method‘11 needs one good quality-50 x 75 mm glass microscope slide, a standard double-edged razor blade (Gillette), a pair of 5 inch hemostats and the use of an inverted microscope (Nikon-Diaphot). The microscope slide (base) was prepared @ scouring the upper surface with 3 urn size diamond particles (Metadi : VWR Scientific, Houston, TX) and a light weight steel wool to produce a slight abrasive surface on the slide, which helped stabilize the embryo when bisected. With Method II, ~0.1 ml of modified PBS holding medium (PBl with 10% FCS) was applied to the surface of the base slide and the embryo placed in the center of the medium pool. Each embryo was viewed under an inverted microscope (100X) while a one-quarter section of the razor blade (held by a pair of hemostats) was carefully lowered (vertically) to manually bisect the embryo (n=50). The edge of the base slide was used as a fulcrum for the hand-held hemostat. Methods I and II were evaluated using intact (A) and ZP-free (B) day 6 to 7 embryos collected non-surgically and allotted to treatments across beef donors. Bisected embryos were then placed in a uterine fibroblast monolayer culture system with Ham's F-10 to evaluate embryo viability. With a little practice, both methods effectively bisected bovine embryos, with Method II being more efficient (>95% bisection success rate). Using Method II, intact and zona-free embryos could be bisected in ~1 minute. Furthermore, Method II had the advantage of being easier to set up and use under field conditions. One should not overlook the use of 2.5% pronase for ZP removal and the use of either of these simplified methods for splitting later-stage bovine embryos, before purchasing a commercial MICM unit.