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A new method of splitting embryos without the use of a commercial micromanipulator unit
THERIOGENOLOGY
Pm NEW METHOD OF SPLITTING EMBRYOS WITHOUT THE USE OF A COMMERCIAL MICROMANIPULATOR UNIT R. W. Rorie,
C. W. McFarland, T. L. Overskei, S. A. Voelkel and R. A. Godke Animal Science Department, LAES, LSU Agricultural Center Louisiana State University, Baton Rouge, Louisiana 70803
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 a standard double-edged razor blade 75 mm glass microscope slide, (Gillette), a pair of 5 inch hemostats and the use of an inverted miThe microscope slide (base) was prepared @ croscope (Nikon-Diaphot). 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 Bisected embryos were then placed in a uterine across beef donors. 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 embryos could be success rate). Using Method II, intact and zona-free 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.
224
JANUARY
1985 VOL. 23 NO. 1
Pm NEW METHOD OF SPLITTING EMBRYOS WITHOUT THE USE OF A COMMERCIAL MICROMANIPULATOR UNIT R. W. Rorie,
C. W. McFarland, T. L. Overskei, S. A. Voelkel and R. A. Godke Animal Science Department, LAES, LSU Agricultural Center Louisiana State University, Baton Rouge, Louisiana 70803
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 a standard double-edged razor blade 75 mm glass microscope slide, (Gillette), a pair of 5 inch hemostats and the use of an inverted miThe microscope slide (base) was prepared @ croscope (Nikon-Diaphot). 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 Bisected embryos were then placed in a uterine across beef donors. 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 embryos could be success rate). Using Method II, intact and zona-free 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.
224
JANUARY
1985 VOL. 23 NO. 1