Superovulation of female rabbits was induced by subcutaneous injection(s) o
f porcine FSH. Zygotes were recovered 17 to 19 hr after hCG injection and w
ere classified into two categories under a microscope equipped with Nomarsk
i interference-contrast optics at x 200 magnification: (A) zygotes with cle
arly visible pronuclei, or (B) zygotes with visualized pronuclei after 10 m
in cenuifugation at 12,000 x g. No significant difference between strains w
as found in the proportion of category-a zygotes (JW 72.6% vs NZW 79.3%). P
ronuclei of category-A zygotes were located in the center of the cytoplasm,
and the pronuclei of category-B zygotes were slightly moved by centrifugat
ion toward the mass of cytoplasmic lipid droplets. Exogenous DNA solution (
5 mug/ml of fusion gene composed of bovine alphaS(1)-casein promoter and hu
man growth hormone structural gene) was microinjected into the pronucleus o
f the JW zygotes. The pronucleus of category-a zygotes with a mean volume o
f 7.4 pi swelled up to 16.6 pi (132% increase), while that of category-B zy
gotes with a mean volume of 6.1 pi swelled up to 15.9 pi (148% increase). N
evertheless, similar proportions of category-A and category-B zygotes devel
oped into offspring after transfer to recipient females (11.1 and 11.2%, re
spectively). The efficiency to produce hGH-carrying transgenic rabbits was
0.9% (2/235) from category-A zygotes and 0.5% (1/215) from category-B zygot
es(P>0.05). To date, transgenic rabbits have been produced without centrifu
gation of pronuclear zygotes. However approximately 25% of fertilized rabbi
t zygotes can be used for DNA microinjection after they have been centrifug
ed to visualize their pronuclei.