Viable embryos and normal calves after nuclear transfer into Hoechst stained enucleated demi-oocytes of cows.
Bovine oocytes were bisected, stained with Hoechst 33342 and observed under a fluorescent microscope to identify nucleated and enucleated demi-oocytes. Other oocytes were bisected but not stained, or bisected and only half of each oocyte stained, and viewed under a fluorescent microscope. The oocytes were then used for nuclear transfer by fusing them with embryonic blastomeres from a 5-6 day bovine embryo. The fusion rate and proportion developing into compact morulae or blastocysts was compared among different types of demi-oocytes. Expt 1 examined the effect of staining and indicated no effect on either fusion rate or embryonic development whether or not the oocytes were stained. In Expt 2, stained and unstained nucleated and enucleated oocytes were compared. As in the first experiment, there were no differences between stained and unstained demi-oocytes. There was no difference between fusion rates of nucleated and enucleated oocytes. However, there was a significant difference in embryonic development between nucleated (10.4%) and enucleated (22.6%) demi-oocytes (P less than 0.05). In a final experiment, stained and unstained enucleated oocytes were used for nuclear transfer and the resulting embryos transferred into recipient cows. There was no difference in pregnancy rates or in the number of normal calves born whether stained or unstained recipient oocytes were used. Results from these experiments indicate that Hoechst staining and fluorescent microscopy can be used to identify enucleated demi-oocytes, and that these can be used for nuclear transfer, and result in viable embryos and normal calves.(ABSTRACT TRUNCATED AT 250 WORDS)
Westhusin
M E
ME
Granada Bioscience Inc., College Station, TX 77840.
Levanduski
M J
MJ
Scarborough
R
R
Looney
C R
CR
Bondioli
K R
KR
D000818:Animals; D001562:Benzimidazoles; D001755:Blastocyst / Q000502:physiology; D001757:Blastomeres / Q000502:physiology; D002417:Cattle / Q000196:embryology / Q000502:physiology*; D002478:Cells, Cultured; D005314:Embryonic and Fetal Development; D005260:Female; D005456:Fluorescent Dyes; D008856:Microscopy, Fluorescence; D009028:Morula / Q000502:physiology; D053652:Nuclear Transfer Techniques; D009865:Oocytes / Q000648:ultrastructure*; D011247:Pregnancy; D013194:Staining and Labeling
D001562:Benzimidazoles; D005456:Fluorescent Dyes; C017807:bisbenzimide ethoxide trihydrochloride