Pregnancies after transfer of aggregated parthenogenetic bovine activated oocytes

Theriogeno/ogy41:166, 1994 PREGNANCIES AFTER TRANSFER OF AGGREGATED PARTHENOGENETIC BOVINE ACTIVATED OOCYTES A. Boediono and T. Suzuki United Graduated School of Veterinary Sciences Yamaguchi University, Yamaguchi 753, Japan Bovine oocytes have been activated either by exposure to ethanol, electrical stimulation or a combination of both treatments. The present study was undertaken to improve the early development of parthenogenetically activated bovine oocytes and assess implantation after transfer to recipients. Follicular oocytes collected from slaughterhouse ovaries were matured in vitro for 30 to 32 h at 38.5°C under 5% CO 2 in air. Following maturation, the oocytes were suspended in culture medium containing 7% ethanol for 10 min to induce parthenogenetic activation. To examine the optimal concentration of cytochalasin for the formation of diploid parthenotes, ethanol-treated oocytes were suspended in culture medium containing 5, 7.5 and 10 #g/ml cytochalasin B (CB) or cytochalasin D (CD). The ethanol-treated oocytes were treated with CB or CD for 5 h to suppress the second polar body extrusion. The oocytes were stained with 1% aceto-orcein to examine activation. The formation of 2 pronuclei in ethanol-treated oocytes was 62%, 63% and 65%, respectively, when treated with 5, 7.5 and 10/~g/ml CB; 59%, 46% and 48%, respectively, when treated with 5, 7.5 and 10 ~g/ml CD. Among activated oocytes, most extruded a second polar body and formed one pronucleus when oocytes were treated with 7% ethanol only [35/43 (81%)]. When the ethanol-treated oocytes were treated with CB or CD (5, 7.5, 10 /~g/ml), overall activation frequency was 309/441 (70%) activated oocytes containing two pronuclei. The development of parthenogenetic oocytes was observed until Day 9 after activation. The cleavage rate was not significantly different between 7% ethanol followed by 5 ~g/ml CB treatment [364/450 (59%)] and 7% ethanol only [174/306 (57%)], but significantly different (P<0.01) than for fertilized embryos [333/456 (73%)]. The blastocyst rate was significantly different (P<0.01) among treatments (7% ethanol + 5 ~g/ml CB was 17%; 7% ethanol was 1%; fertilized was 29%). Karyotypical examination of blastocysts that were produced by ethanol followed by CB or CD treatment showed that almost all of the parthenogenetic oocytes that developed to blastocysts were diploid. To determine implantation rate, four parthenogenetic oocytes (8 cell-stage) were aggregated by hand manipulation under stereo microscope in culture medium. Aggregated parthenogenetic oocytes were then cultured in vitro (without zonae pellucidae) and transferred to 5 recipients (one recipient received 2 aggregated parthenogenetic oocytes). Three of 5 recipients were diagnosed pregnant on day 42. The estrus was prolonged after transfer in 3 recipients which were diagnosed pregnant, but they returned to estrus on day 57, 62 and 67 after the previous estrus. These results suggested that aggregated parthenogenetic oocytes will prolong survival in utero.

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Copyright © 1994 Butterworth-Heinemann