Regardless of the presence of sperm-borne oocyte-activating factors, activation of bovine oocytes with exogenous activation stimuli is required for further development after intracytoplasmic sperm injection (ICSI). The present study was designed to develop a new activation regimen for improving the blastocyst yield after ICSI of bovine oocytes harvested from ovaries stored at 10-12 oC for 24 h. Following ICSI, oocytes were treated with 5 μM ionomycin for 5 min, 7% ethanol for 5 or 10 min, ionomycin followed by ethanol (5 or 10 min), ionomycin followed by 10 µg/mL Cycloheximide for 5 h, or ionomycin followed by 1.9 mM 6-dimethylaminopurine for 3 h. Across the activation regimens, the cleavage rates of ICSI oocytes (45-77%) were higher than those of parthenogenetically activated oocytes (11-21%; P<0.05). Activating the ICSI oocytes with ionomycin plus ethanol improved the blastocyst yield (29-30%) comparing to the non-treated oocytes (12%; P<0.05) but the other regimens did not (9-18%; P>0.05). The higher blastocyst yields were due to increasing the proportion of ICSI oocytes that passed through the early postfertilization events until cleavage. None of the regimens have any adverse effect on the quality of the blastocysts regarding the total cell number or the proportion of the inner cell mass cells. Thus, a new activation regimen composed from two triggers for single calcium increase has been proven effective to improve the blastocyst yield after bovine ICSI using oocytes harvested from stored ovaries.Dear Dr. John P. Kastelic, Thank you very much for prompt reply and accepting our manuscript for publication in Theriogenology. We have replied for each comment from Co-editor and the reviewer-2 in pointby-point basis.Sincerely yours, Shinichi Hochi (Corresponding author)Hany Abdalla (First author)Co-editor 1-P value has been added to the Abstract (Lines 27,29).2-The number of the references in Introduction section has been reduced.
Reviewer #21-Regarding omitting the two groups (5 min ethanol group and ionomycin followed by 5 min ethanol group); We do not think that the presence of these two groups causes any difficulty to recognize the superiority of ionomycin and ethanol combination. Since activation of 7% ethanol for 5 min 4 hr after ICSI is a commonly applied method for bovine ICSI, presence of such group must be important to declare the superiority of the new combination (ionomycin and ethanol) over the methods of ethanol alone under the same experimental circumstance regarding oocyte quality, injection skill, culture condition, and so on. For the ionomycin followed by 5 min ethanol, the efficiency of this method to improve the cleavage and the blastocyst yield was similar to that of ionomycin followed by 10 min ethanol, making it possible to choose the method in which oocytes are exposed to minimum exogenous stimuli. Tables 1 and 2.
2-The time of 2 nd polar body detection has been added in footnote of
15Running head: Bovine oocyte activation after ICSI
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AbstractRegardless of the pres...
