A combined treatment of ionomycin with ethanol improves blastocyst development of bovine oocytes harvested from stored ovaries and microinjected with spermatozoa

Abstract

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 * Manuscript 2 AbstractRegardless of the pres...

Vitrification of ICSI- and IVF-derived bovine blastocysts by minimum volume cooling procedure: effect of developmental stage and age

“…Then, ICSI was performed using a piezo-driven micromanipulator (PMAS-CT150; PrimeTech, Ibaraki, Japan), as described previously [24]. …”

Procedure for Bovine ICSI, not Sperm Freeze-drying, Impairs the Function of the Microtubule-organizing Center

“…(1) For control ICSI, a Percoll-washed sperm pellet was treated with 5 mM DTT in mBO medium for 20 min at 37.0 C and then washed twice with mBO medium at 300 g for 5 min each. The ICSI was performed with a piezo-driven micromanipulator (PMAS-CT150; Prime Tech, Ibaraki, Japan) in M2 medium [29] containing 8% polyvinylpyrrolidone (PVP), as described previously [21]. The ICSI oocytes were first treated with 5 μM ionomycin in Ca 2+ /Mg 2+ -free PBS for 5 min and incubated in TCM199+5% FBS for 4 h. Next, the oocytes were treated with 7% ethanol in TCM199+1 mg/ml PVP for 5 min.…”

“…Twenty μl of the sperm suspension was added to 80 μl of the fertilization medium containing 10-12 matured oocytes (final sperm concentration, 5 × 10 6 sperm /ml) and kept for 6 h at 38.5 C under 5% CO2 in air. Developmental potential of FD-ICSI, ICSI and IVF oocytes into blastocysts was investigated according to the method described previously [21] as a preliminary part of this experiment. Briefly, ≤30 presumptive zygotes (4 h after FD-ICSI/ICSI or 6 h after IVF) were transferred to 250-μl microdrop of modified synthetic oviduct fluid (mSOF) [30] supplemented with 30 μl/ml essential amino acids solution (× 50, Gibco-11130), 10 μl/ml nonessential amino acids solution (× 100, Gibco-11140) and 5% FBS and cultured at 39.0 C under 5% O2, 5% CO2 and 90% N2 for 8 days.…”

“…Only one blastocyst was obtained from culture of 176 FD-ICSI zygotes for 7 days (<1%), while 21 and 43% of the control ICSI-and IVF-derived zygotes developed into blastocysts during the culture period, respectively. Although no analyses for karyotype or ploidy were performed with these blastocysts, the supplementary activation regimen for FD-ICSI and control ICSI oocytes employed here (ionomycin plus ethanol at a 4-h interval) was unlikely to produce parthenogenetically developing blastocysts [21]. An alkaline comet assay indicated that the DNA fragmentation index (moment: length of comet tail × % DNA liberated) was not significantly different between fresh and FD spermatozoa, while a significant increase of the index was detected in H2O2-treated positive control spermatozoa (Table 2).…”

Microtubule assembly crucial to bovine embryonic development in assisted reproductive technologies

“…To overcome this problem, bovine ICSI oocytes have been treated to induce intracellular calcium spike with a direct current pulse(s) (Hwang et al 2000), calcium ionophore (Keskintepe & Brackett 2000), ionomycin (Rho et al 1998; Galli et al 2003; Oikawa et al 2005; Abdalla et al 2009) or ethanol (Horiuchi et al 2002; Oikawa et al 2005; Abdalla et al 2009). Because these stimuli do not induce long‐lasting oscillations, they have been combined with other chemicals, such as cycloheximide (Galli et al 2003) or 6‐dimethylaminopurine (Rho et al 1998; Oikawa et al 2005) that interfere with either the re‐synthesis or the activation of the metaphase‐promoting factor (MPF), respectively.…”