Bovine embryo development following ICSI: effect of activation, sperm capacitation and pre-treatment with dithiothreitol

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

“…This blastocyst yield is comparable to or even higher than those in many reports where bovine oocytes harvested from freshly collected ovaries were used for ICSI [8,10,11,[13][14][15][16]. Simultaneously with the ICSI experiments in the present study, high blastocyst yield (40 ± 3%, 90/223) was obtained after routine 225 IVF protocol, suggesting that oocytes harvested from stored ovaries carry sufficient in vitro developmental potential after IVF and ICSI if they were subjected to appropriate activation regimen.…”

“…Ionomycin + CHX [13]; Injected oocytes were first treated with ionomycin immediately after ICSI as described above, and followed by incubation in culture medium containing 10 µg/mL CHX for 5 h. (6) Ionomycin + 6-DMAP [24]; Injected oocytes were first treated with ionomycin immediately after ICSI as described above, and incubated in the TCM-199/FBS medium for 3 h. Next, the 150 oocytes were incubated in culture medium containing 1.9 mM 6-DMAP for an additional 3 h. The presence of the second polar body was recorded immediately before transferring the oocytes into culture medium (4 h after ICSI), except for the oocytes treated with ionomycin + CHX (5 h after ICSI when the oocytes were released from the CHX treatment) and ionomycin + 6-DMAP (3 h after ICSI when the oocytes were exposed to the 6-DMAP-containing medium). 155…”

“…The ICSI procedure has resulted in birth of live offspring in many species [5]. Since the first attempt of bovine ICSI [6], the developmental potential of the ICSI 45 oocytes into blastocysts in vitro or live calves in vivo still low, unstable and far from satisfactory regardless of the numerous efforts [7][8][9][10][11][12][13][14][15][16]. Beside the technical difficulties caused by darkness of the ooplasm, large size of the sperm heads, and elasticity and toughness of the oolemma, the inability of the mechanical stimulation and/or the injected spermatozoon itself to induce proper oocyte activation after ICSI [17] may be responsible for this low blastocyst yields.…”

“…This hypothesis is supported by the low blastocyst yields after ICSI without any exogenous activation stimuli [8,10,11,[13][14][15][16]. To improve 55 the blastocyst yields after bovine ICSI, additional exogenous activation stimuli that induce intracellular calcium spike such as direct current [19], calcium ionophore [20], ionomycin [8,13,14,16,17] or ethanol [9][10][11]15,16] have been applied. However, the monotonic calcium increase triggered by these stimuli [21,22] was insufficient to completely inactivate the MPF due to re-accumulation of the cyclin B [23] and brought the oocytes to arrest again at the M-III stage 60 [17,23,24].…”

“…Cycloheximide (CHX) as a protein synthesis inhibitor [13] or 6-dimethylaminopurine (6-DMAP) as a protein kinase inhibitor [8,14,16] are often used for this purpose.…”