Sperm and Oocyte Treatments to Improve the Formation of Male and Female Pronuclei and Subsequent Development Following Intracytoplasmic Sperm Injection into Bovine Oocytes1

Abstract

This study assessed pronuclear formation, the chromosomal constitution, and the developmental capacity of bovine zygotes formed by intracytoplasmic injection of oocytes with sperm, treated or not with dithiothreitol (DTT). Oocytes were matured in vitro for 22-24 h and then centrifuged so that sperm, prepared by swim-up in the presence or absence of 5 mM DTT, could be injected into the cleared area of the ooplasm. Injected oocytes were activated by treatment with 5 microM ionomycin (5 min) and, after a 3-h interval, with 1.9 mM 6-dimethylaminopurine (DMAP) for 3 h. They were then cocultured with bovine oviductal epithelial cells in M199. Sperm treatment resulted in a significantly higher proportion of male pronucleus formation 16 h after injection (40% vs. 11%; p < 0.0001) and a significantly higher rate of blastocyst development (24% vs. 10%; p < 0.005). Sixty-one percent of blastocysts produced with treated sperm were diploid. Of 12 blastocysts produced with treated sperm and sexed by a polymerase chain reaction, 4 were male and 7 female, and in one a definite diagnosis could not be made. Embryo transfer (2 embryos per heifer) resulted in pregnancies in 6 of 16 recipients at Day 49, but none was carried to term. These results show that the efficiency of bovine intracytoplasmic sperm injection can be improved by sperm pretreatment with DTT and by oocyte activation with ionomycin plus DMAP, although the developmental capacity of the resulting embryos remains limited.

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

“…The blastocyst yield in non-treated group (12%) is higher than those (0-8%) reported previously by several laboratories [8,10,11,[14][15][16], but comparable to or even lower than those (15-23%) in other reports [12,13]. However, blastocysts/ 235 cleavage rate in our study (30%) is comparable to that reported by Wei and Fukui [12] (32%) which indicates that a failure in the early post-fertilization events -from fertilization until cleavage-is the main determiner to the successful development after bovine ICSI.…”

“…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.…”

“…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.…”