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 study was undertaken to determine the relationship between chromosome composition and embryo development. Bovine cumulusoocyte complexes were matured in vitro and exposed to semen from one of three different bulls, one of which was a 1/29 Robertsonian translocation carrier. There were no significant differences among the three bulls in their sperm penetration or in the cleavage or developmental rates of resulting embryos, which were subjected to chromosome analysis on Day 2 (40-44 h postinsemination [hpil) and Day 5 (120-124 hpi) of development. No difference was detectable in the growth rates of embryos of different chromosomal composition on Day 2. On Day 5, a total of 343 embryos were obtained from all three bulls, of which 158 embryos could be karyotyped and assessed for cell numbers. Cell numbers for the Day 5 embryos showed that the mean numbers for the individual chromosome compositions (leastsquares means ± SEM) were 7.9 + 6.0 for haploids, 7.9 6.0 for polyploids, 16.8 4.3 for aneuploids, 23.4 4.0 for mixoploids, and 30.0 1.7 for diploids, indicating a significant reduction in the growth rate of embryos with chromosomal abnormalities (p < 0.001). It was concluded that development rates (as evidenced by cell numbers) were slowest in haploid and polyploid embryos, intermediate in aneuploid embryos, and fastest in mixoploid and diploid embryos.

Activation of bovine oocytes to produce a single haploid pronucleus in preparation for intracytoplasmic sperm injection (ICSI) has been investigated with various combinations of ionomycin and 6‐dimethylaminopurine (DMAP). Effects were evaluated by immunocytochemical staining, chromosomal analysis and assessment of development in vitro. Oocytes matured in vitro were exposed to: ionomycin alone (single or repeated treatments, Groups 1 and 2 respectively), ionomycin followed by DMAP (immediately or after a 3‐h delay, Groups 3 and 4), or no treatment (control, Group 5). They were then co‐cultured in M199 with bovine oviductal epithelial cells. Activation rates were not significantly different among groups but significantly fewer oocytes in Group 3 extruded a second polar body than in Groups 1, 2, and 4. Most parthenotes (60% to 80%) in Groups 1, 2, and 4 were haploid, whereas 82% in Group 3 were mixoploid or polyploid. Most of the parthenotes (88%) in Group 4 formed a single pronucleus besides extruding the second polar body and were therefore more suitable for ICSI than those of Groups 1 and 2 in which condensed chromosomes predominated. The respective rates of oocyte cleavage in Groups 1 to 4 were 24%, 36%, 70%, and 75%; corresponding blastocyst rates were 1%, 5%, 17%, and 8%. There were significantly fewer cells in the parthenotes of Groups 1, 2, and 4 than of Group 3, or of embryos produced by in vitro fertilization. Thus, delaying the addition of DMAP after ionomycin decreases chromosomal abnormalities and produces a high proportion of activated oocytes suitable for ICSI. Mol. Reprod. Dev. 50:485–492, 1998. © 1998 Wiley‐Liss, Inc.

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Effects of elevated in vitro temperature on in vitro produced early bovine embryos were analysed in order to determine its impact on the expression of heat shock protein 70 (hsp70). In vitro matured bovine oocytes, 2-cell and 8-cell embryos, and day 9 hatched blastocysts subjected to control and elevated temperature conditions were analysed by semiquantitative reverse transcription polymerase chain reaction methods for hsp70 mRNA expression. Results revealed the expression of hsp70 mRNA under control conditions and that early embryos can respond to heat stress by transcribing hsp70 mRNA. Confocal laser scanning microscopy used to localise the hsp70 protein in oocytes and embryos revealed that the distribution of hsp70 in the ooplasm of immature and mature oocytes is unaffected by exposure to elevated temperatures and that this protein was closely associated with the meiotic spindle, indicating its possible role in stabilising this structure. In 8-cell embryos derived under control conditions, hsp70 was evenly distributed in the cytoplasm but appeared as aggregates in some embryos exposed to elevated temperature. In heat-stressed hatched blastocysts, a more even distribution was noted following heat stress relative to corresponding controls, indicating their competence to respond to elevated temperature.