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This research was designed to improve our understanding of oocyte maturation and acquisition of developmental competence of oocytes using prepubertal heifers as a model. Oocytes were collected by ultrasound-guided transvaginal oocyte retrieval from 20 age-matched calves at 5, 7, 9, and 11 mo of age that had or had not received gonadotropin stimulation. Numbers of oocytes recovered from unstimulated heifers decreased with age, being 15 +/- 2, 12 +/- 1, 7 +/- 1, and 7 +/- 1 (for 5-, 7-, 9-, and 11-mo-old calves, respectively). Corresponding numbers for the gonadotropin-stimulated heifers were 18 +/- 2, 16 +/- 2, 13 +/- 1, and 15 +/- 2. Cumulus-oocyte complexes were graded from A to D on the basis of their morphology, and the better-grade A and B oocytes were used for in vitro maturation and fertilization. A higher proportion of grade A and B oocytes was found for the stimulated vs. unstimulated prepubertal calves at 5 mo of age (92% vs. 49%, p < 0.05) and 7 mo of age (96% vs. 63%, p < 0.05), but the improvement of this parameter by stimulation was not significant for peri- and postpubertal calves at 9 (53% vs. 38%, p > 0.05) and 11 (53% vs. 38%, p > 0.05) mo of age. Embryo development to morula and blastocyst stages was poorer (0-11%, p < 0.05) for oocytes collected from unstimulated calves at 5-9 mo of age than for those from the age-matched but gonadotropin-stimulated groups (10%, 39%, and 31%, at 5, 7, and 9 mo of age, respectively). In calves 11 mo of age, embryo development to morula and blastocyst stages was similar with and without gonadotropin stimulation (48% vs. 40%, p > 0.05) and was comparable to that of adult cow oocytes (45%, p > 0.05). The data suggest that the acquisition of oocyte competence for normal embryo development in prepubertal calves is influenced by animal age and hormonal treatment.

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Bovine oocytes matured in vitro (IVM) for 20 hr vs. 40 hr were treated for activation with 7% ethanol in Dulbecco's phosphate-buffered saline for 5 min followed by incubation in M199 + 7.5% fetal calf serum containing cycloheximide (10 micrograms/ml). Treated IVM oocytes and the controls (no ethanol and cycloheximide exposures) were fixed after 0, 1, 2, 3, 4, 5, 7, 10, and 20 hr of incubation and stained 24 hr later with 1% acetoorcein to examine nuclear events. Different stages of nuclear development of the activated oocytes were identified on the basis of nuclear and chromosomal morphology. Pronuclear development was classified into four stages (PN I, II, III, and IV) according to pronuclear progression in chromatin decondensation, nucleoplasm appearance, and nuclear size. The results demonstrated that the combined activation treatment effectively drove the IVM oocytes, both young (20 hr) and aging (40 hr), out of metaphase arrest. The activation rates for young oocytes examined immediately after 0, 1, 2, 3, 4, 5, 7, 10, and 20 hr of incubation with cycloheximide were, respectively, 7%, 24%, 77%, 96%, 92%, 97%, 98%, 93%, and 98%. For aging oocytes (40 hr) the corresponding activation values at the same time intervals were 6%, 84%, 100%, 100%, 100%, 100%, 98%, 100%, and 100%, respectively. These values were significantly higher than those for the corresponding controls. The activated aging oocytes achieved peak activation response more rapidly than did young oocytes. In addition, nuclear events in aging oocytes proceeded faster than those in young ones. Spontaneous activation rates of the aging oocytes were also higher (6-57%) than those of the young ones (0-14%).

This research was undertaken to improve development of parthenogenetic embryos following various combined treatments of ethanol and cycloheximide. In Experiment 1 in vitro matured oocytes (IVM, 24 hr) were treated with 7% ethanol for 5 min followed by incubation in 10 micrograms/ml cycloheximide in Medium 199 for 0 (control), 5, 10, and 20 hr. Development to 2-8 cells following culture for 3 days was similar among treated groups (32-41%; P > 0.05), which was higher than that of controls (6%; P < 0.05). Experiment 2 compared pre-ethanol exposures for 0, 1, 2.5, and 5 min, followed by 5 hr cycloheximide treatment on activation development. One- to 5-min groups resulted in 42-44% cleavage contrasted to 1-12% for controls (P < 0.05). Experiment 3 examined the effect on oocyte development of ethanol and different concentrations of cycloheximide (0, 1, 5, and 10 micrograms/ml). Cleavage to 2-8 cells was similar among the 5 and 10 micrograms/ml cycloheximide groups (36% and 42%, P > 0.05) but lower (P < 0.05) for the 1 micrograms/ml group (24%) and the controls (2-13%). When 5 micrograms/ml cycloheximide was used (Experiment 4), pre-exposure to ethanol (1, 2.5, and 5 min) resulted in more oocytes cleaved (38-41%) than in the cycloheximide alone group (0%) or the control (0%, P < 0.05). Experiment 5 tested blastocyst development of the activated oocytes with or without cytochalasin B treatment.(ABSTRACT TRUNCATED AT 250 WORDS)