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Two experiments were conducted to determine the effect of insemination time on number of accessory sperm per embryo (ovum), fertilization rate, and embryo quality. Semen was collected from three fertile Holstein bulls and cryopreserved in egg yolk-citrate-glycerol. In experiment 1, cows were continuously monitored for behavioral estrus by the HeatWatch estrous detection system and were artificially inseminated (AI) with one 0.5-ml straw (25 x 10(6) sperm) at the onset of estrus (AI 0 h), 12 h after onset (AI 12 h), or received natural service at 0 h (Nat 0 h) from one of three bulls. From 150 inseminations, 115 embryos and ova (AI 0 h: n = 39; AI 12 h: n = 39; Nat 0 h: n = 37) were recovered 6 or 7 d after insemination. Fertilization rates differed between treatments (AI 0 h: 67%; AI 12 h: 79%; Nat 0 h: 98%). Median accessory sperm per embryo (ovum) also differed (AI 0 h: 1; AI 12 h: 10; and Nat 0 h: 27) and paralleled the fertilization rate. Embryo quality was not affected by insemination time or natural service. In experiment 2, cows received AI at 0, 12, or 24 h (AI 24 h) after the onset of estrus as determined by HeatWatch. From 154 inseminations, 117 embryos and ova (AI 0 h: n = 39; AI 12 h: n = 39; AI 24 h: n = 39) were recovered 6 or 7 d after insemination. Fertilization rates did not differ in experiment 2 (AI 0 h: 66%; AI 12 h: 74%; AI 24 h: 82%); however, a trend toward a higher fertilization rate accompanied AI 24 h. Median accessory sperm values increased from AI 0 h (1) to AI 24 h (4). Embryo quality declined with AI at increasing intervals after onset of estrus, as percentages of excellent and good, fair and poor, and degenerate embryos were as follows: 77, 15, 8; 52, 38, 10; and 47, 19, 34 for the 0-, 12-, and 24-h inseminations, respectively. Results indicate AI 12 h after the onset of estrus provides a compromise between potential fertilization failure (AI 0 h) and embryo failure (AI 24 h), despite increased accessory sperm per embryo (ovum) after AI 24 h. Artificial insemination 12 h after onset of estrus should optimize fertility of dairy cattle through an acceptable fertilization rate, number of accessory sperm per embryo, and desirable embryo quality.

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This experiment was conducted to determine whether use of fresh or frozen semen at either 20 x 10(6) (low) or 100 x 10(6) (high) sperm per dose affects the number of accessory sperm and fertilization status/embryo quality as determined from ova/embryos recovered nonsurgically 6 d after insemination. Ejaculates of four bulls were split and prepared for use as fresh or frozen semen at either the high or low dose. From 129 inseminations to normally cycling cows, 98 ova/embryos were recovered. To reduce male effects, ova/embryos used were randomly balanced across treatments, by ejaculate within bull for evaluation of frozen vs fresh semen (n = 80) and by bull for evaluation of high vs low dosage treatments (n = 76). Distribution of accessory sperm was highly skewed downward; thus, median values were more meaningful than means. Freezing semen had no significant effect on fertility status/embryo quality or number of accessory sperm at either dosage. Increasing dosage improved the number of accessory sperm per ovum or embryo (median value) and fertility status/embryo quality (P < .05). Mean +/- SD and median values for accessory sperm were 37.8 +/- 38.3 and 27.5; 28.9 +/- 62.8 and 3.0 for the high and low dose, respectively. Percentage of unfertilized ova, degenerate embryos, and embryos classified poor to fair and good to excellent were 3, 5, 24, 68, and 21, 16, 18, 45, for the high and low dose, respectively. We conclude that number of accessory sperm and fertility status/embryo quality respond favorably to increased dosage of semen and that freezing semen in this study was not detrimental to these parameters.

The present study was carried out to 1 ) evaluate the viability of in vitro fertilized zygotes after microinjection of DNA, 2 ) assess the influence of oocyte quality upon the development rate of embryos when injected with DNA, and 3 ) determine the integration frequency of green fluorescent protein DNA into microinjected embryos. Oocytes were aspirated from ovaries of nine nonlactating Holsteins and were categorized into grades A, B, C, and D. At 16 h after in vitro fertilization, approximately half of the pronuclear stage presumptive zygotes were classified as having 1 pronucleus or 2 pronuclei, and they were microinjected with DNA constructs. A potential predictor of DNA integration frequency at d 10 was assessment of the incidence of green fluorescing embryos. The proportion of cleaved embryos that developed to morulae or blastocysts was not different between groups with 1 pronucleus injected (45%), 1 pronucleus uninjected (64%), or 2 pronuclei injected (49%). However, the development of morulae or blastocysts was higher in the group with 2 pronuclei uninjected (69%). The overall developmental score of green fluorescent protein-positive embryos was higher for grade A oocytes (1.3 ± 0.1) than for grade B (0.8 ± 0.1), C (0.6 ± 0.1), or D (0.3 ± 0.1) oocytes. The results show that production of transgenic bovine blastocysts can occur from the microinjection of a presumptive zygote having only one visible pronucleus. Initial oocyte quality is an important factor in selection of oocytes suitable for microinjection of DNA and for preimplantation development to produce bovine transgenic embryos. ( Key words: bovine oocytes, in vitro fertilization, ovum pickup, green fluorescent protein) Abbreviation key: CMV-EGFP = cytomegalovirus enhanced green fluorescent protein, GFP = green fluorescent protein, IVF = in vitro fertilization, IVM = in vitro maturation, PCR = polymerase chain reaction, 0PN = no pronucleus, 1PN = 1 pronucleus, 2PN = 2 pronuclei, WAP6FIX = whey acidic protein factor IX.