Nuclear transfer to produce cattle is inefficient because 1) donor cells are not easily synchronized in the proper phase of the cell cycle, 2) the nucleus of these cells is not effectively reprogrammed, 3) the rate of attrition of late-term pregnancies is high, and 4) the health of early postnatal calves is compromised. The cyclin dependent kinase 2 inhibitor, roscovitine, was used to maximize cell cycle synchrony and to produce cells that responded more reliably to nuclear reprogramming. Roscovitine-treated adult bovine granulosa cells (82.4%) were more efficiently synchronized (P < 0.05) in the quiescent G0/G1 phase of the cell cycle than were serum-starved cells (76.7%). Although blastocyst development following nuclear transfer was elevated (P < 0.05) in the serum-starved group (21.1%) relative to the roscovitine-treated cells (11.8%), the number of cells in the blastocysts derived from roscovitine-treated cells was higher (P < 0.05) than those derived from the serum-starved group (roscovitine-treated group: 142.8 +/- 6.0 cells; serum-starved group: 86.8 +/- 14.5 cells). The resulting fetal and calf survival after embryo transfer was enhanced in the roscovitine-treated group (seven surviving calves from six pregnancies) compared with serum-starved controls (two calves born, one surviving beyond 60 days, from five pregnancies). Roscovitine culture can predictably synchronize the donor cell cycle and increase the nuclear reprogramming capacity of the cells, resulting in enhanced fetal and calf survival and increased cloning efficiency.

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This study examined bovine cloning strategies that may be used for gene targeting in animals of known phenotypic traits. Fibroblast cells derived from an adult and a fetus of the same genotype were transfected with a plasmid (pEGFP-N1) containing the enhanced green fluorescence protein and neomycin-resistant genes. After transfecting 2 x 10(5) cells, 49 adult and 35 fetal cell colonies were obtained. Green fluorescence expression was observed in 35 out of 49 (71.4%) adult clones and in 30 out of 35 (85.7%) fetal clones. Developmental rates to the blastocyst stage following nuclear transfer (NT) did not differ among nontransfected cell lines (adult, 20.0%; NT fetal, 18.3%), whereas developmental rates were significantly lower for adult and fetal cell lines expressing enhanced green fluorescent protein (EGFP; 11.3% and 6.4%, respectively, P < 0.05). However, there was no decrease in NT developmental rates (19.8%) when donor nuclei from EGFP-transfected cell lines not expressing EGFP but retaining neomycin-resistant gene expression were used as donor nuclei. NT embryos from adult and fetal cell lines had similar morphology, cell number, and ploidy. The results indicated that adult and NT fetal cells (identical genotype) can complete clonal propagation, including transfection and selection, and can be used to produce transgenic NT embryos; however, a possible deleterious effect of EGFP on embryo development should be considered in future gene targeting studies.

The interrelationships between the FSH surge that initiates a follicular wave and the follicles in the wave were examined in heifers. In experiment 1, > or = 5-mm follicles were ablated 5 days after ovulation and heifers (n = 6/group) received a total dosage of 0, 37.5, 75, or 150 units of porcine FSH. Half of the FSH dosage was administered 24 h after ablation followed by the other half 12 h later. Blood samples were taken after the initial FSH injection for FSH assay, and ovaries were examined daily with ultrasound to monitor follicle growth. There were progressively higher FSH concentrations at the mean peak (8 h after initial injection in all groups) as the dosage increased (interaction of dose and time; p < 0.001). Compared to values in controls, the highest dosage (150 units) approximately doubled the number of 5- and 6-mm follicles; this then progressed into a 4- to 7-fold increase in the number of 7- and 8-mm follicles. In experiment 2, either all (controls; n = 6), two (n = 11), one (n = 6), or zero (n = 6) follicles of the first wave of an estrous cycle were retained and the remaining were ablated upon reaching 5 mm. Scanning and blood sampling were performed every 8 h for 72 h after the initial ablation. Mean FSH concentrations during 0 to 72 h decreased (p < 0.004) as the number of retained follicles increased. In heifers in the one-follicle group, the randomly chosen 5-mm follicle developed the characteristics of a dominant follicle. The following conclusions were made: 1) the number of follicles that advanced into a follicular wave was increased by exaggerating the height of the FSH surge, 2) all > or = 5-mm follicles of a wave contributed to the declining portion of the FSH surge, and 3) any 5-mm follicles at the emergence of a wave were capable of becoming the dominant follicle.