67 PREMATURE CHROMOSOME CONDENSATION IS NOT ESSENTIAL FOR BOVINE SOMATIC NUCLEAR REPROGRAMMING
Bibliographic record
Abstract
The objective of this study was to determine the effect of donor nuclear exposure in MII oocyte cytoplasm on nuclear reprogramming events and subsequent development of cloned embryos in cattle. Somatic nuclear transfer (NT) was performed by electrofusion of the enucleated MII oocytes with cultured cumulus cells by ovum pickup. Donor cell-cytoplast pairs were fused by applying two direct current pulses at 2 kV/cm for 10 µs. Fused NT embryos were randomly divided in to Treatment A (immediate activation) and Treatment B (delayed activation, 4-h exposure in MII cytoplast before activation). In both treatment groups, the activation protocol was identical and consisted of incubation in cycloheximide (10 µg/mL) plus cytochalasin D (2.5 µg/mL) in M199 + 7.5% FBS for 1 h, followed by culture in cycloheximide (10 µg/mL) for an additional 4 h. Reconstructed embryos in both groups were subsequently cultured in CR1aa in 5% CO2, 5% O2, and 90% N2 at 39°C. Samples from both treatments were fixed at 0, 1, 2, 4, 6, 12, 18, 24, 30, 36, and 44 h after fusion. All fixed samples were double stained for tubulin and DNA, and observed with a laser-scanning confocal microscope for changes in nuclei and microtubules. The experiment was replicated three times. Cleavage rate and blastocyst rate were recorded and analyzed by Studen's t-test (SPSS 11.0, Chicago, IL). The staining revealed an absence of premature chromosome condensation (PCC) in all embryos in Treatment A. However, delayed activation (Treatment B) resulted in a high incidence of PCC, probably due to high levels of MPF in the MII cytoplasts. Chromosome condensation was observed in Treatment B at 4 h (82%, n = 17), 6 h (80%, n = 10), 12 h (36%, n = 25), 18 h (71%, n = 24), 24 h (50%, n = 16) and 30 h (6%, n = 18) after fusion. Subsequent culture results of these cloned embryos (Table 1) indicated that there were significantly higher cleavage rates and blastocyst development in Treatment A than in Treatment B. This study clearly demonstrated that PCC is not essential to support bovine cloned blastocyst development. Direct exposure of donor nuclei in a MII cytoplast enviroment for a very short time was sufficient for nuclear reprogramming. Table 1. Effect of donor nuclear exposure duration in MII cytoplasm on the development potential of cloned bovine embryos
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How this classification was reachedexpand
Full frame distilled prediction
Teacher imitationNot calibrated prevalence, not ground truth. Human validation pending. Learned from the 10,348 direct Codex labels and 10,348 direct Gemma labels. Candidate is the union of thresholded teacher heads; consensus is their intersection. These outputs are machine_predicted_unvalidated and are not human labels or direct frontier model labels.
Codex and Gemma teacher scores by category
| Category | Codex | Gemma |
|---|---|---|
| Metaresearch | 0.001 | 0.001 |
| Meta-epidemiology (narrow) | 0.000 | 0.000 |
| Meta-epidemiology (broad) | 0.000 | 0.000 |
| Bibliometrics | 0.000 | 0.000 |
| Science and technology studies | 0.000 | 0.000 |
| Scholarly communication | 0.000 | 0.000 |
| Open science | 0.000 | 0.000 |
| Research integrity | 0.000 | 0.000 |
| Insufficient payload (model declined to judge) | 0.000 | 0.000 |
Machine scores (provisional)
The two teacher heads of the student model, read on this work. A score orders the frame for review; it never asserts a category, and the validation status ships verbatim with every row.
Baseline scores from an immature model (maturity gate not passed, 7 training rounds). Scores rank; they never assert a category.
score_only:v0-immature-baseline · verbatim from the scoring run: score_only means the number may rank works, and no category label ships from itClassification
machine, unvalidatedMachine predicted; a candidate call from one teacher head, not a consensus.
How this classification was reached, model by model and score by score, is at the end of the page under "How this classification was reached".