135 LEVELS OF REACTIVE OXYGEN SPECIES (ROS), APOPTOSIS AND CRYORESISTANCE OF BOVINE EMBRYOS PRODUCED IN VITRO IN THE PRESENCE OF ANTIOXIDANTS
Bibliographic record
Abstract
The production of ROS is a normal process that occurs in cellular mitochondrial respiratory chain. However, the increase in ROS due to the high oxygen tension during in vitro production of bovine embryos induces oxidative stress, leading to embryonic development failure. Thus, the aim of this study was to evaluate the effects of supplementation with antioxidants (cysteine – CIST; ß-mercaptoethanol – ßME and catalase – CAT) during in vitro maturation (IVM) and in vitro culture (IVC) on the embryo development, cryoresistance, and quality, as well as the amounts of intracellular ROS produced during embryo culture. Cumulus–oocyte complexes (n = 565) were IVM in medium TCM-199 supplemented with 0.6 mM CIST, 100 µM ßME, 100 UI CAT, or without antioxidants (Contr). After fertilization, zygotes were IVC in SOF medium during the first 72 h (up to Day 3) with or without addition of the same antioxidant used for IVM, and then all the embryos were transferred to SOF medium without antioxidants. All cultures were conducted at 38.5°C in 5% CO2 in air. The cleavage and blastocysts rates were evaluated, respectively, at 72 and 168 h post-insemination. Part of obtained blastocysts (n = 133) was vitrified (Ingámed®, Maringá, PR, Brazil). The remaining was stained (n = 46) with 5 µM of the fluorescent probe 6-carboxy-2'7'-dichlorodihydrofluorescein diacetate (H2DCFDA; Molecular Probes, Invitrogen, Oakville, Canada) or stained (n = 62) for TUNEL (In situ Cell Death Detection Kit, Fluorescein, Roche Applied Science, IN, USA). Stained embryos were immediately evaluated under an epifluorescence inverted microscope and the images of embryos stained with H2DCFDA were analyzed by Q-Capture Pro image software (QImaging, Surrey, BC, Canada) for determining the fluorescent intensity. The vitrified embryos were thawed and cultured for 24 h to evaluate the re-expansion rates. The difference between groups was compared by ANOVA followed by Tukey test, and re-expansion rates by chi-square test (P < 0.05). The cleavage rates were 82.9% ± 3.2a (Contr), 80.4% ± 2.5a (CIST), 87.0% ± 1.8a (ßME) and 81.2% ± 3.0a (CAT). The blastocyst rates were 48.7% ± 3.4ab (Contr), 34.4% ± 4.2b (CIST), 36.0% ± 5.6ab (ßME), and 56.8% ± 7.5a (CAT). The re-expansion rates were 76.0%ab (Contr), 66.7%b (CIST), 50.0%b (ßME), and 66.1%b (CAT). The fluorescent intensity was 1.0 ± 0.07a (Contr), 0.8 ± 0.05bc (CIST), 0.6 ± 0.05c (ßME) and 0.7 ± 0.07bc (CAT). The total number of cells was 85.7 ± 3.5a (Contr), 89.6 ± 4.8a (CIST), 99.5 ± 5.4a (ßME), and 81.5 ± 4.3a (CAT), and the rate of apoptotic cells was 4.3 ± 1.2a (Contr), 1.5 ± 0.4b (CIST), 1.6 ± 0.4b (ßME), and 2.4 ± 0.6ab (CAT). Supplementation with CAT improved embryo development, compared with CIST (P < 0.05). The fluorescent intensity was lower for all embryos produced with antioxidants (P < 0.05) and the rate of apoptosis was reduced in CIST and ßME (P < 0.05). In conclusion, supplementation with antioxidants is an interesting strategy to improve the embryo quality since it reduces the cell death caused by oxidative stress. However, such improvement in embryo quality did not affect embryo cryotolerance. Financial support was provided by CNPq and FAPESP 01/18257-2.
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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 | 0.003 | 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".