Timing of The First Zygotic Cleavage Affects Post-Vitrification Viability of Murine Embryos Produced In Vivo.

BACKGROUND: Timing of the first zygotic cleavage is an accurate predictor of embryo quality. Embryos that cleaved early (EC) have been shown to exhibit higher develop- mental viability compared to those that cleaved at a later period (LC). However, the vi- ability of EC embryos in comparison to LC embryos after vitrification is unknown. The present study aims to investigate the post-vitrification developmental viability of murine EC versus LC embryos. MATERIALS AND METHODS: In this experimental study, female ICR mice (6-8 weeks old) were superovulated and cohabited with fertile males for 24 hours. Afterwards, their ovi- ducts were excised and embryos harvested. Embryos at the 2-cell stage were catego- rized as EC embryos, while zygotes with two pronuclei were categorized as LC embryos. Embryos were cultured in M16 medium supplemented with 3% bovine serum albumin (BSA) in a humidified 5% CO2atmosphere. Control embryos were cultured until the blastocyst stage without vitrification. Experimental embryos at the 2-cell stage were vitri- fied for one hour using 40% v/v ethylene glycol, 18% w/v Ficoll-70 and 0.5 M sucrose as the cryoprotectant. We recorded the numbers of surviving embryos from the control and experimental groups and their development until the blastocyst stage. Results were analyzed using the chi-square test. RESULTS: A significantly higher proportion of EC embryos (96.7%) from the control group developed to the blastocyst stage compared with LC embryos (57.5%, P<0.0001). Similarly, in the experimental group, a significantly higher percentage of vitrified EC embryos (69.4%) reached the blastocyst stage compared to vitrified LC embryos (27.1%, P<0.0001). CONCLUSION: Vitrified EC embryos are more vitrification tolerant than LC embryos. Prese- lection of EC embryos may be used as a tool for selection of embryos that exhibit higher developmental competence after vitrification.

Antioxidant activity characterization, phytochemical screening, and proximate analysis of Cermela Hutan (Phyllanthus gomphocarpus Hook. F) roots and leaves.

BACKGROUND: Roots and leaves of the Cermela Hutan (Phyllanthus gomphocarpus Hook. F) plant were studied to determine antioxidant activity, phytochemical compounds, proportion of carbohydrate, crude protein, moisture, ash, fat, total phenolic content (TPC), and total flavonoid content (TFC). MATERIAL/METHODS: Ten percent (10%) aqueous extract from both Phyllanthus gomphocarpus roots (PGR) and leaves (PGL) were used in this study. Antioxidant activity characterization by TPC, TFC, Ferric Reducing Antioxidant Power (FRAP), 1,1-diphenyl-2-picrylhydrazyl (DPPH) scavenging activity, and phytochemical screening, as well as proximate analysis from both extracts were analyzed in this study. RESULTS: Phyllanthus gomphocarpus roots (PGR) and leaves (PGL) tested positive for flavonoid, saponin, tannins, and terpenoids, but PGR showed negative result for anthraquinones. In average weight of 100.0 g dry sample, the carbohydrates, protein, moisture, ash, fat, and energy content in PGR and PGL were 80.9%, 5.5%, 7.8%, 3.4%, 2.4%, and 367 Kcal/100g, and 66.5%, 14.8%, 10.7%, 6.5%, 1.5%, and 399 Kcal/100 g, respectively. Antioxidant assessments using FRAP and DPPH assay showed that PGL extracts possessed higher antioxidant capacity by reducing the ferric ion-TPTZ complex by 0.14 mg/ml ±0.0018 and higher scavenging activity, 83.83% ±0.54 as compared to PGR, 0.07 mg/ml ±0.0035 for FRAP and 62.87% ±1.33 for DPPH, respectively. The total phenolics content was significantly higher in PGL (208.77 mg GAE/g ±3.79) as compared to PGR (27.53 mg GAE/g ±0.42). However, there was no significant different in the total flavonoid contents for PGR (34.8 mg QE/g ±3.12) and PGL (32.43 mg QE/g ±3.92). CONCLUSIONS: Further investigations are suggested to isolate and characterize the other active constituents from this plant in combatting diseases.

Cytoskeletal alterations in different developmental stages of in vivo cryopreserved preimplantation murine embryos.

BACKGROUND: This study aimed to investigate the effects of vitrification and slow freezing on actin, tubulin, and nuclei of in vivo preimplantation murine embryos at various developmental stages using a Confocal Laser Scanning Microscope (CLSM). MATERIAL/METHODS: Fifty female mice, aged 4-6 weeks, were used in this study. Animals were superovulated, cohabitated overnight, and sacrificed. Fallopian tubes were excised and flushed. Embryos at the 2-cell stage were collected and cultured to obtain 4- and 8-cell stages before being cryopreserved using vitrification and slow freezing. Fixed embryos were stained with fluorescence-labelled antibodies against actin and tubulin, as well as DAPI for staining the nucleus. Labelled embryos were scanned using CLSM and images were analyzed with Q-Win software V3. RESULTS: The fluorescence intensity of both vitrified and slow-frozen embryos was significantly lower for tubulin, actin, and nucleus as compared to non-cryopreserved embryos (p<0.001). Intensities of tubulin, actin, and nucleus in each stage were also decreased in vitrified and slow-frozen groups as compared to non-cryopreserved embryos. CONCLUSIONS: Cryopreservation of mouse embryos by slow freezing had a more detrimental effect on the actin, tubulin, and nucleus structure of the embryos compared to vitrification. Vitrification is therefore superior to slow freezing in terms of embryonic cryotolerance.