98ddebe243fb955b57a80430cd395c05 pmc_33579034.pdf d9d96113d3f9b2e37c7a43decc17e4fe2bffceeb pmc_33579034.pdf 0b34d8ab5d4bd41bf560135bac2f6ba161793f0eae4e93d332df64128f2aeaa4 pmc_33579034.pdf Title: A Shorter Equilibration Period Improves Post-Warming Outcomes after Vitrification and in Straw Dilution of In Vitro-Produced Bovine Embryos Subject: This study was designed to the optimize vitrification and in-straw warming protocol of in vitro-produced bovine embryos by comparing two different equilibration periods, short equilibrium (SE: 3 min) and long equilibrium (LE: 12 min). Outcomes recorded in vitrified day seven (D7) and day eight (D8) expanded blastocysts were survival and hatching rates, cell counts, apoptosis rate, and gene expression. While survival rates at 3 and 24 h post-warming were reduced (p < 0.05) after vitrification, the hatching rates of D7 embryos vitrified after SE were similar to the rates recorded in fresh non-vitrified blastocysts. The hatching rates of vitrified D8 blastocysts were lower (p < 0.05) than of fresh controls regardless of treatment. Total cell count, and inner cell mass and trophectoderm cell counts were similar in hatched D7 blastocysts vitrified after SE and fresh blastocysts, while vitrified D8 blastocysts yielded lower values regardless of treatment. The apoptosis rate was significantly higher in both treatment groups compared to fresh controls, although rates were lower for SE than LE. No differences emerged in BAX, AQP3, CX43, and IFN gene expression between the treatments, whereas a significantly greater abundance of BCL2L1 and SOD1 transcripts was observed in blastocysts vitrified after SE. A shorter equilibration vitrification protocol was found to improve post-warming outcomes and time efficiency after in-straw warming/dilution. Keywords: cow; cryopreservation; expanded blastocyst; total cell number; inner cell mass; trophectoderm; SOX2; TUNEL; apoptosis; gene expression Author: Iris Martínez-Rodero, Tania García-Martínez, Erika Alina Ordóñez-León, Meritxell Vendrell-Flotats, Carlos Olegario Hidalgo, Joseba Esmoris, Xabier Mendibil, Sabino Azcarate, Manel López-Béjar, Marc Yeste and Teresa Mogas Creator: LaTeX with hyperref Producer: pdfTeX-1.40.21 CreationDate: Fri Feb 26 02:18:28 2021 CET ModDate: Fri Feb 26 02:40:46 2021 CET Custom Metadata: no Metadata Stream: no Tagged: no UserProperties: no Suspects: no Form: none JavaScript: no Pages: 15 Encrypted: no Page size: 595.276 x 841.89 pts (A4) Page rot: 0 File size: 1320712 bytes Optimized: no PDF version: 1.7 name type encoding emb sub uni object ID ------------------------------------ ----------------- ---------------- --- --- --- --------- DUFXDP+VnURWPalladioL Type 1 Custom yes yes no 10 0 OCDMAF+URWPalladioL-Ital Type 1 Custom yes yes no 15 0 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Title: Materials and Methods Destination: section.2 Children: Item: Title: Chemicals and Suppliers Destination: subsection.2.1 Item: Title: In Vitro Production of Bovine Blastocysts Destination: subsection.2.2 Item: Title: Embryo Vitrification and Warming Destination: subsection.2.3 Children: Item: Title: Vitrification Protocol Destination: subsubsection.2.3.1 Item: Title: Warming Protocol Destination: subsubsection.2.3.2 Item: Title: Differential Staining and TUNEL Destination: subsection.2.4 Item: Title: RNA Extraction and Relative Quantification of mRNA by Real-Time Reverse Transcription PCR Destination: subsection.2.5 Item: Title: Statistical Analysis Destination: subsection.2.6 Item: Title: Results Destination: section.3 Children: Item: Title: A Shorter Time of Exposure of Embryos to the Equilibrium Solution Leads to Improved Embryo Development (Experiment 1) Destination: subsection.3.1 Item: Title: Different Exposure Times to the Equilibration Solution Modify TCN, ICM Cell Numbers, TE Cell Numbers and Apoptosis Rates at 24 h Post-Warming (Experiment 2) Destination: subsection.3.2 Item: Title: Different Times of Exposure to the Equilibration Solution Modify Gene Expression Patterns in Warmed Expanded Blastocysts Vitrified Using the VitTrans as the Cryodevice (Experiment 3) Destination: subsection.3.3 Item: Title: Discussion Destination: section.4 Item: Title: Conclusions Destination: section.5 Item: Title: References Destination: section.5 Info: Title: A Shorter Equilibration Period Improves Post-Warming Outcomes after Vitrification and in Straw Dilution of In Vitro-Produced Bovine Embryos Author: Iris Martínez-Rodero, Tania García-Martínez, Erika Alina Ordóñez-León, Meritxell Vendrell-Flotats, Carlos Olegario Hidalgo, Joseba Esmoris, Xabier Mendibil, Sabino Azcarate, Manel López-Béjar, Marc Yeste and Teresa Mogas Subject: This study was designed to the optimize vitrification and in-straw warming protocol of in vitro-produced bovine embryos by comparing two different equilibration periods, short equilibrium (SE: 3 min) and long equilibrium (LE: 12 min). Outcomes recorded in vitrified day seven (D7) and day eight (D8) expanded blastocysts were survival and hatching rates, cell counts, apoptosis rate, and gene expression. While survival rates at 3 and 24 h post-warming were reduced (p < 0.05) after vitrification, the hatching rates of D7 embryos vitrified after SE were similar to the rates recorded in fresh non-vitrified blastocysts. The hatching rates of vitrified D8 blastocysts were lower (p < 0.05) than of fresh controls regardless of treatment. Total cell count, and inner cell mass and trophectoderm cell counts were similar in hatched D7 blastocysts vitrified after SE and fresh blastocysts, while vitrified D8 blastocysts yielded lower values regardless of treatment. The apoptosis rate was significantly higher in both treatment groups compared to fresh controls, although rates were lower for SE than LE. No differences emerged in BAX, AQP3, CX43, and IFN gene expression between the treatments, whereas a significantly greater abundance of BCL2L1 and SOD1 transcripts was observed in blastocysts vitrified after SE. A shorter equilibration vitrification protocol was found to improve post-warming outcomes and time efficiency after in-straw warming/dilution. 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