A Shorter Equilibration Period Improves Post-Warming Outcomes after Vitrification and in Straw Dilution of In Vitro-Produced Bovine Embryos
Abstract
:Simple Summary
Abstract
1. Introduction
2. Materials and Methods
2.1. Chemicals and Suppliers
2.2. In Vitro Production of Bovine Blastocysts
2.3. Embryo Vitrification and Warming
2.3.1. Vitrification Protocol
2.3.2. Warming Protocol
2.4. Differential Staining and TUNEL
2.5. RNA Extraction and Relative Quantification of mRNA by Real-Time Reverse Transcription PCR
2.6. Statistical Analysis
3. Results
3.1. A Shorter Time of Exposure of Embryos to the Equilibrium Solution Leads to Improved Embryo Development (Experiment 1)
3.2. 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)
3.3. 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)
4. Discussion
5. Conclusions
Author Contributions
Funding
Institutional Review Board Statement
Informed Consent Statement
Data Availability Statement
Conflicts of Interest
References
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Symbol | Primer Sequences (5′-3′) | Amplicon Size (bp) | GenBank Accession No. |
---|---|---|---|
BCL2 associated X apoptosis regulator (BAX) | F: ACCAAGAAGCTGAGCGAGTG | 116 | NM_173894.1 |
R: CGGAAAAAGACCTCTCGGGG | |||
BCL2-like 1 (BCL2L1) | F: GAGTTCGGAGGGGTCATGTG | 211 | NM_001166486.1 |
R: TGAGCAGTGCCTTCAGAGAC | |||
Superoxide dismutase 1 (SOD1) | F: ACACAAGGCTGTACCAGTGC | 102 | NM_174615.2 |
R: CACATTGCCCAGGTCTCCAA | |||
Aquaporin 3 (AQP3) | F: GTGGACCCCTACAACAACCC | 222 | NM_001079794.1 |
R: CAGGAGCGGAGAGACAATGG | |||
Connexin 43 (CX43) | F: TGGAATGCAAGAGAGGTTGAAAGAGG | 294 | NM_174068.2 |
R: AACACTCTCCAGAACACATGATCG | |||
Interferon tau (IFNτ) | F: CTGAAGGTTCACCCAGACCC | 197 | AF238612 |
R: GAGTCTGTTCATTCGGGCCA | |||
Peptidylprolyl isomerase A (PPIA) | F: CATACAGGTCCTGGCATCTTGTCC | 108 | NM_178320.2 |
R: CACGTGCTTGCCATCCAACC | |||
H3.3 histone A (H3F3A) | F: CATGGCTCGTACAAAGCAGA | 136 | NM_001014389.2 |
R: ACCAGGCCTGTAACGATGAG |
Day 7 Blastocysts | Day 8 Blastocysts | |||||||
---|---|---|---|---|---|---|---|---|
n | Survival (%) (3 h) | Survival (%) (24 h) | Hatching Rate (%) (24 h) | n | Survival (%) (3 h) | Survival (%) (24 h) | Hatching Rate (%) (24 h) | |
Control | 86 | 100 a,A | 100 a,A | 35.9 ± 4.0 a,A | 40 | 100 a,A | 100 a,A | 50.0 ± 7.0 a,B |
SE | 86 | 60.6 ± 1.5 b,A | 78.4 ± 2.0 b,A | 31.4 ± 3.7 a,A | 33 | 48.6 ± 5.3 b,B | 63.0 ± 5.5 b,B | 19.9 ± 2.7 b,B |
LE | 83 | 57.5 ±4.0 b,A | 63.1 ± 2.6 c,A | 10.1 ± 2.4 b,A | 36 | 39.4 ± 4.7 b,B | 55.3 ± 5.0 c,B | 8.1 ± 2.7 c,A |
Day 7 Blastocysts | |||||||||
TCN | ICM Cell Number | TE Cell Number | AR | ||||||
n | Expanded | Hatched | Expanded | Hatched | Expanded | Hatched | Expanded | Hatched | |
Control | 30 | 140.3 ± 8.6 a,A | 189.8 ± 4.4 a,B | 24.4 ± 1.6 a,A | 38.6 ± 1.6 a,B | 115.9 ± 7.9 a,A | 151.3 ± 4.1 a,B | 3.7 ± 0.4 a | 4.7 ± 0.7 a |
SE | 23 | 110.0±2.7 b,A | 195.2 ± 3.5 a,B | 22.0 ± 2,1 a,A | 35.1 ± 1.5 a,B | 87.1 ± 2.3 b,A | 160.1 ± 3.1 a,B | 11.5 ± 1.1 b | 9.1 ± 0.9 b |
LE | 21 | 113.2 ± 4.5 b,A | 170.6 ± 1.4 b,B | 23.3 ± 1.3 a,A | 33.2 ± 3.3 a,B | 89.9 ± 4.3 b,A | 135.4 ± 2.8 b,B | 15.2 ± 0.3 c | 13.6 ± 1.2 c |
Day 8 Blastocysts | |||||||||
TCN | ICM Cell Number | TE Cell Number | AR | ||||||
n | Expanded | Hatched | Expanded | Hatched | Expanded | Hatched | Expanded | Hatched | |
Control | 40 | 125.3 ± 5.4 a,A | 206.8 ± 12 a,B | 29.2 ± 1.9 a,A | 43.1 ± 1.2 a,B | 96.0 ± 4.5 a,A | 163.6 ± 5.6 a,B | 5.6 ± 0.4 a | 4.6 ± 0.7 a |
SE | 21 | 128.1 ± 2.8 a,A | 169.7 ± 5.6 b,B | 22.2 ± 1.0 b,A | 32.0 ± 1.2 b,B | 105.9 ± 2.8 a,A | 137.8 ± 4.0 b,B | 15.1±0.6 b | 12.7 ± 0.8 b |
LE | 20 | 108.4 ± 1.3 b,A | 154.6 ± 2.1 b,B | 19.7 ± 0.8 b,A | 29.0 ± 2.0 b,B | 88.7 ± 1.3 b,A | 125.0 ± 2.7 b,B | 25.1 ± 1.5 c | 23.2 ± 2.1 c |
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Martínez-Rodero, I.; García-Martínez, T.; Ordóñez-León, E.A.; Vendrell-Flotats, M.; Olegario Hidalgo, C.; Esmoris, J.; Mendibil, X.; Azcarate, S.; López-Béjar, M.; Yeste, M.; et al. A Shorter Equilibration Period Improves Post-Warming Outcomes after Vitrification and in Straw Dilution of In Vitro-Produced Bovine Embryos. Biology 2021, 10, 142. https://doi.org/10.3390/biology10020142
Martínez-Rodero I, García-Martínez T, Ordóñez-León EA, Vendrell-Flotats M, Olegario Hidalgo C, Esmoris J, Mendibil X, Azcarate S, López-Béjar M, Yeste M, et al. A Shorter Equilibration Period Improves Post-Warming Outcomes after Vitrification and in Straw Dilution of In Vitro-Produced Bovine Embryos. Biology. 2021; 10(2):142. https://doi.org/10.3390/biology10020142
Chicago/Turabian StyleMartínez-Rodero, Iris, 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 et al. 2021. "A Shorter Equilibration Period Improves Post-Warming Outcomes after Vitrification and in Straw Dilution of In Vitro-Produced Bovine Embryos" Biology 10, no. 2: 142. https://doi.org/10.3390/biology10020142