Slow freezing cryopreservation of Korean bovine blastocysts with an additional sucrose pre-equilibration step
IntroductionEmbryo cryopreservation is a valuable technique used for preserving genetic resources for long periods. However, the survival rate of embryos is dependent on the method used. Therefore, in this study, we evaluated the efficiency of slow freezing method but with an additional dehydration...
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Frontiers Media S.A.
2024-04-01
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| Series: | Frontiers in Veterinary Science |
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| Online Access: | https://www.frontiersin.org/articles/10.3389/fvets.2024.1400899/full |
| _version_ | 1797215831119101952 |
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| author | Seungki Jung Seungki Jung Hyeonseok Sul Dongjin Oh Dongjin Oh Yeon-Gil Jung Joohyeong Lee Sang-Hwan Hyun Sang-Hwan Hyun Sang-Hwan Hyun |
| author_facet | Seungki Jung Seungki Jung Hyeonseok Sul Dongjin Oh Dongjin Oh Yeon-Gil Jung Joohyeong Lee Sang-Hwan Hyun Sang-Hwan Hyun Sang-Hwan Hyun |
| author_sort | Seungki Jung |
| collection | DOAJ |
| description | IntroductionEmbryo cryopreservation is a valuable technique used for preserving genetic resources for long periods. However, the survival rate of embryos is dependent on the method used. Therefore, in this study, we evaluated the efficiency of slow freezing method but with an additional dehydration step prior to freezing to overcome the formation of ice crystals.MethodsOocytes collected from the ovaries of native Korean cattle subjected to in vitro fertilization were cultured for 7 days until the formation of expanded blastocysts. Before freezing, the blastocysts were placed in four pre-equilibration media: a control medium with no addition of sucrose, and three experimental media with the addition of 0.1, 0.25, and 0.5 M sucrose, respectively. Then, the pre-equilibrated embryos were frozen. Embryo survival and hatching rates were evaluated morphologically at 24, 48, and 72 h after thawing. Immunofluorescence staining, terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling (TUNEL) assay, and gene expression analysis of the re-expanded blastocytes were examined 24 h after freeze–thawing.ResultsThe survival rate was significantly higher in the 0.1 M group than in the control group (p < 0.05), and the hatching rate at 72 h was significantly higher in the 0.25 and 0.5 M groups than in the control group (p < 0.05). TUNEL-positive cells were significantly lower in the 0.25 M group than in the control group (12.5 ± 0.9 vs. 8.3 ± 0.8; p < 0.05). The gene expression of BCL2 associated X, heat shock protein 70 kDa, and aquaporin 3 in the 0.25 M group was significantly lower than that in the control group (p < 0.05).ConclusionOur study revealed that treatment with 0.25 M sucrose before slow freezing improved the viability of bovine embryos after freeze–thawing. |
| first_indexed | 2024-04-24T11:36:19Z |
| format | Article |
| id | doaj.art-7650820fec1c449194e57657a0c8bb3e |
| institution | Directory Open Access Journal |
| issn | 2297-1769 |
| language | English |
| last_indexed | 2024-04-24T11:36:19Z |
| publishDate | 2024-04-01 |
| publisher | Frontiers Media S.A. |
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| series | Frontiers in Veterinary Science |
| spelling | doaj.art-7650820fec1c449194e57657a0c8bb3e2024-04-10T05:13:24ZengFrontiers Media S.A.Frontiers in Veterinary Science2297-17692024-04-011110.3389/fvets.2024.14008991400899Slow freezing cryopreservation of Korean bovine blastocysts with an additional sucrose pre-equilibration stepSeungki Jung0Seungki Jung1Hyeonseok Sul2Dongjin Oh3Dongjin Oh4Yeon-Gil Jung5Joohyeong Lee6Sang-Hwan Hyun7Sang-Hwan Hyun8Sang-Hwan Hyun9Veterinary Medical Center and College of Veterinary Medicine, Laboratory of Veterinary Embryology and Biotechnology (VETEMBIO), Chungbuk National University, Cheongju, Republic of KoreaET Biotech Co. Ltd., Jangsu, Republic of KoreaET Biotech Co. Ltd., Jangsu, Republic of KoreaVeterinary Medical Center and College of Veterinary Medicine, Laboratory of Veterinary Embryology and Biotechnology (VETEMBIO), Chungbuk National University, Cheongju, Republic of KoreaInstitute of Stem Cell and Regenerative Medicine (ISCRM), Chungbuk National University, Cheongju, Republic of KoreaET Biotech Co. Ltd., Jangsu, Republic of KoreaDepartment of Companion Animal Industry, Semyung University, Jecheon, Republic of KoreaVeterinary Medical Center and College of Veterinary Medicine, Laboratory of Veterinary Embryology and Biotechnology (VETEMBIO), Chungbuk National University, Cheongju, Republic of KoreaInstitute of Stem Cell and Regenerative Medicine (ISCRM), Chungbuk National University, Cheongju, Republic of KoreaGraduate School of Veterinary Biosecurity and Protection, Chungbuk National University, Cheongju, Republic of KoreaIntroductionEmbryo cryopreservation is a valuable technique used for preserving genetic resources for long periods. However, the survival rate of embryos is dependent on the method used. Therefore, in this study, we evaluated the efficiency of slow freezing method but with an additional dehydration step prior to freezing to overcome the formation of ice crystals.MethodsOocytes collected from the ovaries of native Korean cattle subjected to in vitro fertilization were cultured for 7 days until the formation of expanded blastocysts. Before freezing, the blastocysts were placed in four pre-equilibration media: a control medium with no addition of sucrose, and three experimental media with the addition of 0.1, 0.25, and 0.5 M sucrose, respectively. Then, the pre-equilibrated embryos were frozen. Embryo survival and hatching rates were evaluated morphologically at 24, 48, and 72 h after thawing. Immunofluorescence staining, terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling (TUNEL) assay, and gene expression analysis of the re-expanded blastocytes were examined 24 h after freeze–thawing.ResultsThe survival rate was significantly higher in the 0.1 M group than in the control group (p < 0.05), and the hatching rate at 72 h was significantly higher in the 0.25 and 0.5 M groups than in the control group (p < 0.05). TUNEL-positive cells were significantly lower in the 0.25 M group than in the control group (12.5 ± 0.9 vs. 8.3 ± 0.8; p < 0.05). The gene expression of BCL2 associated X, heat shock protein 70 kDa, and aquaporin 3 in the 0.25 M group was significantly lower than that in the control group (p < 0.05).ConclusionOur study revealed that treatment with 0.25 M sucrose before slow freezing improved the viability of bovine embryos after freeze–thawing.https://www.frontiersin.org/articles/10.3389/fvets.2024.1400899/fullsucroseblastocystslow freezingin vitro productionbovine |
| spellingShingle | Seungki Jung Seungki Jung Hyeonseok Sul Dongjin Oh Dongjin Oh Yeon-Gil Jung Joohyeong Lee Sang-Hwan Hyun Sang-Hwan Hyun Sang-Hwan Hyun Slow freezing cryopreservation of Korean bovine blastocysts with an additional sucrose pre-equilibration step Frontiers in Veterinary Science sucrose blastocyst slow freezing in vitro production bovine |
| title | Slow freezing cryopreservation of Korean bovine blastocysts with an additional sucrose pre-equilibration step |
| title_full | Slow freezing cryopreservation of Korean bovine blastocysts with an additional sucrose pre-equilibration step |
| title_fullStr | Slow freezing cryopreservation of Korean bovine blastocysts with an additional sucrose pre-equilibration step |
| title_full_unstemmed | Slow freezing cryopreservation of Korean bovine blastocysts with an additional sucrose pre-equilibration step |
| title_short | Slow freezing cryopreservation of Korean bovine blastocysts with an additional sucrose pre-equilibration step |
| title_sort | slow freezing cryopreservation of korean bovine blastocysts with an additional sucrose pre equilibration step |
| topic | sucrose blastocyst slow freezing in vitro production bovine |
| url | https://www.frontiersin.org/articles/10.3389/fvets.2024.1400899/full |
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