Vitrification of early-stage bovine and equine embryos.
Abstract: The objectives of this study were to: (1) determine an optimal method and stage of development for vitrification of bovine zygotes or early embryos; and (2) use the optimal procedure for bovine embryos to establish equine pregnancies after vitrification and warming of early embryos. Initially, bovine embryos produced by in-vitro fertilization (IVF) were frozen and vitrified in 0.25mL straws with minimal success. A subsequent experiment was done using two vitrification methods and super open pulled straws (OPS) with 1- or 8-cell bovine embryos. In Method 1 (EG-O), embryos were exposed to 1.5M ethylene glycol (EG) for 5min, 7M ethylene glycol and 0.6M galactose for 30s, loaded in an OPS, and plunged into liquid nitrogen. In Method 2 (EG-DMSO), embryos were exposed to 1.1M ethylene glycol and 1.1M dimethyl sulfoxide (DMSO) for 3min, 2.5M ethylene glycol, 2.5M DMSO and 0.5M galactose for 30s, and loaded and plunged as for EG-O. Cryoprotectants were removed after warming in three steps. One- and eight-cell bovine embryos were cultured for 7 and 4.5 d, respectively, after warming, and control embryos were cultured without vitrification. Cleavage rates of 1-cell embryos were similar (P>0.05) for vitrified and control embryos, although the blastocyst rates for EG-O and control embryos were similar and higher (P<0.05) than for EG-DMSO. The blastocyst rate of 8-cell embryos was higher (P<0.05) for EG-O than EG-DMSO. Therefore, EG-O was used to cryopreserve equine embryos. Equine oocytes were obtained from preovulatory follicles. After ICSI, injected oocytes were cultured for 1-3 d. Two- to eight-cell embryos were vitrified, warmed and transferred into recipient's oviducts. The pregnancy rate on Day 20 was 62% (5/8) for equine embryos after vitrification and warming. In summary, a successful method was established for vitrification of early-stage bovine embryos, and this method was used to establish equine pregnancies after vitrification and warming of 2- to 8-cell embryos produced by ICSI.
Publication Date: 2008-09-11 PubMed ID: 18789516DOI: 10.1016/j.theriogenology.2008.08.001Google Scholar: Lookup
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- Journal Article
- Research Support
- Non-U.S. Gov't
Summary
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This research study aims to identify the best method for freezing (vitrification) of bovine and equine embryos at their early stages and use this method to successfully establish equine pregnancies after warming the vitrified embryos.
Embryo Vitrification Experiment Methods and Results
- The first objective of the study was to determine an effective method for the vitrification of bovine embryos. The researchers first attempted to freeze embryos produced through in-vitro fertilization (IVF) using 0.25mL straws, with unfavorable results. Subsequently, they tested two different vitrification methods using super open pulled straws (OPS) with 1-cell or 8-cell bovine embryos.
- The first method (EG-O) involved exposing embryos to various concentrations of ethylene glycol for specific periods, loading them in an OPS, and then submerging them in liquid nitrogen. The second method (EG-DMSO) used both ethylene glycol and dimethyl sulfoxide (DMSO) before loading and submerging.
- After warming, the cryoprotectants were removed in three steps. The embryos were then cultured for several days, with controls cultured without vitrification. They found that the cleavage rates of 1-cell embryos were similar for both vitrified and control embryos. However, the blastocyst rates were higher for the EG-O method and control embryos compared to the EG-DMSO method.
Application of the Optimal Vitrification Method for Equine Pregnancy
- Following the successful determination of a better vitrification method for bovine embryos (EG-O), the researchers used this approach to cryopreserve equine embryos. They obtained equine oocytes from preovulatory follicles and, post-injection, cultured them for 1-3 days.
- The 2-cell to 8-cell embryos produced were vitrified, warmed and then inserted into recipient’s oviducts. This resulted in a successful pregnancy rate of 62% by Day 20 for equine embryos after vitrification and warming, demonstrating a promising application of the vitrification method determined through the earlier bovine experiment.
In Summary
- This research resulted in the successful identification of an effective vitrification method (EG-O), for bovine embryos at the 1-cell and 8-cell stages. Further, this method was successfully applied to create pregnancies in equines after vitrifying and warming 2- to 8-cell embryos. It presents promising potential for embryo preservation and assisted reproduction in livestock.
Cite This Article
APA
Campos-Chillòn LF, Suh TK, Barcelo-Fimbres M, Seidel GE, Carnevale EM.
(2008).
Vitrification of early-stage bovine and equine embryos.
Theriogenology, 71(2), 349-354.
https://doi.org/10.1016/j.theriogenology.2008.08.001 Publication
Researcher Affiliations
- Animal Reproduction and Biotechnology Laboratory, Foothills Campus, Colorado State University, Fort Collins, CO 80523-1683, USA.
MeSH Terms
- Animals
- Cattle / embryology
- Cryoprotective Agents
- Embryo Transfer / methods
- Embryo Transfer / veterinary
- Horses / embryology
- Preservation, Biological / methods
- Preservation, Biological / veterinary
- Zygote / physiology
Citations
This article has been cited 6 times.- Angel-Velez D, De Coster T, Azari-Dolatabad N, Fernandez-Montoro A, Benedetti C, Bogado Pascottini O, Woelders H, Van Soom A, Smits K. New Alternative Mixtures of Cryoprotectants for Equine Immature Oocyte Vitrification. Animals (Basel) 2021 Oct 28;11(11).
- Uchikura A, Matsunari H, Nakano K, Hatae S, Nagashima H. Application of hollow fiber vitrification for cryopreservation of bovine early cleavage stage embryos and porcine morula-blastomeres. J Reprod Dev 2016 Apr 22;62(2):219-23.
- Roberts MA, London K, Campos-Chillón LF, Altermatt JL. Presumed monozygotic twins develop following transfer of an in vitro-produced equine embryo. J Equine Sci 2015;26(3):89-94.
- Ganji R, Nabiuni M, Faraji R. Development of mouse preantral follicle after in vitro culture in a medium containing melatonin. Cell J 2015 Winter;16(4):546-53.
- Nazmara Z, Salehnia M, HosseinKhani S. Mitochondrial Distribution and ATP Content of Vitrified, In vitro Matured Mouse Oocytes. Avicenna J Med Biotechnol 2014 Oct;6(4):210-7.
- Zhang X, Catalano PN, Gurkan UA, Khimji I, Demirci U. Emerging technologies in medical applications of minimum volume vitrification. Nanomedicine (Lond) 2011 Aug;6(6):1115-29.
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