Successful cryopreservation of expanded equine blastocysts.
Abstract: Effective cryopreservation of expanded equine blastocysts (> 300 μm in diameter) has been difficult, perhaps due to the volume of blastocoele fluid or the presence of the equine embryonic capsule. Recently, we reported normal viability of equine embryos after trophoblast biopsy, which resulted in blastocyst collapse. The present study addressed the effect of biopsy and resultant breach of the capsule and blastocyst collapse on survival of expanded equine blastocysts after vitrification. First, non-biopsied, small embryos (< 300 μm) were vitrified in fine-diameter microloader pipette tips using dimethylsulfoxide-containing medium (DM) or ethylene glycol-containing medium (EG). A third group was vitrified with EG, but was warmed using sucrose (EG/s). Embryos in the DM and EG/s treatments grew in culture after vitrification, and established pregnancies after transfer (3 of 12 and 3 of 6, respectively). Expanded blastocysts 300-730 μm in diameter were then biopsied and vitrified; rates of normal pregnancy (detection of embryonic heartbeat) after warming and transfer were 2 of 16 (13%) and 6 of 13 (46%) for DM and EG/s treatments, respectively (P = 0.05). Within the EG/s treatment, it appeared that greater loss of blastocoele fluid after biopsy was associated with higher survival. Therefore, an altered ("Central") biopsy technique was used to aspirate blastocoele fluid, followed by vitrification in EG/s. Pregnancy rates were 1 of 8 (13%) for embryos cultured after warming and 4 of 7 (57%) for embryos transferred immediately after warming (P = 0.1). Finally, expanded blastocysts 407 to 565 μm in diameter were biopsied from the periphery, and blastocoele fluid was removed with gentle suction. After vitrification with EG/s, this resulted in a rate of normal pregnancy of 5 of 7 (71%). These findings demonstrated that blastocoele collapse and vitrification in fine-diameter pipettes allowed successful cryopreservation of expanded equine blastocysts.
Copyright © 2011 Elsevier Inc. All rights reserved.
Publication Date: 2011-03-31 PubMed ID: 21458049DOI: 10.1016/j.theriogenology.2011.01.028Google Scholar: Lookup
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- Evaluation Study
- Journal Article
- Research Support
- Non-U.S. Gov't
Summary
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This research explores the successful cryopreservation of expanded equine blastocysts, potentially due to the collapse of blastocoel fluid and using a dimethylsulfoxide-containing medium or ethylene glycol-containing medium.
Objective of the Research
- The study focuses on better understanding the process and effectiveness of cryopreservation for expanded equine blastocysts which have previously been difficult to cryopreserve successfully. The researchers hypothesize that this difficulty may be due to the volume of blastocoele fluid or due to the presence of the embryonic capsule in equines.
Methodology and Techniques Used in the Study
- The first part of the study was to vitrify non-biopsied small embryos using dimethylsulfoxide-containing medium (DM) or an ethylene glycol-containing medium (EG).
- A third group of small embryos was vitrified using EG, but was warmed using sucrose (EG/s).
- The second part of the study was to observe the survival of expanded (larger than 300 μm in diameter) blastocysts when they were biopsied and vitrified using DM or EG/s, resulting in the collapse of capsule and blastocyst.
- A variation of the biopsy technique, Central biopsy technique, was used to aspirate blastocoele fluid before vitrification of expanded blastocysts.
- In the final experiment, expanded blastocysts of size ranging from 407 to 565 μm in diameter were biopsied from the periphery, and the blastocoele fluid was removed with gentle suction. Then, these were vitrified with EG/s.
Findings of the Study
- Small embryos in DM and EG/s were able to grow in culture after vitrification, and established pregnancies after transfer.
- Expanded blastocysts that were biopsied and vitrified using DM and EG/s treatments resulted in normal pregnancies after warming and transfer.
- The study indicated that the more blastocoele fluid that was lost during biopsy, the higher the survival rate after vitrification. This discovery led to changing the biopsy technique to the central biopsy method.
- Finally, expanded blastocysts of specific sizes that were biopsied from the periphery and had their blastocoele fluid removed resulted in a high rate of normal pregnancies after vitrification with EG/s.
- Overall, the research highlighted that the collapse of blastocoel and vitrification in fine-diameter pipettes facilitated successful cryopreservation of expanded equine blastocysts.
Cite This Article
APA
Choi YH, Velez IC, Riera FL, Roldán JE, Hartman DL, Bliss SB, Blanchard TL, Hayden SS, Hinrichs K.
(2011).
Successful cryopreservation of expanded equine blastocysts.
Theriogenology, 76(1), 143-152.
https://doi.org/10.1016/j.theriogenology.2011.01.028 Publication
Researcher Affiliations
- College of Veterinary Medicine & Biomedical Sciences, Texas A&M University, College Station, Texas 77843, USA.
MeSH Terms
- Animals
- Blastocyst
- Cryopreservation / methods
- Cryopreservation / veterinary
- Embryo Culture Techniques / methods
- Embryo Culture Techniques / veterinary
- Embryo Transfer / veterinary
- Embryonic Development
- Female
- Horses / embryology
- Pregnancy
- Pregnancy Rate
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).
- Benammar A, Derisoud E, Vialard F, Palmer E, Ayoubi JM, Poulain M, Chavatte-Palmer P. The Mare: A Pertinent Model for Human Assisted Reproductive Technologies?. Animals (Basel) 2021 Aug 4;11(8).
- do Nascimento AD, Marques JCC, Cezar ARR, Batista AM, Kastelic JP, Câmara DR. Inhibition of Na(+), K(+) -ATPase with ouabain is detrimental to equine blastocysts. Anim Reprod 2020 Jan 22;17(1).
- Dijkstra A, Cuervo-Arango J, Stout TAE, Claes A. Monozygotic multiple pregnancies after transfer of single in vitro produced equine embryos. Equine Vet J 2020 Mar;52(2):258-261.
- Bartolac LK, Lowe JL, Koustas G, Sjöblom C, Grupen CG. A comparison of different vitrification devices and the effect of blastocoele collapse on the cryosurvival of in vitro produced porcine embryos. J Reprod Dev 2015;61(6):525-31.
- Gambini A, Smith JM, Gurkin RJ, Palacios PD. Current and Emerging Advanced Techniques for Breeding Donkeys and Mules. Animals (Basel) 2025 Mar 29;15(7).
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