Evaluating the use of piezo manipulator, laser or their combination for blastocoel cavity puncture to improve cryopreservation outcomes of large equine embryos.
Abstract: The main difficulty of large equine embryo cryopreservation is the replacement of blastocoel fluid with cryoprotectant solution. The objective of this study was to improve the cryopreservation of large equine embryos with PMAP and/or LAP. Embryos were collected via the non-surgical transcervical procedure and divided into three groups based on their size (A ≤ 300 µm, 300 µm<B 300 µm). However, more research is required to find the best method for embryos ≥700 µm.
© 2021 Wiley-VCH GmbH.
Publication Date: 2021-07-17 PubMed ID: 34254367DOI: 10.1111/rda.13990Google Scholar: Lookup
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- Journal Article
Summary
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This study investigates methods to improve the preservation of large horse embryos by using a piezo manipulator, laser, or a combination of both to replace fluid in the egg cell with a cryopreservative solution. Although the techniques provided positive pregnancy results for embryos up to 700µm in size, further research is necessary for embryos larger than this.
Objectives and Methods of the Study
- The primary aim of this study was to enhance the cryopreservation process of large horse embryos. The overall process involves the replacement of blastocoel fluid with a concoction known as cryoprotectant.
- The researchers collected the embryos through a non-invasive transcervical procedure. These embryos were then categorized into three distinct groups according to their size: Group A, with sizes up to 300µm; Group B, sizes ranging from 300µm to 700µm; and Group C, sizes above 700µm.
- The piezo manipulator (a precision instrument used to modify a microscopic specimen), the laser pulse, or a combination were used to puncture six chosen embryos.
Cryopreservation, Cultivation and Transfer
- Post-puncture, all embryos underwent cryopreservation on a Cryotop®, a simple and efficient tool used to vitrify biological materials at ultra-rapid cooling rates.
- After being frozen and subsequently thawed, the embryos were cultured for three hours before their transfer into the recipient mares.
Pregnancy Diagnosis and Findings
- One week after the transfer, pregnancy was confirmed by executing an ultrasound.
- Out of the six implanted embryos, two resulted in a pregnancy. For Group A and B embryos, there were positive pregnancy results; however, for those in Group C (≥700µm), the results were negative.
- Laser-assisted puncture thus proved beneficial in extracting blastocoel fluid and replacing it with cryoprotectant.
Limitations and Further Research
- The first successful pregnancy using laser puncture-assisted frozen-thawed horse embryo larger than 300µm was reported in this study.
- Although the study showed promising results, the embryos that were 700µm or larger failed to result in a positive pregnancy, indicating a need for further research to determine the most effective method for cryopreserving larger embryos.
Cite This Article
APA
Rajabi-Toustani R, Watanabe H, Tsogtgerel M, Gao Y, Canbo L, Haneda S, Cheong SH, Nambo Y.
(2021).
Evaluating the use of piezo manipulator, laser or their combination for blastocoel cavity puncture to improve cryopreservation outcomes of large equine embryos.
Reprod Domest Anim, 56(10), 1358-1362.
https://doi.org/10.1111/rda.13990 Publication
Researcher Affiliations
- Department of Clinical Veterinary Sciences, Obihiro University of Agriculture and Veterinary Medicine, Hokkaido, Japan.
- Department of Life and Food Sciences, Obihiro University of Agriculture and Veterinary Medicine, Hokkaido, Japan.
- Department of Clinical Veterinary Sciences, Obihiro University of Agriculture and Veterinary Medicine, Hokkaido, Japan.
- United Graduate School of Veterinary Sciences, Gifu University, Gifu, Japan.
- Department of Clinical Veterinary Sciences, Obihiro University of Agriculture and Veterinary Medicine, Hokkaido, Japan.
- Department of Clinical Veterinary Sciences, Obihiro University of Agriculture and Veterinary Medicine, Hokkaido, Japan.
- Department of Clinical Veterinary Sciences, Obihiro University of Agriculture and Veterinary Medicine, Hokkaido, Japan.
- Department of Clinical Sciences, College of Veterinary Medicine, Cornell University, Ithaca, NY, USA.
- Department of Clinical Veterinary Sciences, Obihiro University of Agriculture and Veterinary Medicine, Hokkaido, Japan.
- United Graduate School of Veterinary Sciences, Gifu University, Gifu, Japan.
MeSH Terms
- Animals
- Blastocyst / physiology
- Cryopreservation / methods
- Cryopreservation / veterinary
- Cryoprotective Agents
- Embryo Transfer / instrumentation
- Embryo Transfer / methods
- Embryo Transfer / veterinary
- Embryo, Mammalian
- Female
- Horses / embryology
- Lasers
- Micromanipulation / methods
- Micromanipulation / veterinary
- Pregnancy
Grant Funding
- Livestock Promotional Subsidy from the Japan Racing Association
References
This article includes 13 references
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Citations
This article has been cited 2 times.- Rajabi-Toustani R, Tsogtgerel M, Gao Y, Li C, Sakato M, Haneda S, Cheong SH, Nambo Y. First Kiso pony foal produced via transfer of long-distance shipped fresh embryo to Hokkaido native pony. J Reprod Dev 2023 Apr 3;69(2):125-128.
- Hannan MA, Watanabe H, Takeyama A, Yoshida S, Wudamu D, Lkhagvasuren N, Claes A, Stout TAE, Cheong SH, Haneda S, Nambo Y. In vitro embryo production via ovum pick-up (OPU) and intracytoplasmic sperm injection (ICSI) in pure and crossbred Japanese Hokkaido native ponies. J Reprod Dev 2025 Jun 6;71(3):191-194.
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