Name: Rabbit Embryo Thawing: A Warming Procedure to Recover Cryopreserved Rabbit Embryos for Embryo Transfer
Uploaded: 2023-04-30T14:55:15
Description: Source: Garcia-Dominguez, X. et al., Minimally Invasive Embryo Transfer and Embryo Vitrification at the Optimal Embryo Stage in Rabbit Model. J. Vis. Exp. ...

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All procedures involving animal models have been reviewed by the local institutional animal care committee and the JoVE veterinary review board.
1. Embryo Warming
<ol>
	<li>Thaw the embryos in a single step.
	<ol>
		<li>Place the ministraw horizontally 10 cm from liquid nitrogen vapour for 20-30 s.</li>
		<li>When the crystallization process begins inside the ministraw, immerse the ministraw in a water bath at 25&#176;C for 10-15 s.</li>
		<li>Remove the ministraw plug and cut the cotton plu...

rabbit embryo thawing: a warming procedure to recover cryopreserved rabbit embryos for embryo transfer

Source: Garcia-Dominguez, X. et al., <a href="https://www.jove.com/v/58055">Minimally Invasive Embryo Transfer and Embryo Vitrification at the Optimal Embryo Stage in Rabbit Model.</a> J. Vis. Exp. (2019)
This video demonstrates the procedure for thawing vitrified rabbit embryos and the precautionary measurement steps for optimal embryo recovery.

Rabbit Embryo Thawing: A Warming Procedure to Recover Cryopreserved Rabbit Embryos for Embryo Transfer

Source: Garcia-Dominguez, X. et al., Minimally Invasive Embryo Transfer and Embryo Vitrification at the Optimal Embryo Stage in Rabbit Model. J. Vis. Exp. ...

Embryo thawing is a process in which cryopreserved frozen embryos are warmed and rehydrated, restoring their biological activity.
To thaw cryopreserved rabbit embryos in a sealed cryogenic straw, remove the straw from liquid nitrogen. The embryos, with minimal volume of cryoprotectant-containing media, are physically isolated by air and media segments within the straw. Briefly position the straw in air, facilitating the release of trapped liquid nitrogen vapors, which otherwise cause embryo damage during exposure to warmer temperatures.
The rapid rise from cryogenic liquid nitrogen temperatures to higher freezing temperatures initiates ice crystal formation within the cryogenic straw, risking embryo damage. As ice crystals begin to form, immediately immerse the straw in a temperature-controlled water bath with gentle agitation for even thawing. This step removes the ice crystals, protecting the embryos from ice crystal damage.
Remove the straw plug. Cut the straw&#39;s sealed end; attach a microdispenser to release the contents into a culture plate with sucrose-containing media. The media helps remove cryoprotectants from the embryos; the sucrose avoids excessive swelling of the embryos, restoring their physiological environment during rehydration.
Transfer the embryos into a fresh media-containing plate to completely remove cryoprotectants, ensuring complete rehydration. The thawed embryos are ready for embryo transfer procedures.

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Research

JoVE Encyclopedia of Experiments

Biology

Large Animal Models

Leporine

Isolation and Cryopreservation of Neonatal Rat Cardiomyocytes

Cell culture has become increasingly important in cardiac research, but due to the limited proliferation of cardiomyocytes, culturing cardiomyocytes is difficult and time consuming. The most commonly used cells are neonatal rat cardiomyocytes (NRCMs), which require isolation every time cells are needed. The birth of the rats can be unpredictable. Cryopreservation is proposed to allow for cells to be stored until needed, yet freezing/thawing methods for primary cardiomyocytes are challenging due to the sensitivity of the cells. Using the proper cryoprotectant, dimethyl sulfoxide (DMSO), cryopreservation was achieved. By slowly extracting the DMSO while thawing the cells, cultures were obtained with viable NRCMs. NRCM phenotype was verified using immunocytochemistry staining for &#945;-sarcomeric actinin. In addition, cells also showed spontaneous contraction after several days in culture. Cell viability after thawing was acceptable at 40-60%. In spite of this, the methods outlined allow one to easily cryopreserve and thaw NRCMs. This gives researchers a greater amount of flexibility in planning experiments as well as reducing the use of animals.

The isolation of neonatal rat cardiomyocytes is a time consuming and unpredictable procedure. This study describes methods for cryopreservation and thawing of neonatal rat cardiomyocytes that allows for more efficient use of cells. The thawed NRCMs can be used for various experiments without the need for performing isolations each time.

Cell culture has become increasingly important in cardiac research, but due to the limited proliferation of cardiomyocytes, culturing cardiomyocytes is ...

JoVE Journal

Developmental Biology

Thawing, Culturing, and Cryopreserving Drosophila Cell Lines

There are currently over 160 distinct Drosophila cell lines distributed by the Drosophila Genomics Resource Center (DGRC). With genome engineering, the number of novel cell lines is expected to increase. The DGRC aims to familiarize researchers with using Drosophila cell lines as an experimental tool to complement and drive their research agenda. Procedures for working with a variety of Drosophila cell lines with distinct characteristics are provided, including protocols for thawing, culturing, and cryopreserving cell lines. Importantly, this publication demonstrates the best practices required to work with Drosophila cell lines to minimize the risk of contaminations from adventitious microorganisms or from other cell lines. Researchers who become familiar with these procedures will be able to delve into the many applications that use Drosophila cultured cells including biochemistry, cell biology and functional genomics.

Thawing, Culturing, and Cryopreserving Drosophila Cell Lines

Drosophila cell lines are important reagents for both fundamental and biomedical research. This article provides protocols for thawing, subculturing, and the cryopreservation of commonly used Drosophila cell lines to assist researchers in incorporating the use of these reagents in their research.

There are currently over 160 distinct Drosophila cell lines distributed by the Drosophila Genomics Resource Center (DGRC). With genome engineering, the ...

Human Blastocyst Biopsy and Vitrification

Blastocyst biopsy is performed to obtain a reliable genetic diagnosis during IVF cycles with preimplantation genetic testing. Then, the ideal workflow entails a safe and efficient vitrification protocol, due to the turnaround time of the diagnostic techniques and to transfer the selected embryo(s) on a physiological endometrium in a following natural cycle. A biopsy approach encompassing the sequential opening of the zona pellucida and retrieval of 5-10 trophectoderm cells (ideally 7-8) limits both the number of manipulations required and the exposure of the embryo to sub-optimal environmental conditions. After proper training, the technique was reproducible across different operators in terms of timing of biopsy (~8 min, ranging 3-22 min based on the number of embryos to biopsy per dish), conclusive diagnoses obtained (~97.5%) and live birth rates after vitrified-warmed euploid blastocyst transfer (&#62;40%). The survival rate after biopsy, vitrification and warming was as high as 99.8%. The re-expansion rate at 1.5 h from warming was as high as 97%, largely dependent on the timing between biopsy and vitrification (ideally &#8804;30 min), blastocyst morphological quality and day of biopsy. In general, it is better to vitrify a collapsed blastocyst; therefore, in non-PGT cycles, laser-assisted artificial shrinkage might be performed to induce embryo collapse prior to cryopreservation. The most promising future perspective is the non-invasive analysis of the IVF culture media after blastocyst culture as a putative source of embryonic DNA. However, this potential avant-garde is still under investigation and a reliable protocol yet needs to be defined and validated.

Blastocyst biopsy and vitrification are required to efficiently conduct preimplantation genetic testing. An approach entailing the sequential opening of the zona pellucida and retrieval of 7-8 trophectoderm cells in day 5-7 post-insemination limits both the number of manipulations required and the exposure of the embryo to sub-optimal environmental conditions.

Blastocyst biopsy is performed to obtain a reliable genetic diagnosis during IVF cycles with preimplantation genetic testing. Then, the ideal workflow ...

Rabbit Embryo Thawing: A Warming Procedure to Recover Cryopreserved Rabbit Embryos for Embryo Transfer

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