BASICS
- Vitrification is cryopreservation using:
- High concentrations of cryoprotectants
- Low volumes of medium
- Ultrarapid freezing (plunging in liquid nitrogen)
- Vitrification does not require step-wise cooling or seeding
- The vitrification procedure is faster and simpler than standard slow freezing
- Embryo vitrification can be used to hold embryos in order to allow scheduling of the time of transfer so that the resulting foal is born at the desired time of year
- Mares may be flushed for embryo recovery to produce embryos regardless of season
- Embryos may be produced from ICSI programs regardless of season
- Embryos produced postmortem by ICSI, owing to a mare's untimely death, may be transferred when the foals produced will be most valuable
- Biopsied embryos may be vitrified to hold them while genetic diagnosis is conducted
- When recipient numbers are small, embryos may be vitrified to simplify scheduling of transfer; the embryo is warmed and transferred when the recipient mare is the correct number of days after ovulation:
- Use of vitrification could decrease the number of recipient mares that are needed in an embryo transfer program
- In procedures such as ICSI, from live mares or postmortem, vitrification can be used to preserve embryos in excess of the number of recipients immediately available, or in excess of the number of foals desired for that year
- Embryo vitrification provides a method of shipment of valuable genetics nationally or internationally
- Preserving embryos by vitrification provides a method of genetic conservation; theoretically the half-life in liquid nitrogen is 50 000 years
- Both vitrification and slow freezing are successful with small VVR embryos (<300 μm in diameter) and with IVP embryos
- Collection of small embryos in vivo requires flushing of the uterus at day 6.5 after ovulation, which may be associated with lower embryo recovery rates
- Mares are typically flushed for embryo recovery on day 7 or 8 after ovulation, and the embryo recovered is an expanded blastocyst
- Expanded blastocysts (>300 μm in diameter) have low pregnancy rates after standard slow freezing or vitrification
- Collapse of the blastocele, using micromanipulation, allows successful vitrification of expanded blastocysts, with normal pregnancy rates after warming and transfer
Mares are managed as for standard embryo collection:
- Insemination during estrus
- Ultrasonography per rectum to determine the day of ovulation
- Uterine flush for embryo recovery on day 6.5 after ovulation, if small embryos are desired
- Uterine flush for embryo recovery day 7 or 8 after ovulation, if small embryos are not desired
Embryo Vitrification by the Practitioner
- Use when small embryos are recovered
- Equine embryo vitrification kits are available from several companies
- Kits provide vitrification and warming solutions and instructions
- Requires standard embryo transfer supplies including stereomicroscope, Petri dishes, pipets, and embryo straws
- Requires liquid nitrogen and a Styrofoam container to hold it, and a liquid nitrogen tank
- Open vitrification devices such as the Cryotop or Cryolock can be used successfully with the equine kits, instead of straws:
- Open devices, which are associated with low volume of medium and rapid temperature change, improve embryo viability upon warming in many applications
- Open devices have been used successfully for equine embryo vitrification, but have not directly been compared with straws for vitrification of horse embryos
- Embryos are placed in the different solutions for the instructed period of time, then loaded into the straw or onto the open device, and plunged in liquid nitrogen
- If a straw is used:
- The embryo is loaded in the straw with dilution solution so that the embryo can be warmed in the straw and transferred directly to a recipient
- Some kits recommend placing straws in nitrogen vapor for a period before plunging to limit the possibility of the straw cracking
- Use of nonirradiated polyvinyl chloride straws may limit the possibility of the straw cracking
- On warming, the straw is typically held in the air for a few seconds before being placed in the warm bath to allow evaporation of liquid nitrogen from the surface
- If an open vitrification device is used:
- The medium around the embryo should be aspirated away until only a film of medium is present around the embryo
- To warm, the device is immersed in warming solution. The embryo is located and placed in the holding medium and loaded for transfer
- Straw cracking and other failures are avoided
- Success of vitrification both in straws and on open devices is technician-dependent and may be poor until the practitioner gains experience
Packaging Embryos for Shipment to a Laboratory for Vitrification
- Used when embryos >300 µm diameter are recovered, that need to be collapsed before vitrification, or when the practitioner does not have the expertise or supplies to vitrify embryos
- Notify the receiving laboratory that the embryo has been collected and will be shipped, and ascertain that the person performing the micromanipulation and vitrification will be there to receive and process it
- Embryos are packaged in Equitainers or EquOcyte containers, with all packaging material, isothermalizer, and ballast at room temperature (~22°C)
- The coolant cans are also at room temperature (~22°C)
- Embryos may be shipped by courier, by air, or by overnight delivery
- Currently because of lack of familiarity of many practitioners with methods for embryo warming, warming is done at the vitrification laboratory and the warmed embryos are shipped back to the practitioner for transfer
Collapse and Vitrification of Expanded Embryos (>300 μm in Diameter)
- Embryos are placed on an inverted microscope with micromanipulators
- The embryo is held by a holding pipet
- An injection pipet is inserted through the capsule and into the blastocele
- If a Piezo drill is used, the capsule is breached by placing the pipet against it and using multiple pulses with the Piezo drill at a high setting (speed 6, intensity 7)
- Conventional micromanipulation can be used, with a standard pointed human ICSI pipet. The pipet is inserted through the capsule into the blastocele
- Use of a coaxial system, in which the small diameter aspiration pipet is extruded through a larger holding pipet, has also been successful
- The blastocele fluid is aspirated gently, with the injection pipet held at the periphery of the blastocele cavity. As much fluid as possible is removed without trauma to the embryo
- The embryo is then vitrified using the methods above, or methods specific to the embryo laboratory performing the vitrification:
- A vitrification device used successfully in our laboratory is the microloader pipet tip, which has a small diameter that limits the volume of medium around the embryo
- One drawback to blastocele collapse is the possibility of loss of the capsule upon warming; this currently happens in about 10% of embryos and renders the embryo nonviable
TREATMENT
FOLLOW-UP
MISCELLANEOUS