Minimally Invasive Embryo Transfer and Embryo Vitrification at the Optimal Embryo Stage in Rabbit Model

Assisted reproductive techniques (ARTs) are in continuous evaluation to improve outcomes and reduce the associated risks. This manuscript describes a minimally invasive embryo transfer procedure with an efficient cryopreservation protocol that allows the use of rabbits as an ideal animal model of human reproduction.

The main goal of this experiment is to describe a laparoscopic embryo transfer procedure in rabbit as a model. The main advantage of this technique is that this one works to transfer embryos using an easy, minimally invasive technique. Furthermore, the effective embryo cryopreservation protocol here described also works to long-term storage the rabbit embryos, providing time-flexible logistical capacities and the ability to transport the sample.

As we know, assisted reproductive technologies are in continuous evaluation to improve outcomes and reduce the associated risks. Therefore, using both techniques, rabbit can be used as an ideal animal model of human reproduction to answer key questions in this field, such as the effects of the in vitro embryo manipulation on offspring. Visual demonstration of these matters are critical as the embryo transfer steps are difficult to learn, because it must be done with great precision to ensure the success of the technique.

For embryonic vitrification, immerse the embryos in equilibrating solution for two minutes, followed by a one minute incubation in vitrification solution. At the end of the incubation, load the embryos into a 125 microliter plastic mini-straw, and couple the closed end of the mini-straw with an appropriate one milliliter microdispenser. Aspirate base medium up to one-third of the straw length, followed by a small air bubble.

Next, use a stereomicroscope to aspirate the embryos in 40 microliters of vitrification solution, followed by another small air bubble and enough base medium to move first liquid fraction up to the cotton of the mini-straw. Then close the opened end of the mini-straw with a straw plug, plunge the mini-straw directly into liquid nitrogen to achieve vitrification, and store the mini-straw in a liquid nitrogen dewar. To thaw the embryos for a transfer, place the mini-straw horizontally 10 centimeters from the liquid nitrogen vapor for 20 to 30 seconds.

When the crystallization process begins inside the mini-straw, immerse the mini-straw in the 25 degree celsius water bath for 10 to 15 seconds. Immediately remove the mini-straw and cotton plugs and use a coupled microdispenser to expel the mini-straw contents into a plate containing 25 degrees celsius base medium supplemented with 0.33 molar sucrose solution, in which embryos must remain during five minutes. Then transfer the embryos to a plate of fresh base medium for another five minute incubation.

As many days before as the age of the embryos to be transferred, observe the turgidity and color of the vulvas of the available sexually mature 4.5 month old female rabbits, and induce ovulation in the receptive animals with a single intramuscular injection of one microgram of buserelin acetate regardless of body weight. On the day of the transfer, rinse the fresh or thawed embryos in 37 degree celsius base medium under a stereomicroscope, and attach an appropriately configured 17-gauge epidural catheter to a one milliliter syringe. Aspirate one centimeter of base medium into the catheter, followed by a small air bubble.

Then aspirate five to seven embryos and 10 microliters of base medium, followed by another small air bubble, and a final one centimeter of base medium. Once the embryos have been loaded, place the anesthetized recipient rabbit in a cage on a warming stage, and apply ointment to the animal's eyes. After confirming a lack of response to pedal reflex, shave the fur from the ventral abdomen, clean the surgical area with chlorhexidine gluconate soap, and remove any remaining hair.

Cover the area with a sterile drape with a hole for the surgical area, and insert one endoscopic trocar five centimeters into the abdominal cavity, two centimeters caudal to the zyphoid process, and use a pressure regulating mechanical insufflator to inflate the peritoneal cavity. Insert the endoscope camera through the endoscopic trocar, and insert the 17-gauge epidural needle into the inguinal region, two to three centimeters from the infundibulum. Insert the loaded catheter through the epidural needle into the abdomen, and insert one to two centimeters of the epidural catheter through the infundibulum in the ampulla.

Gently depress the catheter plunger to release the embryos into the oviduct. Both air bubbles must exit from the catheter. Remove the catheter just after the embryos have been released, and aspirate and release the remaining medium to confirm the absence of the embryos.

After confirming a successful transfer of the embryos, remove the epidural needle and the endoscope camera, and release the abdominal carbon dioxide through the endoscopic trocar. Clean the trocar incision with chlorhexidine solution, and close the wound with a micronised aluminum and a plastic dressing. Then treat the animal with the appropriate antibiotics and analgesics, and monitor the animal until full recovery.

Minimally invasive laparoscopic embryo transfer places the rabbit among the best animal models for reproductive studies, with a high percentage of transferred fresh embryos resulting in a pup. Notably, higher values are achieved when the fresh embryo transfer is performed with embryos in either the early or compact morulae stage. Similar results are observed after early and compacted vitrified rabbit morula transfer, particularly with compact morulae, confirming the efficacy and reliability of this technique for transferring fresh in-vitrified embryos in rabbits.

Following this procedure, other methods like conditional Assistive Reproductive Technologies can be performed in order to answer additional questions like how the addition of densities can incur in an analgesic effect later in life. After recent development, this technique paves the way for scientists in the field of reproduction to explore this effect in humans based on the close phylogenetic distance between rabbit and humans. Thus, this technique can provide results easily transferrable to human clinical medicine, as well in other mammal species.

Don't forget that our method offers some hygienic and economic advantages conforming to the concept of three R's of animal welfare, Replacement, Reduction, and Refinement, by intending the goal of improving human treatment of experimental animals.