Name: minimally invasive embryo transfer and embryo vitrification at the optimal embryo stage in rabbit model
Uploaded: 2019-05-16T12:30:00
Description: Watch this Scientific Journal Video about Minimally Invasive Embryo Transfer and Embryo Vitrification at the Optimal Embryo Stage in Rabbit Model at JoVE.com

This work was supported by funds from the Ministry of Economy and Competitiveness of Spain (AGL2017-85162-C2-1-R) and Generalitat Valenciana Research Programme (PrometeoII 2014/036). English text version revised by N. Macowan English Language Service

All experimental procedures used in this study were performed in accordance with Directive 2010/63/EU EEC for animal experiments and reviewed and approved by the Ethical Committee for Experimentation with Animals of the Universitat Polit&#232;cnica de Val&#232;ncia, Spain (research code: 2015/VSC/PEA/00170). XGD, FMJ, MPVC and JSV holds an authorization certificate issued by the Valencian governmental administration to experiment on animals. XGD is authorized to in situ supervise the welfare and care of the animals durin...

Since the first documented live birth case from transferred embryos9, this technique and the rabbit species have become crucial in reproductive studies. Besides, embryo research studies involving manipulation, production, cryopreservation, etc. require as a last step the evaluation of embryo capacity to generate healthy full-term offspring. Therefore, embryo transfer technique is indispensable13,28. Over the years, the surgical me...

With the aims of bypassing human infertility or improving dissemination of livestock of high genetic value and preserving animal genetic resources, a set of techniques collectively termed assisted reproduction technologies, such as superovulation, in vitro fertilization, embryo culture, or cryopreservation, were developed1,2. Currently, hormonal treatments are given to stimulate the ovaries and produce a large number of antral ovarian follicles1. Oocytes collected from these follicles can be matured, fertilized, and developed in vitro until they are either cryopreserved or transferred to surrogate mothers3. However, during these treatments, gametes and zygotes are exposed to a series of non-physiological processes that could require embryo adaptation to survive in these conditions4,5. This adaptation is possible due to early embryo plasticity, which allows embryo changes in gene expression and developmental programming6. However, these modifications can influence the subsequent stages of embryo development until adulthood, and it is now widely accepted that methods, timing, cryopreservation procedure or culture conditions show different outcomes on embryo fate7,8. Therefore, to elucidate the specific induced effects of ARTs, the use of well-characterized animal models is inevitable.
The first documented live birth resulting from transfer of mammalian embryos took place in 18909. Today, embryo transfer (ET) to a surrogate female is a crucial step in studying the ART-induced effects during preimplantation on subsequent embryo development stages10. ET techniques depend on the size and anatomical structure of each animal. In the case of large-sized animal models, it has been possible to perform ET by transcervical nonsurgical ET techniques, but in smaller-size species catheterization of the cervix is more complex and surgical techniques are frequently used11. However, surgical ET can cause hemorrhaging that could impair implantation and embryo development, as blood can invade the uterine lumen, causing embryo death10. Transcervical nonsurgical ET techniques are still applied in humans, baboons, bovine, pigs and mice12,13,14,15,16,17, but surgical ETs are still being used in species such as goats, sheep or other animals which present additional difficulties10,18,19,20,21, such as rabbits (two independent cervices) or mice (small size). Nonetheless, surgical transfer methods tend to have gradually been replaced by less invasive methods. Endoscopy was used to transfer embryos, for example, in rabbits, pigs and small ruminants18,19,20. These minimally invasive endoscopy methods can be used to transfer embryos into the ampulla via the infundibulum, which is essential in rabbits and has demonstrated beneficial effects in some species20. This is based on the importance of the correct dialogue between embryo and mother during early embryo stages in the oviduct. As mentioned above, the embryo remodeling that takes place in rabbits during embryo migration through the oviduct is essential to achieve embryos able to implant22,23.
Larger-size animal models, such as bovine, are interesting because the biochemical and preimplantation features are similar to those in human species24. However, large animals are too expensive to use in preliminary trials, and rodents are considered an ideal model (76% model organisms are rodents) for laboratory research25. Nevertheless, the rabbit model provides some advantages over rodents in reproductive studies, as some reproductive biological processes exhibited by humans are more similar in rabbits than those in mice. Human and rabbits present a similar chronological embryonic genome activation, gastrulation and hemochorial placenta structure. In addition, using rabbits it is possible to know the exact timing of fertilization and pregnancy stages due to their induced ovulation25. Rabbit life cycles are short, completing gestation in 31 days and reaching puberty at about 4-5 months; the animal is easy to handle due to its docile and non-aggressive behavior, and its upkeep is very economical compared to the expense of larger animals. Moreover, it is crucial to mention that rabbits have a duplex uterus with two independent cervixes11,25. This places the rabbit in a preferential position, as embryos from the different experimental groups can be transferred into the same animal, but into a different uterine horn. This allows us to compare both experimental effects, reducing the maternal factor from the results.
Today, nonsurgical ET methods are not in use in rabbit. Some studies carried out in the late 90s using a transcervical ET technique resulted in low delivery rates ranging from 5.5% to 20.0%11,26 versus 50-65% by surgical methods, among them the laparoscopy procedure described by Besenfelder and Brem18. The low success rates of these nonsurgical ET methods in rabbits coincide with the lack of the necessary embryo remodeling in the oviduct, which is avoided in transcervical ET. Here, we describe an effective minimally invasive laparoscopic ET procedure using rabbits as a model organism. This technique provides a model for further reproductive research in large animals and humans.
Because rabbits have a particularly narrow time window for embryo implantation, ET in this species requires a high degree of synchrony between the developmental stage of the embryo at ET and the physiological status of the recipient27. In some cases, after a reproductive treatment that slows embryo development (such as in vitro culture) or alters the endometrial receptivity (such as superovulation treatments), there is no synchrony between the embryo and the maternal uterus. These situations can negatively affect outcome. To respond in these contexts, we describe an effective rabbit morula vitrification protocol that allows us to pause, organize and resume the experiments. This process is logistically desirable for reproductive studies and gives us the capacity for long-term storage of embryos, allowing their transport. The laparoscopic procedure and cryopreservation strategies allow better planning of studies with fewer animals. Thus, our methodology offers hygienic and economic advantages and conforms to the concept of the 3Rs (replacement, reduction and refinement) of animal research with the stated goal of improving human treatment of experimental animals. Thus, with these methods, rabbits constitute an ideal model organism for in vivo reproductive assays.

<table>
	<tbody>
		<tr>
			<td>Bovine Serum Albumin (BSA)</td>
			<td>VWR</td>
			<td>332</td>
			<td></td>
		</tr>
		<tr>
			<td>Buprenorphine hydrochloride</td>
			<td>Alvet Escart&#237;</td>
			<td>626</td>
			<td>To be ordered by a licensed veterinarian.</td>
		</tr>
		<tr>
			<td>Buserelin Acetate</td>
			<td>Sigma Aldrich</td>
			<td>B3303</td>
			<td></td>
		</tr>
		<tr>
			<td>Clorhexidine digluconate soap</td>
			<td>Alvet Escart&#237;</td>
			<td>0265DCCJ500B</td>
			<td></td>
		</tr>
		<tr>
			<td>Clorhexidine digluconate solution</td>
			<td>Alvet Escart&#237;</td>
			<td>0265DCCA500B</td>
			<td></td>
		</tr>
		<tr>
			<td>CO2</td>
			<td>Air Liquide</td>
			<td>99921</td>
			<td>CO2 N48.</td>
		</tr>
		<tr>
			<td>CO2 Incubator</td>
			<td>Fisher scientific</td>
			<td>15385194</td>
			<td></td>
		</tr>
		<tr>
			<td>Dimethyl Sulfoxide</td>
			<td>Sigma Aldrich</td>
			<td>W387509</td>
			<td></td>
		</tr>
		<tr>
			<td>Dulbecco&#8217;s phosphate-buffered saline (DPBS)</td>
			<td>Sigma Aldrich</td>
			<td>D5773</td>
			<td>Without calcium chloride.</td>
		</tr>
		<tr>
			<td>Electric razor</td>
			<td>Oster Golden A5</td>
			<td>078005-140-002</td>
			<td></td>
		</tr>
		<tr>
			<td>Endoscope camera</td>
			<td>Optomic Spain S.A</td>
			<td>OP-714</td>
			<td></td>
		</tr>
		<tr>
			<td>Endoscope trocar with silicone leaflet valve</td>
			<td>Karl Storz Endoscopia Ib&#233;rica S.A.</td>
			<td>30114GK</td>
			<td>Lightweight trocar model.</td>
		</tr>
		<tr>
			<td>Enrofloxacin</td>
			<td>Alvet Escart&#237;</td>
			<td>9993046</td>
			<td>To be ordered by a licensed veterinarian.</td>
		</tr>
		<tr>
			<td>Epicraneal needle 23G</td>
			<td>Alvet Escart&#237;</td>
			<td>514056353</td>
			<td>Smaller needles can be also used.</td>
		</tr>
		<tr>
			<td>Epidural catheter</td>
			<td>Vygon corporate</td>
			<td>187.10</td>
			<td></td>
		</tr>
		<tr>
			<td>Epidural needle</td>
			<td>Vygon corporate</td>
			<td>187.10</td>
			<td></td>
		</tr>
		<tr>
			<td>Ethylene Glycol</td>
			<td>Sigma Aldrich</td>
			<td>102466-M</td>
			<td></td>
		</tr>
		<tr>
			<td>Eye ointment</td>
			<td>Alvet Escart&#237;</td>
			<td>5273</td>
			<td></td>
		</tr>
		<tr>
			<td>Ketamine hydrochloride</td>
			<td>Alvet Escart&#237;</td>
			<td>184</td>
			<td>To be ordered by a licensed veterinarian.</td>
		</tr>
		<tr>
			<td>Laparoscopy equipment</td>
			<td>Karl Storz Endoscopia Ib&#233;rica S.A.</td>
			<td>26003 AA</td>
			<td>Hopkins&#174; Laparoscope, 0&#186;-mm straight-viewing laparoscope, 30-cm length, 5-mm working channel.</td>
		</tr>
		<tr>
			<td>Light source</td>
			<td>Optomic Spain S.A</td>
			<td>Fibrolux 250</td>
			<td></td>
		</tr>
		<tr>
			<td>Liquid Nitrogen</td>
			<td>Air Liquide</td>
			<td>P1505XXX</td>
			<td></td>
		</tr>
		<tr>
			<td>Mechanical CO2 insufflator</td>
			<td>Karl Storz Endoscopia Ib&#233;rica S.A.</td>
			<td>Endoflator&#174;</td>
			<td></td>
		</tr>
		<tr>
			<td>Meloxicam</td>
			<td>Alvet Escart&#237;</td>
			<td>9993501</td>
			<td>To be ordered by a licensed veterinarian.</td>
		</tr>
		<tr>
			<td>Petri dishes, 35-mm</td>
			<td>Sigma Aldrich</td>
			<td>CLS430165-500EA</td>
			<td></td>
		</tr>
		<tr>
			<td>Plastic dressing (Nobecutan)</td>
			<td>IBOR medica</td>
			<td>7140028</td>
			<td></td>
		</tr>
		<tr>
			<td>Plastic Straw 0.25 mL</td>
			<td>IMV - technologies</td>
			<td>6431</td>
			<td></td>
		</tr>
		<tr>
			<td>Povidone iodide solution</td>
			<td>Alvet Escart&#237;</td>
			<td>02656DPYS500S</td>
			<td></td>
		</tr>
		<tr>
			<td>Scissors</td>
			<td>ROBOZ</td>
			<td>RS-5880</td>
			<td>Any regular surgical grade steel small straight scissors will work.</td>
		</tr>
		<tr>
			<td>Silicone tube for insufflator</td>
			<td>Karl Storz Endoscopia Ib&#233;rica S.A.</td>
			<td>20400040</td>
			<td></td>
		</tr>
		<tr>
			<td>Stereomicroscope</td>
			<td>Leica</td>
			<td>MZ16F</td>
			<td>There are cheaper options such as Leica MZ8 or Nikon SMZ-10 or SMZ-2B, to name a few.</td>
		</tr>
		<tr>
			<td>Sterile Gloves</td>
			<td>Alvet Escart&#237;</td>
			<td>087GL010075</td>
			<td></td>
		</tr>
		<tr>
			<td>Sterile gown</td>
			<td>Alvet Escart&#237;</td>
			<td>12261501</td>
			<td></td>
		</tr>
		<tr>
			<td>Sterile mask</td>
			<td>Alvet Escart&#237;</td>
			<td>058B15924B</td>
			<td></td>
		</tr>
		<tr>
			<td>Straw Plug</td>
			<td>IMV - technologies</td>
			<td>6431</td>
			<td></td>
		</tr>
		<tr>
			<td>Sucrose</td>
			<td>Sigma Aldrich</td>
			<td>S7903</td>
			<td></td>
		</tr>
		<tr>
			<td>Syringe, 1-mL</td>
			<td>Fisher scientific</td>
			<td>11750425</td>
			<td></td>
		</tr>
		<tr>
			<td>Syringe, 5-mL</td>
			<td>Fisher scientific</td>
			<td>11773313</td>
			<td></td>
		</tr>
		<tr>
			<td>Urinary catheter</td>
			<td>IMV - technologies</td>
			<td>17722</td>
			<td></td>
		</tr>
		<tr>
			<td>Waterbath</td>
			<td>RAYPA</td>
			<td>BAE-4</td>
			<td></td>
		</tr>
		<tr>
			<td>Xylazine</td>
			<td>Alvet Escart&#237;</td>
			<td>525225</td>
			<td>To be ordered by a licensed veterinarian.</td>
		</tr>
		<tr>
			<td>Rabbits</td>
			<td>Universitat Polit&#232;cnica de Val&#232;ncia</td>
			<td>Line A</td>
			<td>Other maternal lines, such as Line V or Line HP can be used.</td>
		</tr>
	</tbody>
</table>

minimally invasive embryo transfer and embryo vitrification at the optimal embryo stage in rabbit model

Assisted reproductive techniques (ARTs), such as in vitro embryo culture or embryo cryopreservation, affect natural development patterns with perinatal and postnatal consequences. To ensure the innocuousness of ART applications, studies in animal models are necessary. In addition, as a last step, embryo development studies require evaluation of their capacity to develop full-term healthy offspring. Here, embryo transfer to the uterus is indispensable to perform any ARTs-related experiment.
The rabbit has been used as a model organism to study mammalian reproduction for over a century. In addition to its phylogenetic proximity to the human species and its small size and low maintenance cost, it has important reproductive characteristics such as induced ovulation, a chronology of early embryonic development similar to humans and a short gestation that allow us to study the consequences of ART application easily. Moreover, ARTs (such as intracytoplasmic sperm injection, embryo culture, or cryopreservation) are applied with suitable efficiency in this species.
Using the laparoscopic embryo transfer technique and the cryopreservation protocol presented in this article, we describe 1) how to transfer embryos through an easy, minimally invasive technique and 2) an effective protocol for long-term storage of rabbit embryos to provide time-flexible logistical capacities and the ability to transport the sample. The outcomes obtained after transferring rabbit embryos at different developmental stages indicate that morula is the ideal stage for rabbit embryo recovery and transfer. Thus, an oviductal embryo transfer is required, justifying the surgical procedure. Furthermore, rabbit morulae are successfully vitrified and laparoscopically transferred, proving the effectiveness of the described techniques.

Minimally invasive laparoscopic transfer of fresh or vitrified embryos places the rabbit among the best model animals for reproductive studies. Table 1 shows the results of fresh ET at different developmental stages (Figure 4) of transferred embryos. The survival rate at birth (percentage of embryos resulting in a pup) proved the efficacy of the laparoscopic technique described in this paper. The higher values were achieved when the ET was pe...

Watch this Scientific Journal Video about Minimally Invasive Embryo Transfer and Embryo Vitrification at the Optimal Embryo Stage in Rabbit Model at JoVE.com

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.

Assisted reproductive techniques (ARTs), such as in vitro embryo culture or embryo cryopreservation, affect natural development patterns with perinatal ...

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.

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