Name: Author Spotlight: Surgical Methods and Outcomes in Oviductal Cloned Pig Embryo Transfers
Uploaded: 2024-10-18T13:50:00
Description: This protocol aims to demonstrate the surgical technique for transferring cloned pig embryos to the oviduct, a method widely used in the production of ...

We would like to thank the Equine Veterinary Hospital and the Ruminants Veterinary Hospital of the College of Veterinary Medicine, University of Sao Paulo (FMVZ/USP), Sao Paulo, Brazil, FAPESP (grant 2022/11459-3, Sao Paulo Research Foundation), EMS Pharma, CNPq (grant 405254/2022-9), and &#193;gua Branca pig farming, Itu, Sao Paulo, Brazil.

This study was approved by the Ethics Committee on Animal Use in Research of the College of Veterinary Medicine and Animal Science of the University of S&#227;o Paulo, protocol number 6088030523. Nine seven-month-old gilts from the &#193;gua Branca nucleus pig farming, located in Itu city, S&#227;o Paulo state, Brazil, were used right after the second estrus detection9. The details of the reagents and the equipment used in the study are listed in the Table of Materials.
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Optimizing Oviductal Embryo Transfer in Cloned Pigs

<ol>
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The surgical method described has been previously performed by other research groups working with the production of cloned pigs or genetically modified cloned pigs, with reports of births after the implementation of this technique15,16,17,18,19,20. The pregnancy and birth rates of SCNT embryos produced from various cell lines...

Pigs are an excellent experimental model for biomedical research due to their anatomical, physiological, and genetic similarities to humans1. These animals have often been used in research related to xenotransplantation, with the intention of producing organs, cells, or tissues that promote a low risk of rejection when transplanted into humans. Xenotransplantation research aims to increase the organ supply for human transplantation, thus reducing the waiting list of patients2.
The production of pigs for xenotransplantation involves several steps, including the production of clones from genetically edited swine cells. After the in vitro production of genetically modified cloned embryos, the embryos are transferred to the reproductive system of a sow with a synchronized estrous cycle to prepare the uterine physiology for the reception and gestation of the new conceptus3.
Embryo transfer in pigs can be performed by non-invasive or invasive methods4. Among the non-invasive methods is transcervical transfer, which does not require any surgical intervention. However, this method is restricted to transferring embryos at later development stages (i.e., morula or blastocyst stages) and does not allow for the precise determination of the catheter insertion site or embryo deposition5. Laparoscopy and laparotomy are considered invasive methods of embryo transfer. Laparoscopy is less invasive but requires specific and costly equipment, and its efficiency varies considerably (from less than 20% to over 80%) due to various factors such as difficulty in manipulating reproductive structures and the type of catheter used4,6. Therefore, transfer via laparoscopy is still less efficient compared to surgical transfer methods via laparotomy4.
Embryo transfer surgery in sows via laparotomy is a relatively simple and quick procedure, typically taking about 30 min. However, it should be performed in a surgical center equipped with inhalation anesthesia apparatus and a specialized team. For commercial pig strains (such as Landrace, Large White, or their crossbreeds), special equipment like hoists for lifting gilts and a wide, sturdy surgical table are necessary due to the animals&#39; considerable weight (around 130-150 kg).
For the surgery to be successful, the embryos must be evaluated for their stage of development beforehand. Embryos of up to 4 cells are recommended to be transferred into the uterine tube. Embryos at stages beyond 4 cells, such as morulas and blastocysts, should be transferred to the uterine horn7,8.
Although research groups worldwide are performing the production and surgical transfer of genetically modified cloned pig embryos, there are still no well-defined protocols demonstrating this procedure through videos. This approach is crucial for the success of gestation, as the technique requires precise deposition of embryos in the exact location of the oviduct, involving the localization of the tubal ostium and the introduction of the pipette containing the embryos. This technique can be better understood through explanatory videos of the entire procedure. Therefore, this article aims to demonstrate the laparotomy surgery for transferring cloned embryos to the oviduct of gilts, an essential prerequisite for the future production of genetically modified pigs to be used for xenotransplantation or other related purposes.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>0.25 mL straw</td>
			<td>generic</td>
			<td>-</td>
			<td>Surgical material</td>
		</tr>
		<tr>
			<td>1 mL syringe</td>
			<td>Descarpack</td>
			<td>341001</td>
			<td>Surgical material</td>
		</tr>
		<tr>
			<td>10 mL syringe</td>
			<td>Descarpack</td>
			<td>324601</td>
			<td>Surgical material</td>
		</tr>
		<tr>
			<td>20 mL syringe</td>
			<td>Descarpack</td>
			<td>324801</td>
			<td>Surgical material</td>
		</tr>
		<tr>
			<td>3 mL syringe</td>
			<td>Descarpack</td>
			<td>324201</td>
			<td>Surgical material</td>
		</tr>
		<tr>
			<td>5 mL syringe</td>
			<td>Descarpack</td>
			<td>324401</td>
			<td>Surgical material</td>
		</tr>
		<tr>
			<td>60 mL syringe</td>
			<td>Descarpack</td>
			<td>323201</td>
			<td>Surgical material</td>
		</tr>
		<tr>
			<td>9 mm endotracheal tube</td>
			<td>Rusch</td>
			<td>112482-000090</td>
			<td>Surgical material</td>
		</tr>
		<tr>
			<td>Allis forceps</td>
			<td>generic</td>
			<td>-</td>
			<td>Surgical instrument</td>
		</tr>
		<tr>
			<td>Amox LA</td>
			<td>JA Sa&#250;de Animal</td>
			<td>MAPA registration: 8.781/2004</td>
			<td>Pharmaceutical drug</td>
		</tr>
		<tr>
			<td>Bakhaus forceps</td>
			<td>generic</td>
			<td>-</td>
			<td>Surgical instrument</td>
		</tr>
		<tr>
			<td>Catheter 20G</td>
			<td>Descarpack</td>
			<td>362401</td>
			<td>Catheter for intravenous access</td>
		</tr>
		<tr>
			<td>Cetamin</td>
			<td>Agener Uni&#227;o</td>
			<td>MAPA registration: SP-000292-5.000011&#160;</td>
			<td>Anesthetic</td>
		</tr>
		<tr>
			<td>Conductive clinical gel</td>
			<td>RMC</td>
			<td>ANVISA registration: 80122200013</td>
			<td>Surgical material</td>
		</tr>
		<tr>
			<td>Dipyrone D-500</td>
			<td>Zoetis</td>
			<td>MAPA registration: SP0000728-46</td>
			<td>Pharmaceutical drug</td>
		</tr>
		<tr>
			<td>Disposable scalpel n. 22</td>
			<td>Wiltex</td>
			<td>ANVISA registration: 10150470565</td>
			<td>Surgical instrument</td>
		</tr>
		<tr>
			<td>Disposable sterile sponge-brush</td>
			<td>Rioquimica</td>
			<td>7.89778E+12</td>
			<td>Surgical asepsis</td>
		</tr>
		<tr>
			<td>Easy-Scan:Go</td>
			<td>IMV</td>
			<td>ESCG01</td>
			<td>Ultrasound</td>
		</tr>
		<tr>
			<td>Endozime AW Plus</td>
			<td>Ruhof</td>
			<td>34514</td>
			<td>Detergent for surgical instruments</td>
		</tr>
		<tr>
			<td>Fentanil (Fentanest)</td>
			<td>Crist&#225;lia</td>
			<td>ANVISA registration: 1029800810159</td>
			<td>Anesthetic</td>
		</tr>
		<tr>
			<td>Gosset retractor</td>
			<td>generic</td>
			<td>-</td>
			<td>Surgical instrument</td>
		</tr>
		<tr>
			<td>Halstead-mosquito hemostatic forceps</td>
			<td>generic</td>
			<td>-</td>
			<td>Surgical instrument</td>
		</tr>
		<tr>
			<td>Healing ointment - Unguento Pearson</td>
			<td>Pearson SA</td>
			<td>MAPA registration: SP0000094-16</td>
			<td>Pharmaceutical drug</td>
		</tr>
		<tr>
			<td>hydrogen peroxide solution</td>
			<td>Rioquimica</td>
			<td>ANVISA registration: 218690015</td>
			<td>Surgical material</td>
		</tr>
		<tr>
			<td>Isofluorane</td>
			<td>Biochimico</td>
			<td>ANVISA registration: 100630222</td>
			<td>Anesthetic</td>
		</tr>
		<tr>
			<td>IV Drip set extensor</td>
			<td>generic</td>
			<td>-</td>
			<td>Fluid therapy</td>
		</tr>
		<tr>
			<td>IV Macro drip set</td>
			<td>Descarpack</td>
			<td>410301</td>
			<td>Fluid therapy</td>
		</tr>
		<tr>
			<td>Lactofur (ceftiofur)</td>
			<td>Ourofino SA</td>
			<td>MAPA registration: SP0000051-50</td>
			<td>Pharmaceutical drug</td>
		</tr>
		<tr>
			<td>Laringoscope</td>
			<td>-</td>
			<td>-</td>
			<td>Surgical instrument</td>
		</tr>
		<tr>
			<td>Maxicam 2%</td>
			<td>Ourofino SA</td>
			<td>MAPA registration: SP0000051-69</td>
			<td>Pharmaceutical drug</td>
		</tr>
		<tr>
			<td>Micropore adhesive 5 cm x 10 cm&#160;</td>
			<td>generic</td>
			<td>1530</td>
			<td>Surgical material</td>
		</tr>
		<tr>
			<td>Midazolam</td>
			<td>Hipolabor</td>
			<td>ANVISA registration: 1134301430035</td>
			<td>Anesthetic</td>
		</tr>
		<tr>
			<td>Multi-Way IV Infusion Set</td>
			<td>Descarpack</td>
			<td>413201</td>
			<td>Fluid therapy</td>
		</tr>
		<tr>
			<td>Needle 40 mm x 1.2 mm</td>
			<td>Descarpack</td>
			<td>353601</td>
			<td>Sterile needle for applying medicines and anesthetics.</td>
		</tr>
		<tr>
			<td>Needle 40 mm x 1.6 mm</td>
			<td>Wiltex</td>
			<td>ANVISA registration: 10150470664</td>
			<td>Sterile needle for applying medicines and anesthetics.</td>
		</tr>
		<tr>
			<td>Needle holder</td>
			<td>generic</td>
			<td>-</td>
			<td>Surgical instrument</td>
		</tr>
		<tr>
			<td>Nylon 2 suture trhead</td>
			<td>Shalon Medical</td>
			<td>N502CTI40</td>
			<td>Surgical material</td>
		</tr>
		<tr>
			<td>Ordinary pen</td>
			<td>generic</td>
			<td>-</td>
			<td>Regular pen for taking notes</td>
		</tr>
		<tr>
			<td>Physiological solution 0.9% 500 mL bag</td>
			<td>JP Farma</td>
			<td>MS:1.0491.0070</td>
			<td>Fluid therapy</td>
		</tr>
		<tr>
			<td>Polyglycolic acid 2 suture thread</td>
			<td>Atramat</td>
			<td>G4099-75H</td>
			<td>Surgical material</td>
		</tr>
		<tr>
			<td>Potassium chloride</td>
			<td>Samtec</td>
			<td>ANVISA registration: 1559200010139</td>
			<td>Parenteral drug</td>
		</tr>
		<tr>
			<td>Procedure gloves</td>
			<td>Descarpack</td>
			<td>122401</td>
			<td>Personal Protective Equipment (PPE)</td>
		</tr>
		<tr>
			<td>Propofol (Provive)</td>
			<td>Uni&#227;o Qu&#237;mica</td>
			<td>ANVISA registration: 1049714490057</td>
			<td>Anesthetic</td>
		</tr>
		<tr>
			<td>Ringer lactate solution 500 mL bag</td>
			<td>JP Farma</td>
			<td>MS:1.0491.0061</td>
			<td>Fluid therapy</td>
		</tr>
		<tr>
			<td>Riohex 0.5% clorexidine alcohol solution</td>
			<td>Rioquimica</td>
			<td>218690356</td>
			<td>Surgical asepsis</td>
		</tr>
		<tr>
			<td>Riohex 2% clorexidine solution with surfactant</td>
			<td>Rioquimica</td>
			<td>218690356</td>
			<td>Surgical asepsis</td>
		</tr>
		<tr>
			<td>Scalp 21 G</td>
			<td>Descarpack</td>
			<td>421201</td>
			<td>Surgical material</td>
		</tr>
		<tr>
			<td>Shoe covers</td>
			<td>generic</td>
			<td>-</td>
			<td>Personal Protective Equipment (PPE)</td>
		</tr>
		<tr>
			<td>Sterile compresses</td>
			<td>Cremer</td>
			<td>ANVISA registration: 10071150065</td>
			<td>Surgical material</td>
		</tr>
		<tr>
			<td>Sterile gauze pad</td>
			<td>Procitex</td>
			<td>ANVISA registration: 80245210083</td>
			<td>Surgical material</td>
		</tr>
		<tr>
			<td>Sterile surgical drapes 140 cm x 90 cm&#160;</td>
			<td>Venkuri</td>
			<td>7010003</td>
			<td>Surgical material</td>
		</tr>
		<tr>
			<td>Sterile surgical drapes 150 cm x 190 cm&#160;</td>
			<td>PolarFix</td>
			<td>F00208</td>
			<td>Surgical material</td>
		</tr>
		<tr>
			<td>Sterile surgical gloves</td>
			<td>Mucambo</td>
			<td>CA: 39.317</td>
			<td>Personal Protective Equipment (PPE)</td>
		</tr>
		<tr>
			<td>Stethoscope</td>
			<td>generic</td>
			<td>-</td>
			<td>Surgical equipment</td>
		</tr>
		<tr>
			<td>Surgical cap</td>
			<td>Descarpack</td>
			<td>93201</td>
			<td>Personal Protective Equipment (PPE)</td>
		</tr>
		<tr>
			<td>Surgical gown</td>
			<td>Descarpack</td>
			<td>231101</td>
			<td>Personal Protective Equipment (PPE)</td>
		</tr>
		<tr>
			<td>Surgical mask</td>
			<td>Descarpack</td>
			<td>110701</td>
			<td>Personal Protective Equipment (PPE)</td>
		</tr>
		<tr>
			<td>Surgical scissors blunt-blunt</td>
			<td>generic</td>
			<td>-</td>
			<td>Surgical instrument</td>
		</tr>
		<tr>
			<td>Surgical scissors sharp-sharp</td>
			<td>generic</td>
			<td>-</td>
			<td>Surgical instrument</td>
		</tr>
		<tr>
			<td>Surgical stapler</td>
			<td>Tradevet</td>
			<td>-</td>
			<td>Surgical material</td>
		</tr>
		<tr>
			<td>Tekbond super glue</td>
			<td>Tek Bond</td>
			<td>78072720030</td>
			<td>Surgical material</td>
		</tr>
		<tr>
			<td>Thermometer</td>
			<td>generic</td>
			<td>-</td>
			<td>Surgical equipment</td>
		</tr>
		<tr>
			<td>Three Way Stopcock</td>
			<td>Solidor</td>
			<td>374</td>
			<td>Surgical material</td>
		</tr>
		<tr>
			<td>Tramadol hydrocloride</td>
			<td>Agener Uni&#227;o</td>
			<td>MAPA registration: SP-000292-5.000002</td>
			<td>Anesthetic</td>
		</tr>
		<tr>
			<td>Transparent film dressing</td>
			<td>Skinupper</td>
			<td>ANVISA registration: 82307460014</td>
			<td>Surgical material</td>
		</tr>
		<tr>
			<td>Waterproof adhesive 10 cm x 4.5 cm&#160;</td>
			<td>generic</td>
			<td>364828</td>
			<td>Surgical material</td>
		</tr>
	</tbody>
</table>

embryo transfer surgery via laparotomy in gilts

This protocol aims to demonstrate the surgical technique for transferring cloned pig embryos to the oviduct, a method widely used in the production of genetically modified pigs for biomedical research. Nine gilts underwent hormonal synchronization and laparotomy for the transfer of cloned embryos produced by somatic cell nuclear transfer (SCNT) at stages of up to 4 cells on day 2 to the oviduct. Gestational diagnosis was conducted via ultrasound examination 30 days post-transfer surgery. Six out of the nine operated gilts exhibited signs of pregnancy on ultrasound examination. However, as there was no progression in fetal development as assessed by echography, the gilts underwent necropsy at 60 days for the collection of biological material and assessment of the reproductive system. Adhesions were observed in the uterine horns, ovaries, and oviducts. From the uterine lumen of two of the euthanized gilts, one and four embryonic structures with gestational ages ranging between 12 to 20 days were obtained. Despite the absence of live piglets, likely attributed to the low-efficiency rate of transferring cloned pig embryos, which is influenced by various factors, including the number and quality of transferred embryos, the presented surgical technique proved to be rapid and safe.

Author Spotlight: Surgical Methods and Outcomes in Oviductal Cloned Pig Embryo Transfers

Embryo Transfer Surgery via Laparotomy in Gilts

We intend to help other research teams carry out embryo transfer surgeries in gilts, since embryo transfer is a crucial step in producing genetically modified pigs to be used as experimental models. There are other modern methods of surgical transfer of pig embryos, such as laparoscopy. However, as described in our study, laparotomy remains the most reliable method of transferring embryos into the species.The current challenges range from the in vitro production of swine embryos to perfect synchronization between the surrogate gilt and the embryonic stage. Furthermore, the surgical procedure requires manual dexterity from the surgeon so as not to lacerate the ovarian structures and find the appropriate place for embryo deposition. Our protocol addresses the research gap by describing and demonstrating detail all the steps to perform embryo transfer surgery in pigs, mentioning pre-trans-and postoperative care that it's necessary to perform the surgery safely and effectively.It is necessary to expose only the ovaries, which reduces the organ manipulation, minimize the risks of adhesion and pregnancy failure. The use of adhesive instead of sutures to close the skin also makes the procedure faster and less likely to cause stress to the animals when removing the stitches.

This article aims to demonstrate the laparotomy surgery for transferring cloned embryos to the oviduct of gilts. All animals remained in an adequate anesthetic plane, without any intraoperative incidents or complications during anesthesia recovery. The gilts took, on average, 2-3 h to stand up after the surgery ended.
All surgical procedures lasted, on average, 44 min. Nine gilts underwent surgery, with an average of 185 cloned embryos transferred per gilt, totaling 1,664 embryos transferred b...

This protocol describes the application of the surgical technique used to transfer cloned pig embryos via laparotomy in gilts.

This protocol aims to demonstrate the surgical technique for transferring cloned pig embryos to the oviduct, a method widely used in the production of ...

embryo-transfer-surgery-via-laparotomy-in-gilts

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Research

JoVE Journal

Medicine

Surgical Transfer of Cloned Pig Embryos to the Gilt-Oviduct for Production of Genetically Modified Pigs

To begin, prepare the surgical table with the appropriate sterile instruments. After performing surgical field antisepsis in the anesthetized animal, position the surgical drape over the penultimate pair of inguinal mammary glands. Using scissors, create a rectangular opening in a surgical drape and place four backhouse clamps to secure the surgical drape over the animal skin.With a scalpel, create an incision approximately 10 centimeters in length. Achieve hemostasis of subcutaneous vessels with hemostatic forceps and absorbable suture size two. Using fine-pointed scissors or fingers, dissect the subcutaneous tissue, deepening the incision to reach the linea alba in the abdominal musculature.With Alice forceps, grasp and lift the musculature to make a stab incision in the linea alba with a scalpel. Insert the index finger into the incision to assess the presence of adhesions. Extend the incision over the linea alba caudally and cranially, avoiding injury to other organs and underlying structures.Insert one hand into the abdominal cavity to locate and exteriorize the ovaries. If one of the uterine horns is located, gently follow it until reaching the tip of the uterine horn and the ipsilateral ovary. After locating the ovary, gently pull it and carefully expose it.Assess the presence of pre ovulatory follicles, hemorrhagic corpora lutea, cyclic corpora lutea, and corpus albicans to evaluate if the gilt synchronization is as expected. Gently remove the fimbriae of the uterine tube covering the ovary, and divert its mucosa to locate the osteum of the uterine tube. Then carefully insert a tomcat catheter containing the embryos into the osteum of the fimbriae until reaching the ampulla of the uterine tube.Attach a one milliliter syringe to the Tomcat catheter to push the embryo containing fluid into the uterine tube. Cover the ovary with the fimbriae, and return it to the abdominal cavity. If it is difficult to identify the osteum, use a round bodied suture needle to create a pocket on the ampullae of the oviduct.Then, insert the tomcat catheter containing the embryos. Next, reposition the structures inside the abdominal cavity. To prevent adhesions, add one liter of saline or ringer's lactate solution, tempered at 39 degrees Celsius, to the abdominal cavity.Next, suture the abdominal musculature with absorbable suture size two in X interrupted stitches. Close the skin with ethyl cyanoacrylate adhesive. After cleaning the surgical wound, apply rapamycin or Pearson's ointment and cover the area with gauze.Finally, apply the surgical adhesive. Nine gilts underwent surgery with an average of 185 cloned embryos transferred per gilt, totaling 1, 664 embryos transferred bilaterally. No animal showed signs of intense pain after surgery, with an average pain score ranging between 0.33 to 4.67 on the pain scale of zero to 18, performed over the three postoperative days for each of the gilts.