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.
1. Animal preparation
<ol>
	<li>Fast the animals for 12 h and restrict water access for 4 h before the surgical procedure.</li>
	<li>Using a 40 mm x 12 mm needle, draw up 10 mg/kg of ketamine and 0.2 mg/kg of midazolam in the same 50 mL syringe. Attach the syringe to an IV administration set to facilitate medication administration, maintaining a safe distance from the animal and allowing it to move freely.</li>
	<li>Administer the pre-anesthetic medication via intramuscular injection into the trapezius muscle, caudal to the external ear canal.</li>
	<li>Cannulate the auricular vein with a 20 G catheter and connect it to a macrodrip administration set for intraoperative fluid therapy with 0.9% NaCl solution at a rate of 10 mL/kg/h.</li>
	<li>Induce anesthesia with 4 mg/kg of propofol intravenously (following institutionally approved protocols).</li>
	<li>Perform orotracheal intubation of the animal using a laryngoscope and a size 9 orotracheal tube with a cuff10.</li>
	<li>Maintain anesthesia with 1.5% to 2% isoflurane inhalation. Additionally, monitor anesthesia by evaluating physiological parameters such as heart rate, which should be maintained between 100 and 120 beats per minute, oxygen saturation, which should be kept between 99% and 100%, muscle relaxation, absence of movement, and reflexes evaluation. 
	NOTE: Body temperature should also be monitored and controlled, staying between 37.5-38.5 &#176;C. When necessary, in addition to isoflurane, propofol can be used to maintain control of the parameters. Fentanyl hydrochloride should be administered as an analgesic at a dose of 0.02 mg/kg every 20 min. All parameters should be recorded every 5 min.</li>
	<li>Administer perioperative analgesia with a single dose of 0.002 mg/kg of fentanyl intravenously and 4 mg/kg of tramadol hydrochloride intramuscularly into the gluteal or trapezius muscle.</li>
	<li>Place cardiac electrodes on the thoracic region for intraoperative monitoring of heart rate. Record heart rate, rectal temperature, and oxygen saturation every 5 min.</li>
</ol>
2. Surgical procedure
<ol>
	<li>Position the gilt on the surgical table in dorsal recumbency.</li>
	<li>Cover the animal&#39;s legs with procedural gloves to minimize contamination of the surgical field.</li>
	<li>Perform ventral caudal abdominal hair removal between the penultimate pair of mammary glands using a surgical clipper.</li>
	<li>Perform preoperative skin antisepsis with a chlorhexidine-impregnated antiseptic brush, making circular movements from the umbilical scar region to the last pair of mammary glands. Remove chlorhexidine with a clean, moistened compress.</li>
	<li>Ensure that the surgeon and the assistant perform surgical antisepsis and wear sterile surgical gowns and gloves for surgery. 
	NOTE: The animal should be prepared for surgery in a preparation area separate from the surgical site.</li>
	<li>Prepare the surgical table with the appropriate and previously sterilized instruments listed in the Table of Materials.</li>
	<li>Perform final surgical field antisepsis using sterile gauze and chlorhexidine antiseptic, cleaning the region in a fishbone pattern, starting at the incision site and expanding laterally.</li>
	<li>Repeat this procedure three times with chlorhexidine antiseptic, and then repeat the procedure with alcoholic chlorhexidine.</li>
	<li>Completely cover the animal with a sterile surgical drape, and using scissors, create a rectangular opening in the surgical drape, approximately 10 x 20 cm&#178;, if the drape is not fenestrated.</li>
	<li>Position the surgical drape over the penultimate pair of inguinal mammary glands. Place 4 Backhaus clamps to secure the surgical drape over the animal&#39;s skin.</li>
	<li>Incise the skin with a scalpel, creating an incision approximately 10 cm in length.</li>
	<li>Achieve hemostasis of subcutaneous vessels with hemostatic forceps and/or absorbable suture size 2.</li>
	<li>Dissect the subcutaneous tissue with fingers or with the aid of fine-pointed scissors, deepening the incision to reach the linea alba in the abdominal musculature. 
	NOTE: Palpate the midline of the abdomen to feel the linea alba as a more rigid and fibrous area; observe the separation of the rectus abdominis muscles and note the change in tissue consistency while dissecting to identify the linea alba accurately11.</li>
	<li>With Allis forceps, grasp the musculature and lift it to make a stab incision in the linea alba with a scalpel.</li>
	<li>Insert the index finger into the incision to assess the presence of adhesions. Extend the incision over the linea alba caudally and cranially with blunt/blunt or fine/blunt scissors carefully to avoid injuring other organs and underlying structures.</li>
	<li>Make an incision of sufficient length so that the surgeon&#39;s one hand can be inserted into the abdominal cavity to locate and exteriorize the uterine horns and/or ovaries. A Gosset retractor may be used to facilitate entry into the abdominal cavity and suturing of the musculature in a single layer at the end of the procedure.</li>
	<li>If one of the uterine horns is located before the ovaries, gently follow it until reaching the tip of the uterine horn and the ipsilateral ovary. During this procedure, assess the presence of fluid inside the uterus, which may compromise gestation.</li>
	<li>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&#39;s synchronization is as expected.</li>
	<li>Gently remove the fimbriae of the uterine tube covering the ovary, evert its mucosa, and locate the ostium of the uterine tube.&#160; 
	NOTE:&#160;If the surgeon finds it difficult to identify the ostium of the fimbriae, a pocket on the ampulla of the oviduct can be done with a round-bodied suture needle, avoiding puncturing blood vessels, to insert the Tomcat catheter containing the embryos.</li>
	<li>Carefully insert a Tomcat catheter containing the embryos into the ostium of the fimbriae until reaching the ampulla region of the uterine tube.</li>
	<li>Attach a 1 mL 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.</li>
	<li>Locate the contralateral uterine horn and ovary and repeat steps 2.17-2.21.</li>
	<li>Reposition the structures inside the abdominal cavity and add 1 L of saline solution or Ringer&#39;s lactate solution warmed (39 &#176;C) to prevent adhesions before initiating abdominorraphy.</li>
	<li>Suture the abdominal musculature with absorbable suture size 2 in &#34;X&#34; stitches (Sultan) interrupted.</li>
	<li>Approximate the subcutaneous tissue with a continuous mattress suture using absorbable suture size 2.</li>
	<li>Close the skin with size 2 nylon suture in interrupted simple stitches, surgical staples, or ethyl-cyanoacrylate adhesive. In this procedure, we opted for the use of adhesive.</li>
	<li>Clean the surgical wound with gauze soaked in hydrogen peroxide and dry with a compress to enhance the fixation of the surgical adhesive on the surgical wound. Apply rifamycin or Pearson&#39;s ointment, cover with gauze, and finally, apply surgical adhesive. 
	NOTE: The wound should remain closed with a dressing for 5 days.</li>
</ol>
3. Postoperative care
<ol>
	<li>Evaluate the animals for signs of characteristic clinical pain for at least the next 3 consecutive days, according to the scale proposed by Luna et al.12. 
	NOTE: This scale is based on different behaviors exhibited by pigs when experiencing different levels of pain, such as posture, interaction and interest in the environment, activity, appetite, and attention to the affected area. The score ranges from 0 to 3, depending on the type of behavior exhibited, for each parameter evaluated. At the end of the evaluation, the sum of all scores reflects the intensity of pain experienced by the animal, where 0 represents no pain, and 18 represents very intense pain12.</li>
	<li>Administer a single dose of 15 mg/kg of amoxicillin and 0.4 mg/kg of 2% meloxicam both intramuscularly (in the gluteal or cervical muscle) once a day for 3 days as postoperative care to prevent pain, inflammatory processes, and infection. 
	NOTE: If the pain score is equal to or greater than 6 on the pain scale used (score from 0 to 18), administer 5,000 mg/animal of sodium dipyrone intramuscularly (in the gluteal or cervical muscle) once a day in addition to the anti-inflammatory and antibiotic. Alternatively, perform analgesia as outlined in the local IACUC guidelines.</li>
	<li>On the 6th day after surgery, remove the surgical adhesive for wound cleaning with gauze soaked in 2% alcoholic chlorhexidine solution and apply topical Pearson&#39;s ointment until the 10th day, when suture removal or surgical staples removal, if used, is recommended.</li>
</ol>
4. Ultrasonographic gestational diagnosis of pregnancy
<ol>
	<li>Perform transabdominal pregnancy ultrasonographic evaluation with an ultrasound device (frequency range 4.5 to 8.5 MHz) 30 days after embryo transfer surgery.</li>
	<li>Apply ultrasound gel to the probe and position it near the inguinal abdominal region, facing medially. 
	NOTE: Visualization of rounded anechoic embryonic vesicles, due to fluid in the uterine lumen, indicates gestation. Non-visualization of intrauterine fluid, or&#160;presence of intrauterine fluid without delineation of embryonic vesicles, suggests the absence of gestation.</li>
</ol>
5. Euthanasia of the animals
NOTE: &#160;Euthanize the gilts that did not present fetuses of size and appearance corresponding to gestational age on the ultrasonographic examination.
<ol>
	<li>Using a 40 mm x 12 mm needle, draw up 15 mg/kg of ketamine and 2 mg/kg of midazolam in the same 50 mL syringe attached to an intravenous administration set, and administer intramuscularly into the trapezius muscle in the cervical region caudal to the external ear canal, as preanesthetic medication.</li>
	<li>Cannulate the auricular vein with a 20 G catheter and induce anesthesia with 4 mg/kg of propofol intravenously in a bolus until bradycardia occurs (following institutionally approved protocols).</li>
	<li>Subsequently, administer 10 to 20 mL of 19.1% potassium chloride intravenously. Confirm cardiopulmonary arrest by observing the absence of respiratory movements, pale mucous membranes, loss of corneal reflex, and absence of heartbeats by placing a stethoscope over the cardiac region.</li>
	<li>Collect any type of intrauterine content (embryonic tissue, liquid, or mucous secretions) from necropsied females13 and forward it for microbiological analysis to detect possible pathogens and also for genotypic characterization of the clones (if needed). Visually check the presence or absence of corpora lutea, ovarian cysts, abnormal secretions, and the occurrence of adhesions in the uterus, ovaries, and oviducts14.</li>
</ol>

Optimizing Oviductal Embryo Transfer in Cloned Pigs

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None of the authors disclose any conflict of interest

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.

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			<td>Conductive clinical gel</td>
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			<td>Dipyrone D-500</td>
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			<td>MAPA registration: SP0000728-46</td>
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			<td>Fentanil (Fentanest)</td>
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			<td>Gosset retractor</td>
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			<td>Halstead-mosquito hemostatic forceps</td>
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			<td>-</td>
			<td>Surgical instrument</td>
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			<td>Healing ointment - Unguento Pearson</td>
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			<td>hydrogen peroxide solution</td>
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			<td>Laringoscope</td>
			<td>-</td>
			<td>-</td>
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			<td>Maxicam 2%</td>
			<td>Ourofino SA</td>
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			<td>Micropore adhesive 5 cm x 10 cm&#160;</td>
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			<td>1530</td>
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			<td>Midazolam</td>
			<td>Hipolabor</td>
			<td>ANVISA registration: 1134301430035</td>
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			<td>Multi-Way IV Infusion Set</td>
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			<td>Needle 40 mm x 1.2 mm</td>
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			<td>Needle 40 mm x 1.6 mm</td>
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			<td>Procedure gloves</td>
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			<td>Propofol (Provive)</td>
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			<td>Sterile surgical drapes 150 cm x 190 cm&#160;</td>
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			<td>Sterile surgical gloves</td>
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			<td>Stethoscope</td>
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			<td>Surgical scissors blunt-blunt</td>
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		<tr>
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		<tr>
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			<td>Tramadol hydrocloride</td>
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		<tr>
			<td>Transparent film dressing</td>
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		<tr>
			<td>Waterproof adhesive 10 cm x 4.5 cm&#160;</td>
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			<td>364828</td>
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</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.

372 Views.  Universidade de São Paulo.  This protocol describes the application of the surgical technique used to transfer cloned pig embryos via laparotomy in gilts.

Optimizing Oviductal Embryo Transfer in Cloned Pigs

Özet