Embryo Transfer Surgery via Laparotomy in Gilts

Summary

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

Abstract

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.

Introduction

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 used^4,6. 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' 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 horn^7,8.

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.

Protocol

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 São Paulo, protocol number 6088030523. Nine seven-month-old gilts from the Água Branca nucleus pig farming, located in Itu city, São 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

1. Fast the animals for 12 h and restrict water access for 4 h before the surgical procedure.
2. 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.
3. Administer the pre-anesthetic medication via intramuscular injection into the trapezius muscle, caudal to the external ear canal.
4. 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.
5. Induce anesthesia with 4 mg/kg of propofol intravenously (following institutionally approved protocols).
6. Perform orotracheal intubation of the animal using a laryngoscope and a size 9 orotracheal tube with a cuff¹⁰.
7. 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 °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.
8. 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.
9. Place cardiac electrodes on the thoracic region for intraoperative monitoring of heart rate. Record heart rate, rectal temperature, and oxygen saturation every 5 min.

2. Surgical procedure

1. Position the gilt on the surgical table in dorsal recumbency.
2. Cover the animal's legs with procedural gloves to minimize contamination of the surgical field.
3. Perform ventral caudal abdominal hair removal between the penultimate pair of mammary glands using a surgical clipper.
4. 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.
5. 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.
6. Prepare the surgical table with the appropriate and previously sterilized instruments listed in the Table of Materials.
7. 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.
8. Repeat this procedure three times with chlorhexidine antiseptic, and then repeat the procedure with alcoholic chlorhexidine.
9. Completely cover the animal with a sterile surgical drape, and using scissors, create a rectangular opening in the surgical drape, approximately 10 x 20 cm², if the drape is not fenestrated.
10. Position the surgical drape over the penultimate pair of inguinal mammary glands. Place 4 Backhaus clamps to secure the surgical drape over the animal's skin.
11. Incise the skin with a scalpel, creating an incision approximately 10 cm in length.
12. Achieve hemostasis of subcutaneous vessels with hemostatic forceps and/or absorbable suture size 2.
13. 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.
14. With Allis forceps, grasp the musculature and lift it to make a stab incision in the linea alba with a scalpel.
15. 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.
16. Make an incision of sufficient length so that the surgeon'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.
17. 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.
18. 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's synchronization is as expected.
19. Gently remove the fimbriae of the uterine tube covering the ovary, evert its mucosa, and locate the ostium of the uterine tube. 
    NOTE: 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.
20. Carefully insert a Tomcat catheter containing the embryos into the ostium of the fimbriae until reaching the ampulla region of the uterine tube.
21. 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.
22. Locate the contralateral uterine horn and ovary and repeat steps 2.17-2.21.
23. Reposition the structures inside the abdominal cavity and add 1 L of saline solution or Ringer's lactate solution warmed (39 °C) to prevent adhesions before initiating abdominorraphy.
24. Suture the abdominal musculature with absorbable suture size 2 in "X" stitches (Sultan) interrupted.
25. Approximate the subcutaneous tissue with a continuous mattress suture using absorbable suture size 2.
26. 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.
27. 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's ointment, cover with gauze, and finally, apply surgical adhesive.
    NOTE: The wound should remain closed with a dressing for 5 days.

3. Postoperative care

1. 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.
2. 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.
3. 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's ointment until the 10th day, when suture removal or surgical staples removal, if used, is recommended.

4. Ultrasonographic gestational diagnosis of pregnancy

1. Perform transabdominal pregnancy ultrasonographic evaluation with an ultrasound device (frequency range 4.5 to 8.5 MHz) 30 days after embryo transfer surgery.
2. 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 presence of intrauterine fluid without delineation of embryonic vesicles, suggests the absence of gestation.

5. Euthanasia of the animals

NOTE:  Euthanize the gilts that did not present fetuses of size and appearance corresponding to gestational age on the ultrasonographic examination.

1. 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.
2. 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).
3. 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.
4. 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.

Results

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...

Discussion

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 technique^{15,16,17,18,19,20}. The pregnancy and birth rates of SCNT embryos produced from various cell lines...

Materials

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