equipment needed for endotracheal intubation : means of assisted ventilation , slings for opening the mouth , low pressure suction with blunt tip , veterinary laryngoscope with straight blade between 17 and 25 cm long , stylet , syringe with air , stethoscope and adhesive tape . for adult gttingen the minipig is placed on its back and properly supported in order to ensure that the laryngeal passage is kept straight . the tongue is pulled out slightly and the jaw held open by an assistant without overextending the head . rotation of the airways or excessive extension of the head can occlude airways , making the laryngeal opening more difficult to identify and complicating tracheal placement of the tube . the laryngoscope is passed into the pharyngeal cavity and used to depress the tongue , making the epiglottis visible . the tip of the suction is used to displace the epiglottis from the soft palate . the tip of the laryngoscope is then used to press the epiglottis upward toward the base of the tongue , revealing the laryngeal opening . the arytenoids cartilages and vocal cords come into view and the endotracheal tube is advanced gently with a slight rotation , into the trachea during expiration . the tube cuff is inflated according to the specifications of the manufacture while taking care to avoid excess inflation , which may cause swelling and edema of the airways . this is facilitated by using the correct tube size and importantly by fastening the proximal end of the tube at the level of the snout using adhesive tape . when the minipig is properly intubated , a free passage of air must be felt through the tube , and chest auscultation should reveal respiration sounds in both the left and right side of the thorax . upon connecting the ventilator with monitoring system to the minipig , physiological expiratory carbon dioxide values should be measurable and the carbon dioxide curve that follows the respiration pattern decided by the ventilator settings can be observed . the carbon dioxide and oximetry values may initially be affected by the premedication and anesthesia induction . any sign of cyanosis despite artificial - assisted ventilation indicate improper placement of the tube . due to the curvature and size of the external urethral opening , a catheter with stylet is preferable . a nasal speculum can be used in small minipigs whereas a speculum with longer blades can be useful for large animals . necessary equipment also includes a blunt - tip forceps , a syringe with saline , lubricant and a closed system urine drainage bag . in order to best visualize the external urethral orifice during catheterization the minipig is placed on its back with a support under the lumbar region , while an assistant pulls the hind legs cranially . the female external urethral opening is located in the floor of the vagina approximately one - third to one - half of the distance to the cervix ( fig . however , because the animal is placed on the back , the position of the urethral opening will appear to be inverted . the catheter is controlled with the blunt - tip forceps , so the tip of the catheter is in continuation of the forceps . however , slight resistance may initially be felt until the catheter has passed the external urinary sphincter . if this problem persists , this may indicate that the animal is not sedated deeply enough . as the catheter reaches the bladder , remove the stylet , and urine will be seen flowing . inflate the balloon with saline according to the specifications of the manufacture and gently pull the catheter out until the balloon catches on the bladder neck . tape the catheter to the tail to avoid accidental displacement of the catheter when handling the animal . necessary equipment includes : scalpel , blunt - tip surgical scissors , tissue forceps , small blunt - tip surgical forceps , small self - retaining tissue retractor , needle holder , surgical swaps , suture with needle , heparinized saline as a flush - fluid to maintain catheter patency , two 18-gauge iv cannula , two 4-french brite tip sheaths with introducer and seldinger guide wire . the depth of anesthesia should be assessed by testing the interdigital pain reflex before initiating the procedure . the femoral artery and vein are approached with the minipig placed on its back and the rear leg retracted laterally . identify in the skin fold between the gracilis and sartorius muscle where the pulsation of the superficial part of the medial saphenous artery disappears . make a longitudinal superficial skin incision cranial to this point , thereby avoiding inadvertently damaging the medial saphenous vessels .   use a blunt - tip scissors to dissect the underlying subcutaneous tissue . the fascia division of the sartorius and gracilis muscle is divided cranial to the penetration site of the medial saphenous vessels , first with a small blunt - tip surgical forceps and then digitally . the two muscle groups are separated with a small self - retaining tissue retractor while taking care not to damage the femoral nerve and vessels . the artery is isolated for a length of approximately 1 to 2 cm using blunt dissection . bend the needle a little so it will follow the vessel curvature more easily . remember to ensure the needle still can move back and forth with little effort . the artery is punctured , the needle retracted and the seldinger guide wire is inserted through the venflon tube . remove the venflon tube while gently applying pressure on the artery to ensure fixation of the guidewire . the sheath with introducer is inserted over the seldinger wire and advanced to the desired position , after which the introducer and seldinger wire is removed . the femoral vein is located just below and medial to the artery . after isolating the vein using blunt dissection , the vein is cannulated as previously described for the artery .   to verify that both the artery and vein are correctly catheterized , draw blood from both sheaths and compare the color of the samples whenever drawing a blood sample , remember to subsequently flush the catheters with heparinized saline in order to maintain catheter patency . a vessel loop can be placed around the artery and vein in order to fixate the vessels prior to puncture . despite its advantages , we generally avoid this additional step , because it requires excess dissection of the vessels and can increase the risk of damaging the lateral and deep branches . 2 ) , relevant perfusion fluids , scalpel , blunt - tip surgical scissors , bone shears , and two large self - holding forceps of which at least one has a curved tip . a self retaining sternal retractor is not absolutely necessary , but it eases the procedure considerably . transcardial perfusion must be performed in a well ventilated room with a high power fume extraction system and facilities that allow collection of perfusate liquids and blood for subsequent safe disposal . after intravenously injecting a lethal dose of pentobarbital , test the interdigital pain reflex before continuing . make a deep longitudinal skin incision , extending from the manubrium to the xiphoid process of the sternum . gain access to the thoracic cavity by making a small incision in the diaphragm just below the xiphoid process . care must be taken when performing this procedure , because the heart may be adherent at some place to the inside of the sternum . therefore , make certain that the blade of the bone shears rests at all time inside the thoracic cavity against the inside of the sternum . after the sternum has been divided , put in the self - retaining sternal retractor and open the thoracic cavity . if still intact , make a small incision in the pericardium at the apex of the heart and then digitally open the pericardial sac . identify the left ventricle , the right auricle and the aorta together with the superior caval vein ( fig . 1c ) make a small superficial cut near the apex of the heart and access the left ventricle by perforating the myocardium with a blunt - tipped instrument . insert the perfusion cannula inside the left ventricle and move the cannula tip cranially into the aorta . when the cannula is felt between the fingers in the aorta , clamp the cannula into place with the self - holding curved forceps . make an incision in the right auricle and begin injecting the fixative through the perfusion cannula . the flow of perfusate in the aorta can be felt , and blood will pour out through the incised right auricle as the perfusate flow into the minipig vessels continues . notice how systemic perfusion with paraformaldehyde induces hyperextension of the limbs and twitching of the superficial musculature due to aldehyde cross - linkage of nerves and muscles . when using a perfusion system powered by compressed air instead of gravity , make sure to remove the cannula , or clamp the clear silicone tube , before the canister is empty , in order to avoid air from entering the vascular system . a : the perfusion system consists of inlet tubing with an adjustable pressure valve and a lever used to control inlet of compressed air into the container holding the perfusate . clear silicone tubing connects the container to a blunt - tip cannula made from a 4 mm steel pipe . b and c : by compressing the inlet lever , the pressure rises in the container and the perfusate is propelled through the cannula . d : when the clear silicone tubing and cannula are filled with perfusate , the flow is stopped with a clamp and the system is ready for use . equipment needed for endotracheal intubation : means of assisted ventilation , slings for opening the mouth , low pressure suction with blunt tip , veterinary laryngoscope with straight blade between 17 and 25 cm long , stylet , syringe with air , stethoscope and adhesive tape . for adult gttingen the minipig is placed on its back and properly supported in order to ensure that the laryngeal passage is kept straight . the tongue is pulled out slightly and the jaw held open by an assistant without overextending the head . rotation of the airways or excessive extension of the head can occlude airways , making the laryngeal opening more difficult to identify and complicating tracheal placement of the tube . the laryngoscope is passed into the pharyngeal cavity and used to depress the tongue , making the epiglottis visible . the tip of the suction is used to displace the epiglottis from the soft palate . the tip of the laryngoscope is then used to press the epiglottis upward toward the base of the tongue , revealing the laryngeal opening . the arytenoids cartilages and vocal cords come into view and the endotracheal tube is advanced gently with a slight rotation , into the trachea during expiration . the tube cuff is inflated according to the specifications of the manufacture while taking care to avoid excess inflation , which may cause swelling and edema of the airways . this is facilitated by using the correct tube size and importantly by fastening the proximal end of the tube at the level of the snout using adhesive tape . when the minipig is properly intubated , a free passage of air must be felt through the tube , and chest auscultation should reveal respiration sounds in both the left and right side of the thorax . upon connecting the ventilator with monitoring system to the minipig , physiological expiratory carbon dioxide values should be measurable and the carbon dioxide curve that follows the respiration pattern decided by the ventilator settings can be observed . the carbon dioxide and oximetry values may initially be affected by the premedication and anesthesia induction . any sign of cyanosis despite artificial - assisted ventilation indicate improper placement of the tube . french size 8 or 10 is appropriate . due to the curvature and size of the external urethral opening , a catheter with a nasal speculum can be used in small minipigs whereas a speculum with longer blades can be useful for large animals . necessary equipment also includes a blunt - tip forceps , a syringe with saline , lubricant and a closed system urine drainage bag . in order to best visualize the external urethral orifice during catheterization the minipig is placed on its back with a support under the lumbar region , while an assistant pulls the hind legs cranially . the female external urethral opening is located in the floor of the vagina approximately one - third to one - half of the distance to the cervix ( fig . however , because the animal is placed on the back , the position of the urethral opening will appear to be inverted . the catheter is controlled with the blunt - tip forceps , so the tip of the catheter is in continuation of the forceps . carefully insert the catheter in the urethral opening ( ) . however , slight resistance may initially be felt until the catheter has passed the external urinary sphincter . if this problem persists , this may indicate that the animal is not sedated deeply enough . the solution is to wait awhile and let the anesthesia take its effect .   as the catheter reaches the bladder , inflate the balloon with saline according to the specifications of the manufacture and gently pull the catheter out until the balloon catches on the bladder neck . tape the catheter to the tail to avoid accidental displacement of the catheter when handling the animal . necessary equipment includes : scalpel , blunt - tip surgical scissors , tissue forceps , small blunt - tip surgical forceps , small self - retaining tissue retractor , needle holder , surgical swaps , suture with needle , heparinized saline as a flush - fluid to maintain catheter patency , two 18-gauge iv cannula , two 4-french brite tip sheaths with introducer and seldinger guide wire . the depth of anesthesia should be assessed by testing the interdigital pain reflex before initiating the procedure . the femoral artery and vein are approached with the minipig placed on its back and the rear leg retracted laterally . identify in the skin fold between the gracilis and sartorius muscle where the pulsation of the superficial part of the medial saphenous artery disappears . make a longitudinal superficial skin incision cranial to this point , thereby avoiding inadvertently damaging the medial saphenous vessels .   use a blunt - tip scissors to dissect the underlying subcutaneous tissue . the fascia division of the sartorius and gracilis muscle is divided cranial to the penetration site of the medial saphenous vessels , first with a small blunt - tip surgical forceps and then digitally . the two muscle groups are separated with a small self - retaining tissue retractor while taking care not to damage the femoral nerve and vessels . the artery is isolated for a length of approximately 1 to 2 cm using blunt dissection . bend the needle a little so it will follow the vessel curvature more easily . remember to ensure the needle still can move back and forth with little effort . the artery is punctured , the needle retracted and the seldinger guide wire is inserted through the venflon tube . remove the venflon tube while gently applying pressure on the artery to ensure fixation of the guidewire . the sheath with introducer is inserted over the seldinger wire and advanced to the desired position , after which the introducer and seldinger wire is removed . the femoral vein is located just below and medial to the artery . after isolating the vein using blunt dissection , the vein is cannulated as previously described for the artery .   to verify that both the artery and vein are correctly catheterized , draw blood from both sheaths and compare the color of the samples whenever drawing a blood sample , remember to subsequently flush the catheters with heparinized saline in order to maintain catheter patency . a vessel loop can be placed around the artery and vein in order to fixate the vessels prior to puncture . despite its advantages , we generally avoid this additional step , because it requires excess dissection of the vessels and can increase the risk of damaging the lateral and deep branches . 2 ) , relevant perfusion fluids , scalpel , blunt - tip surgical scissors , bone shears , and two large self - holding forceps of which at least one has a curved tip . a self retaining sternal retractor is not absolutely necessary , but it eases the procedure considerably . transcardial perfusion must be performed in a well ventilated room with a high power fume extraction system and facilities that allow collection of perfusate liquids and blood for subsequent safe disposal . after intravenously injecting a lethal dose of pentobarbital , test the interdigital pain reflex before continuing . make a deep longitudinal skin incision , extending from the manubrium to the xiphoid process of the sternum . gain access to the thoracic cavity by making a small incision in the diaphragm just below the xiphoid process . care must be taken when performing this procedure , because the heart may be adherent at some place to the inside of the sternum . therefore , make certain that the blade of the bone shears rests at all time inside the thoracic cavity against the inside of the sternum . after the sternum has been divided , put in the self - retaining sternal retractor and open the thoracic cavity . if still intact , make a small incision in the pericardium at the apex of the heart and then digitally open the pericardial sac . identify the left ventricle , the right auricle and the aorta together with the superior caval vein ( fig . 1c ) make a small superficial cut near the apex of the heart and access the left ventricle by perforating the myocardium with a blunt - tipped instrument . insert the perfusion cannula inside the left ventricle and move the cannula tip cranially into the aorta . when the cannula is felt between the fingers in the aorta , clamp the cannula into place with the self - holding curved forceps . make an incision in the right auricle and begin injecting the fixative through the perfusion cannula . the flow of perfusate in the aorta can be felt , and blood will pour out through the incised right auricle as the perfusate flow into the minipig vessels continues . notice how systemic perfusion with paraformaldehyde induces hyperextension of the limbs and twitching of the superficial musculature due to aldehyde cross - linkage of nerves and muscles . when using a perfusion system powered by compressed air instead of gravity , make sure to remove the cannula , or clamp the clear silicone tube , before the canister is empty , in order to avoid air from entering the vascular system . a : the perfusion system consists of inlet tubing with an adjustable pressure valve and a lever used to control inlet of compressed air into the container holding the perfusate . clear silicone tubing connects the container to a blunt - tip cannula made from a 4 mm steel pipe . b and c : by compressing the inlet lever , the pressure rises in the container and the perfusate is propelled through the cannula . d : when the clear silicone tubing and cannula are filled with perfusate , the flow is stopped with a clamp and the system is ready for use . this presentation provides step - by - step visual instructions on how to perform four frequently used procedures in minipig research . the techniques described may be used with little or no adaptation in landrace pigs and other large research animals such as dogs and sheep . aseptic technique must be employed to avoid infections , when performing the described procedures in survival studies . induction of anesthesia is important for animal welfare and scientific reasons , and should provide absence of corneal reflexes and good muscular relaxation , before endotracheal intubation is attempted . however , because the anaesthetics used for induction are not given in fixed doses , but administered until the desired effect is observed , it is important to closely monitor the animal due to the risk of apnoea , especially if using propofol . for tracheal intubation , the use of xylocaine spray or muscle relaxant can facilitate the introduction of the tube , but the use of neuromuscular blockers should only be done by personnel who are well - trained in such procedures so as to conform with ethical requirements . care should be taken not to traumatize the laryngeal passage during intubation , in order to avoid tracheal swelling and edema . pigs can be intubated from various positions , but having the animal on its back tends to be easier for those who regularly carry out human intubation , as it presents the airway in a similar manner . the most common error during intubation is inadvertent placement of the tube in the esophagus , which is situated just below the laryngeal passage when the animal is placed on its back . note , the tube should never be advanced without first clearly identifying the arytenoids cartilages and vocal cords . transurethral catheterization of the urinary bladder through the urethra is easily done in female pigs , while this procedure is extremely difficult , perhaps impossible , in males . it should be noted that since the female urethral orifice is anterior to the vagina , the larger vaginal opening can be mistakenly catheterized . furthermore , lubricant with local anesthetic effect may ease the catheterization procedure . when performing femoral artery and vein catherization , the choice of vessel catheters and how they are secured differ between non - survival and survival studies , especially when catheters are implanted for an extended period of time . for instance catheters for chronic implantation should be highly biocompatible , have retention beads or cuffs for fixation as well as not traumatize the vessel wall over time , thus inducing thrombosis or erosion . in the present video , we use a closed system powered by compressed air for transcardial perfusion . the advantage of gravity systems in comparison to systems utilizing compressed air is that they are simple , efficient , and present no risk of pressurized air entering the vascular system . however , the hydrostatic pressure exerted by gravity systems changes continuously during perfusion , and this can affect the histological results . depending on which type of histopathological studies one wants to perform , it may be advantageous to perfuse the animal with heparinized saline first . this is particular importantance when performing immunofluorescent staining in order to avoid autofluorescence problems caused by erythrocytes . furthermore , if the organ of interest is the brain , the descending aorta could be clamped to save fixative . however , in pigs , this step entails additional dissecting and will prolong the procedure . all procedures were approved by the danish experimental animal inspectorate , danish ministry of justice and were conducted according to the " principles of laboratory animal care " ( nih publication no .
the emergence of the gttingen minipig in research of topics such as neuroscience , toxicology , diabetes , obesity , and experimental surgery reflects the close resemblance of these animals to human anatomy and physiology 1-6.the size of the gttingen minipig permits the use of surgical equipment and advanced imaging modalities similar to those used in humans 6 - 8 . 
 the aim of this instructional video is to increase the awareness on the value of minipigs in biomedical research , by demonstrating how to perform tracheal intubation , transurethral bladder catheterization , femoral artery and vein catheterization , as well as transcardial perfusion . 
 endotracheal intubation should be performed whenever a minipig undergoes general anesthesia , because it maintains a patent airway , permits assisted ventilation and protects the airways from aspirates . 
 transurethral bladder catheterization can provide useful information about about hydration state as well as renal and cardiovascular function during long surgical procedures . 
 furthermore , urinary catheterization can prevent contamination of delicate medico - technical equipment and painful bladder extension which may harm the animal and unnecessarily influence the experiment due to increased vagal tone and altered physiological parameters . 
 arterial and venous catheterization is useful for obtaining repeated blood samples and monitoring various physiological parameters . 
 catheterization of femoral vessels is preferable to catheterization of the neck vessels for ease of access , when performing experiments involving frame - based stereotaxic neurosurgery and brain imaging . when performing vessel catheterization in survival studies , strict aseptic technique must be employed to avoid infections6 . 
 transcardial perfusion is the most effective fixation method , and yields preeminent results when preparing minipig organs for histology and histochemistry2,9 . for more information about anesthesia , surgery and experimental techniques in swine in general 
 we refer to swindle 2007 . supplementary information about premedication and induction of anesthesia , assisted ventilation , analgesia , pre- and postoperative care of gttingen minipigs are available via the internet at http://www.minipigs.com10 . for extensive information about porcine anatomy 
 we refer to nickel et al . 
 vol . 1 - 511 .