In this protocol, a model of porcine orthotopic liver transplantation after static cold storage of donor organs for 20 h without the use of a veno-venous bypass during engraftment is described. The approach uses a simplified surgical technique with minimization of the anhepatic phase and sophisticated volume and vasopressor management.
Liver transplantation is regarded as the gold standard for the treatment of a variety of fatal hepatic diseases. However, unsolved issues of chronic graft failure, ongoing organ donor shortages, and the increased use of marginal grafts call for the improvement of current concepts, such as the implementation of organ machine perfusion. In order to evaluate new methods of graft reconditioning and modulation, translational models are required. With respect to anatomical and physiological similarities to humans and recent progress in the field of xenotransplantation, pigs have become the main large animal species used in transplantation models. After the initial introduction of a porcine orthotopic liver transplant model by Garnier et al. in 1965, several modifications have been published over the past 60 years.
Due to specifies-specific anatomical traits, a veno-venous bypass during the anhepatic phase is regarded as a necessity to reduce intestinal congestion and ischemia resulting in hemodynamic instability and perioperative mortality. However, the implementation of a bypass increases the technical and logistical complexity of the procedure. Furthermore, associated complications such as air embolism, hemorrhage, and the need for a simultaneous splenectomy have been reported previously.
In this protocol, we describe a model of porcine orthotopic liver transplantation without the use of a veno-venous bypass. The engraftment of donor livers after static cold storage of 20 h – simulating extended criteria donor conditions – demonstrates that this simplified approach can be performed without significant hemodynamic alterations or intraoperative mortality and with regular uptake of liver function (as defined by bile production and liver-specific CYP1A2 metabolism). The success of this approach is ensured by an optimized surgical technique and a sophisticated anesthesiologic volume and vasopressor management.
This model should be of special interest for workgroups focusing on the immediate postoperative course, ischemia-reperfusion injury, associated immunological mechanisms, and the reconditioning of extended criteria donor organs.
Liver transplantation remains to be the only chance for survival in a variety of different diseases leading to acute or chronic hepatic failure. Since its first successful application in mankind in 1963 by Thomas E. Starzl, the concept of liver transplantation has evolved into a reliable treatment option applied worldwide, mainly as a result of advancements in the understanding of the immune system, the development of modern immunosuppression, and the optimization of perioperative care and surgical techniques1,2. However, aging populations and a higher demand for organs have resulted in donor shortages, with increased use of marginal grafts from extended criteria donors and the emergence of new challenges in the past decades. The introduction and widespread implementation of organ machine perfusion is believed to open up an array of possibilities with regard to graft reconditioning and modulation and to help mitigate organ shortages and reduce waiting list mortality3,4,5,6.
In order to evaluate these concepts and their effects in vivo, translational transplant models are necessary7. In 1983, Kamada et al. introduced an efficient orthotopic liver transplant model in rats that has since been extensively modified and applied by workgroups around the globe8,9,10,11. The orthotopic liver transplant model in mice is technically more demanding, but also more valuable in terms of immunological transferability, and was first reported in 1991 by Qian et al.12. Despite advantages regarding availability, animal welfare, and costs, rodent models are limited in their applicability in clinical settings7. Hence, large animal models are required.
In recent years, pigs have become the main animal species used for translational research due to their anatomical and physiological similarities with humans. Furthermore, current progress in the field of xenotransplantation might further increase the importance of pigs as research objects13,14.
Garnier et al. described a liver transplant model in pigs as early as 196515. Several authors, including Calne et al. in 1967 and Chalstrey et al. in 1971, subsequently reported modifications, ultimately leading to a safe and feasible concept of experimental porcine liver transplantation in the decades to follow16,17,18,19,20,21.
More recently, different work groups have provided data with regard to current issues in liver transplantation using a technique of porcine orthotopic liver transplantation, almost invariably including an active or passive veno-venous, i.e., porto-caval, bypass19,22. The reason for this is a species-specific intolerance to the clamping of the vena cava inferior and the portal vein during the anhepatic phase due to a comparatively larger intestine and fewer porto-caval or cavo-caval shunts (e.g., lack of a vena azygos), resulting in increased perioperative morbidity and mortality23. Vena cava inferior-sparing transplant techniques applied in human recipients as an alternative are not feasible as the porcine vena cava inferior is encased by hepatic tissue23.
However, the usage of a veno-venous bypass further increases technical and logistical complexity in an already demanding surgical procedure, therefore possibly preventing workgroups from attempting implementation of the model altogether. Apart from the direct physiological and immunological effects of a bypass, some authors have pointed out the significant morbidity such as blood loss or air embolism during shunt placement and the need for a simultaneous splenectomy, potentially affecting short- and long-term results after engraftment24,25.
The following protocol describes a simple technique of porcine orthotopic liver transplantation after static cold storage of donor organs for 20 h, representing extended criteria donor conditions without the usage of a veno-venous bypass during engraftment, including donor liver procurement, back-table preparation, recipient hepatectomy, and anesthesiological pre- and intraoperative management.
This model should be of special interest for surgical workgroups focusing on the immediate postoperative course, ischemia-reperfusion injury, the reconditioning of extended criteria donor organs, and associated immunological mechanisms.
This study was performed at the Laboratory for Animal Science of Hannover Medical School after approval by the Lower Saxony regional authority for consumer protection and food safety (Niedersächsisches Landesamt für Verbraucherschutz und Lebensmittelsicherheit [LAVES]; 19/3146)
1. Donor liver procurement
NOTE: The liver donors were female domestic pigs (Sus scrofa domesticus), aged 4-5 months old and with an average body weight of approximately 50 kg, which had already been in quarantine at the animal research facility for a minimum of 10 days prior to surgery.
2. Back-table preparation of the liver
3. Recipient hepatectomy, donor liver engraftment, and perioperative management
NOTE: As liver recipients, female domestic pigs (Sus scrofa domesticus) aged 4-5 months old and with an average body weight of approximately 50 kg, were used. Analogously to the liver donors, the recipients had been in quarantine at the animal research facility for a minimum of 10 days prior to transplant.
The technique presented in this protocol has provided reliable and reproducible results in terms of hemodynamic stability and animal survival throughout the procedure, as well as graft function in the postoperative course.
Most recently, we applied the model for the study of ischemia-reperfusion injury and therapeutic interventions mitigating detrimental effects in the immediate postoperative course. Upon retrieval and 20 h of static cold storage, liver grafts (with a mean weight of 983.38 g) were implanted in the described manner. The experiments were terminated 6 h after portal-venous reperfusion and sampling of blood and bile as well as liver and bile duct tissue at defined intervals. All recipients survived the engraftment and the subsequent 6 h follow-up under general anesthesia until euthanasia.
Since the focus of this protocol lies in the feasibility of a porcine orthotopic liver transplant model without the use of a veno-venous bypass, the results presented here are limited to the intraoperative vital parameters and the application of vasopressors (Figure 2), as well as graft performance, defined by conventional laboratory parameters, i.e., serum concentrations of lactate, aspartate transaminase (AST), alanine transaminase (ALT), and glutamate dehydrogenase (GLDH), bile production (Table 1), and the liver maximum function capacity (LiMAx) test as described previously in a model of porcine liver resection (Figure 3)26. The LiMAx test is based on the real-time metabolism of intravenously injected 13C-methacetin by the liver-specific CYP1A2 system. Before and after injection, the ratio of 13CO212CO2 in the exhaled air is determined to quantify the individual hepatic function27.
As expected, recipients required increased concentrations of norepinephrine immediately before and throughout the anhepatic phase in order to stabilize the mean arterial pressure (MAD) at ≥60 mmHg. Low concentrations of epinephrine were simultaneously used to additionally increase cardiac output in this vulnerable time period. Upon portal-venous reperfusion, the need for vasopressors quickly declined and even more so during temporary clamping of the abdominal aorta for completion of the aortic anastomosis. After engraftment, the MAD and required doses of vasopressors remained stable.
Mean operation time, defined as the time from skin incision to completion of all vascular anastomosis and reperfusion, was 103.50 min, including a mean anhepatic phase of 27.13 min. Of note, only two recipients underwent an anhepatic phase of more than 30 min. All recipients showed declining lactate serum concentrations 4 h after portal-venous reperfusion, and LiMAx values obtained 6 h after portal-venous reperfusion were comparable to the values measured in the liver donors before organ procurement in all but one recipient (anhepatic phase of 34 min).
Figure 1: Graft and recipient preparation. (A) The figure depicts the back-table preparation of the celiac axis and the aortic segment. (B) This figure shows the recipient in a supine position with extended monitoring, including a central venous catheter (blue) in the left internal jugular vein and an arterial catheter (red) in the right internal carotid/cervical artery. Please click here to view a larger version of this figure.
Figure 2: Mean arterial pressure and concentrations of vasopressors required during engraftment. The figure depicts the mean arterial pressure (MAD in mmHg) measured and the concentrations of norepinephrine and epinephrine (in µg/kg/h) during defined time periods throughout the procedure in all eight recipients. Values are presented as mean ± SEM. Please click here to view a larger version of this figure.
Figure 3: Values of the liver maximum function capacity (LiMAx) test obtained from the donors prior to liver procurement and from the recipients 6 h after engraftment. The figure depicts box plot data (mean and standard error of the mean) from the liver maximum function capacity (LiMAx) test from the donors prior to liver procurement and from the recipients 6 h after engraftment (n = 8). Please click here to view a larger version of this figure.
Experiment | Graft weight | Recipient weight | GRWR | Operation time | Anhepatic phase | Lactate (mmol/L) | Peak AST | Peak ALT | Peak GLDH | Bile volume | ||
No. | (g) | (kg) | (%) | (min) | (min) | 2 h | 4 h | 6 h | (U/L) | (U/L) | (U/L) | (mL) |
1 | 1082 | 48.8 | 2.22 | 115 | 25 | 5.8 | 4.7 | 3.7 | 677 | 122 | 39 | 48 |
2 | 946 | 51.4 | 1.84 | 125 | 34 | 6.6 | 5.9 | 5.2 | 1207 | 109 | 268 | 15 |
3 | 957 | 57.6 | 1.66 | 110 | 30 | 8.3 | 5.8 | 8.1 | 742 | 125 | 143 | 73 |
4 | 825 | 49.2 | 1.68 | 87 | 22 | 7.6 | 6.7 | 6.5 | 675 | 99 | 113 | 35 |
5 | 1045 | 53.4 | 1.96 | 101 | 25 | 7.9 | 6.8 | 5.6 | 919 | 86 | 129 | 25 |
6 | 924 | 45.2 | 2.04 | 105 | 32 | 6.7 | 4.6 | 3.7 | 414 | 90 | 114 | 75 |
7 | 785 | 48.2 | 1.63 | 95 | 24 | 6.8 | 4.8 | 4.1 | 557 | 70 | 110 | 1.5 |
8 | 1303 | 54.6 | 2.39 | 90 | 25 | 12.7 | 12.2 | 9.8 | 1011 | 87 | 94 | 10 |
MEAN | 983.38 | 51.05 | 1.93 | 103.50 | 27.13 | 7.80 | 6.44 | 5.84 | 775.25 | 98.50 | 126.25 | 35.31 |
SEM | 57.59 | 1.41 | 0.10 | 4.57 | 1.52 | 0.76 | 0.88 | 0.78 | 90.79 | 6.73 | 23.00 | 9.87 |
Table 1: Perioperative graft and recipient variables. The table summarizes graft and recipient weight, as well as the graft-to-recipient weight ratio (GRWR) and the length of the operation (skin incision to completion of all vascular anastomosis and reperfusion) and of the anhepatic phase. Variables indicating graft function, such as conventional laboratory parameters, i.e., serum concentrations of lactate, aspartate transaminase (AST), alanine transaminase (ALT), and glutamate dehydrogenase (GLDH), and bile production are provided for each of the eight transplants performed.
Recent technical developments such as the introduction of machine perfusion have the potential to revolutionize the field of liver transplantation. In order to translate graft reconditioning or modification concepts into clinical settings, reproducible transplant models in large animals are inevitable.
After the initial introduction of porcine orthotopic liver transplantation, several authors have worked on the improvement of these techniques over the past five decades. Differences within the reported surgical approaches are often minor and concern vascular and biliary anastomoses, anesthesia, and perioperative management. Nonetheless, in contrast to the current situation in clinical liver transplantation in which the usage of veno-venous bypass is still common but optional 28, an active or passive porto-caval bypass during the anhepatic phase in pigs is regarded as a necessity to reduce intestinal congestion and, thus, subsequent intestinal ischemia with hemodynamic instability and perioperative mortality, as described in a well-elaborated work by Esmaeilzadeh et al.25.
Apart from the implicated additional costs and technical challenges of a veno-venous bypass, e.g., catheters, a pump device, the need for additional anticoagulation, and potential complications such as air embolism or hemorrhage, and depending on the chosen approach, the need for a simultaneous splenectomy has prompted groups to describe modified techniques without veno-venous bypasses25,29,30.
Torres et al.31 observed severe hemodynamic instability in animals undergoing engraftment without the use of a veno-venous bypass in comparison to recipients with a passive porto-caval shunt and, thus, performed temporary clamping of the supraceliac aorta in these animals, which was also described by others in models of porcine liver auto-/allo-transplantation23,31,32. However, the induction of warm ischemia by cross-clamping of the recipient aorta bears the risk of relevant release of pro-inflammatory molecules and tissue damage upon reperfusion and should, therefore, be avoided at all costs in order to produce reliable scientific results, especially when evaluating ischemia-reperfusion injury. Furthermore, this approach does not resemble clinical practice in humans, which, hence, limits the translation of results obtained in these models.
To avoid such detrimental supporting measures, we believe that two points are crucial. (1) The anhepatic phase should be kept at an absolute minimum, i.e., below 30 min, as has already been shown in the early phases of porcine liver transplantation by Battersby et al.33. We believe that running sutures (double-armed) and a maximum of one supporting thread are sufficient to create a simple and safe anastomosis for both the suprahepatic vena cava inferior and the portal vein. Obviously, the portal-venous reperfusion should commence before anastomosing the infrahepatic vena cava inferior. (2) Anesthetic management should be performed by an experienced anesthesiologist, ideally familiar with liver surgery or transplantation in human patients34. Sophisticated volume management and therapy with vasopressors, i.e., norepinephrine and epinephrine, in combination with a simplified surgical technique are the basis for the successful implementation of this model.
Interestingly, only a small number of surgical groups have provided data on successful porcine orthotopic liver transplantation without veno-venous bypass and concomitant supraceliac aortic clamping. To our knowledge Oike et al., Heuer et al., and, most recently, Fondevila et al. were the only groups to report their (promising) results, with survival rates of 87%, 80%, and 100%, respectively35,36,37. The median anhepatic time in our cohort was 25 min and was, thus, identical to the data presented by Heuer et al.36. During the anhepatic phase, Oike et al.35 reported a 50%-60% reduction in the arterial blood pressure, similar to the observations made in this cohort, leading to increased doses of vasopressors to avoid a decrease in the MAD below 60 mmHg. Heuer et al.36 did not mention the use of catecholamine therapy in their publication but non-specifically mentioned transfusion of whole blood to improve hemodynamic stability. The latter was not required in this model. Fondevila et al., who reported a mean anhepatic time of less than 20 min, solely relied on the administration of crystalloid solutions and did not apply vasoactive substances during engraftment37.
Of note, as opposed to recent publications applying end-to-end anastomosis from the donor to the recipient hepatic artery19,22, this model includes an end-to-side anastomosis with a Carrel patch of the donor aorta being anastomosed to the supraceliac aorta of the recipient. Especially for experimental settings, with the use of organs fulfilling extended donor criteria, e.g., prolonged cold ischemic time, it might be favorable to rule out any problems with the arterial perfusion of the graft. The application of a Carrel patch will help to avoid stenosis of the arterial anastomosis that might become functionally relevant in the case of concomitant peripheral vasospasms frequently observed following reperfusion. Nevertheless, this approach will be more time-consuming than the conventional end-to-end anastomosis due to the more elaborated access of the aorta.
As our representative experiments focused on the immediate postoperative phase and ischemia-reperfusion injury, recipients were kept under anesthesia and were euthanized 6 h after reperfusion. Although beneficial with respect to animal welfare, this constitutes a significant limitation to the validation of our technique regarding graft and recipient survival. We believe, however, that on the basis of the vital parameters, hemodynamics, lactate clearance, bile production, and especially the real-time liver-specific CYP1A2 metabolism (LiMAx test) observed throughout the follow-up, the long-term application of our model should be feasible, especially as the grafts used within the experiments underwent static cold storage for 20 h prior engraftment, in contrast to the successful application of comparable transplant models by others35,36,37. Furthermore, the mentioned previous reports demonstrated that perioperative mortality was exclusively observed during and up to 6 h after surgery, except for one recipient dying from a pulmonary embolism on the first postoperative day in the study by Oike et al.35.
In this work, we demonstrate that a simplified approach to porcine orthotopic liver transplantation without the use of a veno-venous bypass during engraftment can be performed safely and is more cost-effective, without significant hemodynamic alterations or intraoperative mortality even after prolonged static cold storage of the donor organ. Such a model should be of special interest for (surgical) workgroups focusing on the immediate postoperative course, ischemia-reperfusion injury, the reconditioning of extended criteria donor organs, and associated immunological mechanisms.
The authors have nothing to disclose.
The authors thank Britta Trautewig, Corinna Löbbert, Astrid Dinkel, and Ingrid Meder for their diligence and commitment. Furthermore, the authors thank Tom Figiel for producing the picture material.
Abdominal retractor | No Company Name available | No Catalog Number available | |
Aortic clamp, straight | Firma Martin | No Catalog Number available | |
Arterial Blood Sampler Aspirator (safePICOAspirator) 1.5 mL | Radiometer Medical ApS | 956-622 | |
Atropine (Atropinsulfat 0.5 mg/1 mL) | B.Braun | 648037 | |
Backhaus clamp | Bernshausen | BF432 | |
Bipolar forceps, 23 cm | SUTTER | 780222 SG | |
Bowl 5 L, 6 L, 9 L | Chiru-Instrumente | 35-114327 | |
Braunol Braunoderm | B.Braun | 3881059 | |
Bulldog clamp | Aesculap | No Catalog Number available | |
Button canula | Krauth + Timmermann GmbH | 1464LL1B | |
Calcium gluconate (2.25 mmol/10 mL (10%)) | B.Braun | 2353745 | |
Cell Saver (Autotransfusion Reservoir) | Fresenius Kabi AG | 9108471 | |
Central venous catheter 7Fr., 3 Lumina, 30 cm 0.81 mm | Arrow | AD-24703 | |
Clamp | INOX | B-17845 / BH110 / B-481 | |
Clamp | Aesculap | AN909R | |
Clamp, 260 mm | Fehling Instruments GMbH &Co.KG | ZAU-2 | |
Clip Forceps, medium | Ethicon | LC207 | |
Clip forceps, small | Ethicon | LC107 | |
CPDA-1 solution | Fresenius Kabi AG | 41SD09AA00 | |
Custodiol (Histidin-Tryptophan-Ketogluterat-Solution) | Dr.Franz Köhler Chemie GmbH | 2125921 | |
Dissecting scissors | LAWTON 05-0641 | No Catalog Number available | |
Dissecting scissors, 180 mm | Metzenbaum | BC606R | |
Endotracheal tube 8.0 mm | Covetrus | 800764 | |
Epinephrine (Adrenalin 1:1000) | InfectoPharm | 9508734 | |
Falcon Tubes 50ml | Greiner | 227 261 L | |
Femoralis clamp | Ulrich | No Catalog Number available | |
Fentanyl 0.1mg | PanPharma | 00483 | |
Forceps, anatomical | Martin | 12-100-20 | |
Forceps, anatomical, 250 mm | Aesculap | BD052R | |
Forceps, anatomical, 250 mm | Aesculap | BD032R | |
Forceps, anatomical, 250 mm | Aesculap | BD240R | |
Forceps, surgical | Bernshausen | BD 671 | |
Forceps, surgical | INOX | B-1357 | |
G40 solution | Serag Wiessner | 10755AAF | |
Gelafundin ISO solution 40 mg/mL | B. Braun | 210257641 | |
Guidewire with marker | Arrow | 14F21E0236 | |
Haemostatic gauze ("Tabotamp" 5 x 7.5 cm) | Ethicon | 474273 | |
Heparin sodium 25,000IE | Ratiopharm | W08208A | |
Hico-Aquatherm 60 | Hospitalwerk | No Catalog Number available | |
Infusion Set Intrafix | B.Braun | 4062981 L | |
Intrafix SafeSet 180 cm | B.Braun | 4063000 | |
Introcan Safety, 18 G | B.Braun | 4251679-01 | |
Isofluran CP | CP-Pharma | No Catalog Number available | |
Large-bore venous catheter, 7Fr. | Edwards Lifesciences | I301F7 | |
Ligaclip, medium | Ethicon | LT200 | |
Ligaclip, small | Ethicon | LT100 | |
Material scissors | Martin | 11-285-23 | |
Methylprednisolone (Urbason solubile forte 250 mg) | Sanofi | 7823704 | |
Monopolar ERBE ICC 300 | Fa. Erbe | No Catalog Number available | |
NaCl solution (0.9%) | Baxter | 1533 | |
Needle holder | Aesculap | BM36 | |
Needle holder | Aesculap | BM035R | |
Needle holder | Aesculap | BM 67 | |
Neutral electrode | Erbe Elektromedizin GmbH Tübingen | 21191 – 060 | |
Norepinephrine (Sinora) | Sintetica GmbH | 04150124745717 | |
Omniflush Sterile Filed 10 mL | B.Braun | 3133335 | |
Original Perfusorline 300 cm | B.Braun | 21E26E8SM3 | |
Overhold clamp | INOX | BH 959 | |
Overhold clamp | Ulrich | CL 2911 | |
Pentobarbital sodium(Release 500 mg/mL) | WDT, Garbsen | 21217 | |
Perfusers | B.Braun | 49-020-031 | |
Perfusor Syringe 50 mL | B.Braun | 8728810F | |
Petri dishes 92 x 17 mm | Nunc | 150350 | |
Poole Suction Instrument Argyle flexibel | Covidien, Mansfield USA | 20C150FHX | |
Potassium chloride (7.45%) | B.Braun | 4030539078276 | |
Pressure measurement set | Codan pvb Medical GmbH | 957179 | |
Propofol (1%) | CP-Pharma | No Catalog Number available | |
S-Monovette 2.6 mL K3E | Sarstedt | 04.1901 | |
S-Monovette 2.9 mL 9NC | Sarstedt | 04.1902 | |
S-Monovette 7.5 mL Z-Gel | Sarstedt | 11602 | |
Sartinski clamp | Aesculap | No Catalog Number available | |
Scalpel No.11 | Feather Safety Razor Co.LTD | 02.001.40.011 | |
Scissors | INOX | BC 746 | |
Seldinger Arterial catheter | Arrow | SAC-00520 | |
Sodium bicarbonate (8.4%) | B.Braun | 212768082 | |
Sterilization Set ("ProSet Preparation Kit CVC") | B.Braun | 4899719 | |
Sterofundin ISO solution | B.Braun | No Catalog Number available | |
Suction | Dahlhausen | 07.068.25.301 | |
Suction Aesculap Securat 80 | Aesculap | No Catalog Number available | |
Suction catheter | ConvaTec | 5365049 | |
Sultamicillin (Unacid: 2000 mg Ampicillin/1000 mg Sulbactam) | Pfizer | DL253102 | |
Suprapubic urinary catheter, "bronchialis", 50 cm | ConvaTec | UK 1F02772 | |
Suprasorb ("Toptex lite RK") | Lohmann & Rauscher | 31654 | |
Suture Vicryl 3-0 | Ethicon | VCP 1218 H | |
Suture Vicryl 4-0 | Ethicon | V392H | |
Suture, Prolene 4-0 | Ethicon | 7588 H | |
Suture, Prolene 5-0, double armed | Ethicon | 8890 H | |
Suture, Prolene 5-0, single armed | Ethicon | 8720 H | |
Suture, Prolene 6-0, double armed | Ethicon | 7230 H | |
Suture, Prolene 6-0, single armed | Ethicon | EH 7406 H | |
Suture, Prolene: blau 3-0 | Ethicon | EH 7499H | |
Suture, Safil 2/0 | Aesculap | C 1038446 | |
Suture, Terylene 0 | Serag Wiessner | 353784 | |
Syringe 2 mL, 5 mL, 10 mL, 20 mL | B.Braun | 4606027V | |
TransferSet "1D/X-double" steril 330 cm | Fresenius Kabi AG | 2877101 | |
Ultrasound Butterfly IQ+ | Butterfly Network Inc. | 850-20014 | |
Ventilator "Oxylog Dräger Fl" | Dräger Medical AG | No Catalog Number available | |
Yankauer Suction | Medline | RA19GMD | |
Zoletil 100 mg/mL (50 mg Zolazepam, 50 mg tiletamin) | Virbac | 794-861794861 |