Fixed Volume-Intercept Approach for Serial Contractility Assessment with Closed-Chest Pressure-Volume Loops in a Porcine Model

Summary

This protocol aims to provide a feasible, closed-chest porcine protocol to serially quantify load-independent LV contractility by fixing V₀ and computing single-beat Ees, eliminating repeated IVC occlusions to enhance safety and stability in hemodynamically fragile, acute experiments.

Abstract

The end-systolic pressure–volume relationship (ESPVR) is widely regarded as the gold standard, load-independent descriptor of left-ventricular (LV) contractility. Practically, ESPVR is often approximated as a straight line defined by its slope - end-systolic elastance (Ees), a direct index of contractile state - and its volume-axis intercept (V₀), the theoretical LV volume at zero pressure. In vivo, ESPVR is classically derived from serial inferior vena cava (IVC) occlusions performed with simultaneous LV pressure–volume (PV) catheter monitoring. However, in disease states such as cardiogenic shock, the requisite preload depletion may provoke reflex tachyarrhythmias and transient or sustained hemodynamic instability, limiting feasibility and interpretability. Thus, methods for in vivo assessment of inotropy during hemodynamically fragile states are warranted.

This protocol aims to describe a practical experimental model in which V₀ is determined once under stable baseline conditions and then held fixed for subsequent, single-beat Ees estimation from LV PV loops. This approach, detailed step-by-step for use in acute large-animal studies, allows continuous serial assessment of contractility without repeated IVC occlusions. In a porcine cardiogenic shock model, it reliably tracked load-independent changes in myocardial performance while avoiding arrhythmias and hemodynamic instability otherwise provoked by preload reduction. The method is therefore well suited for acute, closed-chest experiments in which the ventricular geometry remains unchanged, and contractility primarily modulates the ESPVR slope rather than its intercept. Nonetheless, the fixed-V₀ assumption represents a methodological simplification that introduces deliberate systematic error and should only be applied when repeated preload manipulation is impractical or unsafe. When structural remodeling, profound LV dilation, or marked thoracic pressure changes occur, V₀ must be re-estimated. Overall, this protocol provides a feasible and physiologically sound approach for serial contractility assessment in unstable large-animal models, balancing methodological rigor with experimental safety and practicality.

Introduction

Left ventricular (LV) contractility is the intrinsic ability of myocardium to generate pressure independent of loading conditions^1,2,3. It is a critical determinant of cardiac stroke volume, cardiac output, and blood pressure. Hence, when suffering from acute myocardial ischemia, the heart may quickly lose contractility⁴. Thus, impaired myocardial contractility may cause acute heart failure and cardiogenic shock (CS)⁵. CS results in hypotension and inadequate organ perfusion, leading to high mortality (30%–50% in-hospital) despite aggressive therapy^5,6. Common medical therapies involve inotropic agents such as norepinephrine, dobutamine, and milrinone that increase contractility but may also directly affect the vascular tonus⁷. Assessment of contractility is of particular interest when investigating new pharmacological treatments, when evaluating disease severity, or when working with experimental models.

Traditional metrics like LV ejection fraction or dP/dt(max) are load-dependent and can misrepresent the true contractile state when preload or afterload changes. This underscores the value of load-independent indices of contractility such as end-systolic elastance (Ees), which can more reliably track myocardial performance¹.

In vivo methods for assessing contractility have therefore been developed, with pressure–volume (PV) analysis considered the gold standard in experimental research⁸. The classical in vivo approach to quantify Ees is to perform an inferior vena cava (IVC) occlusion to gradually and transiently reduce preload. By recording concomitant end-systolic pressure (ESP) and end-systolic volume (ESV) points during this preload reduction, a regression line of ESPVR can be fitted, and its slope, Ees, determined, with the x-intercept of that line defined as V₀ - the theoretical volume at zero pressure generation. However, in fragile states, such as CS, even a brief IVC occlusion can cause severe hypotension, arrhythmias, or further compromise end-organ perfusion in an already critically low cardiac output state, limiting feasibility and interpretability.

In the present study protocol, a practical alternative to the traditional IVC occlusion method for use in acute, closed-chest experiments is presented, where a single IVC occlusion is performed under stable baseline conditions to determine the volume-axis intercept V₀. This value is then assumed constant for subsequent single-beat Ees estimations. This approach builds on evidence from isolated and intact animal studies showing that acute inotropic changes primarily alter the ESPVR slope (Ees)while the intercept remains largely stable^2,9,10. By avoiding repeated occlusions, the method minimizes arrhythmias and hemodynamic instability while preserving precise, load-independent assessment of LV contractility. In this protocol, this strategy is applied in a closed-chest porcine model of CS using an admittance-based PV catheter, based on experiences from previous studies^{4,8,11,12,13,14,15,16}. While this fixed-intercept approach is intended for acute, closed-chest experiments with stable ventricular geometry, its applicability is limited in settings with evolving chamber size, altered thoracic pressures, or structural remodeling; these practical constraints should be considered when evaluating the method's suitability for a given study. Unlike prior single-beat or model-based approaches, this protocol determines V₀ empirically under stable baseline conditions and then applies it throughout the experiment, offering a safer and more feasible alternative for serial, load-independent contractility assessment in hemodynamically fragile large-animal models.

Protocol

This protocol is approved by the Danish Animal Research Inspectorate (permit no: 2023–15–0201–01466, issued on 19/06–2023). Female Danish Landrace pigs of approximately 60 kg were used. The reagents and the equipment used are listed in the Table of Materials.

1. Anesthesia and ventilation

1. Pre-anesthetize the pig with intramuscular injection of anesthetic mix 1 mL/kg, containing 25 mg/mL tiletamine, 25 mg/mL zolazepam, 2.5 mL butorphanol (10 mg/mL), 1.25 mL veterinary ketaminol (100 mg/mL), and 6.25 mL veterinary xylazine (20 mg/mL).
   NOTE: Pre-anesthetic administration will reduce stress, nociception, and catecholamine release during transport.
2. Transport the animal from farm facilities to research facilities.
3. Establish intravenous (I.V.) access in a marginal ear vein using an 18-20 G venous catheter (Figure 1). Fixate the I.V. catheter using appropriate tape or plaster material. Flush with 10 mL saline to ensure correct placement.
   NOTE: A subcutaneous bulge may appear following a saline flush if the catheter is not correctly placed. Consider placing an additional back-up I.V. access in the opposite ear in case of displacement.
4. Attach a pulse oximetry sensor for early monitoring of arterial oxygen saturation.
   NOTE: In case of hypoxia with oxygen saturation below 90%, administer 100% oxygen via facemask over the snout of the pig until normoxia is restored. Ensure sufficient oxygen saturation before moving on to step 5.
5. Place the pig in supine position on the operating table. Intubate the pig with a 7.5–8.0 mm internal diameter tracheal tube using direct laryngoscopy. Confirm correct tube placement by observing persistent expiratory carbon dioxide on the ventilator. Inflate the cuff on the endotracheal tube and secure the tube to the snout.
6. Adjust the ventilator settings and begin positive-pressure ventilation using the following settings:
   1. Pressure-controlled, volume-gated ventilation setting.
   2. Tidal volume 8 mL/kg.
   3. Positive end-expiratory pressure of 5 cmH₂O.
   4. Titrate inspired fraction of oxygen to normoxia, e.g., arterial oxygen saturation >94%.
   5. Titrate respiratory rate to normocapnia, e.g., arterial CO₂ partial pressure 4.5–5.5 kPa.
7. Initiate and maintain general anesthesia through the I.V. access using propofol at 3.5–4.5 mg/kg/h and fentanyl at 12.5–15.5 µg/kg/h. Assess anesthetic depth by the absence of corneal and ciliary reflexes and lack of response to noxious stimulation. CAUTION: Do not leave animals unattended at any point. Ensure that all experiment staff have the correct education and can perform correct monitoring of anesthetic depth.
8. Attach a 3-lead ECG (right forelimb, left forelimb, left hindlimb).
9. Place a urinary catheter in the urinary bladder and connect the external end to a sampling bag.
10. Monitor core temperature using a rectal probe.
    ​NOTE: The normal body core temperature in an adult pig is 38.5–39.5 °C¹⁷. Hypothermia should be corrected using active warming devices, e.g., using heated blankets to prevent hypothermia-induced modification of hemodynamics or arrhythmias.

2. Ultrasound-guided intravascular access

1. Establish the following intravascular accesses using ultrasound guidance and the Seldinger technique. These accesses will allow for a single beat Ees assessment.
   1. Insert an 8 Fr sheath in a common carotid artery.
      NOTE: This sheath will be used to introduce the LV PV catheter.
   2. Insert a 12 Fr sheath in a femoral vein.
      NOTE: This sheath will be used to introduce the IVC occlusion balloon.
2. Establish the following intravascular accesses using ultrasound guidance and the Seldinger technique. These accesses will allow for hemodynamic monitoring.
   1. Insert a 7 Fr sheath in a femoral artery.
      NOTE: This sheath will be used for invasive blood pressure monitoring. Invasive arterial blood pressure may also be measured elsewhere, such as a limb artery.
   2. Insert an 8 Fr sheath in an external jugular vein.
      ​NOTE: This sheath will be used for the insertion of a pulmonary artery catheter and for the assessment of pulmonary artery pressures.
3. Establish intravascular accesses needed for the desired disease model. In this case, the following intravascular access is established.
   1. Insert an 8 Fr sheath in the opposite common carotid artery as in step 1.1. This sheath will be used for coronary catheterization and embolization, which will induce CS.
      NOTE: Further accesses can be established according to the study protocol.
4. To ensure correct placement of intravascular sheaths, draw blood from each sheath using a 10 mL syringe. A correctly placed sheath will have no resistance when blood is aspirated nor when flushed with saline.
5. Suture all sheaths to the skin and connect all sheaths to relevant monitoring systems.
   NOTE: Sheaths should be frequently flushed to avoid blood clotting inside the sheaths. Alternatively, depending on the study protocol, pigs may be heparinized.
6. Following pressure-calibration according to the manufacturer’s instructions, place the LV PV catheter as previously described⁸ (Figure 1).

3. Left ventricular pressure-volume catheter and inferior vena cava balloon placement

1. Before insertion of the PV catheter, perform blood resistivity calibration for each animal.
   1. Draw blood into a 0.5 mL cryogenic vial. Perform a blood resistivity measure using the calibration probe immediately upon blood collection.
2. Insert LV PV catheter through an 8 Fr carotid sheath. During low-dose fluoroscopic guidance, follow the pigtail of the LV PV catheter to the aortic valve. Advance through the aortic valve during systole.
   NOTE: There should be no resistance at any point during advancement of the LV PV catheter.
3. Place the tip of the LV PV catheter as close to the LV apex as possible.
4. Follow the protocol from the manufacturer to choose the relevant number of recording volume segments to optimize the LV PV catheter positioning based on the recorded phase and magnitude signals.
   NOTE: The catheter's location is very important for data collection. The location can be confirmed using fluoroscopic guidance. However, confirmation of correct positioning may also be done using the phase values. The signal should have a mean value <10° and a periodic shape. During PV catheter placement, the correct segment should be selected. In the initial configuration (segment 1), electrodes 1 through 4 are active. In this position, the measured volume is often underestimated, typically because the electrodes lie too far from the aortic valve. In segment 2, electrodes 1, 2, 4, and 5 are active; depending on ventricular dimensions, this configuration can still produce low-volume signals. When advancing to segment 3, electrodes 1, 2, 5, and 6 are used, which, in adult pigs, generally yield physiologic volumes and appropriately shaped PV loops. Further advancement to segment 4 (electrodes 1, 2, 6, and 7) may extend the sensing field beyond the ventricular length, producing inaccurate signals in smaller hearts.
5. Insert the IVC balloon through the 12 Fr femoral vein sheath and advance the balloon to the diaphragm level guided by fluoroscopy.
   ​NOTE: The experiment can be paused at this level to stabilize hemodynamics before the research protocol begins. Abnormal hemodynamic, ventilatory, and electrolyte values may be corrected at this step. A stabilization period of 1–2 h is often sufficient^13,14,15,16.

4. Baseline measurements and determination of V₀

1. In a healthy baseline, following instrumentation as described, perform calibration of all equipment, including the LV PV catheter as described by the manufacturer, and as thoroughly described elsewhere⁸.
   ​NOTE: Both pressure and volume should be calibrated depending on whether the catheter uses admittance- or conductance-based technologies. Pressure calibration should be performed prior to insertion of the catheter. In brief, calibration involves the following steps:
   1. Perform pressure calibration prior to in vivo insertion of the LV PV catheter.
   2. Ensure stable sinus rhythm.
   3. When using admittance technology-based catheters, use stroke volume derived from the pulmonary artery catheter for volume calibration.
      NOTE: Baseline volume-calibration scan should be performed during transient end-expiratory apnea.
2. Perform a transient end-expiratory breath hold for the duration of 10–15 cardiac cycles for obtaining baseline values.
   ​NOTE: Following each end-expiratory breath hold, let the animal recover until normalization of hemodynamic values such as heart rate and blood pressure, as well as respiratory values such as arterial oxygen saturation and end-expiratory CO₂.
3. For determination of V₀, perform the following steps:
   1. Do another end-expiratory breath-hold and wait a few heartbeats before slowly inflating the IVC balloon until the LV pressure is below 50 mmHg. Deflate the balloon quickly and resume ventilation.
      NOTE: This procedure will progressively decrease the LV preload, and PV loops must demonstrate progressive leftward and downward shift during preload reduction^1,2,3 (Figure 2A and Figure 3A).
   2. Allow animals to recover, following each IVC occlusion and breath-hold until normoxia, normocapnia and normotension is restored. This usually takes 2–3 min. Perform an additional two rounds of IVC occlusion. Ensure stable sinus rhythm.
      ​NOTE: Occasionally, the inflation of the IVC balloon to decrease preload may induce arrhythmias or ventricular extrasystoles, which may disturb blood flow and thus create difficulties in data analysis. In this case, perform a slower inflation of the IVC balloon. Three independent occlusions without arrhythmias should be performed.
   3. Following the three occlusions, no further IVC occlusions are necessary. The IVC balloon catheter may be removed.
4. In LabChart 8 Pro, open the built-in PV Loop Module. In Analyze, select Occlusion. Manually select sequential loops during preload reduction for analysis. Exclude ectopic beats and adjacent cycles. Apply linear or quadratic fit using the dropdown menu.
   ​NOTE: V₀ may be calculated directly after the baseline measurements, or during post hoc data analysis. For each retained cycle during the preload reduction, ESP and ESV are identified using the module’s standard definitions.
   1. Use the built-in function of the software to analyze the selected cardiac cycles. 10 loops should be analyzed during progressive preload reduction. Screen all heartbeats and manually exclude ectopic beats or artifacts and retain only monotonic, smoothly evolving loops. If preferred, automated data cleaning methods have been described¹⁸.
      NOTE: During IVC occlusion, the LV is prone to arrhythmias. In case of supraventricular or ventricular ectopic beats, these beats should be removed together with the prior and the following heartbeat (Figure 2B). Furthermore, dependent on heart rate, IVC occlusion may trigger baroreceptor reflexes, which can influence inotropy.
   2. For retained heartbeats, manually ensure that the built-in function of the software correctly identifies the end-systolic pressure and volume.
   3. Fit the ESPVR line.
      NOTE: Primarily, a linearly fitted ESPVR line may be used^1,2 (Figure 2A). However, the line of ESPVR may be curvilinear^1,19,20,21 (Figure 3). If the ESPVR is visibly curvilinear, apply an accepted nonlinear fit to the ESPVR, e.g., quadratic regression using the dropdown menus^19,21,22. Analysis software such as LabChart 8 Pro allows for multiple options. When the ESPVR visually follows a straight line, a linear least-squares fit should be applied; when curvilinearity is evident, a quadratic fit may be used. CAUTION: Selection of linear versus quadratic ESPVR fitting should be guided by both objective goodness-of-fit from the built-in module and visual inspection of the ESPVR shape. Predefined decision rules should be applied, e.g., a quadratic model may be preferred if it improves the adjusted R² by ≥0.05 compared with the linear fit (Figure 4)²³; however, visual inspection of loop morphology and residual patterns must always be performed to confirm physiologically plausible curvature. Small numerical improvements in R² without consistent visual evidence of curvilinearity should not justify the use of nonlinear fitting.
   4. Compute the V₀ from the fitted ESPVR line, linear or curvilinear, as the intercept with the X-axis.
      NOTE: Model form and goodness-of-fit should be documented. When significant convexity of the ESPVR is observed, linear fits can underestimate true elastance at higher pressures and overestimate it at lower pressures (Figure 4). In these cases, quadratic fitting is recommended for V₀ determination as described in step 4.3. Although single-beat Ees estimation is necessarily linear, awareness of ESPVR curvature is essential, as nonlinear behavior may influence serial measurements, particularly during large afterload changes.
   5. Repeat the approach across the three independent occlusions and take the mean of the three V₀ measurements.
      NOTE: Negative V₀ does not imply a physically meaningful negative ventricular volume; rather, it reflects the limitations of the linear ESPVR model and the underlying nonlinearity of ventricular mechanics, especially at low volumes and pressures. The occurrence of negative V₀ is more frequent in ventricles with high contractility and especially when linear fits are applied over higher-load ranges.
5. Calculate Ees as the slope of the line between the V₀ and the mean end-systolic pressure and volume point from step 2.
   ​NOTE: At this point, baseline measures have been performed, and the relevant disease model may now be applied. The experiment can be paused at this level to ensure normal hemodynamics, ventilatory parameters, and electrolytes.

5. Induction of ischemic cardiogenic shock

NOTE: This protocol describes how Ees may be estimated without additional IVC occlusions in a model of ischemic CS, as described^{4,11,12,13,14}. A detailed protocol for this disease model is beyond the scope of this protocol and may be found elsewhere²⁴. In brief, CS is induced as follows:

1. Produce a stock suspension of embolization particles by mixing 0.125 g microspheres with 5 mL iodinated contrast and 5 mL 0.9% saline, yielding a total volume of 10 mL. Gently agitate the suspension before and during use, and inject in 1 mL aliquots through the coronary guide catheter (Figure 5).
2. Place a coronary guide catheter (5–7 Fr, e.g., JL 3.5) through an arterial sheath in a carotid artery (see step 2) into the ostium of the left coronary artery.
3. Induce myocardial ischemia and CS by stepwise intracoronary microembolization of the left main coronary artery using commercial polyvinyl alcohol embolization particles. After each injection, allow hemodynamics to stabilize for 2–5 min, and repeat injections until either thermodilution cardiac output or mixed venous oxygen saturation is decreased by 30% relative to baseline (Figure 6).
   ​NOTE: Successful induction of CS is confirmed by narrowing of PV loops and rightward shift of end-systolic volume, reflecting an abrupt decrease of contractility^4,9.

6. Serial single-beat determination of Ees throughout the study

NOTE: PV data may be analyzed using LabChart 8 Pro or another similar software that includes a dedicated PV-loop analysis module. All recordings should be visually inspected to ensure stable sinus rhythm and the absence of signal drift. Individual cardiac cycles are automatically segmented by the software and should be subsequently reviewed manually. Ventricular extrasystoles, as well as the beat immediately preceding and the beat immediately following the ectopic cycle, should be excluded, as the loading conditions are transiently altered across all three beats.

1. After defining a subject-specific V₀ at baseline (see prior section), perform single-beat estimation of Ees without additional IVC occlusions.
2. At each timepoint, perform three consecutive end-expiratory breath holds for 10–20 cardiac cycles while recording PV loops. For each time point, based on the breath-hold periods, the mean ESP and ESV are defined.
   NOTE: Ensure a stable sinus rhythm during the transient apnea periods. Each individual loop morphology should be carefully inspected. Loops should be included only if they represent a physiologically plausible ventricular cycle with stable loading conditions and realistic ventricular pressure generation. Ectopic beats, beats immediately preceding and following ectopy, should not be included in the analysis (Figure 2). For detailed protocols on beat selection, please see published methodological guidance^8,18.
   CAUTION: A high PEEP may influence venous return and loading conditions¹². Ensure that loops are falling on top of each other, indicating equal loading conditions. Although approximately 10 loops are generally sufficient for ESPVR analysis, a longer IVC-occlusion recording may occasionally be required to confirm that no further leftward shift of the end-systolic points occurs while pressure continues to decrease (Figure 2, Figure 3, and Figure 4).
3. Calculate Ees for each time point using the fixed V₀ and mean ESP and ESV values from the breath hold PV loops² 
   NOTE: For the rest of the protocol, V₀ is held constant while ESP and ESV may vary between timepoints according to study protocol and interventions. Thus, no effect of loading or contractile state can influence V₀, only Ees.

Results

The instructions above present a protocol to derive Ees on a single-beat basis, keeping the theoretical volume of zero pressure generation, V₀, constant. This section presents data from two studies conducted in our lab. First, a separate pilot study was conducted to examine the development of V₀ and Ees following CS induction. Second, in a large dataset of 30 pigs included in previously published studies, the ischemic model of CS is presented⁴.

Discussion

This protocol describes a pragmatic, reproducible, and feasible strategy for serial, load-independent assessment of LV contractility, viable even in acute cardiac illness such as ischemic CS. The protocol uses a fixed volume-axis intercept, V₀, to estimate Ees from single beats, avoiding repetitive IVC occlusions and thus lowering the risk of arrhythmias and hypotension during vulnerable cardiovascular conditions. The methodological novelty of the present protocol lies in the explicit combination of empiricall...

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
0.9% saline	Fresenius Kabi, Germany	921046	Microsphere suspension and flush
10 mL syringes	BD, USA	BD Plastipak 302143	Microsphere injection
12 Fr femoral venous sheath	Terumo, Japan	Radifocus Introducer II	For IVC occlusion balloon insertion
20 G venous catheter	BD, USA	BD Insyte 381223	Ear vein IV access
7 Fr femoral arterial sheath	Terumo, Japan	Radifocus Introducer II	Invasive arterial pressure monitoring
8 Fr carotid arterial sheath	Terumo, Japan	Radifocus Introducer II	Access for LV PV catheter
8 Fr jugular venous sheath	Terumo, Japan	Radifocus Introducer II	Pulmonary artery catheter access
Contour PVA microspheres	Boston Scientific, USA	Contour 355–500 µm	Induction of ischemic cardiogenic shock
Coronary guide catheter	Boston Scientific, USA	JL 3.5 / JR 4.0	Coronary access for microsphere injection
ECG electrodes (3-lead)	Ambu, Denmark	BlueSensor P	Continuous ECG monitoring
Endotracheal tube 7.5–8.0 mm	Smiths Medical, UK	Portex ETT	Tracheal intubation
Fentanyl (IV)	Hameln Pharma, Germany	Fentanyl Hameln	Continuous IV analgesia
Fluoroscopy system	-	-	Catheter guidance
Heated blankets	3M, USA	Bair Hugger Warming Unit	Maintenance of normothermia
Heparin	Leo Pharma, Denmark	Innohep	Anticoagulation if used
Iomeron contrast agent	Bracco Imaging, Italy	Iomeron 350	Coronary angiography and microsphere suspension
IVC occlusion balloon catheter	Fogarty / Edwards Lifesciences, USA	Fogarty Occlusion Balloon	Temporary preload reduction
LabChart 8 Pro software	ADInstruments, New Zealand	LabChart 8 Pro	PV loop acquisition and analysis
LV PV catheter (admittance)	Millar Inc., USA	MPVS Ultra PV Loop Catheter	Admittance-based LV pressure–volume measurement
Mechanical ventilator	GE Healthcare, USA	Datex Ohmeda S/5	Positive pressure ventilation
MPVS Module	ADInstruments, New Zealand	MPVS Ultra/Duo	Interface for PV catheter
PowerLab data acquisition system	ADInstruments, New Zealand	PowerLab 16/35	Signal acquisition
Propofol (IV)	Fresenius Kabi, Germany	Diprivan / Propofol-Lipuro	Continuous IV anesthesia
Pulmonary artery catheter 7.5 Fr	Edwards Lifesciences, USA	Swan-Ganz 131HF7	Thermodilution CO and PA pressure
Pulse oximeter	-	-	Peripheral oxygen saturation monitoring
Rectal temperature probe	-	-	Core temperature monitoring
Seldinger guidewires	Terumo, Japan	Radifocus Guide Wire M	Vascular access
Sterile saline flushes	Fresenius Kabi, Germany	0.9% NaCl	Sheath flushing
Suture material	Ethicon, USA	Vicryl 3-0	Fixation of sheaths
Syringe pumps	Becton Dickinson (BD), USA	BD Alaris	Continuous anesthetic infusion
Ultrasound system	-	-	Vascular access guidance
Urinary catheter	Teleflex, USA	Rüsch Foley catheter	Bladder drainage
Zoletil (tiletamine/zolazepam)	Virbac, France	Zoletil 100	Used for pre-anesthesia; mixed with butorphanol, ketamine, xylazine according to protocol