Comprehensive Echocardiographic Protocol for Pigs with Emphasis on Diastolic Function: Advantages over MRI Assessment

Abstract

Swine are increasingly utilized in cardiovascular research due to their anatomical and physiological similarities to humans, particularly for studying diastolic dysfunction. While MRI offers excellent structural imaging, echocardiography provides superior real-time assessment of diastolic parameters. To address the lack of standardized methods and reduce variability across studies, we present a comprehensive guide for performing echocardiography in Yorkshire pigs, detailing anatomical considerations, equipment requirements, and technical approaches.

We describe systematic approaches for obtaining and optimizing right parasternal long and short-axis views, apical four-chamber, and subcostal imaging windows, with specific attention to anatomical variations from human cardiac orientation and standard clinical transducer positioning. These tomographic views enable comprehensive assessment of systolic and diastolic function, including ventricular volumes, wall thicknesses, chamber dimensions, ejection fraction, and Doppler measurements of blood flow and tissue velocities.

This standardized methodology for echocardiographic images acquisition enhances data reliability in cardiovascular pig models, improving the interpretation of preclinical study results and strengthening translational research outcomes. The protocol also provides consistency for veterinary applications, making echocardiography a preferred modality for longitudinal studies in this valuable translational model.

Graphical Abstract

NEW AND NOTEWORTHY

Anatomical Positioning

• Porcine heart is more central and caudal than human heart.

• Apical views obtained at 6th-7th intercostal space.

• Right parasternal views are most reliable in pigs.

Technical Requirements

• Standardized sedation protocol is essential.

• Specialized veterinary procedure table.

• Integrated ECG monitoring for timing.

Clinical Value

• Excellent translational model for cardiovascular research.

• Regular protocol adjustment based on animal size.

INTRODUCTION

Echocardiography has emerged as a valuable non-invasive tool for assessing cardiac structure and function in humans and various animal models, including pigs¹. Pigs have been increasingly used in cardiovascular research due to their anatomical and physiological similarities to humans, making them an ideal model for translational studies². ³. As cardiovascular research and the wide-spread use of echocardiography for non-invasive assessment of cardiac structure and function in pigs continues to expand^2,4, it is crucial to establish standardized echocardiographic techniques to ensure accurate and reproducible results.

Heart Failure with Preserved Ejection Fraction (HFpEF) has become a major cardiovascular challenge. The condition disproportionately affects women, who represent 62.5% of HFpEF hospitalizations, with Black females experiencing a concerning 8.2% annual increase in cases. HFpEF lacks effective therapeutic options, standard heart failure medications become less effective as ejection fraction increases. Its growing prevalence and limited treatment options make it an urgent public health concern, particularly for older adults and women. Echocardiography serves as the cornerstone for diagnosing and monitoring HFpEF through standardized assessment of diastolic function. Following established guidelines and protocols ensures accurate evaluation of left ventricular filling pressures, tissue velocities, and structural parameters. This systematic approach enables reliable diagnosis, appropriate classification of severity, and evidence-based clinical decision-making^5,6.

Pigs and humans share many similarities in terms of cardiac anatomy and physiology, including heart size relative to body weight, metabolic rate, coronary artery distribution, and electrophysiological properties^7,8. Pigs have become increasingly valuable as translational research models due to their significant similarities with humans in cardiac anatomy and physiology. These similarities include heart size relative to body weight, metabolic rate, coronary artery distribution, and electrophysiological properties^7,8. Their manageable size, consistent growth patterns, and ease of handling make them particularly suitable for research settings³. Pigs offer significant advantages over traditional rodent models, which often fail to produce relevant phenotypes for certain conditions^9,10. Recent advances in gene editing techniques have greatly improved the efficiency of creating specific pig models for research. Additionally, pigs present fewer ethical concerns than nonhuman primates and face fewer regulatory restrictions than canine models⁹. As previous limitations in swine-specific expertise, reagents, and facilities continue to diminish, pig models have become more attractive and feasible for translational studies^9,10. These combined advantages have established pigs as a preferred large animal model that effectively bridges the gap between basic research and human applications ^11–15.

However, there are notable differences that should be considered when performing echocardiography in pigs. Pigs have a more cylindrical heart shape compared to the conical shape in humans, and their chest wall is thicker, which can affect image quality^7,16. The porcine left atrium received only 2 pulmonary veins, whereas 4 orifices were generally observed in man^8,16,17. The sweep between the inlet and outlet components of the porcine right ventricle is less marked than in man, and a prominent muscular moderator band is situated in a much higher position within the porcine right ventricle compared with that of man^8,16,17. The apical components of both porcine ventricles possessed very coarse trabeculations, much broader than those observed in the human ventricles. In general, aortic-mitral fibrous continuity is reduced in the outlet component of the porcine left ventricle, with approximately two-thirds of the aortic valve being supported by left ventricular musculature^8,16,17.

The position of the heart within the thoracic cavity differs between pigs and humans, necessitating adaptations to standard echocardiographic views^16,17 (Figure 1). In humans, the heart is situated in the mediastinum, with approximately two-thirds of its mass to the left of the midline ^7,17 and lies obliquely with its long-axis angled from the right shoulder to the left hip¹⁸. In contrast, the porcine heart is located more centrally within the thorax and has a perpendicular orientation regarding its body axis (Figure 1)^7,8. The pig heart is also situated more cranially relative to the lungs compared to the human heart^7,16. These differences in cardiac position and orientation necessitate modifications to the standard echocardiographic views used in humans to optimize image quality and consistency in pigs.

Figure 1. Comparison of Cardiac Axis Orientation in Humans and Pigs.

A) Representative frontal chest X-ray images of a human and a normal porcine thorax, with the cardiac silhouette (highlighted in pink) indicating the heart’s position in both species. The images on the right show 3D anatomical representations of the respective hearts and thoracic vasculature. Created in BioRender. Shehadeh, L. (2025) https://BioRender.com/r88n361. Swine X-rays were adapted from Gromann, L.B., De Marco, F., Willer, K. et al. In-vivo X-ray Dark-Field Chest Radiography of a Pig. Sci Rep 7, 4807 (2017). https://doi.org/10.1038/s41598-017-05101-w.

B) Representative lateral chest X-ray images of a human and a porcine thorax, demonstrating the more vertical orientation of the cardiac long axis in swine compared to humans. The cardiac silhouette is highlighted in pink. The images on the right display the corresponding thoracic anatomy of the hearts in 3D. Created in BioRender. Shehadeh, L. (2025) https://BioRender.com/k27z716.

C) Schematic illustrations showing the cardiac long-axis orientation (black arrows) in humans and swine, alongside their respective 3D anatomical representations. These highlight the key anatomical differences in cardiac positioning between species. Illustrations were created in BioRender. Shehadeh, L. (2025) https://BioRender.com/q38b400, and three-dimensional representations were created using licensed Biosphera Software (Biosphera S.L., Barcelona, Spain). All generated images have undergone expert veterinary review to ensure anatomical accuracy and proper procedural representation.

Noteworthy, the parasternal long-axis view, which is commonly used in human echocardiography, requires slight adjustments in probe placement and angle to account for the more central and caudal position of the pig heart^7,16. In human echocardiography, the apical views are typically obtained by placing the transducer at the point of maximal cardiac impulse, which is usually located in the 5th intercostal space, slightly medial to the left midclavicular line ¹⁸. This allows for visualization of the heart from the apex to the base, which permits assessment of ventricular structure (i.e. volumes, chamber dimensions, and valvular sufficiency) and function (mitral inflow velocities, tissue doppler and ejection fraction). However, in pigs, the heart is situated more caudally within the thorax compared to humans^8,16. As a result, the point of maximal cardiac impulse in pigs is typically located in a more caudal intercostal space, usually around the 6th or 7th intercostal space⁷. This difference in cardiac position requires the sonographer to adjust the transducer placement accordingly to obtain the apical views in pigs. By placing the transducer in a more caudal intercostal space, the sonographer can better align the ultrasound beam with the long-axis of the porcine heart, allowing for clearer visualization of the cardiac chambers and valves from the apical perspective.

Recognizing and adapting to species-specific differences in cardiac anatomy and position is crucial for obtaining accurate and reproducible echocardiographic images in pigs.

Issues of reproducibility in preclinical research have become an increasing ethical and economic concern, with particularly low rates of 11–25% reported in cancer and cardiovascular disease research^19,20. While some discrepancies stem from inadequate study design, such as insufficient blinding and randomization, technical variability due to informal research training and inadequate protocols are a significant yet often overlooked factor²⁰. Echocardiography has been shown to be responsive to standardized training interventions in clinical settings, highlighting the importance of proper technique standardization in research applications.

The purpose of this paper is to provide a comprehensive guide on the proper techniques for performing echocardiographic image acquisition in pigs. We will discuss animal preparation, sedation, equipment selection, and detailed descriptions of standard echocardiographic views obtained in pigs. Furthermore, we will address common technical issues encountered during pig echocardiography and propose solutions based on recent literature. By establishing a standardized approach to echocardiography in Yorkshire pigs, we aim to improve the quality and consistency of cardiovascular research using this animal model.

MATERIAL AND METHODS

Animal Preparation and Sedation

While anesthesia enables reliable data acquisition during echocardiography by ensuring animal sedation and immobility, it is important to recognize that it significantly impacts cardiovascular function ²¹. Yorkshire pigs (females, 8 months old, mean weight of 70 kilograms/154 pounds) underwent transthoracic echocardiography.

Despite the importance of understanding potential differences in cardiac function between male and female pigs, there are relatively few studies that have addressed this issue.

In a retrospective single-center study by Meissner et al.²², the cardio-aortic dimensions and left ventricular function of German Landrace pigs were assessed using cardiac MRI. The study found no significant differences between male and female pigs, except that male had a smaller end-diastolic left ventricular volume and, there was a moderate correlation between body weight and the aortic annulus diameter, suggesting that body size may influence certain cardiac dimensions.

However, given the limited number of studies addressing this issue, more research is warranted to fully understand the potential impact of sex on porcine cardiovascular health and to guide the design and interpretation of preclinical studies using pigs as animal models.

Prior to anesthesia, pigs were fasted for 12 hours. Pre-anesthetic sedation was initiated with an intramuscular injection of ketamine (100 mg/kg). Anesthesia was maintained with face mask supplied with isoflurane anesthesia (1.5%–2.5%; inhaled) supplemented with 100% oxygen.

One of the most common drugs for premedication is ketamine. As an NMDA receptor antagonist, it provides sedation, analgesia, and dissociation from the environment by blocking glutamate neurotransmission. In healthy animals, ketamine has a good analgesic effect and only slightly modifies heart rate ²³. When ketamine is administered alone, the ability of the swallowing reflex is unaffected, avoiding the need for endotracheal intubation. These combined characteristics make it an ideal choice for many procedures. Its use, either alone or in combination with other drugs, allows for safer and more effective management of animals undergoing various medical procedures ²⁴.

Anesthesia can be maintained by administering intravenous anesthetics, inhaled anesthetics, or a combination of both. The choice of technique depends on factors such as the procedure type and duration, and the operator’s preference and experience.

Inhalant anesthetics are the most used agents in research settings involving pigs. Isoflurane is considered the inhalant anesthetic of choice for swine due to its low blood-gas solubility, which allows for faster anesthetic recoveries and the ability to quickly adjust the depth of anesthesia². It is known to greatly affect heart rate, blood pressure, and cardiac contractility, as well as cardiovascular homeostatic control systems. Isoflurane produces a decrease in arterial BP and total peripheral resistance (TPR) and an increase in HR²⁵.

Despite causing dose-dependent cardiovascular depression, isoflurane has the highest margin of safety among the currently used inhalation anesthetics ².

Once sedated, pigs were placed in right lateral recumbency on a procedure table (Figure 2A). The right hemithorax was shaved, and conductive gel was applied to the skin to facilitate echocardiographic imaging.

Figure 2. Yorkshire Pig Positioning for Echocardiography.

A) AI-generated visualization of Yorkshire pig positioning for echocardiography on a specialized examination table for an echocardiogram procedure. The pig is lying on its right side, with its front limbs extended forward and its hind limbs positioned back.

B) 3D anatomical representation of this pig’s position.

C) Placement of electrocardiogram (ECG) monitoring electrodes for synchronized echocardiographic imaging.

Illustrations were created in BioRender. Shehadeh, L. (2025) https://BioRender.com/m27t409. Three-dimensional representations were created using licensed Biosphera Software (Biosphera S.L., Barcelona, Spain). Anatomical and positioning illustrations were created using PromeAI Studio (PromeAI Technologies, Berlin, Germany) under a professional license, with all generated images undergoing expert veterinary review to ensure anatomical accuracy and proper procedural representation.

Echocardiographic Equipment

For the echocardiographic assessments in Yorkshire pigs, we utilized a General Electric Versana Active for veterinary use, version R1, equipped with a General Electric 3Sc-RS probe. The 3Sc-RS is a wideband phased array ultrasound probe specially designed for a broad spectrum of applications, including cardiac, pediatric, cardiology, coronary, fetal heart, adult cephalic, abdominal, and renal diagnostics.

The key specifications of the 3Sc-RS probe are:

• Frequency range: 5–1 MHz

• Scan angle: 120 degrees FOV

• Field of view: Wide

This transducer offers the versatility and performance required for obtaining high-quality echocardiographic images in this pig model. The wide frequency range and scan angle allow for optimal visualization of the porcine cardiac anatomy and assessment of cardiac function. Echocardiographic equipment and transducer selection is crucial for capturing accurate and reproducible data in pigs, ultimately supporting the reliability and translational relevance of the research findings.

Electrocardiogram Monitoring

To enhance the precision and reliability of our echocardiographic evaluations in the Yorkshire pig model, we utilized an integrated electrocardiogram (ECG) monitoring system. This system was used in conjunction with the echocardiography equipment. Specifically, we employed the GE Cable Assy ECG 3 Lead w/Grab AHA, which allowed us to simultaneously record the pigs’ cardiac electrical activity alongside the obtained ultrasound images. This integration proved invaluable, as it enabled us to time the cardiac cycle phases with the corresponding structural changes visualized on the echocardiographic images.

This gating technique helped to minimize variability and improve the reproducibility of our echocardiographic assessments, which is crucial when evaluating subtle changes in cardiac function within the Yorkshire pig model (Figure 2C).

Tomographic Views

Parasternal Long-Axis (PLAX) View

The right PLAX view of the left ventricle is obtained from the right hemithorax, generally from the 3rd-5th intercostal space, caudal to the elbow with the transducer notch directed dorsally, slightly pointing toward the pig’s head. The transducer should be perpendicular to the long-axis of the heart (Figure 3A,B,C).

Figure 3. Transducer Positioning for Parasternal Long-Axis View in Pigs.

(A) AI-generated visualization of Yorkshire pig positioning for the long-axis view. The pig is positioned in right lateral recumbency, with the ultrasound transducer placed over the left hemithorax, just caudal to the sternum (labeled). This alignment directs the ultrasound beam along the long axis of the porcine heart, with key structures labeled: right ventricle (RV), right atrium (RA), left ventricle (LV), and left atrium (LA).

(B) 3D anatomical representation of a pig’s heart showing the orientation of the transducer in relation to the heart.

(C) Anatomical diagram of the heart showing three cross-sectional levels (1–3) corresponding to the ultrasound views:

1. Parasternal long-axis view (PLAX): Displays RV, LV, LA, and aorta (Ao).

2. Left ventricular outflow tract view: Shows LV, LA, and Ao.

3. Right ventricular outflow tract view: Displays RV, pulmonary valve, and pulmonary artery.

Created in BioRender. Shehadeh, L. (2025) https://BioRender.com/i79w930.

Lower panel:

4. M-mode of the aortic root and surrounding structures: The M-mode cursor intersects key cardiac structures. The first reflector is the right ventricular outflow tract (RVOT), followed by the aortic root, where the aortic valve leaflets can be seen. The left atrium is visible distal to the aortic root.

5. M-mode of the mitral valve leaflets: Visualizes and measures the movements of the anterior and posterior mitral valve leaflets.

6. M-mode of the left ventricular walls: Enables measurement of ventricular wall thickness during diastole and systole.

Illustrations were generated using BioRender.com (BioRender, Toronto, Canada) under an academic license, and three-dimensional representations were created using licensed Biosphera Software (Biosphera S.L., Barcelona, Spain). Anatomical and positioning illustrations were created using PromeAI Studio (PromeAI Technologies, Berlin, Germany) under a professional license, with all generated images undergoing expert veterinary review to ensure anatomical accuracy and proper procedural representation.

This view divides the left ventricle in longitudinal fashion and bisects the interventricular septum (Figure 3.1).

The key anatomic structures seen in this view are:

• Right ventricle: located superior to the interventricular septum (IVS). The moderator band can be observed on the right border of the IVS.

• Tricuspid valve

• Right atrium

• Interventricular septum

• Left ventricle: The cavity is delineated by the anterior septal and inferior lateral basal and mid segments (the apical segments may not be visible, and a pseudo-apical region may appear). Inside, we can see the papillary muscles and the mitral sub-valvular apparatus, as well as the left ventricular outflow tract (LVOT), between the anterior mitral leaflet and the IVS.

• Left atrium: Located in the inferior right quadrant, below the aorta, to the right of the mitral valve.

• Mitral valve: With its anterior leaflet positioned as described. The posterior leaflet originates from the atrioventricular junction.

• Aortic Valve (AV): Visualizing the right coronary cusp in the superior position, and the non-coronary cusp inferiorly.

• Ascending Aorta (Ao): The aortic annulus, the sinus portion, the sinotubular junction, and the ascending aorta proper can be seen. The anterior wall of the aorta is continuous with the IVS, and the posterior wall is continuous with the anterior mitral valve leaflet.

A cursor placement at the mitral annulus, with proper rotational alignment of the transducer, allows the length of the left ventricle to be measured from the mitral annulus to the left ventricular apex. The left ventricular length is maximized in this view. If the left atrium is small, the pulmonary veins entering the dorsal aspect of the left atrium may be seen with slight ventral angulation of the transducer.

Right Ventricular Outflow Tract View

The long-axis right ventricular outflow tract (RVOT) view is an important echocardiographic projection that provides valuable information about the structure and function of the porcine right ventricle and its outflow tract (Figure 3.2). To obtain this view, the ultrasound transducer is positioned on the right side of the pig’s chest within the 3rd-5th intercostal space, with the notch of the transducer pointed caudally, towards the pig’s hindlimbs. This orientation of the transducer notch helps to direct the ultrasound beam in the appropriate direction to visualize the RVOT in its longest axis. Importantly, the transducer itself is angled slightly in a cranial and medial direction.

In this view, the key structures that can be visualized include:

1. Right ventricular outflow tract: The RVOT can be clearly seen, allowing assessment of its diameter, flow patterns, and any potential obstructions or abnormalities.

2. Pulmonic valve: The pulmonic valve leaflets and their opening and closing movements can be evaluated to determine valve competence.

3. Main pulmonary artery: The diameter and flow characteristics of the main pulmonary artery can be assessed.

4. Right ventricular free wall: The thickness and contractility of the right ventricular free wall can be evaluated.

This view complements the information gathered from other echocardiographic projections, contributing to a more complete understanding of the porcine cardiovascular system.

Left Ventricular Outflow Tract View

From the right parasternal long-axis view, the transducer is slid about one intercostal space cranially, rotated slightly clockwise, and angled ventrally across the mitral valve to obtain the right parasternal long-axis left ventricular outflow tract view (LVOT) (Figure 3.3).

In this view, the following key structures (described above) should be identified:

• Left atrium

• Mitral valve

• Left ventricular outflow tract and interventricular septum

• Aortic valve

• Proximal ascending aorta (partially visualized)

PLAX Motion modes (M-modes)

The M-mode, or motion-mode, echocardiography is a technique that displays the motion of cardiac structures over time in a one-dimensional format¹⁸ (Figure 3.4–6). By positioning the M-mode cursor perpendicular to the cardiac structures of interest in the PLAX view, the sonographer can obtain these quantitative measurements in a precise and reproducible manner¹⁸. These comprehensive M-mode measurements, when combined with the two-dimensional echocardiographic assessment, contribute to a thorough evaluation of cardiac structure and function in the Yorkshire pig model.

When applied to the PLAX view, the following key M-mode measurements can be obtained:

• 1
  Left ventricular dimensions:

  • Left ventricular end-diastolic diameter (LVEDD).
  • Left ventricular end-systolic diameter (LVESD).

These measurements provide insights into left ventricular size and can be used to calculate derived parameters, such as left ventricular fractional shortening, which is an indicator of systolic function.

• 2
  Left ventricular wall thicknesses:

  • Interventricular septal thickness in systole and diastole (IVSs/IVSd)
  • Left ventricular posterior wall thickness in systole and diastole (LVPWs/LVPWd)

Evaluating the thickness of the left ventricular walls and septum can help identify changes in cardiac remodeling.

M-mode of Ao/LA

Place the cursor so that it transects the closure line of the aortic valve.

At this level, two critical cardiac structures can be evaluated simultaneously (Figure 3.4):

1. The aortic root, which appears as parallel lines displaying systematic motion throughout the cardiac cycle. Its measurement is obtained during end-diastole.

2. The left atrium, visualized as a posterior structure that can be measured from its anterior to posterior walls. Its diameter is typically measured at end-systole when the atrium is at its largest dimension.

M-mode of mitral valve

Along the PLAX view, place the cursor so that it transects the tips of both the anterior and posterior mitral valve leaflets¹⁸. The anterior mitral leaflet demonstrates distinctive characteristics. It exhibits a characteristic “M” pattern during the cardiac cycle and appears more echogenic and thicker compared to its posterior counterpart. Additionally, the anterior mitral leaflet shows a notably greater excursion. In contrast, the posterior mitral leaflet presents as a parallel line situated inferior to the anterior leaflet, creating a mirror image “W” configuration, though with notably less excursion than the anterior leaflet¹⁸. The clinical significance of M-mode evaluation extends to two primary applications: it enables detailed assessment of mitral valve motion and provides valuable information about left ventricular function ¹⁸ (Figure 3.5).

Right Parasternal Short-Axis View

From the right parasternal short-axis view (PSAX), the transducer is rotated 90 degrees clockwise such that the notch is directed toward the left elbow, and the beam is oriented perpendicular to the long-axis of the heart. Ensure that the left ventricle appears as a circular structure with symmetrical wall thickness. Slight ventral or dorsal angulation yields short-axis views of the left ventricle at the following levels (Figure 4.1–4):

Figure 4.

Transducer Positioning for Parasternal Short-Axis View in Pigs

A) AI-generated visualization of Yorkshire pig positioning to obtain the short-axis view of the heart.

B) 3D anatomical representation of a pig’s heart showing the orientation of the transducer in relation to the heart.

C) Anatomical diagram of the heart showing four cross-sectional levels (1–4) corresponding to the sequential short-axis views obtained - (1) Great vessels level, (2) Mitral valve level, (3) Mid-ventricular level, and (4) Apical level.

Illustrations were created in BioRender. Shehadeh, L. (2025) https://BioRender.com/i79w930. Three-dimensional representations were created using licensed Biosphera Software (Biosphera S.L., Barcelona, Spain). Anatomical and positioning illustrations were created using PromeAI Studio (PromeAI Technologies, Berlin, Germany) under a professional license, with all generated images undergoing expert veterinary review to ensure anatomical accuracy and proper procedural representation.

1. Great Vessels

2. Mitral valve:

3. Papillary muscles

4. Apex

The cursor is placed across the left ventricle, perpendicular to the interventricular septum and left ventricular free wall, below the level of the mitral valve leaflets at the tips of the papillary muscles for measurements of left ventricular dimensions and fractional shortening.

Apical Four-Chamber View

In this view, the ultrasound beam is positioned at the apex of the heart, displaying four cardiac chambers simultaneously, creating a comprehensive visualization of cardiac structure and function (Figure 5). The imaging plane reveals these chambers in their anatomical relationship, with the septum vertically oriented in the center of the screen, providing a natural division between right and left heart chambers.

Figure 5. Subcostal Echocardiographic View and Technique in a Yorkshire Pig.

(A) AI-generated visualization for a subcostal echocardiographic technique demonstrating proper transducer placement below the sternum (labeled).

(B) 3D anatomical representation of a pig’s heart, showing the orientation of the transducer in relation to the heart.

(C)

1. Subcostal echocardiographic view displaying all four cardiac chambers (RV, LV, RA, LA) and their position relative to the liver.

2. Alternative apical four-chamber view obtained from the subcostal position after repositioning the pig from its usual right lateral decubitus to left lateral decubitus. While the apical four-chamber view is typically acquired from the right hemithorax, this alternative subcostal approach with adjusted positioning provides an additional method for visualizing the RV, LV, RA, and LA.

Illustrations were generated using BioRender.com (BioRender, Toronto, Canada) under an academic license, and three-dimensional representations were created using licensed Biosphera Software (Biosphera S.L., Barcelona, Spain). Anatomical and positioning illustrations were created using PromeAI Studio (PromeAI Technologies, Berlin, Germany) under a professional license, with all generated images undergoing expert veterinary review to ensure anatomical accuracy and proper procedural representation.

The view displays distinct anatomical structures with specific screen orientations depending on the direction of the transducer notch: the left ventricle appears on the left side of the screen, while the right ventricle is visualized on the right side. The upper portion of the image shows the left atrium in the left quadrant and the right atrium in the right quadrant. The atrioventricular valves - the mitral valve between the left chambers and the tricuspid valve between the right chambers - are clearly visible. The interventricular septum maintains a vertical orientation through the center, while the interatrial septum appears in the upper portion of the image.

This projection holds significant clinical value in cardiac assessment. It enables detailed evaluation of chamber sizes and relationships, comprehensive assessment of ventricular function, and examination of atrioventricular valve morphology and function. The view is particularly useful for measuring flow patterns across both mitral and tricuspid valves, detecting septal defects, and evaluating right ventricular function. Additionally, it serves as an excellent window for assessing diastolic function and measuring ejection fraction.

When the ultrasound transducer is tilted forward (anteriorly), it shows the aortic valve and root along with all four heart chambers. This view is called the apical five-chamber view (A5C), and it demonstrates the left ventricle, left ventricle outflow tract, ascending aorta and mitral valve¹⁸.

As mentioned, the point of maximal cardiac impulse in pigs is typically located in a more caudal than humans’ intercostal space, usually around the 6th or 7th intercostal space^7,16. By placing the transducer in a more caudal intercostal space, the sonographer can better align the ultrasound beam with the long-axis of the porcine heart, allowing for clearer visualization of the cardiac chambers and valves from the apical ^7,16.

In order to do so, place the transducer on the right hemithorax, near the apex of the heart, with the notch oriented toward the pig’s right shoulder. Adjust the transducer position and angle until all four chambers of the heart (right and left ventricles, right and left atria) are visualized.

Subcostal View

The Subcostal View is obtained through a specific and precise technique. The transducer is positioned on the ventral midline, just caudal to the xiphoid process, with the notch oriented toward the pig’s left side. The examiner then angles the transducer cranially until the heart comes into view, with the right ventricle and atrium appearing closest to the transducer (Figure 5 A, B). Fine adjustments are made to demonstrate the inferior vena cava, hepatic veins, and the relationship of the heart to the diaphragm. This approach becomes particularly valuable when other acoustic windows are limited or suboptimal.

From this position, the examination provides excellent visualization of several cardiac structures. The right heart chambers are particularly well demonstrated, making it an ideal view for assessing the right ventricle size and function, right atrium, and inferior vena cava. The interatrial septum can be clearly visualized. The view also allows assessment of the pericardial space, making it invaluable in evaluating for pericardial effusion or tamponade physiology.

Research observations in Yorkshire pigs have revealed significant challenges in obtaining reliable echocardiographic images, particularly in heavier specimens^1,26–28. In these larger animals, the success rate of acquiring quality apical four-chamber views drops substantially, with failures occurring in up to half of the attempts. This limitation stems from the increased body mass and adipose tissue, which interfere with image acquisition^1,26–28. Consequently, we have found it necessary to modify our approach, focusing primarily on measurements obtained from the right parasternal long and short-axis view.

These findings emphasize the necessity of considering anatomical variations and adapting ultrasound techniques accordingly to ensure reliable data collection.

Spectral Doppler

Continuous Wave (CW) Doppler of Left ventricular outflow tract and aortic root

To obtain the aortic root Doppler view, the ultrasound transducer is positioned in the PLAX orientation, with the cursor aligned to bisect the aortic valve¹⁸. Continued ventral angulation of the transducer in this view eliminates the left atrium and mitral valve such that only the left ventricular outflow tract, aortic valve, and proximal ascending aorta are visible. This positioning allows the sonographer to interrogate the flow patterns through the aortic valve using pulsed-wave or continuous-wave Doppler techniques. The technique for measuring blood flow in the Left Ventricular Outflow Tract (LVOT) using pulsed wave Doppler requires precise positioning of the sample volume just proximal to the aortic valve¹⁸. This specific placement, slightly before the aortic valve in the direction of blood flow, is crucial for accurate measurements.

The Doppler sound beam should be oriented as parallel as possible to the flow, guided both by the 2D image, sometimes assisted by color flow imaging, and the quality of the Doppler recording¹⁸. Small deviations in angle produce mild errors in velocity measurements. Although these errors may be acceptable for low-velocity flows, when Doppler is used to derive pressure gradients, even a small error in velocity measurement can lead to significant underestimation of the gradient because of the quadratic relation between velocity and pressure gradient. Since the apical four-chamber view is often challenging to obtain in Yorkshire pigs^1,26–28, and the parasternal views are more commonly used, angle correction of less than 35 degrees is performed according to convention²⁹.

The technical settings are equally important: using very low gain reduces the amplification of the received signal, resulting in thin spectral envelopes that produce clean, well-defined velocity curves. This reduction in gain minimizes signal artifact and noise, thereby improving measurement accuracy¹⁸.

This precise positioning allows for accurate measurement of several critical parameters including peak and mean blood flow velocities, velocity time integral (VTI), and pressure gradients. These measurements are crucial for calculating cardiac output, stroke volume, and assessing valve function¹⁸. The technique is particularly valuable in evaluating the aortic function and determining valve area. Additionally, this view enables the assessment of systolic function parameters such as ejection time, rate of systolic ejection, and acceleration/deceleration times¹⁸.

Diastolic function assessment

Mitral inflow Doppler:

This assessment provides a wealth of valuable information about left ventricular (LV) diastolic function and filling dynamics. The velocity of blood flow across the mitral valve during diastole represents an intermediate link between the hemodynamic conditions indicated by instantaneous left atrial (LA) and LV pressures, and the overall characteristics of ventricular filling.

The left apical four-chamber view is the best view to record the atrioventricular flows. Using pulsed wave Doppler to record the mitral inflow velocity profile, with the sample volume positioned between the mitral leaflet tips at their narrowest point at the edge of the valvular funnel, where valve noises are minimal, and the flow signal is optimal.

As mentioned, obtaining these measurements can be technically challenging in pigs. The increased thoracic mass and adipose tissue in these larger specimens often compromises the quality of apical four-chamber views^1,26–28, making it preferable to rely on right parasternal long-axis view with angle correction not greater than 35 degrees²⁹, for comprehensive assessment.

Key parameters that can be measured include the peak early diastolic filling velocity (E wave), peak late diastolic filling velocity during atrial contraction (A wave), E wave deceleration time (DT), the E velocity just before atrial contraction (E at A), the duration of the mitral A wave (Adur), and the isovolumic relaxation time (IVRT) (Figure 6-A).

Figure 6. Multiple modalities that provide comprehensive assessment of diastolic function.

A. Pulse-wave Doppler recording of mitral inflow showing E and A waves with corresponding ECG trace (turquoise line). B. Left atrial volume measurement using planimetry (pink overlay) in the apical four-chamber view. Created in BioRender. Shehadeh, L. (2025) https://BioRender.com/s55n768. C. Tissue Doppler imaging of the mitral annulus from both lateral (1) and medial (2) positions, displaying S’ (systolic), e’ and a’ (early and late diastolic) velocities. D. Continuous-wave Doppler assessment of tricuspid regurgitation with color flow imaging, used for estimating right ventricular systolic pressure.

It’s crucial to note that simultaneously recording the electrocardiogram (ECG) during the Doppler assessment is essential, as it helps recognize and time each component of the cardiac cycle accurately, particularly in distinguishing the E and A waves and their relationship to electrical events.

In healthy individuals, the mitral inflow pattern is characterized by a rapid acceleration of blood flow from the LA to the LV after mitral valve opening, manifesting as a prominent E wave with a velocity occurring after the mitral valve opening. This E wave coincides with the maximum pressure gradient between the LA and LV, which depends on factors such as LV relaxation and the relative compliance of the two chambers. The normal E wave shows rapid acceleration and deceleration.

As diastolic function progressively worsens, the mitral inflow pattern evolves through well-defined stages, including grade 1 (abnormal relaxation), grade 2 (pseudo-normalization), and grade 3 (restrictive filling).

Left Atrial Indexed Volume

The left atrial evaluation is a cornerstone in assessing diastolic function and overall cardiac health. The most crucial measurement is the left atrial volume index (LAVI), calculated by obtaining the biplane maximum LA volume and indexing it to body surface area¹⁸. A LAVI greater than 34 mL/m², according to the recent guidelines³⁰, indicates LA enlargement, which often reflects chronic elevation of left ventricular filling pressures and long-standing diastolic dysfunction (Figure 6-B).

The pulmonary vein flow pattern adds another layer of information, particularly when we see prominent atrial reversal waves suggesting elevated left atrial pressures.

Hepatic vein flow patterns analyzed in the subcostal view, provide supplementary information about right atrial pressure and right ventricular diastolic function.

Tissue Doppler of Left Ventricular Annular Velocity

The velocity of the mitral annulus represents the velocity of changes in the LV long-axis dimension. In systole, the mitral annulus moves toward the LV apex. In diastole, it returns to its initial position in two phases generating two waveforms: (1) a rapid filling waveform (e’) dependent on active LV relaxation, elastic recoil, and the lengthening ventricle and (2) an atrial contraction waveform (a’) dependent on the contracting atrial fibers that pull the mitral annulus away from the apex ¹⁸ (Figure 6-C).

The measurement of mitral annular velocities is an important component in interpreting the diastolic filling pattern, estimating LV filling pressures. These velocities are recorded from the apical four-chamber view by placing a 5- to 6-mm sample volume over the lateral or medial portion of the mitral annulus to cover the longitudinal excursion of the mitral annulus in both systole and diastole.

Tricuspid Doppler color flow

The tricuspid valve assessment provides insights into the right heart function and pulmonary pressures. The main measurement is the tricuspid regurgitation (TR) velocity, which we obtain using continuous wave Doppler, often in an apical four-chamber view. This velocity, combined with estimated right atrial pressure, allows us to calculate pulmonary artery systolic pressure – for evaluation of pulmonary hypertension. The tricuspid inflow pattern mirrors the mitral valve assessment but typically shows lower velocities¹⁸ (Figure 6-D).

Comparison of Magnetic Resonance Imaging (MRI) and Echocardiography in Diastolic function assessment

MRI and echocardiography are both valuable imaging modalities for assessing cardiac structure and function in pigs, particularly in the evaluation of diastolic function. Each technique offers distinct capabilities that serve different research and clinical needs, making the understanding of their respective strengths and limitations crucial for appropriate methodology selection. MRI offers superior soft tissue contrast and spatial resolution, providing comprehensive cardiac assessment through multiple techniques, enabling detailed assessment of cardiac anatomy, function, and tissue characterization. Phase contrast imaging enables quantitative measurement of blood flow across cardiac valves, yielding precise transmitral and pulmonary venous flow velocities. Its capabilities allow detailed analysis of myocardial structure and composition. Advanced sequences such as T1 mapping and late gadolinium enhancement effectively identify myocardial fibrosis, a fundamental substrate of diastolic dysfunction^31–33. Myocardial tagging and feature tracking provide sophisticated strain analysis during diastole, while volumetric analysis offers precise measurements of atrial and ventricular volumes throughout the cardiac cycle. These capabilities make MRI particularly valuable for understanding the pathophysiological mechanisms underlying diastolic dysfunction^31–33.

Echocardiography excels in real-time cardiac assessment through various parameters that directly evaluate diastolic function. The technique’s high temporal resolution enables accurate measurement of key diastolic indices, including the E/A ratio, E/e’ ratio, and isovolumic relaxation time (IVRT)¹⁸. Doppler imaging provides instantaneous pressure gradients and flow patterns, crucial for understanding diastolic hemodynamics. Pulmonary venous flow assessment adds valuable information about left atrial pressure and compliance. These measurements make echocardiography particularly suited for evaluating dynamic aspects of diastolic function.

From a practical standpoint, both modalities present distinct advantages and challenges. MRI’s superior reproducibility and comprehensive tissue characterization make it invaluable for detailed mechanistic studies and precise outcome assessment. However, the technique faces limitations in temporal resolution and cannot provide real-time flow dynamics with the same fidelity as echocardiography. The extended examination times, high operational costs, and complex infrastructure requirements can make MRI less practical for serial assessments in research protocols.

Echocardiography offers significant practical advantages for research applications, particularly in pig models. Its accessibility, cost-effectiveness, and minimal setup requirements make it ideal for serial measurements and large cohort studies. The ability to perform bedside examinations with shorter acquisition times proves particularly valuable in research protocols involving large animal models. However, the technique’s dependence on acoustic windows, operator expertise, and angle-dependent measurements can introduce variability in data collection. Despite these limitations, echocardiography’s practical benefits and real-time imaging capabilities establish it as a fundamental tool for diastolic function assessment.

The optimal approach often involves complementary use of both modalities. Initial comprehensive MRI evaluation can provide detailed tissue characterization and baseline measurements, while serial echocardiographic assessments enable practical monitoring of diastolic function throughout the study protocol. This combined approach leverages each modality’s strengths while mitigating their individual limitations, providing a comprehensive evaluation of diastolic function in pig models. The selection between modalities should consider specific research requirements, resource availability, and the need for repeated measurements, ensuring appropriate technique selection for each study’s unique demands.

Table 1 summarizes the main advantages and disadvantages of MRI and echocardiography ^31–33 in Yorkshire pigs.

Table 1: Comparison of Magnetic Resonance Imaging (MRI) and Echocardiography Capabilities in Yorkshire Pig Cardiovascular Assessment.

Comparison of MRI and echocardiography imaging modalities in Yorkshire pig cardiovascular assessment, highlighting their respective strengths and limitations for research applications. IVRT = isovolumic relaxation time.

Modality	Advantages	Disadvantages
MRI	-Superior tissue characterization in both focal and diffuse pathologies. -Enables quantitative assessment of myocardial tissue properties. - Advanced tissue mapping capabilities (T1, T2, late Gadolinium enhancement), for inflammation, fibrosis and infiltrative diseases. -Three-dimensional assessment of cardiac structure. -Precise volumetric measurements of atrial and ventricular chambers. -Phase contrast imaging capabilities for flow assessment. -Early detection of cardiac alterations before functional changes. -Excellent reproducibility for longitudinal studies. -Ability to perform comprehensive myocardial mapping.	-Cannot assess real-time flow dynamics. -Limited temporal resolution for rapid filling events. -Inability to measure instantaneous pressure gradients. -High operational and maintenance costs prohibitive for many research facilities. -Complex setup requiring specialized infrastructure. -Not practical for serial measurements in research protocols.
Echocardiography	-Direct measurement of diastolic parameters (E/A ratio, E/e’ ratio, IVRT). -Real-time assessment of filling patterns and pressure gradients -Immediate evaluation of interventional effects. -Superior temporal resolution for capturing rapid diastolic events. -Cost-effective for longitudinal studies. -Ideal for repeated measurements in research protocols. -Allows assessment of load-dependent changes in diastolic function. -Practical for large animal studies with minimal setup requirements.	-Image quality dependent on acoustic windows (obesity, lung disease, scars and surgeries affect the acquisition). -Requires standardized imaging protocols for research consistency. -Operator expertise influences measurement reliability due to high operator dependence and variability. -Limited tissue characterization and detection of focal lesions. -Some measurements affected by angle dependency, especially doppler assessment. - Flow and velocity measurements need proper alignment. -Lower special resolution, less comprehensive volumetric analysis. - Restricted field of view.

DISCUSSION

The standardized approach to echocardiography in Yorkshire pigs reveals important anatomical and technical considerations that distinguish it from human and veterinary applications, while offering valuable insights for translational research, particularly in heart failure with preserved ejection fraction (HFpEF) studies. Whereas fundamental principles of cardiac ultrasound remain consistent, several key adaptations are necessary for optimal porcine imaging.

The porcine heart’s more central and caudal anatomical position presents the primary challenge, requiring modified transducer placement compared to human echocardiography. This anatomical difference, combined with the pig’s thicker chest wall and cylindrical heart shape, particularly impacts the acquisition of apical views^5,7. The cardiac apex location in the 6th-7th intercostal space, rather than the typical human 4–5th space⁹, necessitates specific adaptations in imaging protocols. These anatomical considerations are particularly relevant when developing standardized protocols, where consistent and accurate measurements of diastolic parameters are crucial.

Several technical challenges emerge when performing porcine echocardiography, particularly in heavier specimens where image acquisition becomes notably difficult. The success rate of apical views drops significantly due to increased thoracic adipose tissue, while the vertical orientation of the porcine heart complicates consistent transducer positioning. Doppler alignment optimization presents additional challenges given the anatomical differences in cardiac position and orientation. Although most ultrasound systems allow correction of the Doppler equation for the angle of incidence, this measurement is difficult to perform accurately because of the three-dimensional (3D) orientation of the blood flow. Since the apical four-chamber view is often challenging to obtain in Yorkshire pigs, and the parasternal views are more commonly used, angle correction of less than 35 degrees is performed according to convention. Furthermore, the standard veterinary table design limits accessibility from underneath the animal, and physical constraints often restrict optimal positioning and approach angles.

However, certain limitations persist, including reduced accessibility of acoustic windows in larger animals and the necessity for sedation, which may affect hemodynamic parameters.

Equipment selection for porcine cardiac imaging must account for several key factors: sufficient frequency range and penetration depth for the thicker chest wall, adequate field of view for complete cardiac visualization, and capability for all necessary imaging modes. Both veterinary-specific and properly configured human clinical systems can provide suitable imaging performance, with selection typically guided by practical considerations such as budget, portability requirements, and existing expertise.

Anesthetic management presents its own considerations. Our protocol employs ketamine for pre-anesthesia followed by isoflurane maintenance, though other successful approaches include tiletamine (Telazol ^®) or combinations of ketamine with either xylazine or dexmedetomidine^34,35. Longer procedures may require intravenous methohexital maintenance with comprehensive monitoring of vital parameters^23,36.

Machine selection ultimately depends on the specific research needs, available budget, requirements for portability, and the imaging capabilities needed for the study protocols. Both veterinary-specific and appropriately configured human clinical echocardiography systems can provide the necessary imaging performance The choice between them often reflects practical considerations like cost, existing expertise, and intended applications rather than strict technical requirements. Whichever system is chosen must enable reliable acquisition of the key cardiac measurements and parameters needed to evaluate heart function in the pig model.

While MRI provides excellent tissue contrast and 3D analysis capabilities, echocardiography emerges as the superior modality for cardiovascular research in Yorkshire pigs. Its real-time imaging capabilities uniquely capture the dynamic nature of cardiac function, particularly crucial for assessing diastolic parameters through various Doppler modalities. Modern echocardiographic systems excel in measuring complex hemodynamic parameters such as E/A ratios, tissue velocities, and instantaneous pressure gradients - measurements that prove essential for understanding cardiac physiology yet remain challenging or impossible with MRI.

Beyond its lower cost and greater accessibility, echocardiography’s efficiency and minimal setup requirements make it ideal for longitudinal studies. The ability to perform rapid, serial assessments at the bedside provides immediate feedback critical for research protocols. While image quality depends on acoustic windows and operator expertise, standardized protocols and advanced imaging technologies effectively address these challenges.

The implications for translational research and veterinary applications are significant and far-reaching. The standardized approach detailed here strengthens the foundation of cardiovascular research using Yorkshire pigs, establishing them as robust translational models for heart diseases. Their anatomical and physiological similarities to human hearts, combined with the ability to accurately assess diastolic function parameters, position these animal models as invaluable platforms for evaluating novel therapeutic interventions before human trials. This is especially crucial in HFpEF research, where precise measurement of diastolic parameters and tissue characteristics can illuminate treatment efficacy. Beyond research applications, these guidelines offer veterinary practitioners a systematic framework for cardiac evaluation in porcine patients. The standardization of these techniques not only improves diagnostic accuracy but also facilitates better communication and consistency across both research and clinical settings.

Limitations

While the Yorkshire pig model offers significant advantages for cardiovascular research, it is essential to acknowledge certain limitations inherent to this large animal model. One major consideration is the need for anesthesia during echocardiographic examinations. Although necessary for immobilization and proper positioning, anesthetic agents can influence hemodynamic parameters and potentially confound the assessment of cardiac function. Researchers must carefully select anesthetic protocols and monitor vital signs to minimize these effects. The technical challenges of performing echocardiography in pigs present another limitation: The anatomical differences in cardiac position and orientation, combined with the thicker chest wall and increased body mass, can impact image quality and make certain views, particularly the apical windows, more difficult to obtain consistently. Specialized operator training and optimized imaging protocols are essential to overcome these challenges.

Furthermore, while advanced imaging modalities such as MRI and angiography offer complementary information and valuable insights into cardiovascular physiology, their availability for large animal research is often limited. These technologies typically require significant financial investments in equipment and infrastructure, as well as specialized personnel, which may restrict their use to larger academic centers and research institutions. The costs associated with purchasing, housing, and maintaining Yorkshire pigs for cardiovascular studies can also be substantial, potentially limiting the feasibility and scale of such projects.

A significant limitation of the present study is the absence of quantitative data validating the described echocardiographic techniques. Specifically, measurements of inter-operator variability and direct comparisons between MRI and echocardiographic metrics would provide valuable validation of these methods. Future studies should address this gap by including comprehensive reliability analyses and correlation data between imaging modalities to strengthen the implementation of these techniques.

CONCLUSIONS

As large animal models become increasingly prevalent in the research environment, it is crucial to establish standardized protocols that ensure the reliability, precision, and translational relevance of the data obtained. This comprehensive guide to echocardiographic examination in pigs represents a significant step towards this goal, providing a detailed, systematic approach that addresses the unique challenges of porcine cardiac imaging.

By meticulously outlining the key modifications required for optimal image acquisition in pigs, including specific transducer positions, anesthetic protocols, and animal handling techniques, this work establishes a foundation for reproducible and high-quality echocardiographic data. The emphasis on understanding species-specific anatomical variations and adapting imaging techniques accordingly underscores the importance of a tailored approach when working with large animal models.

The Yorkshire pig’s close resemblance to human cardiac anatomy and physiology makes it an invaluable translational model for cardiovascular research. However, realizing the full potential of this model requires a keen awareness of the technical challenges involved and a commitment to employing proper imaging techniques. The detailed descriptions of image optimization strategies and the discussion of common pitfalls serve as a valuable resource for researchers aiming to maximize the quality and reliability of their echocardiographic data.

While both MRI and echocardiography offer valuable insights, echocardiography emerges as particularly advantageous for diastolic function assessment. Its superior temporal resolution, ability to measure instantaneous pressure gradients, and practical advantages for serial measurements make it especially suitable for longitudinal studies.

Key technical considerations in porcine echocardiography include careful attention to transducer positioning, Doppler angle correction limitations, and the effects of anesthetic protocols on hemodynamic parameters. Understanding these factors is crucial for obtaining reliable measurements and validating normal reference values specific to porcine physiology.

The standardization of these techniques enhances the reproducibility and reliability of cardiovascular research using pig models, particularly in the context of diastolic function assessment. This approach provides a foundation for future studies, especially those investigating novel therapeutic interventions for cardiac diseases where precise measurement of diastolic parameters is essential.