Color Flow Doppler Echocardiography in Healthy Racing Pigeons (Columba Livia F

veterinary sciences

Article Color Flow Doppler Echocardiography in Healthy Racing Pigeons (Columba livia f. domestica) and the Evidence of Physiological Blood Flow Vortex Formations

Marko Legler *, Lajos Koy, Norbert Kummerfeld and Michael Fehr

Clinic for Small Mammals, Reptiles and Birds, University of Veterinary Medicine Hannover, Foundation, Bünteweg 9, D-30559 Hannover, Germany; [email protected] (L.K.); [email protected] (N.K.); [email protected] (M.F.) * Correspondence: [email protected]; Tel.: +49-(0)-511-9536823

Received: 27 March 2020; Accepted: 29 April 2020; Published: 4 May 2020

Abstract: In avian medicine, Doppler sonographic techniques are used to visualize and estimate blood flow in the heart. In the literature there is a lack of standardized studies of the use of color Doppler flow on healthy avian species. For this purpose, we examined blood flow in the heart in the four-chamber view of clinically healthy awake racing pigeons (n = 43) by color flow Doppler sonography. With this technique the diastolic and systolic blood flow in the heart chambers and the heart valve regions were well visualized. However, the pulse repetition frequency must be adapted to the specific blood flow velocities of the heart region to be measured to reduce aliasing in higher velocities and to visualize blood flow of lower velocities. With the help of color Doppler imaging in the four-chamber view, typical physiological atrial and ventricular blood flow vortex formations were visualized in the avian heart for the first time. In the left ventricle an asymmetric vortex ring in the passive and active ventricular filling, in the right ventricle a great counter-clockwise blood vortex in the active ventricular filling, in the left atrium a vortex clockwise, and in the right atrium counter-clockwise were observed. The knowledge of these physiological blood flow vortices is important to identify pathological blood flow.

Keywords: color Doppler sonography; heart; blood flow vortices; diastole; systole; birds; valva pulmonis

1. Introduction The four-chambered avian heart is functionally comparable to the mammalian heart, with a left and right ventricle and two atria [1–4]. Unique to avian species is the anatomy of the atrioventricular (AV) valves. The left AV valve is formed as a membranous tricuspid AV valve with a longer septal cusp, similar to the anatomical structure of mammalian species. In contrast, the right AV valve is an oblique muscular flap of the right ventricular free wall and consists of atrial and ventricular myocardial parts [1,2,5]. The function of the left AV valve is a passive closure of the AV orifice depending on the pressure gradient. In contrast, the muscular structure of the right AV valve allows an active closure of the right AV orifice and participation of this valve in the pumping function of the right ventricle [5,6]. To fulfil this special function the right valve is separately innervated by an atrioventricular ring bundle of pacemaker and conducting myocytes (formerly known as AV-Purkinje ring) an important part of the conducting system of the avian heart [7–9]. The aortic and pulmonary valves consist of three semilunar cusps that work passively depending on the pressure gradient [1,2]. Doppler flow imaging is an important tool in human and small animal cardiology to visualize and measure blood flow in the heart, to detect turbulent flow, and to diagnose pathological conditions [10–13].

Vet. Sci. 2020, 7, 60; doi:10.3390/vetsci7020060 www.mdpi.com/journal/vetsci Vet. Sci. 2020, 7, 60 2 of 13

In avian medicine Doppler sonographic techniques are also used to visualize and estimate blood flow in the heart [3,4,14–23]. In particular, color flow Doppler imaging is used in avian medicine to visualize heart valve insufficiencies, aneurysm, and atrial or ventricular septal defects [3,4]. Small valvular regurgitations are easier to diagnose in color flow sonography compared to pulse wave (PW) or continued wave (CW) Doppler imaging [23]. In color flow images velocity information is combined with anatomical information. Movements away from the transducer are normally coded in a blue color and movements toward the transducer are colored in red. The use of the color flow Doppler sonography is described in the literature in birds mainly in clinical cases and there is a lack of standardized studies on healthy animals [3,4,14]. However, in the use of Doppler echocardiography it is important to distinguish physiological flow pattern from a pathological turbulent flow. The aim of the present study is to examine the blood flow in the heart of clinically healthy awake racing pigeons by color flow Doppler sonography to verify the visualization of the blood flow and to determine the function of the heart valves.

2. Materials and Methods The study was conducted in accordance with the German animal welfare regulations and with the permission of the relevant German authorities (reference number: 33.12-42502-04-15/1864).

2.1. Experimental Animals Racing pigeons (Columba livia f. dom.; n = 43) of both sexes (male: n = 16; female: n = 27) were used for the investigation. The pigeons were 2.30 SD 1.69 (range: 0.5 to 8) years old and had a ± weight of 468.16 g SD 51.64 (range: 352–577 g) body mass and were trained for racing. The sternal ± length of the pigeons measured from the visible sternocoracoid joint to the end of the sternum in laterolateral radiographic images was 73.4 mm SD 3.1 mm (range: 63.2–81.0 mm). The pigeons were ± housed in indoor aviaries, offered a commercial pigeon seed mix and fresh drinking water ad libitum, and routinely vaccinated by the owner for pigeon avulavirus 1 (paramyxovirus 1) and salmonellosis. All pigeons showed normal feeding and drinking behavior. Prior to the ultrasound examinations the pigeons were acclimatized for two weeks in the new aviaries. In this time a bacteriological and parasitological testing of a composite fecal sample of the pigeons for salmonella and endoparasites of the intestine were negative. A microscopic examination of fresh crop samples revealed in some pigeons a low-grade infestation with Trichomonas gallinae and all pigeons were treated with 10 mg carnidazole (Spatrix®, Elanco Deutschland GmbH, Bad Homburg, Germany). The pigeons were declared healthy by clinical, hematological, and radiological examinations. The maximum width of the cardiac silhouette of the pigeons measured in the ventrodorsal radiograph was 58.8% 3.3 ± (50.3%–65.1%) of the maximum width of the thorax. These results are comparable to results of healthy birds in the literature [3]. The hematocrit (44.9% 1.8; 42.0%–49.0%) and buffy coat (<1%) of the ± pigeons were in the reference values of healthy and normal hydrated pigeons [24]. Egg-laying pigeons were excluded from sonographic examinations to prevent the influence of a higher abdominal pressure.

2.2. Doppler-Sonographic Examination Echocardiographic images were acquired by using a 10 MHz phased-array transducer (GE 10S-RS Probe; B Mode 4.5–11.5 MHz) with a digital ultrasound system (Vivid 7 Dimension BT08, GE Medical Systems) in combination with an electrocardiogram (ECG) according to Einthoven [25–27]. The pigeons were fixed in a semi-upright position for the sonographic examination of the heart from the left and right parasternal approach. Depending on the individual bird, the left and right fenestra or the space behind the last rib through the liver to the heart were chosen as acoustic windows. The 2D echocardiographic images were named and oriented in accordance with Pees et al. [3], Riedel [28], Schulz [29]. The right and left parasternal longitudinal horizontal views (“four-chamber view”) were chosen individually to visualize the left and right heart ventricle, the AV valves, and the atria Vet. Sci. 2020, 7, 60 3 of 13 as well as the aorta with aortic valve and, after horizontal correction of the transducer, the right heart outflow tract and pulmonary artery. The 2D images were overlaid with color flow Doppler information of the ultrasound system. The field with color flow Doppler information was chosen as small as possible to guarantee frame rates above 80 frames per second (125.5 FPS SD 30.2). ± The pulse repetition frequency (PRF) value was adapted individually to the anticipated blood flow velocities between 1 and 11.76 kHz to reduce aliasing. Depending on the PRF, a wall filter of 6.54 to 19.63 cm per second (15.91 cm s 1 SD 2.99) was used. The sample volume setting was − ± 0.4 mm. Flow towards the transducer was color-coded in red and flow away from the transducer was color-coded in blue. The sonographic examinations were stored on the ultrasound system and evaluated in slow motion. The color flow patterns within the chambers and heart valves were analyzed in a qualitative manner relative to anatomic location and timing within the cardiac cycle.

2.3. Statistical Analysis The blood flow in the color Doppler images was described and the results expressed as the percentage of total number of examined pigeons. For the comparison of the blood flow of different anatomical structures the Chi-squared test was implemented. Mean, standard deviation (SD), range (Xmin to Xmax) and median were calculated for the used PRF. The Kolmogorov–Smirnov test was used to test for normal distribution. According to these results the Mann–Whitney U test was chosen for the evaluations. Statistical tests were performed using SPSS® Statistics 26. A significance level of p 0.05 was chosen. ≤ 3. Results Adequate Doppler sonographic examinations of the diastolic and systolic blood flow were possible in all 43 racing pigeons in the heart rate range of 220.5 41.3 heart beats per minute ± (left heart) and 219.1 34.7 heart beats per minute (right heart). A fused diastolic ventricular inflow ± (fused E and A wave, EA wave) was observed in five birds in a heart rate range of 270 to 360 heart beats per minute. The significant differences between the diastolic and systolic blood flow velocities led to different sonographic settings (PRF) for the visualization of these different blood flows. For the examination of the diastolic blood flow of the left heart a PRF of 6.35 kHz SD 1.25 (4.0 to 9.0 kHz; ± 6.0 kHz; color scale: 0.52 m s 1 SD 0.10; 0.33 to 0.74 m s 1; 0.49 m s 1) and of the right heart a PRF of 5.94 − ± − − kHz SD 1.62 (2.0 to 9.0 kHz; 6.0 kHz; color scale: 0.48 m s 1 SD 0.13; 0.16 to 0.74 m s 1; 0.49 m s 1) ± − ± − − were used (no significant difference Mann–Whitney U test p = 0.17). The blood flow in the atria was also examined with these settings. For the examination of the systolic blood flow of the pulmonary artery a PRF of 8.87 kHz SD 1.71 (5.0 to 11.76 kHz; 9.0 kHz; color scale: 0.73 m s 1 SD 0.14; ± − ± 0.41 to 0.98 m s 1; 0.74) and of the aorta a PRF of 9.46 kHz SD 1.77 (6.0 to 11.76 kHz; 10.0 kHz; − ± color scale: 0.79 m s 1 SD 0.15; 0.49 to 0.98 m s 1; 0.82 m s 1) was used (no significant difference − ± − − Mann–Whitney U test p = 0.07). In contrast to this, the PRF values used to visualize the diastolic and systolic blood flow are highly significantly different (p < 0.001; Mann–Whitney U test).

3.1. Left Ventricular Filling Diastolic ventricular inflow was recorded in the atrium, across the AV valve and within the ventricular inlets and outlets. In the early diastole a red signal was recorded within the left atrium and across the mitral valve annulus into the left ventricle. This phase was interpreted as the passive ventricular filling due to left ventricular relaxation (descending part of T wave in ECG). Behind the longer septal part of the left AV valve leaflet and in the left outflow tract a blue signal in this phase of the cardiac cycle simultaneous to the red signal was observed in 42 birds (97.7%; Figure1) and in the area of the lateral smaller parts of the left AV valve only in nine birds (20.9%; significant difference Chi-squared test p 0.001). In the mid diastolic phase in some pigeons the movement of the blood ≤ in the ventricle was observed (Figure2). Within the P wave of the ECG an intense red blood flow Vet. Sci. 2020, 7, 60 4 of 13 Vet. Sci. 2020, 7, 60 4 of 13 Vet. Sci. 2020, 7, 60 4 of 13 signalthe was ventricle evident was in observed the left (Figure atrium, 2). crossing Within the the P wave left AV of the valve ECG and an intense entering red theblood left flow ventricle signal as a sign ofwasthe blood ventricle evident flow inwas the caused observed left atrium, by (Figure atrial crossing 2). contraction. Within the left the AV P This wave valve rapid of and the ventricularenteringECG an intensethe left filling redventricle blood led to asflow a a visiblesign signal of blue was evident in the left atrium, crossing the left AV valve and entering the left ventricle as a sign of signalblood in the flow left caused outflow by tract,atrial behindcontraction. the septalThis rapid part ventricular of the AV valvefilling (97.7%),led to a visible and in blue the somesignal pigeons in blood flow caused by atrial contraction. This rapid ventricular filling led to a visible blue signal in (48.8%;thesignificant left outflowdi tract,fference behind Chi-squared the septal part test ofp the 0.001AV valve) also (97.7%), behind and the in smallerthe some lateral pigeons leaflets (48.8%; of the significantthe left outflow difference tract, behindChi-squared the septal test ppart ≤ 0.001) of th≤ ealso AV behind valve (97.7%), the smaller and lateralin the someleaflets pigeons of the left(48.8%; AV left AV valve simultaneous to the red signal (Figure3). In one pigeon the passive diastolic inflow was valvesignificant simultaneous difference to Chi-squared the red signal test p(Figure ≤ 0.001) 3). also In behind one pigeon the smaller the passive lateral leafletsdiastolic of inflow the left was AV connected with the active ventricular filling (EA wave). connectedvalve simultaneous with the active to the ventricular red signal filling (Figure (EA 3). wave). In one pigeon the passive diastolic inflow was connected with the active ventricular filling (EA wave).

FigureFigure 1. Color 1. Color Doppler Doppler image image ofof thethe early diastolic diastolic filling filling of the of theleft leftventricle. ventricle. The blood The inflow blood in inflow Figure 1. Color Doppler image of the early diastolic filling of the left ventricle. The blood inflow in in thethe left left ventricle ventricle from the the atrium atrium (red (red signal) signal) and andvortex vortex formation formation of the bloo ofd the flow blood behind flow the behindseptal the partthe left of theventricle left atrioventricular from the atrium (AV) (red valve signal) leaflet and andvortex left formation outflow tract of the (blue bloo signal)d flow isbehind visible. the LV: septal left septal part of the left atrioventricular (AV) valve leaflet and left outflow tract (blue signal) is visible. ventricle;part of the HR left heart atrioventricular rate. Electrocardiogram: (AV) valve leaflet P: P andwave, left S: outflow S wave, tract T: T (blue wave. signal) The color is visible. scale LV: on leftthe LV: left ventricle; HR heart rate. Electrocardiogram: P: P wave, S: S wave, T: T wave. The color scale on rightventricle; of the HR image heart is rate. calibrated Electrocardiogram: in m s−1. P: P wave, S: S wave, T: T wave. The color scale on the 1 the rightright of of thethe imageimage is is calibrated calibrated in in m ms−1 s. − .

Figure 2. Color Doppler image of the ventricle in mid diastolic phase. The vortex formation of the FigurebloodFigure 2. Color in 2. the Color ventricle Doppler Doppler is image visible image of(red of the theand ventricle ventricle blue signal). inin mid LV: di diastolicleftastolic ventricle; phase. phase. LA: The left The vortex atrium; vortex formation HR: formation heart of rate. the of the bloodElectrocardiogram:blood in the in ventriclethe ventricle is P: visibleis P visible wave, (red (red S: andS and wave, blue blue T: signal).signal). T wave. LV: LV: The left left colorventricle; ventricle; scale LA: on LA: leftthe left atrium; right atrium; of HR: the HR:heart image heart rate. is rate. Electrocardiogram:calibratedElectrocardiogram: in m s− P:1. P: P wave,P wave, S: S: S S wave, wave, T:T: TT wave.wave. The The color color scale scale on onthe theright right of the of image the image is is calibrated in m s−1. calibrated in m s 1. −

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Figure 3. Color DopplerDoppler imageimage of of the the late late active active filling filling of of the the left left ventricle. ventricle. The The blood blood inflow inflow in the in leftthe ventricleleft ventricle from from the atrium the atrium (red signal) (red signal) and vortex and formationvortex formation of the blood of the flow blood behind flow the behind septal partthe septal of the leftpart AV of valvethe left leaflet, AV valve left outflow leaflet, left tract, outflow and the tract, smaller and lateral the smaller leaflets lateral of the leaflets left AV of valve the left (blue AV signals) valve are(blue visible. signals) LV: are left visible. ventricle; LV: LA: left left ventricle; atrium; RV:LA: rightleft atrium; ventricle; RV:HR: right heart ve ratentricle;. Electrocardiogram: HR: heart rate. 1 P:Electrocardiogram: P wave, S: S wave, P: T: P T wave, wave. S: The S wave, color scale T: T onwa theve. rightThe ofcolor the scale image on is calibratedthe right of in the m s −image. is calibrated in m s−1. 3.2. Left Ventricular Ejection 3.2. LeftLeft Ventricular ventricular Ejection ejection, displayed as a blue flow signal, was observed during systole (ascending part ofLeft S waveventricular to descending ejection, part displayed of T wave as a ofblue ECG) flow in signal, the ventricle, was observed left ventricular during systole outflow (ascending tract, and aorta.part of This S wave blue to flow descending signal was part aliased of T wave in all pigeonsof ECG) especiallyin the ventricle, in the aortaleft ventricular with our settings. outflow Intract, the firstand aorta. part of This the blue systole flow in signal all pigeons was aliased a simultaneous in all pigeons red ventricularespecially in blood the aorta flow with signal our to settings. the much In largerthe first blue part signal of the under systole the in AV all (Figurepigeons4 )a valve simultan waseous observed. red ventricular During the blood closure flow of signal the aortic to the valve much a littlelarger red blue signal signal in under the area the of AV this (Figure heart valve 4) valve was wa realizeds observed. in some During pigeons the closure (n = 11). of the aortic valve a littleIn red the signal preejection in the period, area of between this heart the valve end was of the realized active diastolicin some fillingpigeons and (n the= 11). contraction of the ventricleIn the myocardium, preejection weperiod, found between in twopigeons the end (4.7%) of the a active blue blood diastolic flow filling signal and between the contraction the leaflets of the leftventricle AV valve myocardium, to the atrium, we afound sign ofin an two insu pigeonfficiencys (4.7%) of the a left blue AV blood valve flow in this signal heart between phase. the leaflets of the left AV valve to the atrium, a sign of an insufficiency of the left AV valve in this heart phase.

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FigureFigure 4. Color 4. Color Doppler Doppler image image of of the the left left ventricleventricle in th thee systole. systole. The The blood blood ejection ejection from from the left the left Figure 4. Color Doppler image of the left ventricle in the systole. The blood ejection from the left ventricleventricle in the in aorta the aorta is visible is visible (blue (blue signal). signal). The blueThe blue flow flow signal signal is aliased is aliased in the in aorta the aorta (wrong (wrong direction: ventricle in the aorta is visible (blue signal). The blue flow signal is aliased in the aorta (wrong red insteaddirection: of bluered instead of the colorof blue Doppler of the color signal Doppler in the aorta). signal Thein the movement aorta). The of movement the valve levelof the of valve the heart direction: red instead of blue of the color Doppler signal in the aorta). The movement of the valve and thelevel resulting of the heart blood and flow the resulting are visible blood as aflow red are signal. visible LV: as lefta red ventricle; signal. LV: LA: left left ventricle; atrium; LA: AO: left aorta; level of the heart and the resulting blood flow are visible as a red signal. LV: left ventricle; LA: left RV: rightatrium; ventricle; AO: aorta; HR: RV: heart right rate. ventri Electrocardiogram:cle; HR: heart rate. P:Electrocardiog P wave, S: Sram: wave, P: P T: wave, T wave. S: S wave, The color T: T scale atrium;wave. AO: The aorta;color scale RV: onright the ventri right ofcle; the HR: image heart is calibratedrate. Electrocardiog in m s−1. ram: P: P wave, S: S wave, T: T on the right of the image is calibrated in m s 1. wave. The color scale on the right of the image− is calibrated in m s−1. 3.3. Left3.3. AtrialLeft Atrial Filling Filling 3.3. Left Atrial Filling After the P wave of the ECG in the phase of the relaxation of myocardium of the left atrium, a After the P wave of the ECG in the phase of the relaxation of myocardium of the left atrium, littleAfter blue the filling P wave signal of over the ECGthe AV in valvethe phase was seen of the in therelaxation some pigeons of myocardium (n = 27; 62.8%) of the with left the atrium, used a a little blue filling signal over the AV valve was seen in the some pigeons (n = 27; 62.8%) with the littlesettings blue filling(Figure signal 5). In overall pigeons the AV a valvelater and was faster seen bl inue the inflow some signal pigeons in the (n atrium= 27; 62.8%) in the with time theof the used used settings (Figure5). In all pigeons a later and faster blue inflow signal in the atrium in the time settingsdescending (Figure part 5). Inof allthe pigeons T wave a waslater recorded and faster over blue the inflow AV valve signal to in the the leftatrium part in of the the time atrium of the of the descending part of the T wave was recorded over the AV valve to the left part of the atrium descending(significant part difference of the Chi-squaredT wave was test recorded p ≤ 0.001). over At thethe same AV timevalve a redto thesignal left was part seen of rightthe atriumand (significantleft from di thefference valva Chi-squaredpulmonis in some test ppigeons0.001). (25 Atout the of 43 same birds; time 58.1%; a red Figure signal 6). wasThe position seen right of the and left (significant difference Chi-squared test ≤p ≤ 0.001). At the same time a red signal was seen right and fromleftvalva thefrom valva pulmonis the valva pulmonis was pulmonis almost in some inconstant some pigeons pigeonsin diastole (25 (25 out (Figure out of of 43 7)43 birds; and birds; systole 58.1%; 58.1%; (Figures FigureFigure 5 6 6).and). The The 6). position position of of the the valvavalva pulmonis pulmonis was was almost almost constant constant in in diastole diastole (Figure(Figure7 7)) andand systolesystole (Figures 5 and 66).).

Figure 5. Color Doppler image of the early blood inflow in the left atrium (red and blue signal). LV: left ventricle; LA: left atrium; HR: heart rate. Electrocardiogram: P: P wave, S: S wave, T: T wave. The −1 FigureFigurecolor 5. 5. scaleColor Color on DopplerDopplerthe right ofimage the image of of the the is early calibrated early blood blood in inflow m inﬂow s . in the in theleft atrium left atrium (red and (red blue and signal). blue signal). LV: LV: left left ventricle; ventricle LA:; LA: left left atrium; atrium; HR: HR: heart heart rate. rate. Elec Electrocardiogram:trocardiogram: P: P wave, P: P wave, S: S wave, S: S wave,T: T wave. T: T The wave. −1 1 Thecolor color scale scale onon the the right right of the of theimage image is calibrated is calibrated in m in s m. s− .

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Figure 6. ColorColor Doppler Doppler image of the late blood blood inflow inﬂow in the left atrium atrium (red signal; 1) and blood flow ﬂow vortex formation (2). LV: left ventricle; LA: left atrium; HR: heart rate. Electrocardiogram: P: P: P P wave, wave, −11 S: S wave, T: T wave. The color scale on thethe right of the image is calibrated in m s− . .

(a) (b)

Figure 7. Position of the valva pulmonis (VP) in the diastole: (a) passive and (b) active ventricular 1 ﬁllingFigure (red7. Position signal); of HR: the heart valva rate; pulmon coloris scale (VP) of in the the color diastole: Doppler (a) imagepassive is calibratedand (b) active in m ventricular s− . −1 3.4. Rightfilling Ventricular (red signal); Filling HR: heart rate; color scale of the color Doppler image is calibrated in m s .

3.4. RightThe earlyVentricular passive Filling diastolic inflow in the right ventricle was visible as a red blood flow signal in 42 pigeons (97.7%; descending part of T wave in ECG). In four pigeons the passive diastolic inflow was The early passive diastolic inflow in the right ventricle was visible as a red blood flow signal in connected with the active ventricular filling (EA wave). In the mid diastolic phase of the heart cycle no 42 pigeons (97.7%; descending part of T wave in ECG). In four pigeons the passive diastolic inflow blood flow was recorded. During the active ventricular filling of the right ventricle (P wave of ECG) an was connected with the active ventricular filling (EA wave). In the mid diastolic phase of the heart intense red signal of high velocity was visible in all pigeons. In the active filling of the right ventricle, cycle no blood flow was recorded. During the active ventricular filling of the right ventricle (P wave an intense blue signal behind the muscular right AV valve simultaneous to the larger red signal was of ECG) an intense red signal of high velocity was visible in all pigeons. In the active filling of the detected in all pigeons (Figure8). For some pigeons (25.6%) in this phase of the ventricular filling in right ventricle, an intense blue signal behind the muscular right AV valve simultaneous to the larger the septal area of the blood inflow, a blue signal was also visible (p 0.001; Chi-squared test). red signal was detected in all pigeons (Figure 8). For some pigeons≤ (25.6%) in this phase of the ventricular filling in the septal area of the blood inflow, a blue signal was also visible (p ≤ 0.001; Chi- squared test).

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Figure 8. ColorColor Doppler Doppler image image of of the the late late active active filling filling of ofthe the right right ventricle. ventricle. The The blood blood inflow inflow in the in theright right ventricle ventricle from from the atrium the atrium (red (redsignal) signal) and vortex and vortex formation formation of the of blood the blood flow behind flow behind the right the rightmuscular muscular AV valve AV valve (blue (bluesignal) signal) are vi aresible. visible. LV: left LV: ventricle; left ventricle; LA: left LA: atrium; left atrium; RV: right RV: rightventricle; ventricle; RA: RA:right right atrium; atrium HR:; HR:heart heart rate. rate. Electr Electrocardiogram:ocardiogram: P: P P:wave, P wave, S: S S:wave S wave,, T: T T: wave. T wave. The The color color scale scale on 1 onthe theright right of the of theimage image is calibrated is calibrated in m in s m−1. s− .

3.5. Right Atrial Filling 3.5. Right Atrial Filling The blood flow in the right atrium was observed in 35 pigeons and was difficult to examine with The blood flow in the right atrium was observed in 35 pigeons and was difficult to examine with our settings. After the P wave of the ECG in the phase of the relaxation of the myocardium of the right our settings. After the P wave of the ECG in the phase of the relaxation of the myocardium of the atrium, a blue filling signal in 15 pigeons (34.9%) was visualized. In the second half of the systole right atrium, a blue filling signal in 15 pigeons (34.9%) was visualized. In the second half of the systole (T wave) a late blue filling inflow signal in the atrium was seen in 35 pigeons (81.4%); at this time a (T wave) a late blue filling inflow signal in the atrium was seen in 35 pigeons (81.4%); at this time a blood flow in the liver veins was also observed (Figure9). Simultaneously to the blue inflow signal a blood flow in the liver veins was also observed (Figure 9). Simultaneously to the blue inflow signal a red blood flow signal in the area of the septum in the right atrium was observed (Figure9). red blood flow signal in the area of the septum in the right atrium was observed (Figure 9).

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Figure 9. Color Doppler image of the late blood inflow in the right atrium (blue signal). The vortex FigureFigure 9. Color 9. Color Doppler Doppler image image of of the the late late bloodblood inflowinflow in in the the right right atrium atrium (blue (blue signal). signal). The Thevortex vortex formation of the blood flow in the atrium (red and blue signal simultaneously) and the blood flow in formationformation of the of the blood blood flow flow in in the the atrium atrium (red (red andand blue signal signal simultaneously) simultaneously) and and the blood the blood flow flowin in the liver vein (LV; blue signal in liver tissue) are visible. RV: right ventricle; RA: right atrium; HR: the liverthe liver vein vein (LV; (LV; blue blue signal signal in liver in liver tissue) tissue) are ar visible.e visible. RV: RV: right right ventricle; ventricle; RA: RA: right right atrium; atrium; HR:HR: heart heart rate. Electrocardiogram: P: P wave, S: S wave, T: T wave. The color scale on the right of the rate.heart Electrocardiogram: rate. Electrocardiogram: P: P wave, P: P S: wave, S wave, S: S T:wave T wave., T: T wave. The color The scalecolor scale on the on right the right of the of imagethe is image is calibrated in m s−1. 1 −1 calibratedimage inis calibrated m s− . in m s . 3.6. Right Ventricular Ejection 3.6. Right3.6. Right Ventricular Ventricular Ejection Ejection During systole (ascending part of S wave to descending part of T wave of ECG), a blue flow During systole (ascending part of S wave to descending part of T wave of ECG), a blue flow Duringsignal was systole seen in (ascending the right ventricular part of S waveoutflow to trac descendingt and pulmonary part of Tartery wave (Figure of ECG), 10). aIn blue all pigeons flow signal signal was seen in the right ventricular outflow tract and pulmonary artery (Figure 10). In all pigeons was seenaliasing in the occurred right ventricularin the pulmonary outflow artery tract with and our pulmonary settings. arteryA regurgitation (Figure 10 of). the In allright pigeons AV valve aliasing aliasing occurred in the pulmonary artery with our settings. A regurgitation of the right AV valve occurredwas not in the seen. pulmonary artery with our settings. A regurgitation of the right AV valve was not seen. was not seen.

FigureFigure 10. 10.Color Color Doppler Doppler image image of the the right right ventricle ventricle in th ine systole. the systole. The blood The ejection blood from ejection the right from the Figure 10. Color Doppler image of the right ventricle in the systole. The blood ejection from the right ventricle in the pulmonary artery is visible (blue signal). The blue flow signal is aliased in the rightventricle ventricle in in the the pulmonary pulmonary artery artery is visible is visible (blue (blue signal). signal). The Theblue blueflow ﬂowsignal signal is aliased is aliased in the in the pulmonary artery (wrong direction: red instead of blue of the color Doppler signal in the pulmonary artery). LV: left ventricle; RV right ventricle; PA: pulmonary artery; HR: heart rate. Electrocardiogram: 1 P: P wave, S: S wave, T: T wave. The color scale on the right of the image is calibrated in m s− . Vet. Sci. 2020, 7, 60 10 of 13

4. Discussion The blood flow in the avian heart follows the same principles as in mammals. The diastolic blood flow is characterized by an early or passive diastolic filling (E wave in pulse wave Doppler image; descending part of T wave in ECG) and an active filling during the atrial contraction (A wave in pulse wave Doppler image) or fused (EA wave in pulse wave Doppler image) in higher heart rates [3,4,10] and a systolic blood flow through the aorta and pulmonary artery (ascending part of S wave to descending part of T wave of ECG). This general blood flow was well visualized for the pigeons with color flow imaging in our study and is already described in the literature for other bird species [3,4,14,23,30]. The generally valid color coding used in small animal medicine, the diastolic blood flow color-coded in red, and the systolic blood flow in blue could also be used in the pigeons in the used acoustic windows [10]. However, faster velocities, especially in the aorta and pulmonary arteries show aliasing. In these cases, the used PRF and/or the highest PRF settings of the used ultrasound system were too low. On the other hand, blood flow of lower velocities, for example of the right atrium, is difficult to investigate and explain why these blood flows cannot be shown in all pigeons. The significant differences in the displayability of the blood flow in the areas of the septal and lateral leaflets of the left AV valve can also be explained by different velocities. These results indicate that a general setting for color Doppler echocardiography of all heart regions in pigeons does not exist and an individual manual adaptation corresponding to the region to be examined is necessary. The significant differences between the PRF settings in systole and diastole to visualize the blood flow in our investigations illustrate this necessity. An initial PRF setting can be selected according to the blood flow velocity specifications in the literature for pigeons depending on heart rate or anesthesia used [21–23,31]. Constant simultaneous occurrences of red and blue color in the Doppler images of ventricles and atria show the presence of typical constant blood flow vortex formations in the avian heart, comparable to the mammalian heart [32]. Vortices in the cardiovascular system are supposed to play fundamental roles in normal physiology and provide a proper balance between blood motion and the stresses on the surrounding tissues [32]. In the color Doppler examinations of the pigeon heart in the “four-chamber view”, a vortex clockwise in the left atrium and counter-clockwise in the right atrium were observed. In this context the valva pulmonis of the left atrium is discussed as an important anatomical structure for sealing the atrial cavum [1]. However, the color Doppler images in our investigation show that in diastole (atrial contraction) and systole (atrial filling) the position of the valva pulmonis is very constant. There is no evidence for a motion of this valve which could contribute to the closure of the left atrium (see Figures5–7). The main function of this anatomical structure seems to be to direct blood flow towards the left ventricle (see Figure6). The passive filling of the atria was visible after the P wave of the ECG as a cause of the relaxation of the myocardium and in the time of the end of the T wave of the ECG as a cause of the ventricular pump mechanism by the myocardial contraction in the systole. The findings in the color Doppler sonographic examination of the ventricles allow the conclusion of the presence of an asymmetric vortex ring in the left ventricle in the passive and active ventricular filling with a stronger vortex behind the longer septal part of the left AV valve (see Figures1–3). It is possible that an asymmetric valve shape allows a typical vortex formation (larger vortex in the direction of the outflow tract) that enables a faster blood flow and emptying of the ventricle [33]. Similar vortex formations in the left ventricle are described in the mammalian heart [32,33]. In the right ventricle the active ventricular filling leads to a great counter-clockwise blood vortex in this ventricle. The blood flow behind the right AV valve in the active ventricular filling as well as the active movement of this muscular valve during the closure seem to be important for the development of this blood flow vortex (see Figure8). In connection with the asymmetrical shape of the right ventricle and oblique arrangement of the right AV valve [1], the shape of the blood vortex simplifies the outflow of blood through the pulmonary artery. In avian medicine the color Doppler sonography is mainly used to visualize abnormal blood flow [3,4,14]. Especially for the interpretation of a pathological blood flow the knowledge of Vet. Sci. 2020, 7, 60 11 of 13 physiological blood vortex formations could be important. However, in our study the insufficiency of the left AV valve in the preejection period could be visualized in two birds with this technique. The importance of muscle contraction of the left ventricle and the influence of anesthesia on the function of the left AV valve were previously described and discussed by Legler et al. [23]. Anesthesia with isoflurane leads to an AV valve insufficiency in the preejection period in 93% of pigeons, which was diagnosed with color Doppler sonography [23]. More research is needed to understand the complex vortex formations of blood flow in the avian heart and the physiological benefits. Furthermore, a connection between the vortex formations and distribution of the natriuretic peptides in heart muscle cells as an important part of the cardiac hormone system of birds is still unknown.

5. Conclusions The diastolic and systolic blood flow as well as heart valve insufficiencies can be well visualized in pigeons with color Doppler sonography. However, the device settings must be adapted to the blood flow velocities of the region to be investigated to display the blood flow sufficiently. With the help of color Doppler imaging, typical physiological atrial and ventricular blood flow vortex formations were visualized in the avian heart for the first time. The knowledge of these physiological blood flow vortices is important to identify a pathological blood flow.

Author Contributions: Conceptualization, N.K., M.F., and M.L.; methodology, N.K. and M.L.; validation, L.K. and M.L.; formal analysis, M.L.; investigation, L.K. and M.L.; writing—original draft preparation, M.L.; writing—review and editing, M.L.; visualization, M.L.; supervision, M.F.; project administration, M.L. All authors have read and agreed to the published version of the manuscript. Funding: This research received no external funding. Acknowledgments: The authors would like to acknowledge the pigeon breeders for participating in this study and F. Sherwood-Brock for critical revision of the manuscript. Conﬂicts of Interest: The authors declare no conﬂict of interest.

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