s105 N J.C.J .C.J .C.J .C.J .C. F F F F Ferererererrrrrreireireireireira ,a ,a ,a ,a , F F F F F.S..S..S..S..S. Ignácio & C M Ignácio & C M Ignácio & C M Ignácio & C M Ignácio & C Meireireireireira .a .a .a .a . 2011. 2011. 2011. 2011. 2011. Doppler ultrasonography principles and methods of evaluation of the reproductive tract in mares. Acta Scientiae Veterinariae. 39(Supl 1): s105 - s111. Acta Scientiae Veter inar iae, 2011. 39(Supl 1) : s105 - s111.Acta Scientiae Veter inar iae, 2011. 39(Supl 1) : s105 - s111.Acta Scientiae Veter inar iae, 2011. 39(Supl 1) : s105 - s111.Acta Scientiae Veter inar iae, 2011. 39(Supl 1) : s105 - s111.Acta Scientiae Veter inar iae, 2011. 39(Supl 1) : s105 - s111. ISSN 1679-9216 (Online) 1Programa de Pós-graduação em Medicina Veterinária, FMVZ, Departamento de Reprodução Animal e Radiologia Veterinária, Universidade Estadual Paulista Júlio de Mesquita Filho, Botucatu, SP, Brasil. 2Faculdade de Medicina Veterinária e Zootecnia, Universidade Estadual Paulista Júlio de Mesquita Filho, Departamento de Reprodução Animal e Radiologia Veterinária, Botucatu, SP, Brasil. CORRESPONDÊNCIA: J.C. Ferreira [jaircfvet@yahoo.com.br.]. Departamento de Reprodução Animal e Radiologia Veterinária, Universidade Estadual Paulista Júlio de Mesquita Filho. CEP18608-210 Botucatu, SP, Brasil. Doppler ultrasonography principles and methods of evaluation of theDoppler ultrasonography principles and methods of evaluation of theDoppler ultrasonography principles and methods of evaluation of theDoppler ultrasonography principles and methods of evaluation of theDoppler ultrasonography principles and methods of evaluation of the reproductive tract in maresreproductive tract in maresreproductive tract in maresreproductive tract in maresreproductive tract in mares Jair Camargo FerreiraJair Camargo FerreiraJair Camargo FerreiraJair Camargo FerreiraJair Camargo Ferreira11111, Fernanda Saules Ignácio, Fernanda Saules Ignácio, Fernanda Saules Ignácio, Fernanda Saules Ignácio, Fernanda Saules Ignácio22222 & Cezinande de Meira & Cezinande de Meira & Cezinande de Meira & Cezinande de Meira & Cezinande de Meira22222 ABSTRACT Background: Doppler ultrasonography is a non-invasive real time pulse-wave technique recently used for the transrectal study of the reproductive system hemodynamics in large animals. This technic is based in the Doppler Effect Principle that proposes the change in frequency of a wave for an observer (red blood cells) moving relative to the source of the respective wave (ultrasonic transducer). This method had showed to be effective and useful for the evaluation of the in vivo equine reproductive tract increasing the diagnostic, monitoring, and predictive capabilities of theriogenology in mares. However, an accurate and truthful ultrasonic exam requires the previous knowledge of the Doppler ultrasonography principles. Review: In recent years, the capabilities of ultrasound flow imaging have increased enormously. The current Doppler ultrasound machines offer three methods of evaluation that may be used simultaneously (triplex mode). In B-mode ultrasound, a linear array of transducers simultaneously scans a plane through the tissue that can be viewed as a two-dimensional gray-scale image on screen. This mode is primarily used to identify anatomically a structure for its posterior evaluation using colored ultrasound modes (Color or Spectral modes). Colored ultrasound images of flow, whether Color or Spectral modes, are essentially obtained from measurements of moving red cells. In Color mode, velocity information is presented as a color coded overlay on top of a B-mode image, while Pulsed Wave Doppler provides a measure of the changing velocity throughout the cardiac cycle and the distribution of velocities in the sample volume represented by a spectral graphic. Color images conception varies according to the Doppler Frequency that is the difference between the frequency of received echoes by moving blood red cells and wave frequency transmitted by the transducer. To produce an adequate spectral graphic it is important determine the position and size of the simple gate. Furthermore, blood flow velocity measurement is influence by the intersection angle between ultrasonic pulses and the direction of moving blood-red cells (Doppler angle). Objectively colored ultrasound exam may be done on large arteries of the reproductive tract, as uterine and ovary arteries, or directly on the target tissue (follicle, for example). Mesovarium and mesometrium attachment arteries also can be used for spectral evaluation of the equine reproductive system. Subjectively analysis of the ovarian and uterine vascular perfusion must be done directly on the corpus luteum, follicular wall and uterus (endometrium and myometrium associated), respectively. Power-flow imaging has greater sensitivity to weak blood flow and independent of the Doppler angle, improving the evaluation of vessels with small diameters and slow blood flow. Conclusion: Doppler ultrasonography principles, methods of evaluation and reproductive system anatomy have been described. This knowledge is essential for the competent equipment acquisition and precise collection and analysis of colored ultrasound images. Otherwise, the reporting of inconsistent and not reproducible findings may result in the discredit of Doppler technology ahead of the scientific veterinary community. Keywords: Doppler ultrasound imaging, hemodynamics, blood-red cells, spectrum, color-flow, power-flow. s106 J.C.J .C.J .C.J .C.J .C. F F F F Ferererererrrrrreireireireireira ,a ,a ,a ,a , F F F F F.S..S..S..S..S. Ignácio & C M Ignácio & C M Ignácio & C M Ignácio & C M Ignácio & C Meireireireireira .a .a .a .a . 2011. 2011. 2011. 2011. 2011. Doppler ultrasonography principles and methods of evaluation of the reproductive tract in mares. Acta Scientiae Veterinariae. 39(Supl 1): s105 - s111. I. INTRODUCTION II. COLORED ULTRASONIC IMAGING 2.1 Spectral mode 2.2 Color-flow mode III. ARTERIAL SYSTEM OF THE REPRODUCTIVE TRACT IV. CONCLUSION I. INTRODUCTION In large animals, Doppler ultrasonography is a non-invasive real time pulse-wave technique recen- tly used for the transrectal study of the reproductive system hemodynamics. The introduction of this technology in current researches has allowed reevaluating previously conceptions considered definitive regarding the physiology of reproduction. This method had showed to be effective and useful for the evaluation of the in vivo equine reproductive tract increasing the diagnostic, monitoring, and predictive capabilities of theriogenology in mares [4]. Color-Doppler ultrasonic imaging is based in the Doppler effect principle that proposes the change in frequency of a wave for an observer moving relative to the source of the respective wave (Figure 1). The frequency is constant when the wave source and observer are stationary. However, if there is a moving toward or away from each other, the returning echoes frequency increase and decrease, respectively [7]. At the moment, this principle is used for different applications as temperature, velocity and vibration measurements. In Doppler sonography, the wave source and stationary object are, respectively, the red- blood cells and the transducer. During an ultrasonic doppler exam is possible to perform a qualitatively and/or quantitatively evaluation of specific vessels and tissues. The direction and velocity of red-blood cells can be represented by different types and intensities of colors pixels or as a velocity spectral graphic. However, to select the most appropriated mode of exam first it is necessary to have a complete knowledge of the Doppler ultrasonography principles and methods of evaluation. II. COLORED ULTRASONIC IMAGING There are two specifically approaches for colored ultrasonic imaging evaluation: Color-flow and Spectral modes (Figure 2). Color-flow mode uses Color-Doppler signals superimposes on a B-mode Figure 1. Collection of images representing the Doppler Effect. Wave frequency emitted by a static object (Vsource = 0) relative to the observer is constant, concentric and equally spaced. When the pulse is emitted during relative motion between source and observer, the wave fronts are no longer concentric. Wave frequency of echoes increases and decreases, respectively, during approach and away movements (Vsource < Vsound). When the velocity of the object is similar to the sound speed (Vsource = Vsound), the crests of waves emitted overlap forming a single ridge that reaches the observer simultaneously with the source. Emitted wave reaches the observer after passing the sound source at supersonic speeds (Vsource > Vsom). Font: Personal file. Figure 2. Ultrasonic images collection of female reproductive tract in B, Power, Color and Spectral modes (A, B, C and D, respectively). (A and B) Cross section image, using B-mode and Power-flow Doppler, of a non-pregnant uterine horn in diestrus. (C) Color Doppler evaluation of a pre-ovulatory follicle. (B and C) Presence of colored pixels within uterine tissue (endometrium and myometrium) and follicular wall indicate the red blood cells displacement and have been used to estimate the tissue vascularity. (D) Spectral graph consisting of maximum speeds values of blood flow in mesometrial artery. Source: Personal file. s107 N J.C.J .C.J .C.J .C.J .C. F F F F Ferererererrrrrreireireireireira ,a ,a ,a ,a , F F F F F.S..S..S..S..S. Ignácio & C M Ignácio & C M Ignácio & C M Ignácio & C M Ignácio & C Meireireireireira .a .a .a .a . 2011. 2011. 2011. 2011. 2011. Doppler ultrasonography principles and methods of evaluation of the reproductive tract in mares. Acta Scientiae Veterinariae. 39(Supl 1): s105 - s111. image of a structure to estimate its vascularity, while pulsed-Doppler spectral analysis of blood velocities of a specific artery is done on Spectral mode. Both methods are based on Doppler-shift frequencies, also called Doppler frequency [7]. Doppler frequency is the difference between the frequency of received echoes and frequency transmitted by the transducer (Figure 3). The transducer transmitted frequency is constant while the frequency of the returning echoes varies according to the Doppler Effect principle. Therefore, when the wave source (blood-red cells) is stationary or moving parallel to the transducer there is no difference between transmitted and returning frequencies and colored Doppler signals are not detected. If the blood flow moves toward to the transducer, the returning frequency is greater than the transmitted frequency resulting in a positive Doppler frequency. A negative Doppler signal is produced when the returning frequency is lower than the transmitted frequency or, in other words, when the blood-red cells moves away to the transducer [11]. 1.1 Spectral mode In the spectral mode, blood flow velocity variations are represented as a graphic wave form called spectrum (Figure 4). The spectrum provides maximum velocities values as Peak systolic (PSV), End diastolic (EDV) and Time-average maximum (TAMV) velocities. PSV is the maximum point along the length of the spectrum, while EDV is the ending point of the cardiac cycle. TAMV is the maximum velocity values average. Additionally, the position of the spectrum in relation to the baseline of the monitor indicates the blood flow direction. By convection, wave forms above and under the baseline indicate, respectively, blood-red cells moving toward and away fromthe transducer [4]. To measure blood velocities is necessary to determine the Doppler angle. Doppler angle or angle of insonation is the angle of intersection of the ultra- sonic pulse with the direction of moving blood-red cells (Figure 5). To produce an accurate spectrum is required a doppler angle of 30° to 60°. However, determine the angle of insonation from uterine Figure 3. Illustration of frequency echoes changes during a colored Doppler ultrasonic exam. Doppler frequency varies according to erythrocytes movement in relation to the transducer. (A and B) Frequency of echoes sent and received is similar in cases of stacionary sources or perpendicular motion, resulting in no production of colored pixels. (C) During an approach movement, the received frequency is greater than the emitted, generating positive Doppler signals. (D) Received frequency is lower than emitted frequency when red cells move away from the transducer, producing negative Doppler signals. Fontt: Personal file. s108 J.C.J .C.J .C.J .C.J .C. F F F F Ferererererrrrrreireireireireira ,a ,a ,a ,a , F F F F F.S..S..S..S..S. Ignácio & C M Ignácio & C M Ignácio & C M Ignácio & C M Ignácio & C Meireireireireira .a .a .a .a . 2011. 2011. 2011. 2011. 2011. Doppler ultrasonography principles and methods of evaluation of the reproductive tract in mares. Acta Scientiae Veterinariae. 39(Supl 1): s105 - s111. (mesometrial, endometrial and myometrial) and ovarian (mesovarium, luteal and follicular) arteries is difficult considering their tortuous anatomy. An alternative used in early studies [2,3,9,10] is to calculate the Doppler index. They are good indicators of vascular perfusion of reproductive organs and are independent of the doppler angle [7]. Resistance and Pulsatility indeces (RI and PI, respectively) are calculated using the ratio of the three velocities measu- rements (PSV, EDV and TAMV) and both indices have a negatively correlation with the vascular perfu- sion of the tissue downstream from the sample gate [4]. For providing precisely values of blood velocities, spectrum mode is an objectively method for the evaluation of normal and pathologic blood flow disturbance at reproductive system. However, even showing a great potential for research and clinical purposes it is avoided by equine farm veterinaries in consequence of the difficulty and time Figure 4. Spectral Doppler evaluation of mesometrial attachment artery. The gate (yellow) is placed on an artery of the mesometrial attachment (red) to capture velocities data of uterine blood flow and, posteriorly, produce a spectral graph. Peak systolic (PSV) and end diastolic (EDV) velocities are, respectively, the maximum and ending blood flow velocities of a cardiac cycle. TAMV is the average of an espectral maximum blood flow velocities. Doppler device automatically calculates Pulsatility and Resistance indices (RI and PI, respectively). Spectral graph located above the baseline indicates erythrocytes moving toward to the transducer. Font: Personal file. Figure 5. Cosine values of the Doppler angle (cosO) and relationship between insonation angle and accuracy of measurement of blood flow velocities. Determination of blood flow velocity varies with the Doppler frequency (DF). DF = 2 ft. v. cosθ /c. While the movement of erythrocytes in relation to the transducer is perpendicular (90 °), cosθ is zero and the device do not produce Doppler signals. 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