Comparison of echocardiographic parameters in Santa Inês sheep breed and healthy Brazilian human adultsABSTRACT
The aim of this study was to compare the values of echocardiographic parameters in healthy Santa Inês sheep with standardized values for normal Brazilian human adults. Two-dimensional echocardiography, Movement mode, and pulse wave and color flow Doppler were performed in 39 non-sedated young adult sheep of Santa Inês breed. Dimensions such as the end-diastolic diameter, ejection fraction, and shortening fraction were similar to those of Brazilian human adults. The end-diastolic and -systolic volumes were within the intervals established by the American Society of Echocardiography and the European Association of Echocardiography for normal human adults; guidelines followed in Brazil as references for humans. The normalized end-diastolic diameter exceeded the normal upper limit but approached it when the index was corrected with the cardiac mass/body surface area of sheep in relation to humans. Diastolic parameters were also within normal human intervals, except for a slightly shorter Mitral E-wave deceleration time. We conclude that the echocardiographic parameters of the systolic and diastolic performance of the left ventricle in Santa Inês sheep can be reliably extrapolated to that of Brazilian human adults, thus supporting the use of national sheep models in human heart disease research.
Keywords:
heart disease; sheep models; echocardiographic; Santa Inês
RESUMO
O objetivo deste estudo foi comparar os valores dos parâmetros ecocardiográficos em ovinos Santa Inês saudáveis com valores padronizados para adultos humanos brasileiros normais. Ecocardiografia bidimensional, modo de movimento e Doppler de onda de pulso e fluxo colorido foram realizados em 39 ovinos não sedados da raça Santa Inês. Dimensões como diâmetro diastólico final, fração de ejeção e fração de encurtamento foram semelhantes às dos adultos humanos brasileiros. Os volumes diastólico final e sistólico estavam dentro dos intervalos estabelecidos pela Sociedade Americana de Ecocardiografia e pela Associação Europeia de Ecocardiografia para adultos humanos normais, diretrizes seguidas no Brasil como referência para humanos. O diâmetro diastólico final normalizado excedeu o limite superior normal, mas aproximou-se dele quando o índice foi corrigido com a massa cardíaca/área de superfície corporal de ovinos em relação aos humanos. Os parâmetros diastólicos também estavam dentro dos intervalos humanos normais, exceto por um tempo de desaceleração da onda E mitral ligeiramente mais curto. Concluiu-se que os parâmetros ecocardiográficos do desempenho sistólico e diastólico do ventrículo esquerdo em ovinos Santa Inês podem ser extrapolados de forma confiável para os de adultos humanos brasileiros, apoiando assim o uso de modelos nacionais de ovinos na pesquisa de doenças cardíacas humanas.
Palavras-chave:
cardiopatias; modelos ovinos; ecocardiografia; Santa Inês
INTRODUCTION
The sheep species has been widely used as an experimental model not only in veterinary medicine but also in translational research (Jousset et al., 2012). According to Rabbani et al. (2008) concluded that some anatomical and physiological structures of sheep were similar to those in humans when anatomically studying the species. As a result, in addition to the species’ abundant availability, low maintenance requirements, and favorable size, it became a model for experimental research and comparisons (Lavinsky and Seibel, 2001).
Santa Inês sheep farming takes place throughout Brazil. Among the advantages of the breed are the fact that it does not require shearing or tail docking, it comes into heat at any time of the year, it is less susceptible to ecto and endo parasites, it has a lower incidence of hoof problems, it is prolific, and has good mothering ability (Veríssimo et al., 2010).
The main areas of basic and translational cardiovascular research in which animal models are used are as follows: myocardial regeneration, ischemic heart disease, heart failure, and neovascularization studies. Pigs and sheep are suitable species for this study, since humans have a limited ability to develop collateral circulation. For studies on myocardial regeneration, sheep have an advantage; unlike pigs, sheep cardiac myocytes have only 1-4 nuclei, thus being more similar to the human cardiomyocyte (Locatelli et al., 2011).
Extrapolation of research findings to the clinical scenario requires knowledge of cardiac function, values, and normal cardiographic indexes in sheep (Jousset et al., 2012). The aim of this study was to compare the values of Doppler, B-mode and M-mode echocardiographic parameters of healthy Santa Inês sheep with the standardized values of normal Brazilian human adults.
MATERIAL AND METHODS
All experimental procedures were submitted for evaluation and approved by the Ethics Committee on the Use of Animals (CEUA/UFF) of the Fluminense Federal University (CEUA No. 8708221021). In addition to the guidelines established by the abovementioned Ethics Committee, the experiment also followed the ARRIVE guidelines described by Percie du Sert et al. (2020). The study was carried out in partnership with Fazenda Cardeiros, located in the municipality of São Pedro da Aldeia, RJ.
Thirty-nine adult Santa Ines sheep, average age 1.26 years, weighing on average 40.6±9.8kg (range 20.7-62.3kg) were studied. All sheep included were dewormed and vaccinated. The appropriate health condition was evaluated by a professional veterinary team by clinical examination, laboratory tests such as blood count and fibrinogen analysis and electrocardiographic examination. Animals with heart diseases diagnosed by echo and electrocardiographic were not included in the study. The sheep were mechanically restrained by the usual handler.
A noninvasive transthoracic echocardiographic examination was performed in right and left lateral decubitus of non-sedated sheep. Images and measurements were obtained using the two-dimensional methods, M-mode, and pulse wave and color flow Doppler (Doppler Echocardiograph Siemens® Acuson P500) with the P4-2 transducer with a frequency of 2-4MHz.
In echocardiographic evaluation, by means of the right-sided parasternal projection and cross-sectional area of the left ventricle at the height of papillary muscles, measurements were obtained (shown in Fig. 1) of the left ventricular end-diastolic diameter (LVIDd), left ventricular systolic diameter (LVIDs), left ventricular diastolic wall thickness (LVPWd), left ventricular systolic wall thickness (LVPWs), calculation of the Ejection Fraction (EF) and Shortening Fraction (SF) of the left ventricle, end-diastolic volume (EDV), and end-systolic volume (ESV).
In the left parasternal window, by means of the apical four-chamber cut, the Mitral E-wave velocity (VelEMit), Mitral A-wave velocity (VelAMit), calculation of the relationship between the Mitral E/A waves, E-wave deceleration time (E-wave DT), isovolumetric relaxation time (IVRT), inspection of the Mitral and tricuspid valves morphology, as well as the direction of the flow through color Doppler were obtained.
To establish the reference values, all parameters were normalized per square meter (m2) of body surface area (BSA). The BSA was calculated using the following equation (Guyton et al., 1973): BSA (m2)=0.84×body weight (kg)0.66.
All measurements were performed according to the guidelines of the American Society of Echocardiography (ASE) and the European Association of Echocardiography (EAE), the guidelines also followed by the Brazilian Society of Cardiology (Lang et al., 2005; Nagueh et al., 2009).
The numerical variables were analyzed as arithmetic mean and standard deviation (SD) if normally distributed, and as median and interquartile deviation if otherwise. The normality of the distribution was evaluated by the Kolmogorov-Smirnov test. The comparison of the means was performed by the t-test (normal distribution) or by the Mann-Whitney Test (nonnormal distribution). A p-value of <0.05 was considered to indicate statistical significance. All analyses were performed with the BioEstat 5.3 program (Ayres et al., 2007).
RESULTS
Figure 2 shows two two-dimensional echocardiographic images, an M-mode study of the sheep’s left ventricle. Images of the EF calculation and SF are also shown.
Figure 3 shows pulse wave and color flow Doppler images. Images of the calculation of the relationship between Mitral E and A waves are also shown.
Table 1 lists the values of the dimensions of the left ventricle, left atrium, and aorta, in addition to systolic function indexes of the experimental animals.
Two-dimensional vision, M-mode, right parasternal window, cross-sectional cut, and height of the papillary muscles of the sheep’s left ventricle (left). Two-dimensional vision, M-mode, right parasternal window, cross-sectional cut, and base of the left aorta-atrium heart (right). LVIDd: left ventricular end-diastolic diameter; LVIDs: left ventricular end-systolic diameter; LVPWd: left ventricular diastolic wall thickness; LVPWs: left ventricular systolic wall thickness; IVSd: interventricular septum diastolic thickness; IVSs: interventricular septum systolic thickness; Ao: aorta artery; Ae: left atrium. Photo: André Luiz de Souza Teixeira. São Pedro da Aldeia/RJ, 2021.
Apical four-chamber longitudinal cutting shows the pulse wave Doppler study of the transmitral flow in sheep. Mitral E and A waves and IVRT are observed. Photo: André Luis de Souza Teixeira. São Pedro da Aldeia/RJ, 2021
Table 2 demonstrates diastolic function parameters and Doppler study of pulmonary and aortic flow.
For the diameters and volumes of the LV, the absolute and normalized values are shown. BSA was 1.11±1.91m2.
The mean heart rate was 105±15.36 beats per minute (bpm). Most echocardiographic measurements presented normal statistical distribution, except for the free wall thickness of the left ventricular diastole and systole and the EDV.
In Table 1, the measurements of sheep in the present study were compared with the reference values of a study in the Brazilian population (Ângelo et al., 2007), the Guidelines of the ASE and the EAE. In Tab. 2, the values of the present study are only compared with the guidelines of ASE and EAE (Lang et al., 2005; Nagueh et al., 2009). The blank spaces correspond to values that are not included in the guidelines and references.
DISCUSSION
In the present study, the echocardiographic evaluation of the left ventricular function parameters was performed in 39 young, healthy, and non-sedated adult Santa Inês sheep. It should be noted that the measurements and calculations were based on parasternal incidences, since the morphology of sheep thorax causes apical effort, which makes it difficult to obtain adequate apical views and four chambers for the evaluation of motility and segmental volumes (Locatelli et al., 2011). In addition to the morphology of the sheep thorax, the fact that the tests were performed without sedation (similar to the examination in humans), obtaining necessary images and measures was a challenge. Even so, it was possible to obtain reliable Doppler measurements. Our results show that the two most used LV systolic performance indexes, the EF, and the SF, as well as the normalized volumes of the LV end-diastole (EDV) end-systole (ESV), present a mean within the reference range of healthy humans, according to Ângelo et al. (2007) and the guidelines of the ASE and EAE (Lang et al., 2005; Nagueh et al., 2009), respectively.
The values of cardiac systolic function, LVIDd, EF, and SF, are within the normal range of adult humans, according to Ângelo et al. (2007), which was the study conducted with Brazilians. The values of EDV and ESV, according to the guidelines of ASE and EAE, are also within normal limits compared to the values established for humans (Lang et al., 2005). However, LVIDd was the only mean measure statistically equal to the reference of ASE and EAE. The fact that the function parameters of the left ventricle of healthy sheep are similar to the values in humans does not mean that the sheep model would behave similarly in the scenario of human diseases or physiological disorders. It only provides a solid basis for proposing echocardiographic performance variables as the LV as reliable results in this type of study (Locatelli et al., 2011).
Regarding diastolic function, all values fit within the normal ranges of adult humans, except the deceleration time of the Mitral E-wave. In the comparison of means, only the ratio of the Mitral E and A waves had no significant difference (p > 0.05) between sheep and humans. The values of this parameter were based on Klein et al. (1994) as recommended in the ASE and EAE guidelines (Nagueh et al., 2009).
Echocardiographic values when compared with other studies of sheep, present a significantly lower number of sheep and breeds with lower expression in Brazil than Santa Inês. In eight Dorset sheep, Moainie et al. (2002) reported a LV EF of 41.2%±6.7%, much lower than the 70.21%±7.70% of our sheep. The EF presented by Locatelli et al. (2011) was 56.7%±8.9% in Corriedale sheep. This may be because Moainie sheep are under general anesthesia (1%-2% isoflurane in oxygen), and the Locatelli sheep are only slightly sedated with diazepam, while in our study no type of sedation or anesthesia was used. Therefore, the EF values in the present study were not within the intervals established by the ASE and EAE for normal adult humans, as in studies with African and New Zealand breeds.
Borenstein et al. (2006); Psaltis et al. (2008); Locatelli et al. (2011) evaluated the SF of the LV in sheep. In these cases, the values of this parameter (38.4%±5.7%; 31.6%±1.8%, and 32.0%±8.0%, respectively) were similar to ours (39.43%±6.47%), although in the first two studies, the animals (n=10) were under general anesthesia (isoflurane 2%-3%) and in the third study they were only sedated.
It is noted that while the normalized EDV of sheep is within the normal human range, according to the ASE and EAE, the end-diastolic diameter is within the reference values, only according to Ângelo et al. (2007), because its normalized value (33.48±2.60mm/m²) exceeds the upper limit of the human range (24-31mm/m²), according to the guidelines of the ASE and EAE (Lang et al., 2005; Nagueh et al., 2009). The ratio between left ventricular mass (in gram) and BSA (in m2) is approximately 20% lower in normal human adults than in sheep weighing 30kg (Locatelli et al., 2011). By subtracting 20% of the measured value, the sheep end-diastolic diameter falls within the range established for humans. For the end-diastolic and end-systolic volumes, the standardized ranges for humans are considerably larger than for the end-diastolic diameter (note that there is a 100% variation between the upper and lower limits); thus allowing our measured volume values to be within the corresponding human ranges, regardless of the application of the correction factor.
Regarding wall thickness, the end-diastolic value is higher than the normal range for the humans, according to the Guidelines of ASE and EAE, which also do not report an interval for end-systolic thickness. However, according to Ângelo et al. (2007), the end-systolic wall thickness of the ventricle in sheep in the present study is also higher than the normal range found in humans. Our measured value was 14.00±2.55mm, representing a systolic wall thickening of approximately 34% in relation to the normal human values reported for two-dimensional echocardiography (Feneley and Hickie, 1984). In a study by Locatelli et al. (2011), this value was 10.5±1.3mm, corresponding to 42% thickening.
Diastolic function was evaluated using tissue Doppler imaging by Hashimoto et al. (2004) in eight sheep weighing 35-47kg. The Mitral E/A ratio was 0.99±0.82, lower than that of our sheep (1.20±0.16). Locatelli et al. (2011) reported results higher than ours (1.41±0.38), but still within the reference values for humans. These differences are justified due to the different experimental conditions (in Hashimoto et al. (2004), the examination was performed with general anesthesia, open chest, and epicardial recordings) thus preventing reliable comparisons with the present study. The second study mentioned was conducted in sedated animals.
In our experimental conditions, very similar to the human clinical scenario, all diastolic values, except the deceleration time of the Mitral E-wave (E-wave DT), were within the normal human range between the age group 21-40 years according to the ASE and EAE (Nagueh et al., 2009). Another study, with sedated sheep, found similar results (125.2±36.4) to the present study 129.13±37.53 (Locatelli et al., 2011). These mean values, found in sheep, were only slightly lower than the minimum limit of the normal range of healthy humans. Therefore, this difference should not challenge the validity of sheep as a model of human diastolic function.
In this study, the IVRT in sheep was (77.45±23.23ms) higher than the average in humans, but within the reference value in this species and was also similar to another study in sedated sheep (71.4±9.6ms) (Locatelli et al., 2011). The IVRT varies with heart rate; as the frequency increases, IVRT decreases, and this decreased value reveals a poor coronary perfusion and lower efficacy of cardiac contraction (Boon, 2011).
CONCLUSIONS
The echocardiographic parameters of the systolic and diastolic performance of the left ventricle in healthy, non-sedated adult Santa Inês sheep can be reliably extrapolated to Brazilian human adults, thus supporting the use of the national Santa Inês sheep breed models in heart disease research.
ACKNOWLEDGMENTS
This work was supported by the Coordination for the Improvement of Higher Education Personnel (CAPES, 2023).
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Publication Dates
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Publication in this collection
21 Feb 2025 -
Date of issue
Mar-Apr 2025
History
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Received
14 Dec 2023 -
Accepted
19 Sept 2024
