Echocardiographic reference ranges for sedated healthy peccaries ( Tayassu tajacu, Linnaeus , 1758 )

Peccaries are wild mammals belonging to the Tayassu genus that are found almost everywhere in the Americas and have demonstrated great potential as an experimental model for scientific investigations. Twelve healthy adult animals were sedated to perform echocardiographic examinations in B, M and Doppler mode. The variables that exhibited statistically significant correlation coefficients with weight were LVFWd, LVIDd, LVIDs, E wave, A’RV, MAM, and TAPSE. The HR exhibited a negative relationship with the IVRT. The LA variable showed a positive correlation with the AO. The MAM exhibited correlations with the LVIDd and LVIDs. The TAPSE showed positive correlations with the E’RV and A’RV. The present study provides the first reference values for echocardiographic measurements in B, M and Doppler modes from peccaries anesthetized with ketamine and midazolam. Echocardiography was easy to perform in collared peccaries, and the collected data revealed values that can aid in their clinical management and conservation.


INTRODUCTION
 Peccaries (Tayassu tajacu, Linnaeus, 1758) are wild mammals belonging to the Tayassuidae family and Tayassu genus. They are found experimental models for evaluating the evolution of diseases, peccaries are a good alternative model for scientific investigations (Bezerra et al., 2014).
Echocardiography is a non-invasive and sensitive imaging method used to obtain cardiovascular hemodynamic data. Studies have demonstrated strong correlations between parameters obtained with echocardiography and those obtained with more expensive methods, such as magnetic resonance imaging (Shukan and Hitt, 2011).
Advanced echocardiography techniques have been incorporated into veterinary medicine in experiments using different animal species (Chetboul et al., 2005;Lee et al., 2007). These studies have demonstrated numerous similarities in the parameters of cardiac function between animals and humans.
Peccaries can only be safely handled after the administration of chemical restraint, and all hemodynamic changes promoted by anesthetic or sedative agents should be considered (Bertozzo et al., 2008). Moreover, to date, no study of the hemodynamic profile of this species as evaluated by Doppler echocardiography has been reported.
Ketamine is widely used in the chemical restraint of wild animals because of its low lethality and high margin of safety (Green et al., 1981). The combination of ketamine and midazolam promotes adequate myorelaxation, thus reducing muscular hypertonicity and promoting tranquilization, hypnosis and amnesia, and possesses an anticonvulsive activity (Valadão, 2002). This combined anesthetic protocol is commonly used in procedures with small animals and has been demonstrated to be a good option for work with wild animals (Diniz et al., 2017).
The aim of this study was to characterize the echocardiographic values of measurements of the cardiac chambers and variables related to the cardiovascular hemodynamics of peccaries (Tayassu tajacu, Linnaeus, 1758) after sedation with a combination of ketamine hydrochloride and midazolam.

MATERIAL AND METHODS
Twelve peccaries from the Center for the Study and Preservation of Wild Animals (NEPAS/CCA-UFPI) of the Federal University of Piauí, Teresina-PI were used. The animals were randomly grouped in bails (10x5x3m) and were distributed in two groups consisting of 6 males and 6 females each. All animals were adults with a mean age of three years and weights ranging from 15 to 20kg. The animals were fed twice daily ad libitum with a commercial diet for domestic pigs that was based on soybean meal and contained 18% crude protein and various vegetables as a source of fiber, energy, minerals and water. The animals were submitted to clinical, hematological and biochemical examinations . Auscultation of the pulmonary fields and cardiac sounds was performed to identify valve insufficiencies and cardiac rhythm disturbances. This examination was followed by an electrocardiographic examination (Tilley, 1992) and an echocardiographic screening (Boon, 2011).
The peccaries were previously prepared with a 12-hour solid fast and a 4-hour water fast. Later, the animals were captured using restraint nets (dip nets) and physically restrained with leather gloves for the application of the anesthetic drugs.
Sedation occurred due to the intramuscular application of the combination of 15mg/kg ketamine hydrochloride (5%) and 1.0mg/kg midazolam. Approximately 5 to 10 minutes after the application, the anesthetic action was observed, and the procedures to perform the echocardiographic examinations were initiated. The protocols produced an average anesthetic time of 30 to 40 minutes in all animals, and there was no need to reapply the drugs during the examinations.
Transthoracic echocardiography with continuous monitoring was performed using an M-Turbo system 5 (FUJIFILM ® SonoSite, Washington 21919, USA) equipped with a 4.0-8.0MHz phased-array transducer (Px10, FUJIFILM ® SonoSite, Washington 21919, USA). The hair was clipped between the right fourth and sixth intercostal spaces, and coupling gel (Mercur ® , São Paulo, Brazil) was applied to this thoracic area.
The echocardiographic examinations were performed, and the standard measurements were collected according to previously established protocols for small animals (Boon, 2011). The left ventricular free-wall and interventricular septal thicknesses in diastole and systole (LVFWd, LVFWs, IVSd, IVSs), left ventricular end-diastolic and end-systolic diameters (LVIDd and LVIDs), and the diameters of the aortic root 2D (AO) and left atrium (LA) were measured in the parasternal right projection. The shortening fraction (FS) and the ejection fraction (EF-Simpson's Method) were calculated. Additionally, the mitral E-point septal separation (EPSS), the final diastolic ratio between the aorta and the left atrium (LA/AO) and the flow velocity in the pulmonary artery (Pmax) were also measured ( Figure 1). The heart rate (HR) was obtained from the Doppler tracing of the pulmonary artery. Flow assessments of the mitral, tricuspid and semilunar valves, as well as the large vessels were performed using color and spectral Doppler on each individual valve. The 2D sector size was adjusted to improve image quality as well as calibrating the color gain to demonstrate excellent filling of the investigated chambers and vessels. The highest pulse repetition frequency (PRF) was used to prevent signs of aliasing during normal flows.  Graphpad Prism 7 software was used to analyze the data and to perform non-parametric Wilcoxon-Mann-Whitney (Mann-Whitney U) tests to verify the existence of differences between the variables according to the sex of the animals. The Spearman rank correlation coefficient was used to assess dependence. In these tests, 0.05 was considered as the level of significance. Table 1 presents the echocardiographic parameters of the studied group of animals. Comparisons of the means between the males and females did not indicate significant differences (P> 0.05, P= 0.337). Thus, the other statistical treatments were based on the total sample of 12 animals. The variation reference intervals were determined by the 95% tolerance interval calculations and were designed to cover 99% of all future events. The variables that exhibited statistically significant correlation coefficients with respect to weight are presented in Table 1. The other variables that exhibited correlations are described as follows:

RESULTS
The HR was negatively correlated with the IVRT (r= -0.46). The LA variable was positively correlated with the AO (r= 0.57). MAM was correlated with the LVIDd (r= 0.41) and LVIDs (r= 0.32). The TAPSE was positively correlated with E'RV (r= 0.4) and A'RV (r= 0.43).

DISCUSSION
Echocardiographic measurements were obtained following previously established protocols that ensure safe measurements and diagnostic imaging and have been performed in other studies of wild species, such as wild dogs (Garcia et al., 2016).
In veterinary medicine for domestic animals, echocardiographic parameters are well documented, and standardized values are available for several species including canines, felines, equines and domestic pigs (Lee et al., 2007;Boon, 2011). However, few studies have examined the hemodynamics and cardiac functions of wild animals (Cetin et al., 2005;Mantovani et al., 2012;Diniz et al., 2017). This is the first study to report measurements of the echocardiographic variables of chemically restrained peccaries (Tayassu tajacu).
In the present study, several variables exhibited statistically significant correlation coefficients in relation to weight (LVFWd, LVIDd, LVIDs, E wave, A'RV, MAM, and TAPSE). Corroborating the findings of this study, similar relationships have been described between body weight and LVIDd, LA, AO and SIV in dogs of different breeds (Pellegrino et al., 2007). Additionally, positive correlations of body weight with LVIDd, IVS, LVFW, LA and AO were observed by Bradley and Ross (1987) in sheep.
The EPSS values of peccaries (0.57±0.05cm) were similar to those of wines (0.56±0.03cm) and minipigs (0.56±0.04cm) (Lee et al., 2007) and were within the normal range for dogs (≤0.77cm) (Boon et al., 2011). These results are consistent with the literature because most reports suggest that normal EPSS values are less than 0.6cm for most domestic and wild species (O'Grady et al., 1986;Diniz et al., 2017).
No statistical correlation was found between the FS (%) and weight, as has been described in dogs and humans (O'Grady et al., 1986;Feigenbaum, 1994). For peccaries (26±1.70), this variable presented values similar to those found in minipigs (24.35±0.68) (Lee et al., 2007) and lower than those found in domestic pigs (35.95±4.15) (Lee et al., 2007). A depressant effect of ketamine is associated with a decrease in FS when it is used as the sole agent or in anesthetic combination in cats and mice (Roth et al., 2002).
TDI has been demonstrated to be a sensitive technique for the quantitative evaluation of segmental myocardial movement (Wess et al., 2011) and is reproducible for examinations of basal cardiac function (D'Hooge et al., 2000)) in peccaries. In humans, TDI is useful for the differential diagnosis of heart diseases, the follow-up of cardiac transplantation and the evaluation of left and right ventricular function (Vinereanu et al., 2001). The mean velocity peak of the E' wave of peccaries (8.76±2.11cm/s) was greater than that of the A' wave (5.67±1.29), as has been observed in anesthetized cats (E': 7.2±2.5, A': 4.7±1.5) (Borlini et al., 2009) and dogs (E': 6.9; A': 7.4) (Dickson et al., 2017). Similar to LV TDI, the RV exhibited a negative diastolic velocity curve composed of the E'RV (11.35±2.41) and A'RV (6.11±1.53) waves. These characteristics have been reported for dogs of different breeds (Chetboul et al., 2005). The TAPSE demonstrated positive correlations with the E' RV (r= 0.4) and A' RV (r= 0.43), and these correlations are explainable as the result of normal RV functioning during systole, which influences the RV TDI values measured adjacent to the tricuspid annulus (Kjaergaard et al., 2009).
The IVRT was found to be consistent and to exhibit little variability between the studied animals. The values found for this variable (65±3.20m/s) were similar to findings that have been reported for humans (65±20ms/s) (Graziosi, 1998).
Tachycardia and sympathetic stimulation may exacerbate the IVRT, reduce diastolic filling time and accelerate the early LV diastolic elastic recoil. The partial drainage of the LV caused by chronic mitral valvular insufficiency, along with the activation of the sympathetic system, leads to LV hyperkinesia, an increase in HR and a reduction in the IVRT (Pipers et al., 1981). Indeed, although in the present study the IVRT values were within the normal limits, they exhibited a negative correlation with the HR (r= -0.46).
Some limitations of this study should be considered: although these examinations have been performed by an experienced operator, we believe that the small number of animals may be responsible for the deviation found within each variable. In addition, the action of the studied drugs, which although well characterized in domestic species, still have few references to its application in wild species. Nevertheless, in the authors' knowledge, this is the first study regarding the echocardiographic assessment of peccaries, allowing the preliminary acquisition of knowledge of the hemodynamic effect of drugs most commonly used for sedation and anesthesia in this species.

CONCLUSION
The present study provided the first reference values for B, M and Doppler mode echocardiographic measurements of peccaries (Tayassu tajacu) anesthetized with ketamine and midazolam. Echocardiography of collared peccaries was easy to perform, and the collected data revealed values that can aid in their clinical management and conservation.