Cardiorespiratory Effects of Three Infusion Doses of Adenosine in Conscious Goats: A Preliminary Study

Adenosine (AD) has been implicated in human healthcare as an endogenous signaling nucleotide in both physiologic and pathologic states. The effects of AD on cardiorespiratory parameters in ruminants has not yet been studied. The objective of this study was to evaluate the cardiac and respiratory changes that resulted from an intravenous AD infusion in goats. Six clinically healthy adult goats weighing 28 ± 2 kg were randomly assigned to one of four treatments in a crossover design with a seven day washout period. The goats received a 0.9 % saline solution (SAL treatment) and three AD treatments (AD 50, 100 and 200) intravenously at a dose rate of 50, 100 and 200 μg/kg/min. Cardiorespiratory and key cardiac parameters were measured before the treatment (baseline), during the infusion (dInf) and at 1, 3, 5 and 10 min after each infusion was discontinued. The AD 100 produced a significant increase in HR (p = 0.001) and the AD 200 resulted in significant rises in HR (p = 0.006) and RR (p = 0.001) compared with the baseline. This study concluded that the AD infusion could trigger an increase in HR and RR in a dose-dependent manner in healthy goats.


Introduction
Adenosine (AD) is a purine nucleoside cell signaling agent that exerts its biological effects via four distinct receptors (A 1 , A 2A , A 2B, A 3 ). It has a short half-life ranging from seconds to minutes [1]. AD receptors play a pivotal regulatory role not only in normal cell biology and physiology but also in pathologic states. The binding of AD to A 1 receptors inhibits the catecholamine release and slows the atrioventricular conduction in the heart [2]. Additionally, AD promotes vasodilation in all but renal vasculature [3][4][5].
Not all AD receptors appear to exert the same physiologic effects. The binding of AD to A 2A receptors indirectly increases cardiac contractility in humans by modulating the antiadrenergic effect that AD has when it binds to A 1 [6]. Recent reports within the human medical literature have confirmed that AD has the potential to promote a sympathetic outflow and chemoreceptor activation through this mechanism [7][8][9][10][11].
Goats are increasingly raised as companion animals and maintaining their cardiovascular health is an important part of preventative care. Several cardiac arrhythmias such as sinus tachycardia and respiratory sinus arrythmia have been reported in goats and sheep [12]. In addition, supraventricular (SVT) and ventricular tachycardias have been induced by bupivacaine, a commonly used local anesthetic in small ruminants [13].
AD has been increasingly used in human clinical trials as a diagnostic and antiarrhythmic agent. In patients with paroxysmal SVT, AD could slow down atrioventricular Six clinically healthy goats ranging from 13-15 months of age and a body weight of 28 ± 2 kg were enrolled in this study. For the purpose of this study, the animals were considered physically fit as determined by a comprehensive physical examination (including cardiothoracic auscultation, ECG, echocardiography and testing within the normal reference ranges for packed cell volume (PCV), complete blood count (CBC) and serum biochemical profiles).
The six goats enrolled in this study were selected from a group of thirty goats based upon their handleability. The group was purchased locally and brought into an experimental research unit one week prior to the infusions for daily acclimatization to handling and the environmental design for a duration of thirty minutes per day. This study was approved by the animal experimental ethical inspection of the Laboratory Animal Center, Huazhong Agricultural University (HZAUGO-2019-004).

Study Design
This experimental study used a prospective, randomized crossover design with a seven day washout period. Six adult goats were randomly assigned to one of four treatments using a computer program (www.randomizer.org) (accessed on 11 October 2019) in which all goats received the four treatments in a random order (Figure 1). 100 and 200 μg/kg/min for 2 min using a syringe driver through their jugular catheter. There was a 1 min stabilization period before taking measurements after starting the infusion. AD was diluted to reach a concentration of 600 mg/mL so that the total volume infused ranged from 2 to 10 mL. Identical volumes of saline were infused in the SAL treatment. Figure 1. Different treatment sequence in the randomized crossover design. Each goat received saline (SAL) and adenosine at a dose rate of 50, 100 or 200 μg/kg/min but in a random sequence with a washout period of 7 days.
Cardiorespiratory and key echocardiographic parameters were measured before the treatment (baseline), during the infusion (dInf) and at 1, 3, 5 and 10 min after discontinuing each infusion ( Figure 2).

Figure 2.
Study timeline showing electrocardiogram (ECG) recordings and echocardiographic measurements in goats receiving saline (SAL) and three adenosine (AD) doses at the baseline, during the infusion (dInf) and at 1, 3, 5 and 10 min after discontinuing the infusion.

Cardiorespiratory Parameters and Rectal Temperature
A multi-parameter 3-lead electrocardiogram patient monitor (Mindray MEC-1200 Vet, Mahwah , USA) was used to measure HR, beats/min, hemoglobin oxygen saturation (SpO2, %) and respiratory rate (RR, breaths/min) as well as the rectal temperature (RT, °C). The reflective pulse oximeter probe was placed over the goat's shaved tail. All goats were shaved in the distal one third region between the 3rd and 6th intercostal spaces on their right thoraxes and placed with minimum restraint on right lateral recumbency in preparation for echocardiography. The goat's tail was shaved for the placing of a pulse oximeter probe. A 20-gauge 2.5 cm catheter was then placed in each goat's left jugular vein in preparation for their infusion.
The animals received a 0.9 % saline solution (SAL treatment) and three AD (Adenocor ® 3 mg/mL; Sanofi; Berkshire, UK) treatments (AD 50, 100 and 200) at a dose rate of 50, 100 and 200 µg/kg/min for 2 min using a syringe driver through their jugular catheter. There was a 1 min stabilization period before taking measurements after starting the infusion. AD was diluted to reach a concentration of 600 mg/mL so that the total volume infused ranged from 2 to 10 mL. Identical volumes of saline were infused in the SAL treatment.
Cardiorespiratory and key echocardiographic parameters were measured before the treatment (baseline), during the infusion (dInf) and at 1, 3, 5 and 10 min after discontinuing each infusion ( Figure 2). 100 and 200 μg/kg/min for 2 min using a syringe driver through their jugular catheter. There was a 1 min stabilization period before taking measurements after starting the infusion. AD was diluted to reach a concentration of 600 mg/mL so that the total volume infused ranged from 2 to 10 mL. Identical volumes of saline were infused in the SAL treatment. Cardiorespiratory and key echocardiographic parameters were measured before the treatment (baseline), during the infusion (dInf) and at 1, 3, 5 and 10 min after discontinuing each infusion ( Figure 2).

Figure 2.
Study timeline showing electrocardiogram (ECG) recordings and echocardiographic measurements in goats receiving saline (SAL) and three adenosine (AD) doses at the baseline, during the infusion (dInf) and at 1, 3, 5 and 10 min after discontinuing the infusion.

Cardiorespiratory Parameters and Rectal Temperature
A multi-parameter 3-lead electrocardiogram patient monitor (Mindray MEC-1200 Vet, Mahwah , USA) was used to measure HR, beats/min, hemoglobin oxygen saturation (SpO2, %) and respiratory rate (RR, breaths/min) as well as the rectal temperature (RT, °C). The reflective pulse oximeter probe was placed over the goat's shaved tail.

Cardiorespiratory Parameters and Rectal Temperature
A multi-parameter 3-lead electrocardiogram patient monitor (Mindray MEC-1200 Vet, Mahwah, NJ, USA) was used to measure HR, beats/min, hemoglobin oxygen saturation (SpO 2 , %) and respiratory rate (RR, breaths/min) as well as the rectal temperature (RT, • C). The reflective pulse oximeter probe was placed over the goat's shaved tail.

Echocardiographic Parameters
Guided M-Mode 2D echocardiography was conducted using an ultrasound device (Siemens, X-300, Seongnam, Korea). Cardiac imaging was obtained using the right paraster-nal long axis views. To minimize imprecision, overestimation and intra-observer variability, an experienced cardiac sonographer was tasked with gathering all images. To evaluate the reliability and consistency of the examiner, the intra-observer variability (measurement error) was assessed ([mean difference between measurements/average of measurements] × 100) and expressed as the coefficient of variation in a percent (%). Intra-observer variability was valued by three repeated measurements of the same M-mode image performed by the examiner in six goats. The cardiac scanning technique and linear measurement of the left ventricular internal dimensions during diastole (LVIDd) and systole (LVIDs), the interventricular septum dimension (IVSD) and the left ventricular posterior wall dimension (LVPWD) were standardized in line with recommendations from the American Society of Echocardiography and the European Association of Cardiovascular Imaging [25]. The long axis views were obtained at the mitral valve level. Angled and oblique views were avoided. The measurement values were obtained carefully perpendicular to the left ventricle long axis at the level of the mitral valve leaflet tip. The calibration of the LV internal dimensions was carried out using ultrasound integrated software, which automatically computes the ejection fraction (EF) (%), fractional shortening (FS) (%) and stroke volume (mL). CO (L/min) was calculated by multiplying the ECG-recorded HR by the stroke volume (SV); thus, CO = HR × SV.

Statistical Analysis
Statistical tests were performed using GraphPad Prism software version 8.0 (GraphPad Inc, San Diego, CA, USA). Continuous data of cardiorespiratory and echocardiographic variables were taken at the baseline, during the infusion (dInf) and at 1, 3, 5 and 10 min after discontinuing the AD infusion. All data were expressed as mean ± the standard deviation (SD). After confirming a normal distribution of data using the Kolmogorov-Smirnov test, the analysis of variance (ANOVA) with Dunnett's test post-hoc was used to compare each group with the baseline and also between AD and SAL treatments. The differences were considered significant at p < 0.05.

Results
There were no significant differences in the baseline values of HR, RR, SpO 2 and RT between the SAL and the three AD treatments. During the infusion (dInf), the AD 100 and 200 showed an increase in HR; however, only the AD 200 exhibited a significant rise in HR compared with the SAL (p = 0.003). At the one minute mark following the discontinuation of the infusion, HR rapidly reduced; however, it significantly increased in the AD 200 compared with the SAL (p < 0.001). Compared with the baseline, HR did not change in the SAL and the AD 50 but increased significantly in the AD 100 (p = 0.001) and 200 (p = 0.006) at dInf. At the one minute mark following the discontinuation of the infusion, HR returned to the normal range in the AD 100 but remained significantly higher in the AD 200 (p = 0.035). Moreover, the RR increased significantly in the AD 200 (p < 0.001) at dInf. However, AD 50 and 100 did not exhibit a significant difference compared with the baseline. The AD infusions did not influence the baseline values of SpO 2 and RT (Table 1). In all treatments, non-significant variations occurred in HR, RR, SpO 2 and RT at 3, 5 and 10 min after discontinuing the infusion. No signs of arrythmia and abnormalities in the QRS and P wave observed in the electrocardiograms in all AD treatments ( Figure 3). Table 1. Cardiorespiratory variables and rectal temperature of goats (n = 6) receiving saline (SAL) and three adenosine (AD) dose infusions at the baseline (B), during the infusion (dInf) and at 1, 3, 5 and 10 min after discontinuing the infusion.   In this study, EF, FS and CO were measured as vital indicators for the left ventricular function. Compared with the SAL treatment, all AD treatments did not show significant differences at dInf and at 1, 3, 5 and 10 min after discontinuing the infusion. Even though HR significantly increased at dInf in the AD 100 and 200, the AD infusion did not significantly alter the baseline values for the cardiac parameters within each treatment (   In this study, EF, FS and CO were measured as vital indicators for the left ventricular function. Compared with the SAL treatment, all AD treatments did not show significant differences at dInf and at 1, 3, 5 and 10 min after discontinuing the infusion. Even though HR significantly increased at dInf in the AD 100 and 200, the AD infusion did not significantly alter the baseline values for the cardiac parameters within each treatment (   In this study, EF, FS and CO were measured as vital indicators for the left ven function. Compared with the SAL treatment, all AD treatments did not show sig differences at dInf and at 1, 3, 5 and 10 min after discontinuing the infusion. Even HR significantly increased at dInf in the AD 100 and 200, the AD infusion did not cantly alter the baseline values for the cardiac parameters within each treatment (T   In this study, EF, FS and CO were measured as vital indicators for the left ventricular function. Compared with the SAL treatment, all AD treatments did not show significant differences at dInf and at 1, 3, 5 and 10 min after discontinuing the infusion. Even though HR significantly increased at dInf in the AD 100 and 200, the AD infusion did not significantly alter the baseline values for the cardiac parameters within each treatment (  Table 1. Cardiorespiratory variables and rectal temperature of goats (n = 6) receiving saline (SAL) and three adenosine (AD) dose infusions at the baseline (B), during the infusion (dInf) and at 1, 3, 5 and 10 min after discontinuing the infusion.  In this study, EF, FS and CO were measured as vital indicators for the left ventricular function. Compared with the SAL treatment, all AD treatments did not show significant differences at dInf and at 1, 3, 5 and 10 min after discontinuing the infusion. Even though HR significantly increased at dInf in the AD 100 and 200, the AD infusion did not significantly alter the baseline values for the cardiac parameters within each treatment (Table 2). In this study, EF, FS and CO were measured as vital indicators for the left ventricular function. Compared with the SAL treatment, all AD treatments did not show significant differences at dInf and at 1, 3, 5 and 10 min after discontinuing the infusion. Even though HR significantly increased at dInf in the AD 100 and 200, the AD infusion did not significantly alter the baseline values for the cardiac parameters within each treatment (Table 2). Table 2. Echocardiographic variables of goats (n = 6) receiving saline (SAL) and three adenosine (AD) dose infusions at the baseline (B), during the infusion (dInf) and at 1, 3, 5 and 10 min after discontinuing the infusion.

Discussion
This preliminary study indicated that intravenous infusions of AD in conscious goats produce a transient, dose-dependent, excitatory effect on HR and RR. In humans, AD infusion at a dose rate of 50 and 100 µg/kg/min has been reported to induce positive chronotropy with a non-significant change in systemic blood pressure [26,27]. Another study found that the steady state infusions of AD at a dose rate of 140 µg/kg/min resulted in a significant increase in HR (+30 beats/min) [7]. In this current study, AD infusion in goats produced a modification in HR and RR resembling that occurring in humans. In pregnant ewes, the intravenous injection of AD at a dose of 200 µg/kg was also reported to produce an acceleration in HR [28]. The hemodynamic effect of AD is dependent on its dose, route of administration and if the patient is conscious or anesthetized. Nonsignificant changes in HR, CO and cardiac index were found during a continuous AD infusion at a dose rate of 140 or 280 µg/kg/min in anesthetized dogs [24]. Moreover, minor increases in HR (+9 beats/min) and CO (+2 L/min) were observed with an AD infusion of 0.14 ± 0.04 mg/kg/min in patients undergoing cerebral aneurysm surgery [29]. In contrast to its effects in the anesthetized state, AD infused at a dose rate of 140 µg/kg/min in conscious volunteers initiated a significant rise in HR, thoracic excursion (+200%), partial arterial oxygen pressure (+12 mmHg) and a decrease in partial arterial carbon dioxide pressure (−10 mmHg) [7]. In another study, intravenous boluses of AD at four different doses (80, 100, 120 and 140 µg/kg) resulted in significant increases in both the depth and rate of respiration [30].The effect of an AD infusion seems to be less excitatory and blunted with anesthesia, which may be due to the sympatholysis induced by the anesthetic agents.
In the present study, HR increased in a dose-dependent manner during AD infusion in which a higher significant increase was observed in the AD 200 as opposed to the AD 100. However, the AD 50 showed no effect on the baseline values. Our findings were consistent with prior results found during an AD infusion in conscious humans [7,31]. Furthermore, conscious dogs showed a significant increase in HR and a non-significant alteration in CO following injections of AD at doses of 10, 25, 50, 100 and 250 µg/kg [32]. AD has been well-known to induce cardiac depression and hypotension via the activation of AD A 1 , resulting in the vasodilation of most vascular beds in the body [33]. In this study, the AD-induced tachycardia might be a compensatory reflex caused by hypotension. Moreover, the autonomic innervation and vagal tone of the heart might play a pivotal role in the AD-induced tachycardia because the increase in HR induced by the AD infusion could be completely blocked in patients with an autonomic dysfunction [7]. Additionally, a bilateral vagotomy in normal conscious dogs could prevent the tachycardic response to the AD infusion [34]. The AD infusions were also associated with a dose-dependent increase in the plasma levels of norepinephrine and epinephrine [35]. The activation of a sympathetic outflow is mostly attributable to the AD-induced increases in HR in goats [8,10]. Carotid body chemoreceptor activation could be also induced by AD infusion, which may be involved in the respiratory stimulation [7]. Pulmonary stretch receptors could be activated due to hyperventilation and they tend to produce tachycardia [36]. In this current study, the increase in HR was observed during AD infusion, particularly with AD 200; however, non-significant changes occurred in CO, EF and FS. A decrease in SV was more likely to be the compensatory mechanism of maintaining CO. This finding was supported by a prior study in which a significant increase in HR with no change in CO was associated with intravenous boluses of AD up to 250 µg/kg [32]. The increase in HR may contribute to a decrease in end diastolic volume (EDV) due to the reduced time for ventricular filling during diastole. Another study revealed that an intravenous infusion of AD at a dose rate of 140 µg/kg/min produces a significant increase in HR (+18 beats/min) and a decrease in EDV (−9 mL) with a non-significant alteration in EF [37]. The small sample size in this study may have limited the value of the observations as a true representation of the goat population. In addition, the use of direct arterial blood pressure monitoring and arterial blood gas analysis may have provided a superior assessment of the effect of the AD infusion on the cardiopulmonary status.

Conclusions
In conscious goats, AD produced a dose-dependent increase in HR and RR without altering EF, FS and CO. The higher dose of AD could be used therapeutically as a cardiorespiratory stimulant. Although this preliminary study could provide valuable information about the effect of AD in conscious goats, a more comprehensive study is needed to evaluate the effect of AD in anesthetized animals as well as those with a cardiac compromise. Informed Consent Statement: Not applicable.

Data Availability Statement:
The data presented in this study are available on request from the corresponding author.