Effects of two‐dose intravenous administration of furosemide on clinical variables, electrocardiographic indices and serum electrolytes in dromedary calves

Abstract The intravenous (IV) usage of diuretic agents such as furosemide may cause changes in clinical signs, electrocardiographic (ECG) indices and serum electrolytes (sodium, potassium, chloride, calcium, phosphorus and magnesium) concentrations in dromedary calves. The purpose of this study was to evaluate the clinical, ECG and biochemical effects of two‐dose IV administration of furosemide in dromedary calves. A total of 21 clinically healthy male dromedary calves with the age of 5 (± 1) months and weight of 95 (± 5) kg were studied. The animals were randomly divided into three groups of control (normal saline), low‐dose furosemide (2.5 mg/kg) and high‐dose furosemide (5 mg/kg). Two IV injections with 12‐hr intervals were administered in all animals. The clinical and ECG parameters were measured at 0 (baseline), 2 (T2), 24 (T24) and 48 (T48) hours after drug administration. Serum concentrations of electrolytes were measured at T0, T24 and T48 hr. The results of this study showed no changes in clinical parameters (heart rate, rectal temperature, respiratory rate and Unruminal motility), ECG indices and also no significant changes in serum electrolytes levels. Under conditions with free access to fresh water, two‐dose IV injection of furosemide (2.5 and 5 mg/kg) could be administered in healthy dromedary calves.

There were limited information on the effect of two-dose intravenous (IV) administration of furosemide on clinical variables, electrocardiographic (ECG) indices and serum electrolytes levels in camelide, especially calf-camels (Ali et al., 1998;Chalmeh & Mazrouei Sebdani, 2016;Santamarina et al., 2001). The purpose of this study was to investigate the effects of two-dose IV administration of furosemide on clinical variables, ECG indices and serum electrolytes levels in the dromedary calves.

| Animals
The present study was carried out in the Faculty of Veterinary These animals were kept under the same management conditions and received a diet close to the maintenance and physiological requirements as set by Laudadio et al. (2009). Roughage (containing corn silage and alfalfa hay) supplemented with minerals (0.3%) was considered for diet of animals. At all stages of the study, the feed was distributed ad libitum in two meals at 9 a.m. and 6 p.m. Two months before the start of the experiment, all animals were treated with broad-spectrum antiparasitic agents (by administration of oral albendazole, 15 mg/kg, and subcutaneous ivermectin, 0.2 mg/kg) to control possible contamination of internal and external parasites. At the baseline, the clinical and laboratory status of the animals was evaluated and only clinically and paraclinically healthy animals were allocated in the study. The experiment was carried out in the morning. The ambient temperature and relative humidity during the experiments were 19°C-21°C and 12%-14%, respectively. The experiments were carried out in an outdoor covered 6 × 7 m area. The animals were weighed before each treatment for calculation of drug dosages. The calves were moved to the experiment area and restrained in a quiet sternal position on a soft and comfortable mattress. Three or four calves were studied at any one time. Skin over the left jugular vein was clipped and scrubbed with povidone-iodine for IV administration and blood sampling.

| Clinical findings and ECG indices
HR was evaluated using stethoscope (Classic II SE, Littmann Co) on the left side of chest wall (fourth intercostal space, behind the olecranon), and RR was assessed using direct observation of thoraco-abdominal movement for 1 min. RT was measured using a digital thermometer inserted into the rectum. Ruminal motility (number of audible rumen contraction within 2-min auscultation) clinical variables immediately before (baseline) and up to 48 hr following twodose intravenous administration of low dose (2.5 mg/kg) and high dose (5 mg/kg) of furosemide, and normal saline (control) in dromedary calves (Camelus dromedarius) was assessed using stethoscope placed on the left paralumbar fossa. Two large animal internists who were unaware of the drug evaluated the clinical parameters. ECG was obtained from all animals by bipolar base-apex lead method similar to the method of Samimi et al. (2020).

| Biochemical parameters
Blood samples were taken from all animals via jugular vein and kept in test tube with no anticoagulant. For blood biochemistry, samples were kept at 24ºC for 1 hr and then centrifuged (10 min, 3,000 g). Sera were separated and stored at −20°C for 1 day (before variables measurement). Sera were analysed for sodium and potassium values by flame photometry (FLM, Ontario, Canada). Serum concentrations of chloride and phosphorus were measured using UV spectrophotometer. Atomic absorption spectrophotometry (Shimadzu AA-670) was used to determine calcium and magnesium levels.

| Statistical analysis
Data (clinical variables, ECG indices and serum electrolytes levels) were expressed as mean ± standard deviation (SD). Data normality was analysed by Kolmogorov-Smirnov test before statistical analysis. Data obtained from the same time between different groups were analysed using one-way analysis of variance (ANOVA).  Table 3. There were no significant differences in serum electrolytes concentrations among different groups at any time point. All parameters were not significant changes from baseline in all groups after drug administration.

| D ISCUSS I ON
Electrolyte disturbances due to extensive administration of potent diuretics may lead to cardiac dysrhythmia. Hypokalemia is one of the common electrolyte disturbances as a side effect of diuretics (Wasfi et al., 2017). Changes in ECG after furosemide administration are largely unknown and there have been limited studies in veterinary science (Pourjafar et al., 2013). The dose of furosemide used in the current study was based on studies comparing in other ruminants (Chalmeh & Mazrouei Sebdani, 2016;Constable et al., 2017;Pourjafar et al., 2013). There were no published articles about pharmacological effects of furosemide at higher doses (such as 10 mg/kg) in dromedary calves and it may be associated with side effects and unpredictable complications. The 48-hr time course chosen for present study was based on what is known about the pharmacokinetic characteristic of furosemide in ruminants (Ali et al., 1998;Wasfi et al., 2017).
It should be noted that camelides are 'pseudoruminants' or 'modified ruminants' compared with bovine, caprine and ovine that are true ruminants. Changes in haematobiochemical parameters following furosemide have been reported in humans (Licata et al., 2003), ponies (Houpt & Perry, 2016) and horses (Raidal et al., 2014).
Electrolytes values were within normal physiological range (for this species at this age) at baseline in all study groups. This is in accordance with the finding of Samimi et al. (2019). In our study, no changes in serum electrolytes concentrations were recorded up to 48 hr. A decrease in serum levels of sodium, chloride and potassium was observed in goats 5 hr after IV furosemide (10 mg/kg) administration (Pourjafar et al., 2013). In addition, serum sodium and potassium levels were decreased after IV administration of furosemide (5 and 10 mg/kg) in high-yielding Holstein cows (Chalmeh & Mazrouei Sebdani, 2016). In the study by Ali et al. (1998) Akita et al. (1998) confirmed the ECG changes during furosemide-induced hypokalemia. In addition, prolonged duration of P wave and QRS complex were observed during hypokalaemia in rats (Akita et al., 1998). These results suggest that the myocardial excitabilities in the atria and ventricles may be affected by extracellular potassium level rather than by the atrioventricular conduction system in rat (Akita et al., 1998).
It should be noted that the absence of comparisons of furosemide with other diuretics as well as non-use of higher and consecutive doses can be considered as limitations of the present study.
Another limitation of this study is lack of evaluation of electrolytes levels at urinary excretion for up to 7 days.

| CON CLUS ION
The results obtained from the present study showed no significant changes in the clinical variables (including HR, RR, RT and ruminal motility). In addition, there were no significant differences in serum electrolytes levels and ECG indices after furosemide administration (2.5 and 5 mg/kg). Therefore, two-dose IV injection of furosemide (2.5 and 5 mg/kg) could be administered in healthy dromedary calves under conditions with free access to fresh water. Further studies with more frequent sampling, long-term duration, higher doses of furosemide and urine analysis are required.

CO N FLI C T O F I NTE R E S T
The authors declare that they have no competing interests.

E TH I C A L A PPROVA L
All ethical considerations including animal utilization were considered cautiously. Also, the trial convention was affirmed by the animal welfare committee (which was covered IACUC approval) of the School of Veterinary Medicine, Shahid Bahonar University of Kerman, Kerman, Iran. All applicable international, national, and/or institutional guidelines for the care and use of animals were followed.