BACKGROUND
digoxin is the most commonly used cardiac glycoside  <cit> .
it is a well-tolerated and inexpensive drug which has been in wide use for many years as a treatment for heart failure and arrhythmia.
although a number of sophisticated management options and new therapeutic agents have been developed in recent years for the treatment of patients with heart failure, digoxin is still one of the most frequently prescribed drugs and remains the first choice of treatment  <cit> .
in equidae, digoxin is used in the treatment of naturally-acquired heart failure  <cit> .
treatment with digoxin results in an increase in cardiac contractility and a decrease in heart rate, with increased myocardial oxygen consumption, increased cardiac output and decreased cardiac size  <cit> .
the improvement in cardiac output promotes diuresis and the reduction and elimination of oedema.
the half-life of digoxin in the horse is 17–23 h  <cit> .
the proposed therapeutic, nontoxic range of digoxin in horses is calculated as follows: iv loading  <dig>  iv maintenance  <dig>  oral loading  <dig>  and oral maintenance 35 μg/kg/24 h  <cit> .
cardiac troponins, especially cardiac troponin i , are highly sensitive and specific markers of myocardial injury in veterinary medicine  <cit> .
in horses, ctni is elevated in animals undergoing training and endurance exercise  <cit> , horses with cardiac diseases  <cit> , horses with muscular disease  <cit> , and in horses with monensin intoxication  <cit> .
measurement of ctni concentrations has also been used recently for prognostication in horses undergoing emergency abdominal surgery  <cit> .
the effect of digoxin administration on the serum concentration of ctni has not been reported to date in equidae.
the present study was therefore designed to investigate the influence of the cardiac glycoside digoxin on cardiac cell damage as assessed by ctni, using donkeys  as an equine model.
the second objective was to investigate the effects of digoxin injection on the acid–base and electrolyte balance and on the haematobiochemical profiles in donkeys.
methods
animals, history and physical examination
the experimental protocol and all procedures used in this study were approved by the ethics committee for animal research of the scientific research deanship of qassim university, saudi arabia.
in addition, animals were treated according to the regulations of the laboratory animal control guidelines of qassim university, which basically conform to the guide for the care and use of laboratory animals of the national institutes of health in the usa .
ten male donkeys  were used.
the animals were selected on the basis of absence of any disease.
each donkey had received a full clinical examination, with special attention to the cardiovascular system.
animals were considered healthy based on physical examination, laboratory evaluation , echocardiography and electrocardiography .
administration of digoxin and blood sampling
digoxina was administered as an iv infusion at 14 μg/kg, a dose recommended for horses with heart failure  <cit> .
throughout the experiment, eight blood samples  were obtained from each animal.
the first blood sample  was collected immediately prior to injection of digoxin.
four blood samples were collected at 1 h , 2 h , 3 h  and 4 h  post-injection.
three additional blood samples  were obtained at the following  <dig>   <dig> and 72 h. at each sampling time, 10 ml of jugular blood was collected using sterile vacutainers.
a 2-ml volume of blood was collected into a heparinised tube for determining blood gas parameters, 2-ml in edta tubes for haematological analyses, and the remaining 6 ml of blood was placed in plain tubes to obtain serum for the determination of digoxin, ctni concentrations and other biochemical analytes.
electrocardiogram recording
a lead ii electrocardiographb with a paper speed of 25 mm/s and a sensitivity of 10 mm/mv  was used to constantly monitor the donkeys for the presence of arrhythmias during the entire study period.
alligator clips fixed to the electrocardiographic leads were attached directly to the skin after vigorous application of an electrode paste.
the limb lead placement consisted of four electrodes: two just above the point of the elbow  and two on the stifle .
the forelimbs were kept parallel to each other and perpendicular to the long axis of the body.
the donkeys were kept standing and insulated from the ground by means of a rubber mat.
blood gas analyses
the heparinised blood samples were used immediately to analyse the acid–base and blood gas parameter values in situ using a portable clinical veterinary analyser.c in this way, blood ph, partial pressure of carbon dioxide , oxygen partial pressure , bicarbonate , anion gap base excess , oxygen saturation , sodium, potassium, chloride and lactate were determined immediately in order to prevent changes in the concentrations of these parameters  <cit> .
haematology and serum biochemistry
haematological examinations  were carried out using an automated analyser.d the serum samples were used to determine the concentrations of total protein, albumin, globulin, blood urea nitrogen , calcium, glucose, creatinine and total bilirubin.
the serum activity of γ-glutamyl transferase , aspartate aminotransferase , and creatine kinase  was also measured.
an automated biochemical analyser was used for the measurement of the biochemical parameterse.
cardiac troponin i and digoxin assays
cardiac troponin i was analysed in serum using a point-of-care analyser according to the manufacturer’s instructions.c this analyser employs a two-site enzyme-linked immunosorbant assay.
all results are expressed as nanograms per millilitre  with an intra-assay coefficient of variance of 5%.
the lower limit of detection of ctni for this assay is  <dig>  ng/ml.
the serum concentrations of digoxin were determined using an electrochemiluminescent immunoassay-kitf with a measuring range of  <dig>  -  <dig>  ng/ml.
the intra- and inter-assay coefficients of variance for digoxin were  <dig>  and  <dig> %, respectively.
the coefficients of variance for ctni and digoxin assays were developed in human samples.
statistical analysis
data from the  <dig> donkeys are presented as means ± standard deviation, and comparisons among t0-t <dig> values were conducted using spss program software  <cit> .
the normality of the data was tested by the kolmogorov–smirnov test.
data were analysed using repeated measures analysis, with fisher’s protected least significant difference  as the post-anova test.
the level of significance was tested at p <  <dig> .
RESULTS
none of the donkeys showed clinical abnormalities following injection of digoxin.
the rectal temperature and heart and respiratory rates did not differ significantly after digoxin administration.
in addition, the ecg results showed no cardiac arrhythmias in any of the donkeys by 72 h post-injection of digoxin.
figure  <dig> shows the serum concentration of digoxin in the donkeys before and after digoxin infusion.
serum digoxin concentration significantly increased at many time points after digoxin had been administered intravenously.
at 1 h , the digoxin concentration had increased to  <dig>  ±  <dig>  compared to  <dig>  ±  <dig>  ng/ml pre-injection .
from the time points t <dig> to t <dig>  gradual decreases of digoxin concentrations were observed until they returned to pre-injection values by t <dig>  at all-time points , the serum concentrations of ctni in the donkeys before and after digoxin infusion was ‘below detection limit’ for the assay and below established normal values using this assay.
table  <dig> summarises mean ± sd of acid–base and electrolyte parameters in the donkeys after injection of digoxin.
at t <dig>  there was an increase in blood ph .
at t2-t <dig>  there were decreases in pco <dig>  while at t3-t <dig>  there were increases in po <dig> .
oxygen saturation increased at t3-t <dig> .
at t1-t <dig>  there were decreases in blood lactate .
other blood gas parameters including sodium, potassium, chloride, be, hco3- and anion gap did not differ significantly compared to the pre-injection values .
<dig>
<dig>
3
<dig>
pco <dig>  partial pressure of carbon dioxide; po <dig>  partial pressure of oxygen; be, base excess; hco3¯, bicarbonate; so <dig>  oxygen saturation; ag, anion gap.
t <dig>  immediately prior to injection of digoxin; t1-t <dig>   <dig>   <dig>   <dig>   <dig>   <dig>   <dig> and 72 h after digoxin administration.
bdiffer significantly at p <  <dig>  compared to pre-injection values.
compared to pre-injection values, none of the haematological parameters showed a statistically significant difference after digoxin injection .
table  <dig> shows the biochemical parameters in the donkeys before and for the 72 h post-injection of digoxin.
the serum concentration of glucose remained significantly elevated  at all-time points  after digoxin infusion.
other biochemical parameters did not differ significantly among all time points.
wbc, white blood cells; lym, lymphocyte; mon, monocyte; neu; neutrophil; eos, eosinophil; bas, basophil; rbc, red blood cells; hgb, hemoglobin; hct, hematocrit; mcv, mean corpuscular volume; mch, mean corpuscular hemoglobin; mchc, mean corpuscular hemoglobin concentration; plt, platelets.
t <dig>  immediately prior to injection of digoxin; t1-t <dig>   <dig>   <dig>   <dig>   <dig>   <dig>   <dig> and 72 h after digoxin administration.
ca, calcium; bun, blood urea nitrogen; cre, creatinine; ast, aspartate aminotransferase; ggt, γ-glutamyl transferase; ck, creatine kinase; tbil, total bilirubin, tp, total protein; alb, albumin; glob, globulin.
t <dig>  immediately prior to injection of digoxin; t1-t <dig>   <dig>   <dig>   <dig>   <dig>   <dig>   <dig> and 72 h after digoxin administration.
bdiffer significantly at p <  <dig>  compared to pre-injection values.
discussion
to the authors’ knowledge, this is the first study to evaluate the effect of digoxin on cardiac cell damage as assessed by ctni, acid–base and electrolyte balance and the haematobiochemical profiles in donkeys  as an equine model.
before injecting digoxin, the mean serum ctni in the donkeys was  <dig>  ng/ml, a value ‘below detection limit’ of the assay and below established normal values in healthy horses  using this assay  <cit> .
however, we don’t know for sure whether we can apply the normal values of horses for donkeys.
at no time point after injecting digoxin was ctni significantly different compared to pre-injection values .
cardiac troponin i is a protein found in the myocardial cells.
its serum concentration elevates after acute myocardial injury because of leakage from the damaged myocardial cells  <cit> .
it is the ‘gold standard’ for the non-invasive diagnosis of myocardial injury in humans  <cit> .
similarly, in animals ctni has a high sensitivity and specificity in patients with primary or secondary cardiac disease  <cit> .
persistently increased ctni blood concentration suggests ongoing active and irreversible damage to cardiomyocytes  <cit> .
the degree of increase has been shown to be correlated with the extent of myocardial damage and with survival in human  <cit>  and animal  <cit> .
in humans, the therapeutic and toxic plasma concentrations of digoxin are usually set at  <dig>  -  <dig>  ng/ml and greater than  <dig>  ng/ml, respectively  <cit> .
similar numbers have been derived for animals: e.g., digoxin plasma concentrations of  <dig>  -  <dig>  ng/ml are nontoxic in horses  <cit>  and a  <dig>  ng/ml concentration is nontoxic in cats  <cit> .
in dogs, plasma digoxin concentrations up to  <dig>  ng/ml are nontoxic, but concentrations of  <dig>  -  <dig>  ng/ml are associated with increased probability of toxicosis  <cit> .
in donkeys, however, the therapeutic and toxic plasma concentrations of digoxin have not been reported in the literature.
digitalis intoxication is characterised by several clinical signs varying from mild gastrointestinal upset  to chronic weight loss and life-threatening arrhythmias  <cit> .
increased serum bun and creatinine are interpreted as evidence that digoxin toxicity has compromised renal function.
in this study, serum digoxin concentration had increased in the donkeys at t <dig> to  <dig>  ±  <dig>  ng/ml compared to  <dig>  ±  <dig>  ng/ml pre-injection.
thereafter, digoxin concentration decreased steadily to reach  <dig>  ±  <dig>  ng/ml 4 h post-injection.
during that time and up to 72 h post-injection, none of the donkeys showed signs of digoxin intoxication, cardiac arrhythmias, ecg abnormalities or elevated serum bun and creatinine.
therefore, it can be assumed that the toxic serum digoxin concentration in donkeys would be higher than in humans, horses, dogs and cats.
thus, another study is needed to verify the toxic serum digoxin concentration in donkeys.
the metabolic alkalosis observed 3 h after digoxin injection may have been due to decreases in pco <dig> and lactate.
the decreased pco <dig> and the increased po <dig> and so <dig> after injection may have resulted from the increased respiratory rates.
lactate is known as the end product of anaerobic glycolysis, a pathway that is of key importance during normal metabolic and athletic events.
aside being a waste product during high intensity exercise, lactate is also a valuable substrate that significantly contributes to the energy production of heart, liver, kidneys, non-contracting muscles and even brain  <cit> .
in the present study, lactate concentration decreased during the 4 h  post-injection of digoxin, and this may have been caused by the decrease in the metabolic index due to the inactivity of the animals during these time points.
similarly, in healthy humans, lactate concentration was reported to be stable following digoxin infusion  <cit> .
concerning the biochemical variables, hyperglycaemia was found in the donkeys after digoxin administration.
in human medicine, it is well-known that digoxin therapy increases the adrenaline-induced, and attenuates the insulin-induced hypoglycaemia due to catecholamines release in the acute stage of the action of digitalis glycosides  <cit> .
this study has its own limitations.
one of these limitations is the small sample size; therefore the results of this study are preliminary and should be interpreted with caution.
another limitation of this study is the using of healthy rather than diseased donkeys.
braunwald  <cit>  had reported that there is a direct correlation between the level of myocardial impairment and the effectiveness of digoxin therapy on cardiac function.
although the i- stat® ctni assay was initially designed for use in humans, there is good reason to anticipate cross reactivity based on the highly conserved nature of the cardiac troponins across mammalian species  <cit> .
while it may not be practical in veterinary medicine to develop species-specific assays for all relevant species, this practical assumption should come with an acknowledgement of less than perfect validation of this assay.
in this study, we thought that the i- stat® ctni assay would detect the donkeys’ ctni, because, despite the lack of a known donkey ctni amino-acid sequence, a third limitation of this study, the sequence of ctni is highly conserved among all species.
in addition, the i- stat® analyser has been clinically validated against reference analysers in other species  <cit> .
in addition, the high homology of amino-acid sequences between mammals within the sequence regions detected by ctni analysers allows veterinarians to measure ctni concentrations in many animal species with the same equipment as has been developed for humans, rather than necessitating the development of species-specific technology.
as all the troponin samples in this study were below the detection level of the test, therefore the assay may not have had sufficient sensitively to detect small changes in ctni levels below the detection level.
these small elevations may still be of clinical relevance.
CONCLUSIONS
although the serum digoxin concentrations had increased in the donkeys to  <dig>  ±  <dig>  ng/ml 1 h after injection, the donkeys showed no sign of digoxin intoxication, ecg abnormalities or elevated serum bun and creatinine.
it is therefore assumed that in donkeys, the toxic serum digoxin concentration is higher than in humans, horses, dogs and cats  <cit> .
consequently, another study is warranted to verify the toxic serum digoxin concentration in healthy donkeys and in those with cardiac diseases.
endnotes
alanoxin, glaxosmithkline s.p.a, parma, italy.
bkenz cardico  <dig>  suzuken co., ltd., japan.
cvetscan i-stat  <dig>  abaxis, california, usa.
dvetscan hm <dig>  abaxis, california, usa.
evetscan vs <dig>  abaxis, california, usa.
froche diagnostics, indianapolis, indiana, usa.
competing interests
the authors declare that they have no competing interests.
authors’ contributions
mt initiated and planned the study.
mt and fa carried out the experimental and laboratory work.
mt wrote the manuscript and made the figure and tables and revised the manuscript.
fa read and revised and approved the manuscript.
both authors read and approved the final manuscript.

 digoxin is the most commonly used cardiac glycoside. it is a well-tolerated and inexpensive drug which has been in wide use for many years as a treatment for heart failure and arrhythmia. although a number of sophisticated management options and new therapeutic agents have been developed in recent years for the treatment of patients with heart failure, digoxin is still one of the most frequently prescribed drugs and remains the first choice of treatment. in equidae, digoxin is used in the treatment of naturally-acquired heart failure. treatment with digoxin results in an increase in cardiac contractility and a decrease in heart rate, with increased myocardial oxygen consumption, increased cardiac output and decreased cardiac size. the improvement in cardiac output promotes diuresis and the reduction and elimination of oedema. the half-life of digoxin in the horse is 17–23 h. the proposed therapeutic, nontoxic range of digoxin in horses is calculated as follows: iv loading iv maintenance oral loading and oral maintenance 35 μg/kg/24 h. cardiac troponins, especially cardiac troponin i, are highly sensitive and specific markers of myocardial injury in veterinary medicine. in horses, ctni is elevated in animals undergoing training and endurance exercise, horses with cardiac diseases, horses with muscular disease, and in horses with monensin intoxication. measurement of ctni concentrations has also been used recently for prognostication in horses undergoing emergency abdominal surgery. the effect of digoxin administration on the serum concentration of ctni has not been reported to date in equidae. the present study was therefore designed to investigate the influence of the cardiac glycoside digoxin on cardiac cell damage as assessed by ctni, using donkeys as an equine model. the second objective was to investigate the effects of digoxin injection on the acid–base and electrolyte balance and on the haematobiochemical profiles in donkeys. methods animals, history and physical examination the experimental protocol and all procedures used in this study were approved by the ethics committee for animal research of the scientific research deanship of qassim university, saudi arabia. in addition, animals were treated according to the regulations of the laboratory animal control guidelines of qassim university, which basically conform to the guide for the care and use of laboratory animals of the national institutes of health in the usa. ten male donkeys were used. the animals were selected on the basis of absence of any disease. each donkey had received a full clinical examination, with special attention to the cardiovascular system. animals were considered healthy based on physical examination, laboratory evaluation, echocardiography and electrocardiography. administration of digoxin and blood sampling digoxina was administered as an iv infusion at 14 μg/kg, a dose recommended for horses with heart failure. throughout the experiment, eight blood samples were obtained from each animal. the first blood sample was collected immediately prior to injection of digoxin. four blood samples were collected at 1 h, 2 h, 3 h and 4 h post-injection. three additional blood samples were obtained at the following and 72 h. at each sampling time, 10 ml of jugular blood was collected using sterile vacutainers. a 2-ml volume of blood was collected into a heparinised tube for determining blood gas parameters, 2-ml in edta tubes for haematological analyses, and the remaining 6 ml of blood was placed in plain tubes to obtain serum for the determination of digoxin, ctni concentrations and other biochemical analytes. electrocardiogram recording a lead ii electrocardiographb with a paper speed of 25 mm/s and a sensitivity of 10 mm/mv was used to constantly monitor the donkeys for the presence of arrhythmias during the entire study period. alligator clips fixed to the electrocardiographic leads were attached directly to the skin after vigorous application of an electrode paste. the limb lead placement consisted of four electrodes: two just above the point of the elbow and two on the stifle. the forelimbs were kept parallel to each other and perpendicular to the long axis of the body. the donkeys were kept standing and insulated from the ground by means of a rubber mat. blood gas analyses the heparinised blood samples were used immediately to analyse the acid–base and blood gas parameter values in situ using a portable clinical veterinary analyser.c in this way, blood ph, partial pressure of carbon dioxide, oxygen partial pressure, bicarbonate, anion gap base excess, oxygen saturation, sodium, potassium, chloride and lactate were determined immediately in order to prevent changes in the concentrations of these parameters. haematology and serum biochemistry haematological examinations were carried out using an automated analyser.d the serum samples were used to determine the concentrations of total protein, albumin, globulin, blood urea nitrogen, calcium, glucose, creatinine and total bilirubin. the serum activity of γ-glutamyl transferase, aspartate aminotransferase, and creatine kinase was also measured. an automated biochemical analyser was used for the measurement of the biochemical parameterse. cardiac troponin i and digoxin assays cardiac troponin i was analysed in serum using a point-of-care analyser according to the manufacturer’s instructions.c this analyser employs a two-site enzyme-linked immunosorbant assay. all results are expressed as nanograms per millilitre with an intra-assay coefficient of variance of 5%. the lower limit of detection of ctni for this assay is  ng/ml. the serum concentrations of digoxin were determined using an electrochemiluminescent immunoassay-kitf with a measuring range of -  ng/ml. the intra- and inter-assay coefficients of variance for digoxin were and %, respectively. the coefficients of variance for ctni and digoxin assays were developed in human samples. statistical analysis data from the donkeys are presented as means ± standard deviation, and comparisons among t0-t values were conducted using spss program software. the normality of the data was tested by the kolmogorov–smirnov test. data were analysed using repeated measures analysis, with fisher’s protected least significant difference as the post-anova test. the level of significance was tested at p < .  none of the donkeys showed clinical abnormalities following injection of digoxin. the rectal temperature and heart and respiratory rates did not differ significantly after digoxin administration. in addition, the ecg results showed no cardiac arrhythmias in any of the donkeys by 72 h post-injection of digoxin. figure  shows the serum concentration of digoxin in the donkeys before and after digoxin infusion. serum digoxin concentration significantly increased at many time points after digoxin had been administered intravenously. at 1 h, the digoxin concentration had increased to  ±  compared to  ±   ng/ml pre-injection. from the time points t to t gradual decreases of digoxin concentrations were observed until they returned to pre-injection values by t at all-time points, the serum concentrations of ctni in the donkeys before and after digoxin infusion was ‘below detection limit’ for the assay and below established normal values using this assay. table  summarises mean ± sd of acid–base and electrolyte parameters in the donkeys after injection of digoxin. at t there was an increase in blood ph. at t2-t there were decreases in pco while at t3-t there were increases in po. oxygen saturation increased at t3-t. at t1-t there were decreases in blood lactate. other blood gas parameters including sodium, potassium, chloride, be, hco3- and anion gap did not differ significantly compared to the pre-injection values. 3 pco partial pressure of carbon dioxide; po partial pressure of oxygen; be, base excess; hco3¯, bicarbonate; so oxygen saturation; ag, anion gap. t immediately prior to injection of digoxin; t1-t and 72 h after digoxin administration. bdiffer significantly at p <  compared to pre-injection values. compared to pre-injection values, none of the haematological parameters showed a statistically significant difference after digoxin injection. table  shows the biochemical parameters in the donkeys before and for the 72 h post-injection of digoxin. the serum concentration of glucose remained significantly elevated at all-time points after digoxin infusion. other biochemical parameters did not differ significantly among all time points. wbc, white blood cells; lym, lymphocyte; mon, monocyte; neu; neutrophil; eos, eosinophil; bas, basophil; rbc, red blood cells; hgb, hemoglobin; hct, hematocrit; mcv, mean corpuscular volume; mch, mean corpuscular hemoglobin; mchc, mean corpuscular hemoglobin concentration; plt, platelets. t immediately prior to injection of digoxin; t1-t and 72 h after digoxin administration. ca, calcium; bun, blood urea nitrogen; cre, creatinine; ast, aspartate aminotransferase; ggt, γ-glutamyl transferase; ck, creatine kinase; tbil, total bilirubin, tp, total protein; alb, albumin; glob, globulin. t immediately prior to injection of digoxin; t1-t and 72 h after digoxin administration. bdiffer significantly at p <  compared to pre-injection values. discussion to the authors’ knowledge, this is the first study to evaluate the effect of digoxin on cardiac cell damage as assessed by ctni, acid–base and electrolyte balance and the haematobiochemical profiles in donkeys as an equine model. before injecting digoxin, the mean serum ctni in the donkeys was  ng/ml, a value ‘below detection limit’ of the assay and below established normal values in healthy horses using this assay. however, we don’t know for sure whether we can apply the normal values of horses for donkeys. at no time point after injecting digoxin was ctni significantly different compared to pre-injection values. cardiac troponin i is a protein found in the myocardial cells. its serum concentration elevates after acute myocardial injury because of leakage from the damaged myocardial cells. it is the ‘gold standard’ for the non-invasive diagnosis of myocardial injury in humans. similarly, in animals ctni has a high sensitivity and specificity in patients with primary or secondary cardiac disease. persistently increased ctni blood concentration suggests ongoing active and irreversible damage to cardiomyocytes. the degree of increase has been shown to be correlated with the extent of myocardial damage and with survival in human and animal. in humans, the therapeutic and toxic plasma concentrations of digoxin are usually set at -  ng/ml and greater than  ng/ml, respectively. similar numbers have been derived for animals: e.g., digoxin plasma concentrations of -  ng/ml are nontoxic in horses and a  ng/ml concentration is nontoxic in cats. in dogs, plasma digoxin concentrations up to  ng/ml are nontoxic, but concentrations of -  ng/ml are associated with increased probability of toxicosis. in donkeys, however, the therapeutic and toxic plasma concentrations of digoxin have not been reported in the literature. digitalis intoxication is characterised by several clinical signs varying from mild gastrointestinal upset to chronic weight loss and life-threatening arrhythmias. increased serum bun and creatinine are interpreted as evidence that digoxin toxicity has compromised renal function. in this study, serum digoxin concentration had increased in the donkeys at t to  ±   ng/ml compared to  ±   ng/ml pre-injection. thereafter, digoxin concentration decreased steadily to reach  ±   ng/ml 4 h post-injection. during that time and up to 72 h post-injection, none of the donkeys showed signs of digoxin intoxication, cardiac arrhythmias, ecg abnormalities or elevated serum bun and creatinine. therefore, it can be assumed that the toxic serum digoxin concentration in donkeys would be higher than in humans, horses, dogs and cats. thus, another study is needed to verify the toxic serum digoxin concentration in donkeys. the metabolic alkalosis observed 3 h after digoxin injection may have been due to decreases in pco and lactate. the decreased pco and the increased po and so after injection may have resulted from the increased respiratory rates. lactate is known as the end product of anaerobic glycolysis, a pathway that is of key importance during normal metabolic and athletic events. aside being a waste product during high intensity exercise, lactate is also a valuable substrate that significantly contributes to the energy production of heart, liver, kidneys, non-contracting muscles and even brain. in the present study, lactate concentration decreased during the 4 h post-injection of digoxin, and this may have been caused by the decrease in the metabolic index due to the inactivity of the animals during these time points. similarly, in healthy humans, lactate concentration was reported to be stable following digoxin infusion. concerning the biochemical variables, hyperglycaemia was found in the donkeys after digoxin administration. in human medicine, it is well-known that digoxin therapy increases the adrenaline-induced, and attenuates the insulin-induced hypoglycaemia due to catecholamines release in the acute stage of the action of digitalis glycosides. this study has its own limitations. one of these limitations is the small sample size; therefore the results of this study are preliminary and should be interpreted with caution. another limitation of this study is the using of healthy rather than diseased donkeys. braunwald had reported that there is a direct correlation between the level of myocardial impairment and the effectiveness of digoxin therapy on cardiac function. although the i- stat® ctni assay was initially designed for use in humans, there is good reason to anticipate cross reactivity based on the highly conserved nature of the cardiac troponins across mammalian species. while it may not be practical in veterinary medicine to develop species-specific assays for all relevant species, this practical assumption should come with an acknowledgement of less than perfect validation of this assay. in this study, we thought that the i- stat® ctni assay would detect the donkeys’ ctni, because, despite the lack of a known donkey ctni amino-acid sequence, a third limitation of this study, the sequence of ctni is highly conserved among all species. in addition, the i- stat® analyser has been clinically validated against reference analysers in other species. in addition, the high homology of amino-acid sequences between mammals within the sequence regions detected by ctni analysers allows veterinarians to measure ctni concentrations in many animal species with the same equipment as has been developed for humans, rather than necessitating the development of species-specific technology. as all the troponin samples in this study were below the detection level of the test, therefore the assay may not have had sufficient sensitively to detect small changes in ctni levels below the detection level. these small elevations may still be of clinical relevance.  although the serum digoxin concentrations had increased in the donkeys to  ±   ng/ml 1 h after injection, the donkeys showed no sign of digoxin intoxication, ecg abnormalities or elevated serum bun and creatinine. it is therefore assumed that in donkeys, the toxic serum digoxin concentration is higher than in humans, horses, dogs and cats. consequently, another study is warranted to verify the toxic serum digoxin concentration in healthy donkeys and in those with cardiac diseases. endnotes alanoxin, glaxosmithkline s.p.a, parma, italy. bkenz cardico suzuken co., ltd., japan. cvetscan i-stat abaxis, california, usa. dvetscan hm abaxis, california, usa. evetscan vs abaxis, california, usa. froche diagnostics, indianapolis, indiana, usa. competing interests the authors declare that they have no competing interests. authors’ contributions mt initiated and planned the study. mt and fa carried out the experimental and laboratory work. mt wrote the manuscript and made the figure and tables and revised the manuscript. fa read and revised and approved the manuscript. both authors read and approved the final manuscript. 
blood samples were collected from the donkeys up through 72 h post-injection. it is concluded that administration of digoxin to healthy donkeys did not result in elevations of serum ctni concentration, signs of digoxin intoxication, ecg abnormalities and did not increase serum concentrations of blood urea nitrogen and creatinine. ten clinically healthy donkeys were given an iv infusion of digoxin at a dose of 14 μg/kg. in the other seven animals, the heart and respiratory rates were lower 4 h post-injection. decreases in pco at to 48 h post-injection as well as a decrease in blood lactate at 4 h post-injection were observed. an increase in blood ph was noted at 3 h after digoxin injection. the serum concentration of glucose remained significantly elevated at all-time points after digoxin injection. the effect of digoxin administration on the serum concentration of the cardiac troponin i has not been reported to date in equidae. this study was therefore designed to evaluate the effect of digoxin on cardiac cell damage in donkeys as assessed by ctni, acid–base and electrolyte balance and haematobiochemical profiles. the serum digoxin concentration increased significantly at many time points after injection. there were also increases in po and in oxygen saturation. three of the donkeys exhibited increased heart and respiratory rates post-injection. the serum concentration of ctni did not differ significantly between pre- and post-injection.
 the effect of digoxin administration on the serum concentration of the cardiac troponin i has not been reported to date in equidae. this study was therefore designed to evaluate the effect of digoxin on cardiac cell damage in donkeys as assessed by ctni, acid–base and electrolyte balance and haematobiochemical profiles. ten clinically healthy donkeys were given an iv infusion of digoxin at a dose of 14 μg/kg. blood samples were collected from the donkeys up through 72 h post-injection.  three of the donkeys exhibited increased heart and respiratory rates post-injection. in the other seven animals, the heart and respiratory rates were lower 4 h post-injection. the serum digoxin concentration increased significantly at many time points after injection. the serum concentration of ctni did not differ significantly between pre- and post-injection. an increase in blood ph was noted at 3 h after digoxin injection. there were also increases in po and in oxygen saturation. decreases in pco at to 48 h post-injection as well as a decrease in blood lactate at 4 h post-injection were observed. the serum concentration of glucose remained significantly elevated at all-time points after digoxin injection.  it is concluded that administration of digoxin to healthy donkeys did not result in elevations of serum ctni concentration, signs of digoxin intoxication, ecg abnormalities and did not increase serum concentrations of blood urea nitrogen and creatinine. cardiac troponin idigoxindonkeyheart failureintoxication 