The initial blood pH, after immobilisation, indicated a mild acidaemia (normal ruminant arterial blood pH reference range 7.37 to 7.48 [25]) due to the elevated PaCO2 causing a respiratory acidosis. Thereafter, the PaCO2 increased further and blood pH of the impala significantly decreased over time, regardless of the immobilisation and anaesthetic protocol used. The Henderson-Hasselbalch approach and the quantitative physicochemical Stewart approach were used to interpret the acid-base status of the impala. The profoundly elevated PaCO2 at the end of the anaesthesia would cause a respiratory acidosis, while the rising apparent strong ion difference (SIDa) and waning total weak acids (Atot) contributed to a simultaneous occurring metabolic alkalosis. Furthermore, the progressive elevation of the calculated serum bicarbonate (HCO3
−) and base excess (BE) both indicate an emerging compensatory metabolic response. Therefore the resultant pH values at the end of the anaesthesia are because of a pronounced metabolic compensatory response to the severe respiratory acidosis that resulted in an overall mild acidaemia. Because there are no published reference ranges for acid-base variables in resting impala we used ranges from closely related species (healthy awake goats and calves; Table 1) to interpret our findings.
Unfortunately, more advanced techniques used to calculate compensation, like expected compensatory changes in PaCO2 or bicarbonate ion concentrations, would be difficult to use for interpretation due to the paucity in referenced normal ranges for small wild ungulates. Although the impala were habituated to the boma, they were not tame enough for us to obtain awake control or reference samples. Gaining such samples from an awake wild animal can only be achieved by using remote sampling devices [26]. Such devices are not readily available and need to be custom made per species [27].
We expected respiratory acidosis to be pronounced, especially at the end of the anaesthesia, as PaCO2 was greatly elevated compared to the normal awake range of 35–45 mmHg in mammals [5]. The PaCO2 at the end of the anaesthesia was substantially higher compared to just after induction into immobilisation in our impala, and compared to values measured in other immobilised impala (PaCO2 of 39.1 ± 3.4 to 41.3 ± 5.0 mmHg) [28]. One of the stimuli to take a breath in a healthy awake animal is brought about by the rising PaCO2 level reaching a threshold. The drugs used in this study, especially the potent opioids, either alone or in combination with the other anaesthetic and sedative drugs, are known to cause respiratory-neuronal depression [29, 30] which shifts the carbon dioxide respiratory response curve to the right [31]. Whereby a higher threshold level of PaCO2 is required to stimulate the respiratory centre to initiate a breath. Therefore these drugs ultimately result in hypoventilation (decreased alveolar minute ventilation) which causes the increase in PaCO2. Ventilation is challenging to assess when only subjectively monitoring the respiratory system by counting the respiratory rate and estimating the tidal volume. Often an animal will appear to be ventilating normally, as in the case of these impala that had a normal respiratory rate and tidal volume at Time 1, after dosing with butorphanol (data reported elsewhere) [22, 23], but on closer examination this may not be the case. Thus, more invasive monitoring tools, such as arterial blood gas analysis or capnography, may be required to detect shifts in blood pH that are due to alterations in ventilation [32]. Furthermore, other co-aetiologies should always be considered when there is an obvious respiratory acidosis without overt evidence of simple hypoventilation, such as severe right-to-left pulmonary shunting, large dead-space ventilation or ventilation-perfusion mismatch [5, 23, 30]. Haemoglobin is an important intracellular buffer that will bind reversibly to either carbon dioxide or to the hydrogen ion formed by the bicarbonate buffer system, to transport them from the metabolising tissues to the lungs [4, 5, 8, 33, 34]. With oxygen supplementation an increase in the PaO2 will increase the force for oxygen to bind to haemoglobin as opposed to carbon dioxide or hydrogen ions (Haldane Effect; high PaO2 levels decrease the buffering effects of haemoglobin, therefore hydrogen ions are unbound from haemoglobin to preferentially transport oxygen). Therefore, during anaesthesia, the PaCO2 in the impala most likely increased due to the oxygen supplementation [4, 5, 33, 34]. Furthermore, the haemoglobin concentration (and haematocrit) dropped during general anaesthesia [35], a known phenomenon in patients under general anaesthesia, especially if alpha2-adrenoceptor agonists like medetomidine are used [36], therefore decreasing an important plasma buffering system which could have also contributed to the increase in PaCO2 and hydrogen ion concentration. The emergency treatment of apnoea with butorphanol did result in a regular spontaneous breathing pattern in the impala immobilised with both protocols [22, 23]. A limitation to this study is that we did not take arterial blood samples immediately after recumbency, while apnoeic episodes occurred, especially in P-TMP which required more frequent butorphanol boluses compared to P-EME. Therefore, the effects of butorphanol on the blood pH could not be determined. However, in another etorphine immobilized ungulate, the goat, butorphanol corrected hypoxaemia but not hypercapnia and therefore it may be that it had little influence on blood pH in the impala at Time 1 [37].
Irrespective of the cause of the respiratory acidosis, metabolic compensation, indicated by the significant rise in the bicarbonate ion concentration, occurred within a 120 min. This indicator of compensation is according to the traditional Henderson-Hasselbalch approach used to evaluate blood pH, whereby the body attempts to correct the increased hydrogen ion concentration by elevating the bicarbonate ion concentration to normalise the bicarbonate ion to carbonic acid ratio ([HCO3
−]:[H2CO3] ratio) back to 20:1 [4]. The PaCO2 corresponds to H2CO3 and is merely substituted to simplify the calculation of the compensatory response [12, 17, 28]. Therefore, any rise in PaCO2 should be met by a rise in the HCO3
− ions in uncomplicated respiratory acidosis, as demonstrated in these impala.
A simple change in the HCO3
− does not completely explain the acid-base compensation that occurred in the impala. The apparent strong ion difference (SIDa) was higher and the total weak acids (Atot) were lower than that of published ranges for healthy control goats and calves. Both changes indicate an additional non-respiratory alkalinising effect [5,6,7, 11]. The measured electrolytes (sodium, potassium, calcium, chloride and phosphorus) were within accepted published ranges for impala [38]. Furthermore, all but the potassium and chloride concentration did not significantly change over time. Yet the decrease in the potassium level was not large enough to solely explain the increased apparent strong ion difference (SIDa) value. Therefore, the decrease in lactate ion concentration that occurred most likely contributed the most to the increased apparent strong ion difference (SIDa) at the 120 min measurement. The drop in the chloride concentration could have been due to the increase in plasma bicarbonate, whereby the plasma attempts to maintain electrical neutrality by excreting chloride [5,6,7, 12, 39]. At the rate of administration used it is unlikely that the infused physiological saline increased sodium or chloride concentrations in the plasma of the impala, as reported in healthy dogs [40]. The decreased total weak acids (Atot) over time was attributed to the decrease in albumin and globulin concentrations. A decrease in plasma protein levels has been described in animals undergoing general anaesthesia [35], especially when alpha2-adrenoceptor agonists such as medetomidine are administered [36]. The total weak acids (Atot) levels reported in this study were also lower compared to goats and calves. This difference could be attributed to different measurements of phosphorus, or different calculations used to determine the total weak acids (Atot). Furthermore, the rising bicarbonate ion concentration could cause the negatively charged proteins to move out of the plasma in order to maintain electrical neutrality, a plausible theory requiring further confirmation.
The decrease in the anion gap (AG) over time was attributed to the pronounced increase in the HCO3
− concentration. Overall the anion gap (AG) was substantially lower than those reported for goats and calves [14, 17,18,19,20]. However, the HCO3
− concentrations and BE at the end of the anaesthesia were higher than a generally accepted upper limit of 30 mmol/L and 6.0 to 8.0 mmol/L, respectively for herbivores [4, 5, 8] and the published ranges for healthy goats [14, 17, 19] and calves [18, 20]. Both of these variables demonstrate that a metabolic compensatory response was initiated to correct the respiratory acidosis in the impala.
The worsening acidaemia, and precipitous drop in serum protein concentrations, especially albumin, may alter ionisation and protein binding of drugs, which could have profound effects on drug pharmacokinetics and dynamics [41]. These possible alterations warrant further investigation to gain better clarity of their clinical implications. In other words, did the impala in this study experience more pronounced respiratory depression due to alterations in the pharmacokinetics and dynamics of the drugs used to maintain general anaesthesia by causing a relative overdose?
Furthermore, the clinical implications of acidosis, regardless of cause, are serious and warrant careful consideration. The effects of acidosis on the cardiovascular system include negative inotropy, tachycardia and vasodilation which translates into a decreased blood pressure due to the reduction in cardiac output (decreased stroke volume) and systemic vascular resistance [1, 2]. Oxygen binding to haemoglobin is altered, causing a right shift in the oxygen-haemoglobin dissociation curve (Bohr Effect; acidaemic plasma pH, usually brought about by increasing PaCO2 levels at metabolically active tissue decrease haemoglobins affinity for oxygen, therefore increase its offloading to the tissue) [4, 5]. The right shift translates into a decrease affinity for haemoglobin to bind to oxygen, therefore less is transported to the tissue potentially resulting in tissue hypoxia. Among other causes, the decrease in pH increases the stimulation to breathe which results in increased workload of the respiratory system and therefore global oxygen demand. Furthermore, the decrease in cardiovascular performance decreases oxygen delivery [2]. Animals that cannot initiate compensatory responses to acidosis due to illness or the effects of anaesthetic drugs or both may suffer further physiological derangements which could lead to increased morbidity or mortality.
The shift in blood pH was evident in healthy impala undergoing immobilisation and general anaesthesia using two different drug protocols. The profound increase in PaCO2, despite a seemingly normal respiratory rate for a medium sized ruminant suggests that monitoring for a respiratory acidosis is not reliable when using just the respiratory rate as an indicator of respiratory suppression. The gases found within the alveoli while breathing room air include nitrogen, oxygen and carbon dioxide (originating from the metabolically active tissue and transported to alveoli via the blood). If the amount of carbon dioxide increases to levels above 50 mmHg, as noted in these impala, then there is an increased competition for gases within the alveoli [4, 5]. This competition decreases the amount of oxygen that is available for absorption and can lead to hypoxaemia [4, 5]. Therefore, if these protocols are to be used in the field, oxygen supplementation should be considered mandatory [22, 23]. Despite the substantial increase in PaCO2 over time the acidaemic shift in the blood pH was negligible due to the profound compensatory metabolic responses that we detected. These important responses require normal health and physiological function and therefore caution should be taken, and acid-base status carefully assessed, when immobilising ill or anorexic wild ungulates.