Kulans (Equus hemionus kulan) are a subspecies of the Asiatic wild ass. Anesthesia in kulans can be particularly challenging due to their stoic nature (1). Adaptation to the desert influences fluid and electrolyte balance and accordingly, modifies the metabolism of certain pharmacological agents (2). These physiological alterations also affect pharmacokinetic and pharmacodynamic characteristics of immobilizing agents; hence, increased dosages of anesthetics may be required (3).

The ultrapotent opioid etorphine is the most commonly used agent for the remote immobilization of wild equids (4–12). Small volumes of etorphine can be lethal for humans, and therefore, it is subject to special purchase regulations. Consequently, availability of etorphine in certain countries is sometimes limited, and reliable alternatives are needed (7, 13, 14).

Etorphine, in combination with low doses of detomidine, acepromazine and butorphanol, has been used to successfully capture free-ranging kulans (9, 11). Tachycardia, hypertension, hypoxemia, hypercapnia, metabolic acidosis, muscle rigidity and tremors are common side effects and numerous studies have documented the cardiorespiratory effects of etorphine (7–9, 11, 15, 16). However, the limited availability of etorphine, as well as its high toxicity for humans, has encouraged veterinarians to test other combinations (7). For wild equids, there is only a limited number of studies of anesthesia combinations without ultrapotent opioids (7, 13, 14, 17–20). The combination of medetomidine and ketamine (MK) has been successfully used for remote anesthesia of non-domestic equids (13, 14, 18, 20). Potential side effects of these combinations include ataxia during induction and recovery, bradycardia, hypoxemia and hypertension. However, in some cases, MK failed to induce recumbency and was therefore used rarely (13, 18). A combination of ketamine, medetomidine and butorphanol (KMB) was recommended for routine management procedures of captive zebras (7). This anesthetic regime provided an effective immobilization characterized by a deep plane of anesthesia and excellent muscle relaxation. Side effects of KMB include severe systemic hypertension and moderate hypoxemia. No etorphine-free anesthesia protocols have been reported for kulans.

Recent studies on the white rhinoceros focussed on the attenuating influence of midazolam on peripheral oxygen consumption (21). It has been shown that the excellent muscle relaxation properties of midazolam ensures deep ventilation and thus improves peripheral tissue oxygenation in rhinos (22). Furthermore, midazolam may lower the dosage of medetomidine, and reduce alpha-2 agonist-mediated vascular hypertension (23). We therefore hypothesized that adding midazolam to the KMB protocol may have beneficial effects on cardiorespiratory parameters, such as oxygenation and blood pressure in kulans.

All etorphine-free drug combinations with commercially available ketamine, medetomidine, midazolam and butorphanol (KMMB) resulted in a large volume for darting which potentially limits its use in free-ranging settings (17, 18, 20). Furthermore, the poor solubility of concentrated ketamine preparations also restricts the use of higher concentrations of this drug in small volumes (13). Tiletamine has greater solubility than ketamine (24, 25), which facilitates the application of 3cc darts for remote drug administration. Tiletamine is commercially available as a dry substance in combination with zolazepam (Zoletil® or Telazol®), a benzodiazepine-like tranquilizer (26). The combination of tiletamine, zolazepam and medetomidine (TZM) has been successfully used for distance immobilization of feral horses, where it induces adequate, safe and partially reversible anesthesia (17). With the advantage of high solubility, in the current study, tiletamine and zolazepam were added to the combination of medetomidine and butorphanol (TZMB), in order to provide a highly concentrated alternative to KMMB, which could also be used in free-ranging settings.

This study aims to establish and to evaluate two safe and effective alpha-2 agonist-based protocols for captive kulans and to compare the results with the currently used etorphine-based drug combination (the prevailing gold standard). The cardiorespiratory parameters and benefits of both combinations were determined. As far as the authors are aware, this is the first report on these drug combinations in non-domestic equids. We hypothesize less respiratory depression, better induction quality and muscle relaxation, for the two etorphine-free combinations.

Both treatment protocols effectively induced immobilization provided excellent muscle relaxation and a safe and reliable anesthesia. However, it should be highlighted that there were certain variations between the treatment groups and the control group, especially concerning side effects on the cardiorespiratory system. Evaluation of cardiovascular measurements showed elevated systolic arterial pressure in all three groups, although severe systemic hypertension (> 160 mmHg) was only observed in the kulans immobilized with TZMB (Table 6) (26). Assessment of respiratory function revealed normoxemia in the treatment groups, while the control group maintained hypoxemic over the course of immobilization (Figure 4, Table 7) (26). In addition, PaCO₂ levels indicated normocapnia for the treatment groups, while the control group was hypercapnic (Figure 3).

The transition to recumbency in the kulans was characterized by a consistent pattern corresponding to increasing drug effects. The EADB kulans followed the typical course of etorphine inductions with a hypermetric “hackneyed” gait, stargazing, walking into fences, head pressing and tremors (7, 9, 11), while the treatment groups revealed similar characteristics as reported in previous studies on etorphine-free protocols in wild equids, such as standing with a wide based stance, head down, bottom lip hanging until stumbling and transition to recumbency (7, 13, 14, 19, 20). Inductions of the etorphine-free combinations were longer but smoother, while induction times of the EADB group were similar to the 5.6 min reported for the same dosages of this combination in free-ranging kulans (9). Due to the faster onset of the effect of ketamine, compared to tiletamine, induction times for the KMMB group were shorter than those for the TZMB group (26). Protocols using similar dosages of only ketamine and medetomidine in other equids resulted in longer inductions (13, 20). Due to the synergistic effects of adding butorphanol and midazolam, similar induction times with lower dosages of ketamine and medetomidine could be achieved (20). To the authors knowledge, this is the first comprehensive study incorporating the benzodiazepine midazolam into a total intramuscular anesthesia (TIMA) protocol of wild equids.

Both treatment protocols provided excellent muscle relaxation, while the current study, the control group revealed high EMG values, indicating a state of excitation without sufficient muscle relaxation. Etorphine-induced respiratory depression and thoracic muscular rigidity (31–35) may result in impaired alveolar ventilation. The consequences include respiratory acidosis, reflected in significantly higher PaCO₂ and lower pH levels in the control group. Furthermore, etorphine-induced muscle tension, tremors and seizures (31–35) may result in increasing cellular metabolism, higher lactate levels and consequently a mild degree of metabolic acidosis. However, a greater degree of metabolic acidosis was detected in TG 1, while the high potency of the benzodiazepine zolazepam in TG 2 may have contributed to improved muscle relaxation and consequently lower lactate, a less negative base deficit and significantly higher bicarbonate levels (TP0 = p < 0.0325) (Table 7).

Increased muscle rigidity is a known side effect of etorphine, and severe seizures and continuing tremors were observed with several animals of the control group (26). The immobilization quality in the control group kulans was therefore scored as deep sedation. For the treatment groups, the qualitative assessment indicated a light to deep immobilization plane with maintained palpebral and anal reflexes. There was no evidence of spontaneous arousals as previously reported for Przewalski horses immobilized with medetomidine and ketamine (13). Both treatment groups did not require additional anesthetics until reversal after 60 min.

Systolic arterial hypertension combined with a presumably baroceptor-initiated lower pulse wave was detected in the treatment groups (7). TZMB kulans received a lower medetomidine dosage than KMMB kulans. However, severe systolic arterial hypertension was observed only in TZMB kulans, with a significantly higher systolic arterial pressure than both TG 1 and the control group. These differences indicate that the indirect cardiovascular impact of tiletamine in the TZMB group induces more pronounced hypertensive effects than ketamine in the KMMB group (7, 26). Systolic arterial pressure in both treatment groups was considerably lower than in zebras anesthetized with KMB (7). The addition of midazolam and the consequently have contributed to this improvement of cardiovascular parameters. Etorphine administration in horses enhances sympathetic activity and consequently produces tachycardia and an elevation of systemic blood pressure (33–35). However, in the current study, the EADB kulans showed only transient systemic hypertension decreasing over time to normotension, while sustained compensatory tachycardia was observed.

As a consequence of drug-induced hypoventilation, most immobilizations of wildlife are associated with hypercapnia (16, 36). PaO₂ levels of < 80 mmHg indicate hypoxemia, while profound hypoxemia is of concern when values drop below 60 mmHg (26). PaCO₂ levels in the treatment groups were comparable to awake equids (7, 26), while moderate hypercapnia was detected in the control group (Table 7). Both treatments showed normocapnia and a RR within normal limits. Etorphine reduces the sensitivity of arterial chemoreceptors, inhibiting respiratory response mechanisms to hypoxemia and hypercapnia (31). Hypoxemia (PaO₂ < 80 mmHg) was initially observed only in the control group, where hypoventilation may have contributed to clinically significant hypoxemia and hypercapnia (26). However, although oxygen was administered from TP 10 onwards, hypoxemia did not resolve in the EADB group over the course of immobilization. Also the PaO₂ levels only slightly increased following oxygen supplementation, regardless of the drug combination used (Figure 4). These findings indicate that other drug-induced mechanisms, apart from hypoventilation, may have contributed to the hypoxemia, such as elevated alveolar-arterial oxygen partial pressure gradients (P(A-a) O2) (26). Medetomidine and etorphine are known to cause pulmonary hypertension and consequently intrapulmonary shunting and V/Q-mismatching (36). Pulmonary hypertension leads to the formation of pulmonary congestion with interstitial oedema and decreases the capillary blood flow transit time. These mechanisms may contribute to hypoxemia by hindering gas exchange across alveolar capillary membranes (37). The findings of this study support these considerations with an elevated (P(A-a) O2) in all three groups compared to 10 mmHg reported for healthy horses at rest (Table 7).

Furthermore, medetomidine reduces peripheral oxygen availability, due to peripheral vasoconstriction (38). Observations in zebras immobilized with KMB appeared to be consistent with the above mentioned findings (7). Despite normocapnic PaCO₂ levels, profound hypoxemia was observed in these zebras (7). These results indicate that the addition of midazolam has a dose-reducing effect on medetomidine, which may reduce pulmonary and peripheral hypertension (23).

The recovery times in the control group (EADB 3.5 ± 1.27 min) were significantly shorter than those in the treatment groups (KMMB [6 ± 1.10 min) and TZMB (6.29 ± 1.00 min)] and similar to the recovery times observed in free-ranging kulans with no evidence of re-narcotization (11). The kulans in both study groups had faster recovery times than ketamine-medetomidine immobilized feral donkeys (21 min) and Przewalski horses (13 min). As the administration routes of antagonists is similar, the differences in recovery times may be related to the higher atipamezole-to-medetomidine ratios used in kulans (5 mg/mg) compared to feral donkeys and Przewalski horses (2.5 mg/mg) (13, 20). It should also be taken into consideration that longer recumbency times in KMMB and TZMB kulans (60 min) may improve antagonist efficiency because the residual drug effects decrease over time. It has been reported in feral horses, that following comparable medetomidine dosages in a combination of medetomidine and ketamine, residual drug effects diminished, and horses recovered approximately 70 min after anesthesia induction, without any reversal of anesthetics (18). Recoveries of ketamine–medetomidine–butorphanol-immobilized zebras took 1.5 min to standing after atipamezole was administered (7.5 mg/mg medetomidine), partly intravenous and partly intramuscular (1:1) (7). KMB zebras and KMMB kulans received similar naltrexone dosages intravenously. The faster recovery times for these zebras may be related to higher atipamezole-to-medetomidine ratios (7.5 mg/mg), as well as to different intramuscular-to-intravenous ratios of atipamezole (7). The residual effects of midazolam may also be responsible for the longer recovery times in KMMB kulans. The flumazenil-to-midazolam ratio (0.005 mg/mg) used in this study was fairly low; therefore, further studies should be conducted to determine the influence of residual midazolam effects on recovery time and quality.

Tiletamine–zolazepam–medetomidine-immobilized feral horses needed 31 min to recover from 60 min of recumbency (17). Their immobilization times were comparable to the kulans, but the differing atipamezole-to-medetomidine ratios for feral horses (0.7 mg/mg) may also be responsible for the longer recovery times (17). In conclusion, higher atipamezole-to-medetomidine and flumazenil-to-midazolam ratios may be beneficial, especially for shorter procedures, as the main anesthetic effects are still present.

The recovery qualities for the different study groups were excellent, regardless of the drug combination used. Reports on the immobilization of free-ranging wild equids emphasize preventing rapid attempts to stand up immediately after the administration of antidotes. The aim of these measures is to coordinate the first attempt to stand with sufficient antagonist effects. Physical restraint in a lateral position with fixation of the head was therefore recommended, to facilitate rapid and safe recoveries (11). In a study on feral horses, sternal positioning was mentioned to have beneficial effects on recovery quality (18). The sporadic pre-study use of the KMMB combination (unpublished data) revealed that sternal positioning without any physical restraint may improve the recovery qualities of kulans. This technique was applied in our study and it resulted in controlled, calm, and smooth recoveries and hence reduced stress for the animals. Therefore, we recommend sternal positioning during recovery for other captive equine species anesthetized with the KMMB and TZMB protocol.

In conclusion, the essential benefit of the KMMB and TZMB protocol is an excellent muscle relaxation. Both combinations provided effective and safe inductions, smooth recoveries, and an adequate and reliable depth of anesthesia suitable for prolonged procedures on captive kulans.

Although species-specific differences require further evaluation, the findings in this study indicate that supplementing KMB with midazolam provides excellent muscle relaxation and considerably improves the cardiorespiratory parameters. With regard to the promising results of the non-opioid-based KMMB combination of the current study, this protocol should be further investigated in other wild equids.

The raw data supporting the conclusions of this article will be made available by the authors, without undue reservation.

The animal study was reviewed and approved by the Committee for Ethics and Animal welfare of the Leibniz-IZW.

JB, JP, and FG designed the study protocol, performed anesthesias, and carried out the clinical examinations. DB and EG assisted with clinical examinations. DB performed laboratory diagnostics. HR completed the statistics. JB drafted the manuscript. BE added comments and reviewed blood pressure results. JP, AH, FG, EG, HR, BE, and DB conducted review and manuscript conceptualization. All authors read and approved the final manuscript version.

The publication of this article was funded by the Deutsche Forschungsgemeinschaft (DFG, German Research Foundation) – Projektnummer 491292795.

The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

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