MARINE MAMMAL SCIENCE, 18(2):483-499 (April 2002) 0 2002 by the Society for Marine Mammalogy

USE OF MIDAZOLAMIPETHIDINE AND TILETAMINE/ZOLAZEPAM COMBINATIONS

FOR THE CHEMICAL RESTRAINT OF LEOPARD SEALS (HYDRURGA LEPTONYX)

DAMIEN P. HIGGINS TRACEY L. ROGERS ANDREW D. IRVINE

SOPHIE A. HALL-ASPLAND Australian Marine Mammal Research Centre,

University of SydneyiZoological Parks Board of New South Wales, P. 0. Box 20, Mosman, NSW, 2088, Australia

E-mail: damienh@vetp.usyd.edu.au

ABSTRACT

We immobilized 200-550-kg leopard seals (Hydrurga leptonyx) on sea ice in Prydz Bay, Antarctica (68"25'S, 77'10'E) between November 1997 and February 2000. Midazolam (0.18-0.27 mgikgfi pethidine (1 .O-1.5 mgikg) was administered by dart to 16 leopard seals. Unpredictable immobilization, poor airway maintenance, and our inability to fully assess the suitability of flumazenil (0.003-0.01 mg/kg), naloxone (0.01-0.013 mgikg), and naltrex- one (0.05-0.12 mgikg) as reversal agents limited suitability of midazolam/ pethidine. Tiletamineizolazepam 1:l (0.5-1.5 rng/kg) was, therefore, adrnin- istered to 19 leopard seals. It produced faster induction (19 ? 3 min), more effective and reliable response to dose (rank correlation: r,r = 0.88, n = 18), and better pulmonary ventilation and faster return of cognitive function upon recovery, in comparison to midazolam/pethidine. Best results were achieved with tiletamineizolazepam (1.2-1.4 mgikg) which safely immobilized seven of nine seals for 20-30 min. Entry to the water upon darting was minimized, but not eliminated, by the use of lightweight air-pressurized darts and a thorough knowledge of leopard seal behavior.

Key words: leopard seal, Hydrurga leptonyx, phocid, anesthesia, midazolam, pethidine, tiletamine, zolazepam, antagonist, immobilization.

The leopard seal (Hydmrga leptonyx) is an antarctic phocid of up to 650-kg body mass, which rests on pack or fast ice, usually within 25 m of the water. Collection of blood, biopsies, and morphometric data, and attachment of sat- ellite-linked time depth recorders (SLTDRs) requires reliable, safe, and com- plete chemical restraint with rapid, smooth induction and recovery, and min-

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484 MARINE MAMMAL SCIENCE, VOL. 18, NO. 2, 2002

imal residual effects, minimizing the risk of seals entering the water in a drugged state.

Gales (1989) and Lynch et al. (1999) reviewed sedation or immobilization of a range of phocid species and the anatomical and physiological character- istics relevant to phocid anesthesia, such as pharyngeal folds, a collapsible trachea and the dive response. Most studies of leopard seal anesthesia report the sedation of small numbers of animals with high mortality rates. The largest and most successful study to date (Mitchell and Burton 1991) reported 34 procedures using xylazine, describing variable levels of sedation, 3.5-7-h re- covery periods, a high incidence of vomiting and aspiration, 12% mortality during sedation, and further mortality following recovery. Midazolam (0.13 mg/kg)/pethidine (0.95-1.07 mg/kg) (MP) induced inadequate, but uncom- plicated sedation when administered to three leopard seals' and has also been used successfully for the sedation of southern elephant seals (Mirounga leonina) (Woods et al. 1994,) and crabeater seals (Lobodon carcinophaga).' Effective dose differed among species. Based on the previous success of this combination and our desire for a fully reversible sedative regime, we attempted, unsuccessfully, to develop an effective and safe technique for immobilizing leopard seals using MP as an immobilizing agent and flumazenil and naloxone or naltrexone as antagonists. Tiletamine/zolazepam (TZ), at various doses, has induced reliable immobilization with maintenance of upper airway reflexes and muscle tone in many mammalian species (Short 1987), and a range of other phocid species (Baker et al. 1990, Mitchell and Burton 1991, Phelan and Green 1992, Woods et al. 19946). Mitchell and Burton (1991) administered TZ (2 mg/kg) to one leopard seal which developed respiratory arrest and died. Therefore, in the summer of 1999 we conducted a trial of TZ as an immobilizing agent for leopard seals at dose rates of 0.5-1.5 mg/kg. We also administered flumazenil to three animals during recovery from T Z sedation, in an effort to hasten recovery by antagonizing the zolazepam component of the TZ combination. Here we compare the dose-response and physiological data from the two trials on leopard seals and assess our methods of drug administration and cardio- respiratory support for use in further studies.

METHODS

Animals and Approach

This study was conducted on ice floes and fast-ice edge in the Prydz Bay region, Eastern Antarctica (68"25'S, 77'10'E). Forty-six leopard seals of esti- mated body mass 200-550 kg, were selected for sedation. Seals were located and approached by helicopter to a minimum range of 800 m, personal all- terrain vehicles to 100 m or inflatable boat to 20 m. To reduce risk of the seal entering the water, seals were rejected if they were within 5 m of the water, on a slope declining toward the water, or appeared restless or agitated.

Personal communication from M. A. Tahmindjis, 56 Orange Base Hospital, P. 0. Box 139, Orange, NSW 2800, Australia, August 1997.

HIGGINS ETAL.: CHEMICAL RESTRAINT 485

After allowing the animal to become calm, the final approach was made from behind or beside the animal, walking quietly, slowly, and with an averted gaze.

Intramuscular (IM) injection of sedative and reversal agents was into the lumbar or gluteal musculature. Seals were injected using 10-ml capacity, air- pressurised, plastic darts (Telinject, Germany), charge-pressurized, aluminium darts (Cap-chur, Tetra Systems, Australia) or air-modified aluminium darts (Cap-chur darts modified in the field) with 100-mm batbless needles. Darts were fired from a C0,-powered rifle (GUT 50, Telinject, Germany or Cap- chur CO, long-range projector, Tetra Systems, Australia) from a distance of 12-16 m perpendicular to the long axis of the animal. Distances from the researcher were measured with a prismatic range finder. If the seal responded to approach or darting, the researcher lay down or withdrew, and did not look directly at the seal until it resumed its previous behavior. Responses to dart impact were recorded and compared. Once seals were sufficiently sedated, IM injections of all drugs were by hand using an 18-gauge, 90-mm, spinal needle. Venipuncture for blood collection and intravenous drug administration was via the extradural, intravertebral venous sinus using a 90-mm, 18-gauge spinal needle (Hubbard 1969, Sweeney 1974). Estimation of body mass was based on past experience.

The parasympathetic system blocker, atropine (0.01 5-0.043 mglkg IM) (Atrosine Forte 16 mg/ml, Parnell Laboratories, Silverwater, Australia), was administered in the same dart as all immobilizing drugs to reduce oral and upper respiratory tract secretions and reflex bradycardia (Gales 1989).

M idazolamlPethidine

During November and December 1997 and in November 1999, midazolam (20 mg/ml) and pethidine (150 mg/ml) (MP) (M: P = 1:5.4-1:6.4) were administered at estimated dose rates of 0.18-0.27 mg/kg IM and 1.0-1.5 mg/ kg IM, respectively (Appendix 1). Reversal agents for midazolam (flumazenil, Anexate 0.1 mg/ml, Roche Products Pty Ltd) and pethidine (naloxone, Nar- can, naloxone HC1 injection USP 0.4 mg/ml, Boots Healthcare Australia Pty Ltd. or naltrexone, Nalrrexone HC1 5 0 mg/ml, Wildlife Pharmaceuticals Inc.) were administered by dart (flumazenil 0.003 mg/kg, and naloxone 0.012 mg/ kg or naltrexone 0.05-0.1 mg/kg); by hand (flumazenil 0.01 mgikg IM, and naloxone 0.01 mg/kg IM or naltrexone 0.05-0.12 mg/kg SC); or IV (fluma- zenil 0.008 mg/kg and naloxone 0.013 mg/kg).

TiletaminelZolazepam

Between November 1999 and March 2000, tiletamine/zolazepam (1: 1) (Tel- azol 100 mg/ml, Fort Dodge, Australia) (TZ) was administered at estimated dose rates of 0.5-1.5 mg/kg IM combined substance (Appendix 1). Flumazenil (0.004-0.008 mg/kg IM) was administered to three seals in an effort to hasten their recovery by antagonizing the zolazepam component.

486 MARINE MAMMAL SCIENCE, VOL. 18, NO. 2 , 2 0 0 2

Table 1. Classification of stages of immobilization (adapted from Woods et al. 1994a).

Definition

Stage 0 No effect. Stage 1 Negligible sedation. Stage 2 Stage 3

Visibly sedated but mobile. Sedated. Sluggish movement in response to body or head touch. Loco-

motion may be elicited with repeated stimulus. Hand injection possi- ble.

Stage of immobilization sufficient if not adjacent to water.

Eye may follow passing objects. Ideal for purpose.

muscle tone. Sluggish palpebral response. Excessive.

negligible muscle tone. Negligible palpebral response.

muscle tone. No palpebral response.

Stage 4

Stage 5

Stage 6

Stage 7

Stage 8

Small movements of head or eyes. Locomotion possible but sluggish.

No body movement. Muscle tone variable, dependent on drug used.

No response to vigorous shaking of the tail, body, or head. Very little

As for stage 6 but slightly deeper, with some respiratory depression and

Deep anesthesia. Cardiac or respiratory depression. Complete loss of

Vital Signs and Monitoring

Extent of immobilization was classified to correspond to the system em- ployed by Woods et al. (1994a) (Table 1). Stage 5 was considered ideal for SLTDR and tail tag attachment, swab and blood collection, and collection of blubber biopsies from the shoulder or lumbar region using a biopsy punch of 8-mm diameter. Heart rate was measured by observing incursions of the tho- racic wall. Respiration rate was measured by counting exhalations over 60 sec. Oxygenation was assessed through observation of gingival and lingual colour, gingival capillary refill time, and fluctuation of pupil size. Due to equipment failure and the incomplete immobilization of many animals, pulse oximetry (Model 340, Palco Laboratories, U.S.A.) was employed only on two animals, by applying an enlarged small animal lingual probe to the tongue. Level of cardio-respiratory complication was graded from 0 to 5 (Table 2).

MP seals were marked with oil-based paint and TZ seals were bleach marked and tagged for resighting. Plastic identification tags were applied through the interdigital web of the hind flippers. SLTDRs were attached to the fur of non-molting seals using epoxy cement. Seals were monitored visually at least until they had recovered to stage 2 immobilization, and visual resight- ing of seals was incidental during helicopter, boat, and vehicle based surveys.

Cardio-respiratory Support

Endotracheal intubation (Baker et al. 1990) with a 20-22-mm endotracheal tube was performed on four animals. Oxygen (D size, BOC Gases, Australia) was delivered by intermittent positive pressure ventilation (IPPV) via a 160- litedmin maximum flow, demand/resuscitation head UD Medical Distributing

HIGGINS ETAL.: CHEMICAL RESTRAINT 487

Table 2. Classification of extent of cardio-respiratory complications.

Definition

Level 0 No detectable complication. Level 1 Shallow respiratory pattern, or mild inspiratory stridor with slight

opening of mouth. Very mild gingival cyanosis and occasional com- pensatory tachypnoea.

More shallow respiration, or moderate to severe stridor. Mild to mod- erate gingival cyanosis. Often progresses to level 3 if not corrected.

Marked reduction of respiratory effort or occlusion of airway with col- lapse of thorax on effort to inhale. Moderate to severe cyanosis. In- tubation and IPPV are necessary to arrest progression to level 4.

Respiratory arrest, with or without bradycardia. Severe cyanosis and dilation of pupils. Urgent and aggressive respiratory and cardiac support required for avoidance of cardiac arrest.

Level 2

Level 3

Level 4

, Level 5 Cardiac arrest.

Stridor = a harsh, high pitched respiratory sound such as that made during airway obstruction. Cyanosis = a bluish discoloration of skin and mucous membranes due to excessive concentration of reduced (de-oxygenated) hemoglobin in the blood. Tachy- pnoea = rapid respiratory rate. Apnoea = not breathing. Bradycardia = slow heart rate. IPPV = intermittent positive pressure ventilation.

Co Inc, Phoenix, AZ). Extubation was performed when the seal began to bite the endotracheal tube.

The cardiac stimulant adrenaline (1: 1,000, 0.02-0.06 mYkg) and the re- spiratory stimulant doxapram (Dopram, 20 mg/ml, Wyeth Australia Pty Ltd, Australia, 1.3 mg/kg) were administered as required, endotracheally (Woods et al. 1996) through extension tubing threaded inside the endotracheal tube.

Data Analysij Times are expressed from time of initial administration of sedative. All data

are expressed as mean ?SD. Relationships between dose and response, and between estimated body mass and respiratory rate, were assessed using Spear- man’s rank correlation. Remaining data were assessed for homogeneity of var- iance by plotting studentized residuals against estimated values. One-way AN- OVA was then used to compare the efficacies of flumazeniUnaloxone and flu- mazeniUnaltrexone, and to compare estimated body masses and heart rates between the two sedative groups. Repeated-measures ANOVA was used to compare respiratory rates at 5 min and time of maximum sedative effect (TZ: 20 min, MP: 30 min), between and within the two drug treatments. All statistical calculations were done using Systat Version 9 for Windows (SPSS, Inc.).

RESULTS Animals and Approach

Of the seals selected for darting, three fled to the water during approach for darting and 43 were darted. Another three seals that fled to the water

488 MARINE MAMMAL SCIENCE. VOL. 18, NO. 2, 2002

Table 3. Responses of seals to different darting systems employed in this study.

Response

Aluminum, Plastic, air Aluminum, air charge pressurized pressurized pressurized

darts darts darts

Entered water l(396’0)” 0 (0%) 2 (40%) Moved more than 5 m 3 ( 9 % ) 2 (33%) 2 (40%) Little or no movement 28 (78%) 4 (67%) 1 ( 2 0 % )

a Also harassed by skuas (Catharucta muccormicki) immediately after darting.

upon darting were darted with reversal agents before entry to the water and immediately disappeared from view. The telinject darting system was the least likely to cause animals to flee (Table 3). In wind of 20 kn or more, air tur- bulence made the flight of all darts unpredictable.

Of the 40 seals that remained on the ice after darting, one was darted with a saline dart to test the animal’s response to a new darting system and one animal was abandoned, non-drugged, due to dart malfunction. Immobilization procedures are reported for the remaining 38 seals.

M idazolnmlPethidine

Intramuscular MP was administered to 16 seals (Appendix 1). Times to induction and recovery are presented in Table 4. Induction was smooth with no signs of excitement. Correlation of maximal stage of immobilization to dose rate was r, = 0.62 and r, = 0.75 (n = 16) for midazolam and pethidine, respectively. The four animals that were allowed to recover to stage 2 or less without reversal agents appeared to be restless and disoriented during recovery. No seals entered the water at immobilization stage 3 or greater.

Timing and magnitude of responses to antagonists are reported in Table 4 and Appendix 1, respectively. Intravenous administration of antagonists was associated with severe excitation. Intramuscular administration of antagonists produced mild excitation and restlessness for up to 25 min in all cases, and

TuBIe 4. Time (min) of responses to sedative and reversal drugs.

MP TZ

Time to full effect of initial sedative

Time to first sign of recovery from

Time to recovery to stage 2 or beyond Time to effect of IM flumazenil and

Time to effect of IV flumazenil and

dose

stage 3 or greater

26 ? 6.6 (n = 16)

61 I+_ 27 (n = 4 ) 9 1 -+ 22 ( n = 4 )

5.9 2 0.8 (YJ = 9 )

19 +- 3 (n = 18)

44 I+_ 1 4 ( n = 9) 89 2 16 (n = 9 )

naloxonelnaltrexone

naloxone 2 (n = 1)

HIGGINS ETAL.: CHEMICAL RESTRAINT 489

rapid, apparently disoriented locomotion in the two seals that received the highest doses of naltrexone (0.059 mgikg SC and 0.059 mg/kg IM, and 0.118 mg/kg SC). Renarcotization was not seen in any seal that was reversed from stage 6 or less. A more heavily sedated seal (97/7) was found dead, approxi- mately 3 h after its reversal to stage 2 (Appendix 1). Gross necropsy and histopathological examination failed to reveal the cause of death.

Stridor developed in all but the two least-sedated seals (Appendix 1). Fre- quency and intensity of inhalation efforts were not visibly reduced, but tho- racic expansion was impeded to various degrees. Stridor began at 42 f 14 min ( n = 14) and appeared to be associated with loss of muscle tone in the throat. Head movement temporarily reduced stridor and allowed deeper breaths only in animals at stage 2 or less. Only two seals could be rolled to lateral recumbency and this did not restore adequate airflow. Seals in low stage 5 or less retained sufficient jaw function to prevent intubation, and reversal of sedation was deemed necessary to arrest the deterioration of vital signs in six of these seals. One additional animal (97/7, Table 1) was intubated and supported by IPPV. Cyanosis of four seals became moderate to severe within 1-3 min of the onset of stridor.

In addition to the above seals, one instance of accidental IV injection of MP occurred (97/15). The seal was laterally recumbent when darted and the needle entered the intravertebral, extradural venous sinus via the lumbar dorsal midline. Stage 8 was detected at 12 min and cardio-respiratory support was administered. The seal resumed spontaneous respiration at 50 min, 2 min after the administration of naltrexone (0.05 mg/kg IV) and flumazenil (0.005 mg/ kg IV). Prior IV and IM administration of naloxone, naltrexone, and fluma- zenil to this seal, before the restoration of normal heart rate, had produced no visible response.

Best results with the MP combination were achieved with midazolam 0.20- 0.22 mg/kg IM and pethidine 1.1-1.2 mg/kg IM, which produced stage 4 to 5 immobilization in six of seven animals, with level 1 or level 2 complications, for a period of 15 min. In four of the seals, this working time was limited by the need to administer reversal agents due to further deterioration of vital signs (Appendix 1). The nature of sedation produced by MP was such that at stage 4, noise, movement around the head, or simultaneous placement of an SLTDR and collection of samples, prompted disorientated forward movement which posed a risk to animals adjacent to the water. Attempts to reduce stimulus by placing a blanket over the seal’s head caused further agitation.

TiletaminelZolazepam

Intramuscular TZ was administered to 19 seals (Appendix 1). Seal 99/13 received an unknown initial dose due to dart malfunction. This seal was re- moved from the TZ dose response data, but, as it was subsequently sedated, it was included in resighting data.

Times of induction and recovery are presented in Table 4. Induction was smooth. Occasionally, animals yawned and produced pre-vocal breathing pat-

490 MARINE MAMMAL SCIENCE. VOL. 18. N O . 2. 2002

terns and agonistic vocalizations during induction. Maximal stage of immo- bilization was highly correlated to dose (r; = 0.88, n = 18). Supplementary TZ doses, administered when maximum effect of the first injection was in- adequate for our purpose, produced their effect in 15-20 min, but response was less predictable than administration of an adequate single dose (Appendix 1). Seals that received supplementary TZ doses were not included in recovery- time data.

Most animals produced occasional agonistic vocalizations during recovery. Seal 99/33 vomited and ejected the vomitus from its mouth without difficulty. As recovery was smooth and return of cognition preceded forward mobility, work could continue at the rear of the animal while it was able to move its head, and animals appeared to be completely orientated when they moved to the water.

Two animals failed to maintain deep breaths and pink mucous membranes during the procedure. Seal 99/19 displayed a shallow respiration pattern (level 2) for 10 min while at stage 7 . Pulse oximetry indicated that SpO, increased from 72% to 82% over the following 10 min as gentle rocking of the seal deepened respiration. Seal 99/ 18 developed cardio-respiratory atrest. After the initial agitation of darting, respiratory rate had returned to 12 breathslmin. At 20 min (stage 2, 16 breathdmin, pink gingivae) a supplementary dose was administered. At 42 min (stage 3, 8 breaths/ min, pink gingivae) the animal flinched in response to venipuncture. Three minutes later, respiration and heart contractions ceased simultaneously. Gross necropsy showed that the animal’s stomach was filled with fragmented penguin carcasses, and blood lipid was much higher than in other animals. Histopathology was normal.

Accidental injection, by dart, of TZ into the intravertebral extradural venous sinus of two animals (99124, 99/25), produced stage 7 at 3 and 4 min, re- spectively, Both seals were intubated and ventilated with oxygen by IPPV. Doxapram was administered endotracheally to the seals at 10 and 1 5 min, and respiratory efforts returned 2 min and 15 min following administration, re- spectively. Flumazenil (0.006 mg/kg IM) was administered after restoration of respiratory effort, twice to each animal, and each administration reduced im- mobilization by 2 stages within 10 min. Flumazenil appeared to mildly ac- celerate recovery of intramuscularly sedated animals (n = 3) without inducing excitement.

Best results with TZ followed administration of a single dose of 1.2-1.4 mg/kg, which, in 7 of 9 animals, induced stage 4-6 (sufficient for concurrent sample collection and transmitter attachment), for 20-30 min, without com- promising vital signs. The remaining two animals (9913 1,99133) reached stage 3 and were subsequently adequately immobilized by a supplementary dose.

Vital Signs and Monitoring

There was no significant difference in the heart rates (ANOVA: F,, ,9 = 0.55, P = 0.47) of the MP and TZ groups. Mean heart rate overall was 72 ? 7.5 beats/min (n = 25). Heartbeats were not detected in some shaking or

HIGGINS ETAL. : CHEMICAL RESTRAINT 491

lightly sedated seals. Cross-wind and bright light on snow sometimes made difficult the detection of respiratory sounds and thoracic movements, respec- tively, before onset of sedation permitted closer observation. Rate and depth of exhalation was the most effective indicator of ventilation as nostril move- ment often occurred in the absence of respiration and a succession of small inhalations often contributed to a single inflation of the lung. There was no difference between respiration rates at 5 min and peak effect within the MP or TZ groups (Repeated-measures ANOVA: F1,15 = 0.003, P = 0.956) or between factor interaction (F1,15 = 0.157, P = 0.70). However, respiratory rates at 5 min and peak effect were significantly higher in the TZ group (10 2 2.0 breathdmin) than the MP group (6.1 L 1.4 breathdmin) (F1 ,15 = 12.52, P < 0.01). Estimated body mass also differed between the MP and TZ groups (ANOVA: P,,,, = 30.40, P < 0.01), but the correlation between estimated body mass and respiration rate within groups was not significant (MP: r, = -0.155, E = 12, TZ: r, = 0.667, E = 15).

Data for post procedural monitoring are presented in Appendix 1. No seals entered the water at stage 3 or greater. Resight data are also presented in Appendix 1 ; however, visual resighting was only occasionally effective as seals ate often concealed in the water or behind hummocks of ice, and weather restricted resighting effort. Resight data are not comparable between treat- ments. Paint marks on MP seals washed off, and most MP seals could not be tail tagged due to inadequate sedation and proximity to water, whereas bleach effectively marked TZ seals and most TZ seals were sufficiently immobilized to tail tag. In addition, resights of TZ seals occurred over six months during two consecutive summers with access to floes by boat, whereas resights of MP seals occurred over less than one month, with no resight effort the following summer. Functioning SLTDRs displayed haul-out and movement data consis- tent with a living animaL2 Loss of SLTDR signal may be due to seal death or to technical failure, and SLTDR data from seals in this study suggested that technical failure was likely.2

Cardio-respiratory S.u@ori

Respiratory support was given to seals 97/15, 99/24, 99/25, and 9717. Intubation was successful and IPPV restored normal mucous membrane color in 4-15 min when heart rate was within normal limits. The D-size cylinder was sufficient to ventilate a seal for more than 50 min. Postponing the ad- ministration of reversal agents until the oxygen was 70%-80% exhausted delayed extubation, providing greater opportunity for reversal of oxygen debt, while still allowing time to ensure effective reversal of sedatives. Neither MP seal displayed a gag reflex before head and jaw movement necessitated extu- bation. Following extubation these seals were unable to inhale despite strong respiratory efforts and thrashed their head back and forth with their mouth

Personal communication from T. L. Rogers, AMMRC, P. 0. Box 20, Mosman, NSW, Aus- tralia, May 2001.

492 MARINE MAMMAL SCIENCE, VOL. lS, NO. 2 , 2002

open. Moderate cyanosis returned before the obstruction spontaneously re- solved 5 min after extubation. TZ animals displayed a gag reflex before they regained consciousness and extubation was uneventful.

Administration of adrenaline to seal 97/15 was followed within 4 min by an increase in heart rate from 12 beatslmin to 80 beats/min. Adrenaline and IPPV administered to seal 99/18 with no discernible heartbeat had no effect.

DISCUSSION

Animals and Approuch

The entry of animals to the water following darting remains a risk when immobilizing leopard seals on the sea ice. In addition, the need to maintain distance from the seal during induction impaired monitoring of vital signs in some light and wind conditions. This postponed the detection of complications in seal 97/15. Mitchell and Burton (1991) used herding boards to prevent entry to the water, with some success, however the body masses in their study group were generally smaller. We kept the smallest of our recovering seals (<300kg) from the water for short periods using boards and making aggressive gestures, but this was not successful on mature seals. Minimizing this risk, therefore, requires a thorough understanding of the seal’s response to approach, considering the many variables that we suspect affect their behavior, e.g., age, sex, wind, ambient temperature, presence of other animals. While the data for response to darting were unsuitable for statistical analysis (Table 3), we think that lower dart weight and air-pressure injection were both desirable factors when selecting a darting system for this work.

Intravenous injection of the sedative drugs used in this study caused rapid induction of deep anesthesia and apnoea. Due to air turbulence, we were unable to eliminate this risk in laterally recumbent animals by aiming at the gluteal area, or firing from a standing position to expose more of the flank. However, in the case of TZ, this problem was responsive to rapid implemen- tation of respiratory support. Operators should prepare IPPV equipment before darting to prepare for the eventuality of a dart striking a blood vessel.

lmmobilization

The decision not to weigh animals in this study was due to equipment transport and personnel limitations, and the incomplete immobilization of many animals. Some caution should be employed when comparing dose rates in this study to others, and when applying our recommended dose rates. We chose to estimate body mass directly because of our greater experience with the technique, our imprecision when judging the length of animals in various postures, and the discrepancies reported between different length-mass rela- tionships used in other species (Gales and Burton 1987, Griffiths et ul 1993). We expect that the degree of immobilization achieved with T Z would enable weighing of animals and we plan to attempt to do so in future procedures.

HIGGINS ET AL.: CHEMICAL RESTRAINT 493

Induction and recovery times in this study were similar to those in Weddell (Phelan and Green 1992), gray (Hulichoerus grypw) and southern elephant seals (Baker et ul. 1990, Woods et ul. 19946). Techniques used in this study pro- duced faster recovery, lower mortality under anesthesia, and reduced incidence of vomiting compared to the xylazine procedures reported by Mitchell and Burton (1991). In comparing MP with TZ, the shorter induction time of TZ reduced opportunity for seals to enter the water during induction. The dis- sociative anesthesia induced by the phencyclidinehiletamine, produced more complete immobilization than the sedative-analgesia induced by the benzo- diazepine-opiate combination, MP, and this reduced the risk of arousal and movement or escape during the procedure. The period from first arousal to full mobility was shorter in the MP group, however seals recovering from MP were more active and less orientated than seals recovering from TZ. In addi- tion, most animals immobilized with MP required reversal to restore airway and the resulting excitement, sometimes associated with disorientation, pro- moted their entry to the water earlier than TZ seals. Thus, although not fully reversible, TZ provided less opportunity for seals to enter the water in a disoriented state than did MP.

One seal from each of the groups died. Although the MP seal died after the normal recovery time of MP, it had received the largest dose of any animal and thus renarcotization following reversal must be considered as a likely cause of death. Phocids are hypothesized to sequester oxygen-depleted blood in ab- dominal reservoirs during dives, which may provide a source of non-metabo- lized sedative following a hypoxic episode (Backhouse 1964). As naltrexone had antagonized sedation in this seal, the duration of action of naltrexone in leopard seals may not be as long as in some other mammalian species (Schmitt and Dalton 1987, Haigh 1991). The extended action of naltrexone relies upon its conversion to metabolites with long half-life (Bryson 1989) and nothing is known of this process in either normal or hypoxic phocids. The TZ seal died after the administration of a divided dose that was safe in other animals. Apnoea, bradycardia, excessively deep anesthesia, or deterioration of mucous membrane color did not precede cardiac arrest, as might be expected in the case of overdose, inappropriate dive response, or respiratory compromise. We speculate that the combined physiological and pharmacological effects of di- gestion, profound lipemia, and anesthesia precipitated cardiac arrest.

The incidence of tremors during TZ procedures was similar to that seen in gray seals (Baker et al. 1990), Weddell seals (Phelan and Green 1992), and southern elephant seals (Woods et al. 19946). These tremors could be attri- buted to excitatory effects of tiletamine (as seen with ketamine, a similar drug) (Geraci et al 1981) or to hypothermia, which has been recorded during TZ anesthesia of northern sea lions (Eumetopias jubatus) (Loughlin and Spraker 1989). We plan to monitor body temperature in future procedures.

Vitul Signs and Monitoring Heart and respiratory rates in this study were similar to those reported by

Mitchell and Burton (1991). It appeared that airway restriction during sedation

494 MARINE MAMMAL SCIENCE, VOL. 18, NO. 2, 2002

with MP was associated with loss of muscle tone in the neck region. We saw a similar but less-pronounced obstructive, or stridor, breathing pattern in sleeping non-sedated seals and those seals relieved the obstruction by raising their throat slightly. Placing an endotracheal tube immediately caudal to the larynx during respiratory support did not restore airway unless combined with IPPV, indicating that tracheal collapse contributes to the obstruction. The ability of the leopard seals’ trachea to collapse completely (Kooyman 1981) is a likely explanation for the greater prevalence of obstruction than was reported during MP sedation of southern elephant seals (Woods et al. 1994a), whose trachea is better supported by cartilaginous rings (King and Bryden 1981). It also suggests that similar obstructions are likely to occur when MP is administered to Weddell and Ross (Ommtopboca rossi) seals, whose trachea resembles that of the leopard seal in this regard (Kooyman 1981). We were reluctant to routinely intubate animals sedated with MP because of the response of seals 97/7 and 97/15 to extubation, and the incomplete immobilization induced by MP.

The 1-2-min duration apnoeic periods observed rarely in the leopard seals in this study, and the short: periods reported by Mitchell and Burton (1991), were less frequent than the 8-40-min periods of apnoea reported in southern elephant seals (Mitchell and Burton 1991, Phelan and Green 1992, Woods et al. 19946, Slip and Woods 1996). Southern elephant seals are capable of re- maining submerged for up to 2 h (Hindell 1990), whereas dives of leopard seals recorded to date have not exceeded 10 min.2 It is, therefore, possible that leopard seals have less-developed diving adaptations than the deeper-diving species. When compromised, leopard seals from both groups in this study progressed more rapidly to a state of hypoxia than is suggested by studies of sedated southern elephant seals and Weddell seals. The apparently lower tol- erance to hypoxia of leopard seals relative to southern elephant seals necessi- tates closer observation and more rapid response to changes in exhalation rate and depth, thoracic wall movement, and respiratory sounds than would be appropriate in southern elephant seals. To aid earlier detection of hypoxia we recommend the use of pulse oximetry whenever possible. It is possible that suboptimal oxygenation prior to the detection of stridor contributed to the apparently particularly rapid development of hypoxia in MP-sedated leopard seals, as these seals had slower respiratory rates than those sedated with TZ. It was uncertain whether body mass influenced respiratory rate in this study, but opioids can cause respiratory depression in most mammals (Anderson 1982), including hooded seals (Cystopbora cristata) (Haigh and Stewart 1979), gray seals (Baker and Gatesman 1985), California sea lions (Zalophw califor- nianus) and walrus (Joseph and Cornell 1988, Griffiths et a/. 1993). TZ is usually associated with respiratory depression only during deep anesthesia (Hall and Clarke 1983). It is, therefore, possible that choice of sedative drug was responsible for the lower respiratory rate in the MP group.

Conclusion While midazolam/pethidine (M:P = 0.22: 1.1 mgikg) produces manageable

sedation in leopard seals, this study suggests that tiletamine/zolazepam (1.2-

HIGGINS ET AL.: CHEMICAL RESTRAINT 495

1.4 mg/kg) is the most effective and safe drug to date for chemical restraint of leopard seals in the field. The advantages of greater reliability, fewer respi- ratory complications, and more complete return of cognitive function upon recovery outweighed the reversibility of the MP combination, especially as the duration of action and the ability of antagonists to restore cognition have not yet been determined in this species. Thorough knowledge of leopard seal be- havioral cues and the use of low-impact darting systems are crucial t o reduce the risk of seals entering the water during darting. Further study needs to be directed to this aspect and to the assessment of reversal agents for preventing the onset of anesthesia in phocid species. I n addition, postprocedural moni- toring of captive or readily accessible wild leopard seals would help to establish long-term survival of seals following these procedures.

ACKNOWLEDGMENTS

The Animal Care and Ethics Committees of the University of Sydney and the Aus- tralian Antarctic Division approved these research activities. This project was funded by the Antarctic Science Advisory Committee, Australian Research Council, Committee for Research and Exploration, National Geographic, and Sea World Research and Res- cue Foundation. The authors wish to thank the members of the Australian National Antarctic Research Expeditions (A.N.A.R.E.) 1996-2000, in particular Peter Earnshaw and Doug Watts of Helicopters Australia for their assistance and support. We also thank Colin Southwell and Julie Barnes of the Australian Antarctic Division and Clair Holland for their assistance in the field and the use of their equipment, Sea World Australia and Currumbin Sanctuary, Australia, for their support with equipment, Mich- ele Odewahn for assistance with the manuscript, and Karrie Rose, Taronga Zoo, for examining histological sections. Tiletamine/zolazepam (Telazol) was kindly donated by Fort Dodge, Australia, flumazenil (Anexate) by Roche Products, Australia, and nal- oxone (Narcan) by Boots Healthcare Australia Pty Ltd. We would also like to thank Tetra Systems, Australia, for their support. The constructive advice of the journal referees is also much appreciated.

LITERATURE CITED

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BACKHOUSE, K. M. 1964. Anaesthesia in marine mammals. Page 79 in G. Jones, ed. Small Animal Anaesthesia. Pergamon Press, London.

BAKER, J. R., AND T. J. GATESMAN. 1985. Use of carfentanil and a ketamine-xylazine mixture to immobilize wild grey seals (Halicboerza gqJpus). The Veterinary Record 116:208-210.

BAKER, J. R., M. A. FEDAK, S. S. ANDERSON, T. ARNBORN AND R. BAKER. 1990. Use of a tiletamine-zolazepam mixture to immobilize wild grey seals and southern elephant seals. Veterinary Record 126:75-77.

BRYSON P. D. 1989. Comprehensive review in toxicology. Aspen Publications. Rock- ville, MD.

GALES, N. J. 1989. Chemical restraint and anaesthesia of pinnipeds: A review. Marine Mammal Science 5 : 2 2 8-2 5 6.

GALES, N. J., AND H. R. BURTON. 1987. Prolonged and multiple immobilizations of the southern elephant seal using ketamine hydrochloride-xylazine hydrochloride

496 MARINE MAMMAL SCIENCE, VOL. 18, NO. 2, 2002

or ketamine hydrochloride-diazepam combinations. Journal of Wildlife Diseases 2 3:6 14-6 18.

GERACI, J. R., K. SKIRNISSON AND D. J. ST AIJBIN. 1981. A safe method for repeatedly immobilizing seals. Journal of the American Veterinary Medicine Association 179: 1192-1 193.

GRIFFITHS, D., 0. WIIG AND I. GJERTZ. 1993. Immobilization of walrus with etorphine hydrochloride and zoletil. Marine Mammal Science 9:250-257.

HAIGH, J. C. 1991. Immobilization of wapati with carfentanil and xylazine and opioid antagonism with diprenorphine, naloxone and naltrexone. Journal of Zoo and Wildlife Medicine 22:318-323.

HAIGH, J. C., AND R. E. A. STEWART. 1979. Narcotics in hooded seals (Cy.rtophora rristata): A preliminary report-. Canadian Journal of Zoology 57946-949.

HALL, L. W., AND K. W. CLARKE. 1983. Veterinary anaesthesia. 8th edition. Balliere Tindall, London.

HINDELL, M. A. 1990. Population dynamics and diving behaviour of a declining pop- ulation of southern elephant seals. Ph.D. thesis, University of Queensland, Bris- bane. 192 pp.

HUBBARD, R. C. 1969. Chemotherapy in captive marine mammals. Bulletin of the Wildlife Diseases Association 5:2 18-230.

JOSEPH, B. E., AND L. H. CORNELL. 1988. The use of meperidine hydrochloride for chemical restraint in certain cetaceans and pinnipeds. Journal of Wildlife Diseases 24:69 1-694.

KING J. K., AND M. M. BRYDEN 1981. Southern elephant seal-Mivozlnga leonina. Page 310 in R. J. Harrison and S. H. Ridgeway, eds. Handbook of marine mammals Volume 2. Seals. Academic Press, New York, NY.

KOOYMAN, G. L. 1981. Leopard seal- Hydnwga &onyx. Page 261 in R. J. Harrison and S. H. Ridgeway, eds. Handbook of marine mammals. Volume 2. Seals. Ac- ademic Press, New York, NY

LOUGHLIN, T. R., AND T. SPRAKER. 1989. Use of Telazola to immobilize northern sea lions (Ezlmetopias jzlbdtus) in Alaska. Journal of Wildlife Diseases 25:353-358.

LYNCH, M. J., M. A. TAHMINDJIS AND H. GARDNER. 1999. Immobilization of pinniped species. Australian Veterinary Journal 77:181-185.

MITCHELL, P. J., AND H. R. BURTON. 1991. Immobilization of southern elephant seals and leopard seals with cyclohexamine anaesthetics and xylazine. Veterinary Record 129332-336.

PHELAN, J. R., AND K. GREEN. 1992. Chemical restraint of Weddell seals (Leptonychotes weddellii) with a combination of tiletamine and zolazepam. Journal of Wildlife Diseases 28:230-235.

SCHMITT, S. M., AND W. J. DALTON. 1987. Immobilization of moose by carfentanyl and xylazine and reversal by naltrexone, a long acting antagonist. Alces 23:195-2 19.

SHORT, C. E. 1987. Dissociative anaesthesia. Pages 158-172 in C. E. Short. ed. Principles and practice of veterinary anaesthesia. Williams and Wilkins, Baltimore, MD.

SLIP, D. J., AND R. WOODS. 1996. Intramuscular and intravenous immobilization of juvenile southern elephant seal. Journal of Wildlife Management 605302-807.

SWEENEY, J. C. 1974. Procedures for the clinical management of pinnipeds. Journal of the American Veterinary Medicine Association 16531 1-814.

WOODS, R., S. MCLEAN, S. NICOI. AND H. BURTON. 1994a. Use of midazolam, pethi- dine, ketamine and thiopentone for the restraint of southern elephant seals (Mir- ounga leonina). Veterinary Record 135: 572-577.

WOODS, R., S. MCLEAN, S. NKOL AND H . BURTON. 1994b. A comparison of some cyclohexamine based drug combinations for chemical restraint of southern ele- phant seals (Mirozlnga leonina). Marine Mammal Science 10:4 12-429.

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HIGGINS ETAL.: CHEMICAL RESTRAINT 497

respiratory stimulant doxapram in southern elephant seals (Mivoungu leonind). Vet- erinary Record 138:514-517.

Received: 5 June 2000 Accepted: 17 July 2001

Appe

ndix

1.

Estim

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bod

y m

ass

(kg;

BM

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max

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leve

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and

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ital

sign

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b

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