Capture-induced exertional rhabdomyolysis in the Shortfin Mako Shark, Isurus oxyrinchus

Nicholas M. Otway

New South Wales Department of Primary Industries, Port Stephens Fisheries Institute, Taylors Beach, NSW, Australia

Correspondence
Nicholas M. Otway, New South Wales Department of Primary Industries, Port Stephens Fisheries Institute, Taylors Beach Road, Taylors Beach, NSW, 2316, Australia. Email: [email protected]

Abstract
Background: Shortfin Mako sharks (Isurus oxyrinchus) are top-order predators in oce- anic food chains. They are captured worldwide by commercial and recreational fish- eries, but little is known about the effects that fishing has on the homeostasis and longevity of these animals.

Objective: This study aimed to assess the health of Shortfin Mako sharks captured by recreational fishers off eastern Australia.
Methods: Twenty-four sharks were captured, and the gender, length, weight, repro- ductive maturity, and stage were recorded. After blood and urine collection, serum analytes were quantified using standard biochemical methods, whereas urine was analyzed using semi-quantitative reagent strips, microscopic examination, centrifu- gation, and ammonium sulfate precipitation tests.

Results: Six Makos presented with red-brown urine. The means of notable serum an- alytes were as follows: sodium 276 mmol/L, potassium 15.6 mmol/L,
inorganic phos- phate 10.6 mmol/L, magnesium 3.3 mmol/L, urea 325 mmol/L, creatinine 52 μmol/L,
AST 2806 U/L, CK 240938 U/L, lactate 44.4 mmol/L, osmolarity 1160 mmol/L, and pH 7.13. These analytes differed from the respective sand tiger shark reference inter- val, which was used as a proxy for Makos. The red-brown urine was due to myoglobin and had a mean pH of 5.76 that, when combined with red-brown casts, led to a diag- nosis of fishing-induced exertional rhabdomyolysis that occurred secondary to lactic acidosis, hypoxia, and hypovolemia. It was further exacerbated by hyperkalemia and acute renal failure, serious complications that often lead to mortality.

Conclusions: Practitioners caring for sharks and rays should consider collecting urine from free-living or aquarium animals when they are captured for examination and/or treatment, particularly at times with maximal seawater temperatures.

K E Y WO R D S
blood, capture myopathy, CK, myoglobin, urine

1 | INTRODUC TION

The Shortfin Mako shark, Isurus oxyrinchus (Family Lamnidae—mack- erel sharks) and hereafter referred to as Mako(s), has a circumglobal
distribution and primarily inhabits tropical and temperate waters.1 They have been recorded to depths of 866 m while undertaking ex- tensive oceanic migrations exceeding 2000 km.2-4 Like many elas- mobranchs, Makos are predominantly piscivorous, but also include

© 2020 American Society for Veterinary Clinical Pathology

Vet Clin Pathol. 2020;00:1–19.

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squid in their diet.5,6 They grow to ≈4.00 m total length (TL) and exhibit a late onset of reproduction with ogives having a median age (TL) at maturity of ≈7-9 years (1.80-1.95 m TL) and ≈18-21 years (2.75-2.85 m TL) for males and females, respectively.7,8 They have a triennial reproductive cycle with a relatively small fecundity of 4-16 pups (0.67-0.82 m TL, 2.25-3.25 kg total weight [TW]) born over spring following a 15-18-month oviphagous, embryonic develop- ment.8,9 While the estimated longevity is 29-32 years,10,11 it is likely that the maximum age is greater, given the triennial reproductive cycle and median age at maturity.
In contrast to other elasmobranchs, the Mako and other lamnids are regionally endothermic and conserve metabolic heat of the ep- axial muscle, gut, associated viscera, eyes, and brain by using lateral cutaneous, suprahepatic, and orbital retia that enable maintenance of body temperatures at 7-10°C above ambient seawater tempera- tures.12-15 Regional endothermy enables lamnids to maintain high metabolic rates with increased swimming performance necessary for respiration given that they are all obligate ram ventilators.16-20 The increased aerobic capacity associated with endothermy in Makos and other lamnids also leads to greater enzyme activities in the locomotor and myocardial muscles21,22 and the digestive tract resulting in increased rates of digestion and food processing.23

Makos have been captured in commercial fisheries worldwide as a target species and/or by catch,24-26 and are extremely popular with recreational fishers, and have been targeted worldwide be- cause of their strong fighting abilities and the superior taste of their meat.3,5,27,28 An ecological risk assessment of the Atlantic Ocean fisheries has placed Makos at high risk of pelagic longlines because of their limited productivity and postcapture mortalities.26 As a result they have been declared globally “vulnerable” by the International Union for the Conservation of Nature (IUCN) Red List29 and in- cluded on Appendix II of the Convention on International Trade in Endangered Species of Wild Fauna and Flora (CITES).

The inaccessibility of Makos and the difficulties associated with trying to research these sharks are primarily responsible for the absence of clinically important data. Thus, it is not sur- prising to find that limited serum biochemical and hematologic data regarding these animals are available.30-32 While a subset of biochemical analytes has been used to examine the response of Makos to capture stress,33-35 this has prevented an accurate health evaluation prior to release. Moreover, much-needed serum biochemistry and/or hematology reference intervals (RI) currently do not exist for Makos, and it is unlikely that their determination will be forthcoming because of the rapidity and force with which the shark responds to capture.
Off the coast of eastern Australia, a well-organized recreational fishery has been operating since the late 1930s with Makos as one of the target species.6,27,28,36 Following the listing of the Mako on CITES Appendix II, the species was listed (January 2010) as a mi- gratory species under the
Commonwealth EPBC Act (1999), and all targeted commercial fishing was prohibited. A subsequent amend- ment to the EPBC Act (July 2010) enabled targeted recreational fishing, and the retention of captured Makos in Commonwealth
waters to continue. While a large proportion of the Makos cap- tured in fishing tournaments are tagged and released,28 only 2.2% had been recaptured from 1977 to 2013,3 and the fate of the re- maining Makos is currently unknown. Importantly, these fishing tournaments have also provided access to the Mako population off eastern Australia and enabled intermittent sampling of blood and urine to assess the effects of capture stress on the health of these animals.

2 | MATERIAL S AND METHODS

This study was performed under a scientific research permit (Permit No. P01/0059[A]) and two Animal Research Authorities (95/7 – Cronulla and 99/14 – Port Stephens) issued by the New South Wales (NSW) Department of Primary Industries (Fisheries NSW) Animal Care and Ethics Committee. The study commenced in 2002, and free-living Makos were caught by recreational fishers in the NSW offshore waters between 32°17.5ʹS and 34°10.0ʹS from January to April each year. This coastal region is subjected to the Eastern Australian Current (EAC) that has a predominant southerly flow of two knots, an austral summer NE sea-breeze reaching 20-30 knots by the afternoon, variable onshore winds in autumn, a prevailing 1-2 m south-easterly swell, wind-driven upwellings, daily fluctua- tions in the sea surface temperature within a 14-26°C mean annual range, and a thermally stratified water column in summer/autumn periods with a 7-10°C thermocline starting at 30 m and finishing at 50 m, respectively.37,38
The Makos were caught using a fishing rod, reel, line of various
breaking strains (usually 10-24 kg), and terminal tackle comprising a length (≈ 4 m) of plastic-coated, 2.0 mm stainless steel wire trace swaged onto a baited hook. Chumming with macerated fish was used to bring the sharks to the surface while slowly trolling the fish- ing line near the sea surface. Once hooked, Makos frequently jump several meters clear of the sea surface10 and this could be followed by a rapid descent into deeper waters.39 Depending on the size of the shark, the angler might then need several hours to bring the ani- mal close enough to the boat for capture.

Following capture, phlebotomies were performed before all other sampling which was achieved in three stages, ensuring sample qual- ity in the preanalytic phase.40 A 10-mL blood sample was collected by caudal venipuncture41 with the shark in dorsal recumbency using an 18G × 90 mm styletted spinal needle (Terumo) to prevent needle occlusion by the hemal arch cartilage.38 Blood pH was then recorded by transferring 1-mL of whole blood via a Discofix C stopcock sys- tem (B. Braun) into a 5-mL syringe with the plunger replaced by a glass pH electrode and sealed using O-rings. The probe was attached to a hand-held, waterproof pH/temperature meter (Model 300 pH meter; Oakton Instruments) with pH measurements accurate to 0.01 pH units (range = −2.00 to 16.00) with optional (automatic/manual) temperature compensation. The needle-like temperature probe (range = −10.0 to 110.0°C, accuracy = ±0.5°C) was placed in the ven- tral muscle mass adjacent to the anal fin of each shark.

The remaining whole blood was then transferred into a gradu- ated, conical centrifuge tube without anticoagulant (Sarstedt) and four replicate 75 mm micro-capillary tubes were filled and spun at 10 000g for 3 minutes in a Clements Microhematocrit Centrifuge (Model CEN 96222) to quantify the PCV. The remaining whole blood was capped, stored in darkness on ice, and allowed to clot (≈2 hours after collection). An initial health assessment was accomplished an- temortem and immediately after phlebotomy. It included recording eye, skin color, teeth, and gingival appearance, muscle tone, any spi- nal deformities, the presence of scars, open wounds, external par- asites (and associated lesions), and the location of the embedded hooks. As the sharks caught were 2-3 m in length, each animal was allowed to cease struggling (for human safety) prior to euthanasia using physical methods (ie, blunt force trauma to the head, pithing) according to accepted guidelines42,43 as the carcasses are donated by the fishers to a local bible college (Tahlee, Port Stephens) for human consumption. The remaining sampling was performed post- mortem following return to port around dusk ≈1.5 hours later.
On landing, the shark’s TW was recorded to the nearest 0.1 kg
using an electric chain hoist fitted with an electronic balance. While on the hoist, each shark was photographed and then scanned with a hand-held metal detector (Ranger Security Detectors Inc) to de- termine whether any additional hooks had been retained from prior fishing interactions. Then, a partial necropsy was done, compris- ing external and internal examinations. The external examination focused on recording the TL with the caudal fin in the depressed position44; fork length (FL); precaudal length (PCL); the pecto- ral-pelvic space (PPS), and a further 50 standard measurements38 to the nearest millimeter. Then, the cloaca was flushed with fresh seawater to remove the liquid fecal stream, the anus occluded with a rolled absorbent bandage, and the cloaca wiped with a 4% solu- tion of chlorhexidine gluconate to remove the naturally occurring bacteria.45 A urine sample (≈6 mL) was then collected from the dor- sal urinary sinus via the urinary papilla using a Covidien 3.5 FR/Ch (1.2 mm × 56 mm) polypropylene suction catheter (Medtronic) and a 10-mL disposable syringe. Urine pH was determined as described for whole blood (see above) using a separate glass pH probe and insertion of the temperature probe into the dorsal muscle mass su- perior to the origin of a pelvic fin of each shark. Urine color was then recorded prior to transferring the urine into two graduated conical centrifuge tubes (≈3.5 and 1.5 mL) which were capped and placed into separate, sealed plastic bags and stored in darkness on ice. In the absence of species-specific information concerning the urine of Makos, the subsequent analyses were performed following stan- dard protocols used with mammals.46

A gross examination of the abdominal organs was executed via a
medial incision extending 20 cm cranially from the pelvic girdle and tissue samples, or any obvious lesions, when required, were retained and preserved in 10% neutral buffered formalin (NBF) for histopa- thology. Reproductive maturity in females was assessed visually using standard methods7,47 and included the TL and PPS, oviducal gland diameter, the degree of expansion of the uteri, and the presence/ absence of uterine hymen on cloacal palpation. Female reproductivestages were estimated via the presence/absence of mating scars, and the ovarian and follicle sizes. Sexual maturity in males was assessed using: TL, clasper length, and the degree of clasper calcification,7,47 whereas the reproductive stage was estimated via the presence of viable sperm/spermatophores in the seminal vesicles.48 A sample of skeletal muscle was also preserved in 95% ethanol for genetic stud- ies.3 Finally, the anatomic location of the fishing hooks was confirmed and any hooks from prior fishing interactions were assessed visually in the jaws, gills, and buccal cavity. Any hooks present in the esoph- agus and lower gastrointestinal tract were located by palpation. The pericardial cavity was then exposed via a left or right lateral incision extending 10 cm cranially from the pectoral girdle. A sample of peri- cardial fluid was collected in a disposable syringe, and its color was recorded before transferring into a sealed graduated conical centri- fuge tube for possible biochemical analysis.

A gross examination of the heart was then done, and when required, a sample of the ventricle was retained and preserved in 10% NBF for histopathology.
On completion of the partial necropsy (≈20 minutes), the urine
was allowed to warm (≈25°C), and the initial analysis was done on- site. The color and clarity were recorded under light from a water- proof LED head torch (300 lumens) and urine specific gravity (USG) was determined from both noncentrifuged tubes using a digital refractometer (USG range = 1.000-1.050, accuracy = ±0.001) with automatic temperature compensation (Bellingham and Stanley). A semi-quantitative reagent strip (Multistix 10 SG; Siemens) was im- mersed in the tube with a smaller urine volume to measure the pH, glucose, bilirubin, blood, red blood cells (RBCs), and protein. A re- agent strip was also immersed in de-ionized water as a control. The urine remaining in this tube was centrifuged at 500g for 5 minutes, and the color of the supernatant noted. The urine volume was re- duced to 0.5 mL using a pipette to enable microscopic examination following return to the laboratory.

Any reasonable excess was saved in a clean, graduated conical centrifuge tube and placed on ice for possible biochemical analysis. Then, a second tube of urine was cen- trifuged at 2000g for 5 minutes, the supernatant was pipetted into a clean tube, its color was noted, and the sample was retained for further analysis on return to the laboratory.
On completion of field sampling, the biochemistry sam- ples were placed in a portable freezer at −5°C (Model CF-40AC; WAECO Pacific) and transported, together with the remaining samples, by road (≈30 minutes) to the laboratory. In the labora- tory, serum separation was performed via centrifugation at 3000g for 6 minutes (Model 7112; APSCAN) with—three to four replicate 2-mL aliquots transferred into capped plastic vials with a variable volume pipette (Model 1000; Eppendorf). All biochemistry sam- ples were then transferred to a chest freezer (−20°C) for storage until submitted for analysis, which was completed within 3 days of collection. The retained urine supernatant was subjected to an ammonium sulfate precipitation test (80% saturated solution) to discriminate between hemoglobin and myoglobin.49 This was followed by microscopic analysis of the urine using standard pro- tocols46,50 with the mean number of RBC, and white blood cell (WBC) counts quantified using n = 10 replicate high power fields(hpf), and the mean number of crystals and casts quantified using n = 10 replicate low power fields (lpf).

Biochemical analyses were performed using ion-selective elec- trode techniques (sodium, potassium, and chloride) and automated bichromatic spectrophotometry (inorganic phosphorus, total cal- cium, magnesium, glucose, urea, ALP, ALT, AST, CK, total protein, albumin, cholesterol, triglyceride, creatinine, total bilirubin), with a Beckman Coulter AU680 clinical biochemistry analyzer (Beckman Coulter) following standard methods.38 Lactate was quantified using the pyruvate-hydrogen peroxide method on a Vitros E250 ana- lyzer (Ortho Clinical Diagnostics). Serum osmolality was quantified using freezing point depression with a micro-osmometer (Advanced Osmometer, Model 330; Advanced Instruments Inc).
Analytical quality assurance of the serum biochemical results that had values within the limits set by the IDEXX laboratory and were similar to previous studies was maintained following standard operating procedures.38,51 Duplicates of sera from five Makos were blindly and randomly selected from all sharks sampled (n = 24) and submitted contemporaneously to assess quality control. The results showed that the within-sample variation was <2% for sodium, chlo- ride, urea, and osmolality and was <5% for the remaining biochemi- cal analytes. Calibrations of the pH meter (six solutions of known pH) and refractometer (one solution) were completed at each sampling occasion prior to use ensuring analytical quality.50
All statistical analyses were accomplished with a nominal Type I (α) error rate of P = .05 using readily available statistical software in- cluding
Data Desk Version 6.0 (Data Description Inc), Pop-Tools, ver- sion 3.2.5 (Greg Hood, CSIRO), and Sigma Plot version 11.0 (Systat Software Inc) software. Raw data were examined using standard procedures to test for the presence of outliers (Dixon, Tukey tests), normality (Shapiro-Wilk test), and homoscedasticity (Bartlett's test).

All data were transformed as required. Several statistically significant linear and curvilinear regressions were developed from prior necrop- sies of Shortfin Mako sharks collected throughout the year, the signif- icance of which were tested with ANOVA and two-tailed t tests for regression slopes with H0: b = 0 (Table 1). These relationships were used to assist with the assessments of reproductive maturity and nor- mal TW. As TL, FL, and PCL are not reduced in cachectic sharks38 all three can provide predictions of normal TW (ie, TWTL, TWFL, and
TWPCL). Hence, the observed TW of each Shortfin Mako shark wascompared to the predicted (ie, normal) TWTL via a least-squares linear
regression of log10(TW) on log10(TL) (Table 1) using a two-tailed t test.

Importantly, initial evidence of cachexia was identified when the observed TW was significantly less than the predicted TWTL. Cachexia can be seen due to the retention of fishing hooks in the gas- trointestinal tract,38 which causes muscle atrophy, or other chronic illnesses. However, if the observed TW was significantly greater than the predicted TWTL there was evidence of obesity; however, obesity has never been seen in free-living sharks that are pelagic and mi- grate over large oceanic distances,2,4 and obesity was not observed in any of the sharks examined over the period from 2002 to 2017. Unbalanced t tests were used to compare mean analyte values be- tween the Makos with red-brown urine and the remaining 18 Makos. When a RI was not available from the STS, an “unstressed” analyte value from the literature was compared with the mean of the six Makos using a one-tailed asymmetrical t test.53 Possible differences in individual analytes/variables among replicate sharks in the urine anal- ysis were examined using ANOVA with significant differences among the means identified using Student-Newman-Keuls (SNK) post hoc tests.54 Correlations between biochemical analytes and/or physical variables were examined using the Pearson product-moment correla- tion coefficient (r). Finally, age was estimated using FL in conjunction with a Schnute model 3 growth curve.8

3 | RESULTS

Over the austral summer-autumn period from 2002 to 2017, 84 Shortfin Mako sharks were captured on circle or “J” hooks with hook locations varying from the jaw to the cardiac stomach. Once landed, each shark was sampled consistently, and the overall catch (Table 2) exhibited substantial gender bias (8 males, 76 females and χ2 = 55.05, P < .001) with almost 58% of the catch exceeding 2.5 m TL and 100.0 kg TW. Of these, 24 Makos with similar gender bias (2 males, 22 females and χ2 = 16.67, P < .001) were sampled for blood and/or urine intermittently from 2006 to 2017. A serum biochemis- try panel comprising 23 analytes was carried out in 10 individuals (1 male, 9 females) and then combined with urine analysis in another 14 individuals (1 male, 13 females) (Table 2). Six of the 14 Makos in the latter group exhibited conspicuously red-brown urine (RBU) that was correlated with distinctly elevated serum CK and AST activities com- pared with the remaining 18 sharks (Tables 3 and 5) and was markedly

TA B L E 1 Morphometric relationships used to assess total weight, body condition, age and sexual maturity of free-living Shortfin Mako sharks (Isurus oxyrinchus) captured by recreational fishers over the austral summer/autumn months in east Australian offshore coastal waters. Relationships derived from prior necropsies of free-living sharks captured throughout the year in east Australian waters

Relationship Equation N r2 F1, 156 df P
TW on TL TW = 7.2083(TL3.0843) 158 .97 4976.08 <.001
TL on FL TL = 1.1395(FL) – 0.0288 158 .99 7164.36 <.001
TL on PCL TL = 1.2476(PCL) + 0.0100 158 .99 26 439.90 <.001
PPS on TL PPS = 0.2944(TL) – 0.0673 158 .96 3954.14 <.001
Abbreviations: FL, fork length in meters; N, number of individuals; PCL, precaudal length in meters; PPS, pectoral-pelvic space in meters; TL, total length in meters; TW, total weight in kilograms.

TA B L E 2 Sampling and biological information for free-living Shortfin Mako sharks (Isurus oxyrinchus) captured by recreational fishers over the austral summer/autumn months in east Australian offshore coastal waters for the period 2002 to 2017 inclusive

Total population sampled Population sampled for blood and urine
N TL TW N TL TW
Life-history stage Min-Max Min-Max All Blood and urine Blood only Min-Max Min-Max
Adult male 8 2.04-3.10 66.5-245.0 2 1 1 2.04-2.20 66.5-83.5
Juvenile female 27 1.85-2.68 52.1-168.0 14 6 8 2.10-2.65 70.0-169.0
Adult female 49 2.71-3.72 155.0-420.0 8 7 1 2.86-3.22 174.5-274.5
All individuals 84 2.04-3.72 52.1-420.0 24 14 10 2.04-3.22 70.0-274.0
Abbreviations: Max, maximum; Min, minimum; N, number of individuals; TL, total length in meters; TW, total weight in kilograms.

TA B L E 3 Summary of serum biochemical values for 18 free-living Shortfin Mako sharks (Isurus oxyrinchus) with urine normal in appearance and a comparison of the minimum/maximum values with the respective biochemical reference interval for the Sand Tiger shark (Carcharias taurus) or the mean (±SD) when not determined

Descriptive statistics for Shortfin Mako
shark Sand Tiger shark Comparison—SMS vs STS
Analyte Unit N Mean SD Median Min–Max RI Mean (±SD) Min Max
Sodium mmol/L 18 294 20 285 268-339 249-267 258 (4) Just above URL Above URL
Potassium mmol/L 18 10.4 4.8 9.4 5.1-20.5 4.3-5.7 5.0 (0.3) Within RI Above URL
Chloride mmol/L 18 273 19 267 242-311 229-250 242 (7) Within RI Above URL
Inorganic phosphorus mmol/L 18 6.0 2.8 4.9 0.8-12.0 1.7-2.0 1.8 (0.1) Below LRL Above URL
Total calcium mmol/L 18 4.5 1.2 4.2 3.4-8.8 3.3-4.4 3.9 (0.3) Within RI Above URL
Magnesium mmol/L 18 2.4 1.0 2.2 1.4-5.4 1.6-2.2 1.9 (0.2) Below URL Above URL
Glucose mmol/L 18 3.5 2.4 3.6 0.8-11.8 2.2-3.2 2.7 (0.2) Below LRL Above URL
Urea mmol/L 18 360 55 371 181-413 360-394 377 (8) Below LRL Above URL
Creatinine µmol/L 18 30 20 20 20-120 ND 32 (10) Below mean ± SD Above mean ± SD
ALP U/L 18 37 76 12 4-329 8-31 20 (6) Below LRL Above URL
ALT U/L 18 2 1 2 0-3 ND 3 (1) Below mean ± SD Below mean ± SD
AST U/L 18 284 219 235 20-798 13-45 29 (8) Below LRL Above URL
CK U/L 18 615 995 30 10-3018 5-79 42 (18) Within RI Above URL
Total protein g/L 18 22 4 30 16-30 24-36 30 (3) Below LRL Within RI
Albumin g/L 18 5 1 45 1-7 ND ND Mean similar to values literature
Globulin g/L 18 20 4 17 13-25 ND ND Mean similar to values literature
Triglyceride mmol/L 18 0.5 0.3 0.3 0.1-1.1 0.1-0.6 0.3 (0.1) Within RI Above URL
Cholesterol mmol/L 18 0.8 0.3 0.7 0.3-1.5 0.9-2.1 1.4 (0.3) Below LRL Within RI
Total bilirubin µmol/L 18 0.2 0.5 0.0 1.0-2.0 ND 1.5 (0.5) Below mean ± SD Above mean ± SD
Lactate mmol/L 18 27.2 11.5 24.6 8.2-52.4 ND 0.5 (0.7) Above URL Above URL
Osmolarity mmol/L 18 1127 26 1125 1085- 1027- 1082 (26) Within RI Above URL
1215 1136
Note: Bolded text denotes that the value of the biochemical analyte from the Shortfin Mako shark provides a clinically reasonable reason for using the Sand Tiger shark reference interval38 in subsequent analyses of Shortfin Mako shark serum biochemistry.
Abbreviations: LRL, lower reference limit38; Max, maximum; Min, minimum; N, number of individuals; ND, not determined; RI, reference interval; SD, standard deviation; SMS, Shortfin Mako shark; STS, Sand Tiger shark, URL, upper reference limit.381 dfdiscrepant from the normal clear, colorless to pale yellow urine evi- dent in the other eight sharks. A preliminary examination of the serumbiochemical analytes from the 18 remaining Makos also indicated that a range of individuals exhibited marked differences in biochemical val- ues from those reported in other shark species (Table 3). However, a detailed analysis of these 18 individuals will be reported elsewhere as precedence in this study was given to the six Makos with RBU. That said, 71.4% of the serum biochemical analytes examined in the 18 re- maining Makos (Table 3) had minimum values that were either within or below the RIs determined by the author for free-living Sand Tiger sharks (STS) Carcharias taurus, or when RIs could not be determined, were less than/equal to the mean values for the STS.38 In the absence of RIs for Makos, these results indicated that the STS RIs38 would pro- vide a clinically reasonable approximation and are, therefore, used in line with ASVCP guidelines55 to enable a more detailed analysis and interpretation of the serum biochemical results. Furthermore, neither TL nor TW of the six Makos with RBU differed significantly from the mean of the remaining 18 Mako sharks (Tables 3 and 5, t5,17 df = 1.91 and 1.26, P = .07 and .22, respectively) and there was a similar gender bias toward females in both groups (χ2 = 16.88, P < .001).

The six Makos that presented with RBU were captured using whole fish as bait over February to March in offshore waters with a sea surface temperature of 25-26°C and at sites up to 40 nautical miles apart after “fight-times” of 1.0 to 3.0 hours with the 14/o offset J-shaped hooks becoming embedded in the proximal or distal esoph- agus (Table 4). The sharks exhibited a similar gender bias compared with the overall catch and comprised one male (2.20 m TL, 83.5 kg TW) and five females (2.42-3.22 m TL, 96.5-274.5 kg TW) (Table 4). The six sharks exhibited normal TW that was within natural variation (Table 4, all two-tailed t tests, P > .10) and comprised one sexually mature, precopulatory male; four sexually mature, preovulatory fe- males in the second year of the triennial reproductive cycle; and one sexually immature female requiring a further 8 years of growth prior to sexual maturity (Table 4).

Apart from the obvious hook injuries in the esophagus, the sharks with RBU appeared reasonably healthy, as evidenced by their typical indigo blue colored dorsal skin with no pallor or blotchiness. Three individuals (Table 4, sharks 1, 3, and 6) had a few (<10/shark) copepod parasites (Pandarus cranchi, ≤5 mm ceph- alothorax length) on the caudal margin of the paired fins that were associated with very faint, shadow-like, underlying lesions. Another individual (Table 4, shark 4) had three copepod parasites (Brachiella thynni, ≤4 mm cephalothorax length) on the superior surface of the distal tongue. All six sharks exhibited normal ocular appearance, pupillary reflexes, gingiva, and teeth, some of which were broken (Table 4). All of the sharks had good body condition with mean (± SD, range) muscle temperatures of 28.5°C (0.6, 28.0- 29.3) and 26.9°C (1.0, 25.5-28.0) near the pelvic and anal fins, respectively. Also, there were no large skin lesions, spinal defor- mities, prior fishing-related injuries, or rigidity of the caudal pe- duncle muscles (Table 4).

No additional hooks were identified with the metal detec- tor, and palpation of the gastrointestinal tract of the six Makos
confirmed this result. Internally, the hooks in four sharks perfo- rated the proximal, dorsal esophagus, and passed through the folded mucosa. The hooks then continued cranially through the submucosa and the reduced lymphomyeloid tissue of the organ of Leydig,56 and finally became embedded in the outer striated mus- cle (Table 4, sharks 1-3 and 6). The remaining two hooks (Table 4, sharks 4 and 5) perforated the distal, dorsal esophagus and passed through the folded mucosa, submucosa, and the organ of Leydig exiting the outer striated muscle medially. This led to the points of the hooks lying parallel and inferior to the peritoneum of the dor- sal abdominal cavity and millimeters from the liver and posterior cardinal sinus.

Excluding the bait, three individuals with RBU (Table 4, sharks 1, 4, and 5) had consumed varying amounts of fish (Centroberyx af- finus, Cybiosarda elegans, Trachurus mccullochi, and Trachurus novae- zelandiae) and/or squid (Sepioteuthis australis) with the Pomatomus saltatrix notably absent. The cardiac stomachs of the three remaining Shortfin Mako sharks (Table 4, sharks 2, 3, and 6) were empty with no bait remnants and appeared very “clean.”
The sera of the six Makos with RBU exhibited no evidence of hemolysis and were clear and almost colorless. Almost half (47.6%) of the serum biochemistry analytes examined exhibited marked departures from the STS RI with values in all individuals consis- tently above or below (Table 5). As expected, the sodium ion con- centrations were less than those of seawater, but all six sharks were hyponatremic with a mean (± SD, range) concentration of 276 (8.5, 268-291) mmol/L (Table 3), which was significantly less than
the mean of the remaining 18 Makos (Table 5, t5,17 df = −2.21, P = .04). Hyperkalemia was also present, and concentrations in each shark exceeded 7.0 mmol/L with an overall mean (± SD,range) of 15.6 (4.3, 10.9-23.8) mmol/L, which was ~ 2.7 times the STS upper reference limit and significantly greater than the mean of the remaining 18 Makos (Tables 3 and 5, t5,17 df = 2.35, P = .03).

In contrast, all of the chloride concentrations were within the STS
RI and had a mean (± SD, range) of 243 (7.5, 229-250) mmol/L, which was significantly less than the mean of the remaining 18 Mako sharks (Tables 3 and 5, t5,17 df = −2.21, P < .001). Interestingly, this suggests a very active chloride (Hamburger) shift in the eryth- rocytes of the six Makos, probably promoted by a reduced serum pH. All six sharks exhibited hyperphosphatemia with a mean (± SD, range) inorganic phosphorus concentration of 10.6 (3.5, 6.6-16.3) mmol/L, ≈5.3 times the STS upper reference limit, and significantly greater than the mean of the remaining 18 Makos (Tables 3 and 5, t5,17 df = 3.27, P = .004). Total calcium concentrations were more variable, with values in four sharks within the STS RI, and two sharks (Table 4, sharks 1 and 3) above the STS RI and combined, produced a mean (± SD, range) total calcium concentration of 4.3 (0.5, 3.7-5.0) mmol/L (Table 5) which did not differ significantly from the mean of the remaining 18 Makos (Table 3, t5,17 df = −0.39, P = .70). The serum the calcium-phosphorus product (Table 5) was elevated in all six sharks, with a mean (± SD, range) of 45.4 (13.0, 29.0-60.3), exceeding the level where soft tissue calcification is possible in two Makos (Table 5, sharks 1 and 5); however, the level

TA B L E 4 Capture, biological, and clinical information for free-living Shortfin Mako sharks (Isurus oxyrinchus) exhibiting red-brown urine following capture by recreational fishers over the austral summer/autumn months in east Australian offshore coastal waters for the period 2002 to 2017 inclusive

Shortfin Mako shark
Factor Variable 1 2 3 4 5 6
Capture Month February February March March February February
SST (°C) 25 25 26 26 26 26
Duration (h) 2.00 1.50 2.00 1.00 3.00 2.50
Hook
Size 14/o 14/o 14/o 14/o 14/o 14/o
Location Right PE Left PE Left PE Right DE Left DE Right PE
Bait Kingfish Australian salmon Kingfish Sea mullet Australian salmon Australian salmon
Biologic TL 2.20 2.90 2.93 3.22 3.22 2.42
FL 2.00 2.53 2.56 2.85 2.84 2.11
PCL 1.83 2.26 2.33 2.60 2.58 1.88
PPS 0.58 0.80 0.80 0.89 0.83 0.70
Age (y) 10 17 17 20 20 12
TW
Observed 83.5 175.5 168.0 260.0 274.5 96.5
Predicted 82.0 192.3 198.5 265.6 265.6 110.1
Two-tailed t test t = 0.13, P > .10 t = −0.68, P > .10 t = −1.24, P > .10 t = −0.16, P > .10 t = 0.24, P > .10 t = −0.98, P > .10
Assessment Normal TW Normal TW Normal TW Normal TW Normal TW Normal TW
Difference (1.83% ↑) (8.74% ↓) (15.37% ↓) (2.11% ↓) (3.35% ↑) (12.35% ↓)
Reproduction
Gender Male Female Female Female Female Female
Maturity Mature Mature virgin Mature virgin Mature nonvirgin Mature nonvirgin Immature virgin
Stage Precopulatory Preovulatory Preovulatory Preovulatory Preovulatory –
Diet
Fullness 33% full Empty Empty 50% full 10% full Empty
Prey Fish (1, 2) – – Fish (4), squid (5) Fish (3, 4) –
Clinical Skin
Color Indigo blue Indigo blue Indigo blue Indigo blue Indigo blue Indigo blue
Appearance No pallor or
blotchiness No pallor or
blotchiness No pallor or
blotchiness No pallor or
blotchiness No pallor or
blotchiness No pallor or blotchiness
Parasites ND 7 Copepods on right PECDPM 4 Copepods on left PELDPM ND ND 5 Copepods on left PECDPM
Ocular status
Appearance Clear Clear Clear Clear Clear Clear
Pupillary reflex Equal and
reactive Equal and reactive Equal and
reactive Equal and reactive Equal and reactive Equal and reactive

Oro-branchial cavity
Teeth White in color,
unbroken
Gingiva Cream in color, no parasites
Tongue Cream in color, no parasites

White in color,
unbroken
Cream in color, no parasites
Cream in color, no parasites

White in color,
unbroken
Cream in color, no parasites
Cream in color, no parasites

White in color,
unbroken
Cream in color, no parasites
Cream in color, 3 copepods

White in color, 4 broken – ULJ
Cream in color, no parasites
Cream in color, no parasites

White in color, 3 broken
– LRJ
Cream in color, no parasites
Cream in color, no parasites

Gills Bright red, no Bright red, no Bright red, no Bright red, no Bright red, no Bright red, no parasites
parasites parasites parasites parasites parasites
Muscle mass
Condition Good Good Good Good Good Good
(Continues)

TA B L E 4 (Continued)

Shortfin Mako shark
Factor Variable 1 2 3 4 5 6
Rigidity ND ND ND ND ND ND
Spinal deformity ND ND ND ND ND ND
Temp (°C)
PELO 28.1 28.0 29.0 28.3 29.3 28.0
AFO 26.0 25.5 27.2 27.5 28.0 27.1
Abdomen
Hook perforation No No No Yes Yes No
Penetrating
trauma No No No No No No
Hemorrhage No No No Very minor Very minor No
≈4 mL blood ≈2 mL blood
Note: Dietary contents of fish: 1, Centroberyx affinus; 2, Cybiosarda elegans; 3, Trachurus mccullochi; 4, Trachurus novaezelandiae; and squid: 5,
Sepioteuthis australis.
Abbreviations: ↑, increase; ↓, decrease; AFO, anal fin origin.; DE, distal esophagus; FL, fork length in meters; ND, not detected; PCL, precaudal length in meters; PE, proximal esophagus; PECDPM, pectoral fin dorsal posterior margin; PELDPM, pelvic fin dorsal posterior margin; PELO, pelvic fin origin; PPS, pectoral-pelvic space in meters; SST, sea surface temperature; TL, total length in meters; TW, total weight in kilogramsof the product was less relevant because of the acute nature of the disturbance (ie, fishing capture). Hypermagnesemia was also evi- dent in all six Makos, with a mean (± SD, range) concentration of3.3 (1.0, 2.3-5.1) mmol/L, that was approximately 1.5 times the STS upper reference limit, but it did not differ significantly from the mean of the remaining 18 Makos (Tables 3 and 5, t5,17 df = 1.77,
P = .09). Serum glucose concentrations were more variable (as ex-
pected), with values within the STS RI for four sharks, and below the STS RI in two sharks (Table 5, sharks 3 and 6). Overall, a mean (± SD, range) concentration of 2.1 (0.9, 0.5-3.1) mmol/L was found, which was significantly less than the mean of the remaining 18 Makos (Table 3, t5,17 df = −2.12, P = .04). Serum urea concentrations in these Makos were all below the STS RI (ie, ≈11% below the STS lower reference limit), producing a mean (± SD, range) of 325 (16, 297-343) mmol/L (Table 5), which was also significantly less than the mean of the remaining 18 Makos (Table 3, t5,17 df = −2.43, P =
.02). All six Makos exhibited elevated serum creatinine concentra- tions resulting in a mean (± SD, range) concentration of 52.0 (8, 40-60) µmol/L that was significantly greater than the mean of 32 µmol/L in the STS (Tables 3 and 5, two-tailed asymmetrical t test, t5 df = 26.00, P < .001). The mean creatinine concentration of these six Makos was also significantly greater than the mean of the remaining 18 Makos (Tables 3 and 5, t5,17 df = 3.23, P = .004).

The serum urea: creatinine ratio declined by 44%, on average, across the six sharks, and was driven by simultaneous decreases in serum urea and increases in serum creatinine. Detectable ALP, ALT, AST, and CK enzyme activities were also present in the sera of the six Makos. Serum ALP activity was similar across the six sharks, within the STS RI, and had a mean (± SD, range) of 14 (4, 9-21) U/L that did not differ significantly from the mean of the remaining 18 Makos (Tables 3 and 5, t5,17 df = −1.39, P = .21). Serum ALT and GGT activ- ities in these six Makos were identical and just above the limits ofdetection for the analyzer (ie, 1 U/L) producing mean (± SD, range) activities of 2.6 (1.5, 2-6) U/L in both enzymes (Table 5). Moreover, serum ALT and GGT activities were not significantly different from the STS means (ie, 3 and 2 U/L, respectively and Table 3, two- tailed asymmetrical t tests, both t5 df = 1.40, P > .10), and did not differ significantly from the means of the remaining 18 Makos (Tables 3 and 5, t5,17 df = 1.06 and 1.29, P = .34 and .25, respec- tively). Serum AST and CK activities were markedly elevated in the six Makos with means (± SD, range) of 2806 (1996, 1188-5944) U/L and 240 938 (318 975, 12 220-808 200) U/L, respectively, and had approximately 62- and 3050-fold increases above the respective STS upper reference limit. Serum AST and CK activities were also significantly greater than their respective mean activities in the remaining 18 Makos (Tables 3 and 5, t5,17 df = 3.09 and 7.02, P < .03 and .001, respectively). Serum total protein concentrations were variable, with the values in two sharks (Table 5, sharks 2 and 4) below the STS RI, whereas the remaining sharks were within the STS RI, yielding a mean (± SD, range) of 25 (2.8, 22-30) g/L, which did not differ significantly from the mean of the remaining 18 Mako sharks (Tables 3 and 5, t5,17 df = 2.05, P = .06). Serum albumin concentrations were similar across the six Makos with a mean (± SD, range) of 6.8 (1.5, 5-9) g/L, a significantly greater concentra- tion than the mean of the remaining 18 Makos (Tables 3 and 5, t5,17 df = 3.70, P = .001). Serum globulin concentrations were variable with concentrations of 16 g/L or below in three sharks (Table 5, sharks 2, 4 and 5) and a mean (± SD, range) of 18.2 (4.1, 14-25) g/L, which did not differ significantly from the mean of the remaining 18 Makos (Tables 3 and 5, t5,17 df = 0.65, P = .52). Serum triglyceride concentrations varied among the six Shortfin Mako sharks, with one shark (Table 5, shark 5) markedly above the STS RI, and com- bined this yielded a mean (± SD) of 0.6 (0.3) mmol/L, which did not differ significantly from the mean of the remaining 18 Makos

TA B L E 5 Results of serum biochemistry analyses for free-living Shortfin Mako sharks (Isurus oxyrinchus) exhibiting red-brown urine following capture by recreational fishers over the austral summer/autumn months in east Australian offshore coastal waters from 2002 to 2017

Shortfin Mako shark
Analyte Unit STS RI 1 2 3 4 5 6 Mean SD
Sodium mmol/L 249-267 291 273 279 274 269 268 276 8.5
Potassium mmol/L 4.3-5.7 12.3 15.8 14.8 10.7 23.0 17 15.6 4.3
Chloride mmol/L 227-257 229 241 248 250 245 242 243 7.5
Inorganic mmol/L 1.7-2.0 11.4 11.6 10.0 6.60 16.3 7.60 10.6 3.5
phosphorus
Total calcium mmol/L 3.3-4.4 5.0 4.20 4.6 4.4 3.7 4.1 4.3 0.5
Ca × Pia mmol2/L2 56.5 57.0 48.7 46.0 29.0 60.3 31.2 45.4 13.0
Magnesium mmol/L 1.6-2.2 5.1 3.1 3.1 2.3 3.6 2.30 3.3 1.0
Glucose mmol/L 2.2-3.2 3.1 2.4 0.5 2.4 2.5 1.9 2.1 0.9
Urea mmol/L 360-394 297 319 343 325 328 337 325 16
Creatinineb µmol/L 32 60 60 50 40 52 50 52.0 8
Urea: 11 781:1 4950:1 5317:1 6860:1 9425:1 6308:1 6740:1 6600:1 –
creatinine
ratioc
Δ Urea/
creatinine % 0.0 58.0 ↓ 54.9 ↓ 41.8 ↓ 20.0 ↓ 46.5 ↓ 42.8 ↓ 44.0 ↓ –
ALP U/L 8-31 18 11 12 21 9 11 14 4
GGTb U/L 2 2 2 2 2 6 2 2.6 1.5
ALTb U/L 3 2 2 2 2 6 2 2.6 1.5
AST U/L 13-45 1188 4492 2616 1188 5944 1409 2806 1996
CK U/L 5-79 33 030 432 200 41 180 12 220 808 200 118 800 240 938 318 975
Multiplier above URL 1× 418× 5471× 521× 155× 10 230× 1504× 3050× –
Total protein g/L 24-36 26 22 25 23 24 30 25 2.8
Albumind g/L 5.0 6 8 6 7 9 5 6.8 1.5
Globulinse g/L 19-31 20 14 19 16 15 25 18.2 4.1
Triglyceride mmol/L 0.1-0.6 0.7 0.3 0.6 0.6 1.1 0.3 0.6 0.3
Cholesterol mmol/L 0.9-2.1 1.8 0.5 1.0 0.5 0.4 1.5 1.0 0.6
Total bilirubinf µmol/L 1.5 0 0 0 0 0.5 0.5 0.17 0.26
Lactate-Lg mmol/L 0.5-1.0 44.9 31.8 45.3 31.1 60.8 52.4 44.4 11.6
Osmolality mmol/kg 1027-1136 1150 1139 1182 1137 1207 1143 1160 28
Abbreviations: Bolded text, above/below reference value; Ca, total calcium; Pi, inorganic phosphate; SD, standard deviation; STS RI, Sand Tiger shark (Carcharias taurus) reference interval38; URL, upper reference limit.
aThe comparative value of the Ca × P product is the level where soft tissue calcification is possible under chronic conditions (>70 mg/dL).46
bReference interval not determined and thus mean for Sand Tiger shark used.
cMean values for urea and creatinine obtained for Sand Tiger shark used to calculate reference ratio; ↓, decrease.
dReference interval not determined for albumin for Sand Tiger shark and most prevalent mean in the literature used; maximum value used instead.
eRange estimated by subtraction from total protein.
fReference interval not determined for total bilirubin and mean for Sand Tiger shark used.
gReference interval not determined and range of values from minimally-stressed Sand Tiger sharks used (Otway, unpublished data).

(Tables 3 and 5, t5,17 df = −0.28, P = .78). Serum cholesterol concen- trations also varied, with three sharks below the STS RI, producing an overall mean (± SD, range) of 1.0 (0.6, 0.4-1.8) mmol/L that did not differ significantly from the mean of the remaining 18 Makos (Tables 3 and 5, t5,17 df = 0.94, P = .36). A total bilirubin concentra- tion of 0.5 µmol/L was only evident in two Makos (Table 4, sharks
5 and 6) and was below the level of detection in the remaining sharks resulting in a mean (± SD) of 0.17 (0.26) µmol/L that was not significantly different from the mean of 1.5 µmol/L in the STS (one-tailed asymmetrical t test, t5 df = 0.89, P > .10), and not signifi- cantly different from the mean of the remaining 18 Mako sharks (Tables 3 and 5, t5,17 df = 0.00, P = 1.00). As expected, serum lactate

ImageOTWAY
10 |

TA B L E 6 Summary of PCV, pH, bicarbonate, and anion gap for the six free-living Shortfin Mako sharks (Isurus oxyrinchus) exhibiting red-brown urine following capture by recreational fishers over the austral summer/autumn months in east Australian offshore coastal waters from 2002 to 2017
5
46 (1.6)
6.51
4.0
+ 53.0
6
41 (0.8)
7.01
4.3
+ 38.7
Mean
40
7.13
4.4
46.1
SD
1.1
0.34
0.3
13.8
concentrations in all six Makos were markedly elevated and re- sulted in a mean (± SD, range) of 44.4 (11.6, 31.1-60.8) mmol/L that was significantly greater than the mean of the STS (0.5 mmol/L, Table 3 andtwo-tailed asymmetrical t test, t5 df = −3.04, P < .001) and was also significantly greater than the mean of the remaining 18 Makos (Tables 3 and 5, t5,17 df = 3.16, P = .005). The fight-times were also positively correlated with serum lactate concentrations (Table 4, r2 = .98, P < .01), whereas the pH was negatively cor- related with serum lactate concentrations (Table 4, r2 = 0.94, P < .01). Finally, the osmolality exceeded the STS RI in the six Makos, and had a mean (± SD, range) of 1160 (28, 1137-1207) mmol/kg, which was also significantly greater than the mean of the remaining 18 Makos (Tables 3 and 5, t5,17 df = 2.28, P = .03).
Shortfin Mako shark
1
39 (0.8)
7.22
4.5
+ 69.8
2
43 (1.6)
7.40
4.2
+ 43.6
3
38 (0.8)
7.21
4.5
+ 41.3
4
35 (2.5)
7.45
4.8
+ 29.9
The results of the PCV, pH, bicarbonate concentration, and the anion gap for the six Makos with RBU are summarized in Table 6 and were in contrast with literature values from minimally stressed sharks. The PCV in all six sharks was elevated with the overall mean (± SD, range) of 40% (1.1, 35-46%), which was significantly greater than the literature mean of 29% (Table 6 and two-tailed asymmetrical t test, t5 df = 19.38, P < .001). The pH and bicarbonate concentration declined in the six Makos with the respective means (± SD, range) of 7.13 (0.34, 6.51-7.45) and 4.2 (0.3, 3.8-4.6) mmol/L, significantly less than the mean literature values of 7.80 and 5.5 mmol/L, respectively (Table 6 and two-tailed asymmetrical t tests, t5 df = −11.55 and −9.70, both P < .001). The anion gap was elevated in all six Makos, and the overall mean (± SD, range) of 46.1 (13.8, 29.9-69.8) mmol/L was sig- nificantly greater than the mean of 15.5 mmol/L in the STS (Table 6 and two-tailed asymmetrical t test, t5 df = 12.42, P < .001).
Minimally stressed literature values
Analyte
PCV (±SD)
pH
HCO3-
Anion gapa
Unit
%
—
mmol/L mmol/L
Min–Max
28-30
7.6-8.0
5.0-6.0
12-19
Mean
29.0
7.8
5.5
+ 15.5
Authors
13,32,33
33-35,70
38,51,70,71,85
38

Abbreviation: SD, standard deviation.
aRange and mean from Sand Tiger shark used.
Results of urine analyses for eight Shortfin Mako sharks with normal, pale-yellow urine, and the six Shortfin Mako sharks with RBU, are summarized in Tables 7 and 8, respectively. The eight Makos (2.10-3.17 m TL, 70.0-232.5 kg TW) with normal, pale-yellow urine were all female and comprised three sexually mature, precopu- latory individuals, and five immature animals. Moreover, each Mako had good body condition and normal TW that was within natural variation (Table 7, all one-tailed t tests, P > .10).

Excluding hook in- juries, each shark appeared healthy, as evidenced by their typical indigo blue colored dorsal skin, no pallor, blotchiness, or caudal pe- duncle rigidity. Their urine composition exhibited a marked degree of similarity, but four individuals also had trace amounts of protein and nonhemolyzed blood (Table 7). The urine of these eight individuals contrasted markedly with the six Shortfin Mako sharks with RBU (Table 8), which also showed a great degree of consistency in urine composition among individuals for many (76.5%) of the variables ex- amined. The clear, red-brown color suggested the presence of hema- turia, hemoglobinuria, or myoglobinuria. The urine had a mean (± SD, range) specific gravity of 1.026 (0.6, 1.025-1.030), and was acidic in all six sharks, with a mean (± SD, range) pH of 5.76 (0.08, 5.65-5.85) obtained with the pH meter. Urine pH was negatively correlated with serum lactate concentrations and fight-times (r2 = .99 and .97, respectively, both P < .01). In contrast, the reagent dipsticks pro- vided an indicative mean (± SD, range) pH of 5.83 (0.41, 5.00-6.00). Glucose, ketones, and bilirubin were not detected, but the reagent

Shortfin Mako Shark
1 2 3 4 5 6 7 8
TL 2.10 2.32 2.36 2.53 2.61 2.86 2.96 3.17
TW 70.0 85.0 88.0 170.0 123.5 174.5 171.0 232.5
Analysis Variable Unit Sex F F F F F F F F
Visual Color – Pale-yellow Pale-yellow Pale-yellow Pale-yellow Pale-yellow Pale-yellow Pale-yellow Pale-yellow
Clarity – Clear Clear Clear Clear Clear Clear Clear Clear
Electronic USG, Mean (±SD) – 1.030 (0.001) 1.030 (0.000) 1.029 (0.000) 1.031 (0.000) 1.030 (0.001) 1.029 (0.000) 1.030 (0.000) 1.030 (0.000)
pH – 6.15 6.65 5.75 5.85 5.15 6.70 6.00 5.54
Reagent dipstick pH – 6.00 6.50 6.00 6.00 5.00 6.50 6.00 5.50
Glucose mg/dL ND ND ND ND ND ND ND ND
Ketones mg/dL ND ND ND ND ND ND ND ND
Bilirubin – ND ND Trace ND ND ND ND ND
Protein mg/dL Trace ND ND 1+ Trace ND Trace 1+
Blood – Trace (NH) ND ND Trace (NH) ND ND Trace (NH) Trace (NH)
Microscopic RBC
Mean (±SD) No/hpf 0.2 (0.42) ND ND 0.3 (0.48) ND ND 0.2 (0.42) 0.5 (0.53)
(Min-Max) No/hpf (0-1) ND ND (0-1) ND ND (0-1) (0-1)
WBC
Mean (±SD) No./
hpf ND ND ND ND ND ND ND ND
(Min-Max) No/hpf ND ND ND ND ND ND ND ND
Casts – ND ND ND ND ND ND ND ND

ImageOTWAY
TA B L E 7 Results of urine analyses for eight free-living Shortfin Mako sharks (Isurus oxyrinchus) exhibiting normal, pale-yellow urine following capture by recreational fishers over the austral summer/autumn months in east Australian offshore coastal waters from 2002 to 2017
Abbreviations: HPF, high power field of view; Max, maximum; Min, minimum; ND, not detected; NH, nonhemolyzed; SD, standard deviation; TL, total length in meters; TW, total weight in kilograms; USG, urine specific gravity.

Image|
12
TA B L E 8 Results of urine analyses for six free-living Shortfin Mako sharks (Isurus oxyrinchus) exhibiting red-brown urine following capture by recreational fishers over the austral summer/ autumn months in east Australian offshore coastal waters from 2002 to 2017

Analysis Variable Unit 1 2 3 4 5 6
Visual Color – Red-brown Red-brown Red-brown Red-brown Red-brown Red-brown
Clarity – Clear Clear Clear Clear Clear Clear
Electronic USG, mean (±SD) – 1.026 (0.001) 1.028 (0.001) 1.030 (0.000) 1.029 (0.000) 1.025 (0.001) 1.027 (0.001)
pH – 5.75 5.85 5.75 5.85 5.65 5.70
Reagent dipstick pH – 6.00 6.00 6.00 6.00 5.00 6.00
Glucose mg/dL ND ND ND ND ND ND
Ketones mg/dL ND ND ND ND ND ND
Bilirubin – ND ND ND ND ND ND
Protein mg/dL 1+ 1+ 1+ 1+ 1+ 1+
Blood – 3+ 3+ 3+ 3+ 3+ 3+
Microscopic RBC
Mean (±SD) No/hpf ND ND 0.4 (0.5) ND 0.5 (0.7) 0.3 (0.5)
(Min – Max) No/hpf ND ND (0-1) ND (0-2) (0-1)
WBC
Mean (±SD) No./hpf 0.2 (0.4) ND ND 0.1 (0.3) ND 0.2 (0.4)
(Min-Max) No/hpf (0-1) ND ND (0-1) ND (0-1)
Casts
Color – Red-brown Red-brown Red-brown Red-brown Red-brown Red-brown
Appearance – Smooth Granular Smooth Smooth Smooth Granular
Mean (±SD) No/lpf 2.5 (1.0) 2.9 (0.9) 2.1 (0.6) 1.7 (0.7) 3.3 (0.5) 2.5 (0.5)
(Min-Max) No/lpf (1-4) (2-4) (1-3) (1-3) (3-4) (2-3)
Crystals – ND ND ND ND ND ND
Centrifugation Supernatant color – Red-brown Red-brown Red-brown Red-brown Red-brown Red-brown
Precipitation # Precipitate – None None None None None None
Centrifugation Supernatant color – Red-brown Red-brown Red-brown Red-brown Red-brown Red-brown
Reagent dipstick Blood – 3+ 3+ 3+ 3+ 3+ 3+
OTWAY
Abbreviations: #, formation of precipitate with 80% saturated solution of ammonium sulfate49,86; HPF, high power field of view; lpf, low power field of view; Max, maximum; Min, minimum; ND, not detected; SD, standard deviation; USG indicates urine specific gravity.

dipsticks inferred proteinuria in the six sharks. Microscopic examina- tion indicated that the RBCs and WBCs were present in extremely low numbers in the urine, which varied among sharks (Table 8). Three Makos (Sharks 3, 5, and 6) had means of 0.3-0.5 RBC/hpf and were significantly greater (ANOVA, F5, 54 df = 3.1, P = .02) than the three remaining sharks with no RBCs in their urine. Similarly, three sharks had no WBCs present, whereas the remaining three (Sharks 1, 4, and 6) had means of 0.1-0.2 WBC/hpf, but overall there was no significant difference in the mean number of WBCs among sharks (ANOVA, F5, 54 df = 1.3, P > .29). The microscopic examination also revealed the presence of casts but no crystals. The casts were evi- dent in the urine of all six sharks, had a faded red-brown color and a smooth appearance in four sharks, and granular appearance in the remaining two (Table 8, Sharks 2 and 6). The number of casts var- ied significantly among the sharks (ANOVA, F5, 54 df = 6.40, P < .001), with Sharks 2 and 5 having more casts than the remaining sharks (SNK test, P < .05).
Following centrifugation of the second (ie, larger) urine sample from the six Makos, the color did not clear and remained red-brown, eliminating a diagnosis of hematuria (Table 8). Following the addition of ammonium sulfate and further urine centrifugation, hemoglobin did not precipitate, and the supernatant did not clear, and the urine remained red-brown in all six sharks, which eliminated a diagnosis of hemoglobinuria. Only myoglobinuria was left as a possibility, which was further confirmed using another reagent dipstick test of the urine supernatant49 that showed a consistent 3 + result for blood (ie, myoglobin) in all six Makos (Table 8).

4 | DISCUSSION

The TL and TW ranges in the Shortfin Mako sharks of this study were remarkably similar to those obtained by recreational fishers over a 20-year period from 1961 to 1990,27 and again from 1979 to 19826 off the coast of eastern Australia. The female-dominated bias observed in this study was likely due to the temporal focus (austral summer/autumn) compared with the year-round sampling reported in the previous studies,6,27 The length-weight relation- ships developed for this study did not differ from those previously established for Makos in east Australian waters,6 off the coast of New Zealand,7 the east coast of South Africa57 and in the US waters of the western North Atlantic.58 The size/age at sexual maturity of the six Makos with RBU were more similar to the median size/ age at maturity for sharks from New Zealand waters,7,10 than those more generally provided for the southern hemisphere.8 While only three of the Makos with RBU had dietary items present in their cardiac stomach, the ingested fish and squid species were consist- ent with previous dietary studies in east Australian waters,6 off the coast of New Zealand,25 the east coast of South Africa,57 and off the east coast of the USA.5 The clean, empty cardiac stomachs in these three sharks were most likely due to cardiac stomach ever- sion which is a normal/natural process in Shortfin Mako and other shark species.59,60 Additionally, an underwater video has provided
irrefutable evidence that stomach eversion in Makos can be trig- gered in response to hooking,60 presumably to try and remove the offending object. Stomach eversion would also account for a pro- portion of the empty stomachs observed in previous studies fol- lowing capture by hook and line.5,6,57 Lastly, the trauma of capture by hook and line was attributed as the direct causal mechanism for reductions in cardiac stomach temperature normally maintained at 6-8°C above ambient seawater by the paired supra-hepatic retia13 in contrast to modulated retial heat exchange efficiency.61 The similarity in biological traits exhibited between the Makos in the earlier studies6,27 and the six individuals with RBU, indicates that they are a representative (albeit small) subsample of the Shortfin Mako shark population off the coast of Eastern Australia.
Small copepod ectoparasites were evident in four of the sharks with RBU, and occurred on the tongue and the fins, but not in and around the teeth and gingiva. The parasitic copepod Anthosoma cras- sum has been documented in the jaws of lamniform sharks, includ- ing Makos, and can produce deep lesions in the jaw tissues and can cause severe, subacute necrotizing stomatitis in the mucosa and re- active lymphocytic infiltration of the submucosal skeletal muscle.62 Complications can include the loss of teeth, deep inflammation, nervous tissue involvement, and the hollowing out of the tongue.63 While this species was not present, three Lernaeopodid copepods (Brachiella thynni) were found on the tongue of one of the sharks ex- amined, and this microhabitat was consistent with prior descriptions of this parasitic copepod.64 No copepod parasites were observed on the gills of these six sharks, but copepods (Genus Brachiella and Nemesis) inhabiting the gills can cause respiratory distress in sharks with large infestations.65 Finally, the copepod Pandarus cranchi was found on the posterior margins (trailing edges) of the fins of three Makos was entirely consistent with previous descriptions, as the Genus Pandarus has a ubiquitous global distribution with various species occurring in restricted niches on the fins of Makos through- out the shark's circumglobal range.66,67 However, this ectoparasite has minimal effects on the health of its host67 and has previously been observed over many years in varying numbers on the fins of Makos captured off the coast of eastern Australia.

The initial health assessments were primarily designed to detect possible physiological, metabolic, and/or bacterial distur- bances in sharks using visual cues, palpation, and some physical measurements.38,51,68 There was little overt physical evidence of capture stress in the six Makos with RBU, apart from the unsighted internal hook wounds and some broken teeth. All of the sharks appeared healthy, as evidenced by their normal skin color, eye ap- pearance, and pupillary reflexes, teeth, and gums. Furthermore, there was no skin pallor or blotchiness, rigidity of the caudal pe- duncle muscles, large skin lesions, or spinal deformities. There was also no evidence of prior fishing interactions, as no additional hooks were found by the metal detector or palpation of the diges- tive tract.
Following hooking, catecholamines would have been secreted as part of the primary stress response33,39 and provoked the rapid onset of hyperglycemia (secondary response) in the six Makoswith RBU.39,69,70 While four sharks had glucose levels within the STS RI, it is probable that they were on the decline, as the remain- ing two sharks were hypoglycemic because of the long fight-times. Importantly, with hyperactivity and ongoing stress, a hyperglyce- mic shark can become hypoglycemic, and this can ultimately lead to death.70,71 Overall, the observed variation in glucose levels in the six Makos with RBU was consistent with previous studies and indicated that glucose might not be an efficacious stress indicator in sharks.
As expected, the serum lactate concentrations in all six Makos with RBU were markedly elevated as a result of metabolic (lactic) acidosis produced by anaerobic metabolism because of hypoxia, and in line with previous studies.30,31,33,35 The lactic acidosis and resulting acidemia were also more evident in the sharks that ex- perienced longer fight-times. The extreme physical exertion in the six Makos also produced increases in serum sodium concentra- tion, PCV, anion gap, and osmolality, and these changes were in line with previous studies of capture stress in various shark spe- cies.31,70,71 Combined, these results indicated that the Makos were also experiencing hypertonic dehydration and associated hypovo- lemia at the time of capture.

It was also important to consider the rectal gland function in the six Makos with RBU because it is controlled by body fluid volume, and reduced secretion also occurs with stress and associated circulating catecholamines.72 The rectal gland secretes sodium and chloride ions at concentrations equivalent to or exceeding those in seawater to counter their continuous influx through the gills.73 Fortuitously, previous research with the Sand Tiger shark38 has shown that rectal gland dysfunction caused marked, simultaneous increases in serum sodium and chloride levels (ie, both ions ≥30% above the STS URL). The absence of such changes in the six Makos indicated that rectal gland dysfunction was unlikely, but that reductions in secretion rate were possible.72,73

Similarly, there was also no evidence of hepatobiliary disease in the six sharks with RBU, as the serum activities of the inducible en- zymes ALP and GGT and the leakage enzyme ALT were very low, and the serum total bilirubin concentrations were normal, and all were in line with other sharks.31,38,51,74 The apparent hypocholesterolemia evident in three of the sharks could be of limited clinical significance and simply represent a past fasting state75 and/or natural fluctua- tions in the levels of cholesterol and other lipids that occur with re- productive status, season, and gender in various sharks, including lamnids.76 It is also likely that the elevated triglyceride levels in two of the sharks occurred for similar reasons.

The hyperkalemia evident via muscle leakage70,71,77 in the six Makos with RBU (ie, mean = 15.6 mmol/L and 274% above the STS URL) should be considered to be life-threatening per se,78-80 but it did not cause cardiac arrest as evidenced by the struggling and er- ratic movements of the sharks on landing in the boat. However, the levels were well above that known (ie, 7.0 mmol/L) to cause cardiac arrhythmias in sharks,70,75 which would have led to cardiac insuf- ficiency and leakage of CK and AST enzymes.31,38,51,70 The associ- ated prerenal hypotension would have led to renal hypoperfusion
and acute renal failure (ARF), leading to possible increases in serum urea and creatinine levels during the early stages of the capture. However, neither heart rate via ultrasonography,81 nor serum car- diac troponins80 or the CK-MB (cardiac) isoenzyme78 were quanti- fied during this study to confirm cardiac insufficiency and should be considered and/or measured in future studies to permit an as- sessment of the applicability and/or use of the latter biochemical indicators with sharks.

While glomerular filtration rate (GFR) is the best indicator of renal function in mammals due to its direct relationship with renal mass/ number of functioning nephrons,46 decreases indicating azotemia occur only after a 75% reduction in nephron function, and thus, GFR is not useful as an early indicator of renal dysfunction and is rarely used in marine mammals owing to the complexities of measurement. However, serum urea and creatinine levels can provide indirect ev- idence of renal function. And, while this relationship has not been confirmed in sharks, it is reasonable to assume that similarities exist given the complex structure and physiological functioning of shark kidneys.82,83 In contrast with mammals, sharks maintain high serum urea levels, which contribute markedly to serum osmolarity.38,51,83 While urea synthesis occurs in the liver, it is achieved via an ornithine cycle that also differs from mammals.84 As expected, the kidneys regulate serum electrolytes and urea levels with ≈95% of the filtered urea reabsorbed in the renal tubules.38,51,82 Under normal conditions, shark kidneys produce dilute urine that is hypo-osmotic to seawa- ter with sodium and chloride concentrations iso-osmotic to serum. Finally, the GFR has only been quantified in a few, small shark species. The average GFRs of ≈4.0 mL/kg/h (range = 0.2-12.0 mL/ kg/h) and elevated GFRs were seen with increases in the number of functioning nephrons similar to what is seen in mammals.82,83 Importantly, and in the absence of liver dysfunction in sharks, renal failure (from renal causes) is characterized by falling serum urea levels68,85 and increas- ing creatinine levels, a pattern that differs from azotemia in mam- mals. With this in mind, the six Makos with RBU exhibited an ≈14% reduction in serum urea concentration (ie, 52 mmol/L below the STS LRL), on average, together with average increases of 62.5% in serum creatinine (ie, 20 µmol/L above STS mean), 530% for inorganic phos- phate (ie, 8.6 mmol/L above the STS URL), and 50% for magnesium (ie, 1.1 mmol/L above STS URL) which provided clear evidence of ARF. The resulting clear, red-brown urine was caused by myoglobin, and the increased acidity in all six sharks was negatively correlated with serum lactate concentrations and fight-times. Microscopic ex- amination of the urine also revealed faded red-brown casts in all six sharks, providing further evidence of ARF. These results were also in great contrast to the eight Makos with normal, pale-yellow urine, that included four individuals with trace amounts of protein and nonhe- molyzed blood that was most likely caused by the extremely strenu- ous physical activity during their capture. However, the postcapture urinary catheterization could also have produced trace amounts of blood and needs to be considered.

The extreme exertion of the six Makos with RBU during their 1.0 to
3.0-hour fight-times in water temperatures of 25-26°C, together with the markedly elevated serum CK, myoglobinuria, and red-brown casts,confirmed the diagnosis of fishing-induced exertional rhabdomyolysis complicated by ARF. Once leaked from muscles, serum myoglobin is freely filtered by the glomeruli, but myoglobinuria is not grossly visible until the urine concentration is greatly elevated (>18 µmol/L) when it becomes visible86,87 by imparting the red-brown discoloration that was evident in the six Makos in this study, and with other animals and humans.46,78,88,89 Moreover, patients with red-brown urine attribut- able to myoglobin have a high risk of developing ARF.90 In acidic urine, myoglobin dissociates into ferrihematite and globin, which can exert a nephrotoxic effect through pigment glomerulopathy and obstruc- tive tubulopathy.80,87 The precipitation of myoglobin with Tamm- Horsfall (uromodulin) protein91 forming intra-tubular casts, and the subsequent renal tubule obstruction is supported by morphological and histopathological studies.92-95 The presence of casts and more acidic urine in the six Makos with RBU indicated that ARF was likely occurring through obstructive tubulopathy and nephrotoxic effects. Moreover, when urine pH is 6.50, only 4% of the myoglobin precipi- tates, whereas at pH <5.00, 73% of the myoglobin precipitates,95 and hence urine pH can greatly affect prognosis. Finally, ferrihematite is also directly nephrotoxic through the inhibition of nitric oxide syn- thase leading to vasoconstriction and renal hypoperfusion.80,95 Given that urine pH varied among the six Makos, it is very likely that the degree of obstructive tubulopathy and tubulotoxic effects also dif- fered among the sharks. This was also supported by the significantly greater number of casts in the two Makos (Sharks 5 and 6) that had the longest fight-times, greatly elevated serum CK activities and lac- tate levels, and reduced serum and urinary pH.

From an analytical perspective, the relatively rapid ammonium
sulfate precipitation test86 used in this study was also pH dependent and, in the absence of accurate pH measurements, could have led to false-negative results and the need to consider alternative tests including HPLC, electrophoresis, and immunoassay79,90 potentially causing additional diagnostic delays.

In rhabdomyolysis, CK activity rises over 12 hours, peaks at 24-72 hours, and has a half-life of 36 hours, whereas AST activity peaks in 24-48 hours, and has a half-life of 17-22 hours.80,87,96 In con- trast, myoglobin tends to normalize 6-8 hours after the stress event and has a half-life of 2-4 hours. Given the 1- to 3-hour fight-times, the muscle necrosis would have still been active when the six Makos with RBU were landed, and thus, it is very likely that the serum CK activities and myoglobin concentrations would have continued to increase had these sharks been released. Serum CK activities ex- ceeding 5000 U/L are also thought to be closely associated with the development of ARF,80,87 and enzyme activities greatly exceeding this value (ie, ≈2.5-162 fold increases) were evident in all six Mako sharks. Importantly, patients with nontraumatic rhabdomyolysis (in- cluding exertional forms) that develop ARF have greater mortality rates, with 31-59% in humans,79,80 and 70%-86% in animals.93,96,97
Exertional rhabdomyolysis treatment requires aggressive fluid
therapy combined with sodium bicarbonate infusions to reduce aci- demia and cast formation associated with urinary acidity in humans,80 terrestrial and marine mammals,93,96,97 and sharks.98 The use of so- dium bicarbonate is particularly important in sharks as normal serum
bicarbonate levels are maintained at 5-6 mmol/L,38,51,85 providing a minimal reserve under compromised physiological conditions. Treatment of Makos is made more difficult, as there is little clini- cal information for the species, although some advances have been made with blood/tissue biochemistry18,21,22 and respiratory physi- ology.20,99 For example, the oxygen dissociation curve for whole blood collected from Makos appears to be relatively unaffected by changes in CO2 and pH (ie, minimal Bohr and Root effects) 30 but can be affected by changes in temperature under normal (unstressed) conditions.99 Moreover, it is not clear what happens to the oxygen dissociation curve under stressful conditions such as those during capture. Thus, several clinical and logistic difficulties are present when treating Makos. Fortuitously, Makos have been restrained on research vessels and artificially ventilated for up to 15 minutes while telemetry equipment was fitted.13 As Makos are active swim- mers and rely on ram ventilation for respiration,20 it is not known whether they can withstand restraint and artificial ventilation over longer periods that would enable the administration of sufficient fluids to restore renal function without causing additional adverse effects. Nevertheless, the placement and maintenance of juve- nile Makos (0.77-1.25 m TL, 4.4-13.6 kg TW) in swim tunnels for up to 96 hours16,19,61 provides an avenue for further investigation. Whether this would be logistically feasible is a different, but import- ant question that needs consideration, as four of the sharks with rhabdomyolysis and almost 58% of the entire catch over the austral summer-autumn period from 2002 to 2017 exceeded 2.5 m TL and
100.0 kg TW. Furthermore, under current recreational fishing prac- tices, there is minimal capacity to provide any onboard treatment, and thus, a positive prognosis for sharks with exertional rhabdomy- olysis complicated by ARF is extremely unlikely at present.

Shortfin Mako sharks also appear capable of regulating their re- gionally endothermic body temperatures,10,15 but experiments ex- amining temperature regulation,61 using juvenile Makos (0.9-1.25 m TL, 5.0-13.6 kg TW) in a swim tunnel, were only able achieve maximal temperatures of up to 3°C less than those of Shortfin Mako sharks (1.36-2.40 m TL and 28-180.0 kg TW) landed on the stern of re- search vessels following longline capture.10,13-15 In the current study, the muscle temperatures measured immediately postcapture (28.0- 29.3°C) mirrored those of earlier studies10,13 and suggested that any temperature regulation could be disrupted under extremely stressful situations, such as caused by hook and line fishing. Consequently, fishing with long fight-times might cause pronounced hyperther- mia (ie, heatstroke) in Mako sharks, and their normal regional endo- thermy might predispose them and the other endothermic sharks (ie, Great White, Porbeagle, Salmon and Long-finned Mako sharks) to rhabdomyolysis during the summer/autumn periods when seawater temperatures peak. Moreover, as documented causes of rhabdo- myolysis in humans and animals include overexertion and/or heat- stroke,78,88,89,93,97 this potentially life-threatening condition could also become more prevalent along the east Australian coast under climate change with predictions of ocean warming by an average of 1°C and a strengthening EAC by 2030 under a regime with low CO2 emissions.100 Likely alterations to ocean temperatures due to climatechange will also affect NW Atlantic waters off the east coast of the USA that is bathed by the Gulf Stream, where the vertical movements of Mako sharks are strongly influenced by seawater temperatures.4

Exertional rhabdomyolysis complicated by ARF in Makos will be further exacerbated by the anatomic location of the hook injuries, and the degree of tissue trauma. The hooks in the six Makos with RBU became embedded in the dorsal wall of the esophagus, and thereby, serendipitously avoided causing internal hemorrhage by damage to the paired supra-hepatic retia immediately inferior to the esophagus13 or the posterior cardinal sinus superior to the esopha- gus.56 Nevertheless, the points of the hooks in the two sharks that perforated the distal dorsal esophageal wall were both within mil- limeters of the liver and posterior cardinal sinus. Had these sharks been released and survived, any subsequent hook migration would have likely caused hepatic damage56 and/or internal hemorrhage due to perforation of the posterior cardinal sinus and likely mortality.
Depending on the anatomic location of retained hooks, Makos can survive following release but can succumb to a disease arising from bac- terial infections101-103 or become cachectic through systemic inflam- mation, enhanced proteolysis, and excessive oxidative stress.101,104 For example, a Mako captured off the coast of NSW (34°34ʹS, 150°32ʹE) on December 5, 2010, using lighter fishing gear was released following tagging and removal of the fishing line, but not the embedded hook. At first capture, the shark had an estimated TW of 34 kg, but 65 days later, the shark was recaptured dying immediately after capture. At death, the shark's TW was 10 kg, a loss of 24 kg overall.

To my knowledge, this is the first description of fishing-induced exertional rhabdomyolysis in Shortfin Mako sharks that occurred sec- ondary to metabolic (lactic) acidosis, hypoxia, and hypovolemia, and was further exacerbated by ARF, which have been shown to be serious yet common complications in animals93,96,97 and 33%-50% of human patients80 often leading to mortality. It can be readily diagnosed by greatly elevated serum CK activity, red-brown urine, and associated casts. These findings have led practitioners to conclude that serum CK activity is the gold standard for ARF diagnoses, whereas serum myoglobin level is the gold standard for ARF patient prognoses.80 However, neither analyte can be quantified using point-of-care ana- lyzers (eg, VETSCAN, i-STAT, and ASVCP guidelines105), which have previously been used in field situations with terrestrial animals,106,107 seals,108 turtles,109,110 and sharks.34,111-113 Thus, future diagnostic con- firmation will still need to rely on laboratory-based serum biochemis- try analyses and associated time lags. Most importantly, if the urine had not been collected from the Shortfin Mako sharks, it is unlikely that rhabdomyolysis would have been considered, given the absence of overt symptoms in the initial health assessment. As the triad of symptoms for rhabdomyolysis includes muscle weakness, muscle pain, and RBU,78,80,88 the initial health assessment results could also be con- sidered to be misleading, especially because there was no evidence of muscle pain or weakness in the animals examined. However, many animals with exertional rhabdomyolysis exhibit no signs of illness but die 2-4 days after onset.93 With this in mind, I strongly encourage vet- erinary practitioners that care for sharks and rays to ensure that urine is collected from free-living or aquarium animals (size permitting) if an
animal is stressed after capture for examinations or treatments, partic- ularly in the season(s) of maximal seawater temperature.

ACKNOWLEDG MENTS
This research would not have been possible without the cooperation and assistance willingly provided by many recreational fishers. Discussions with J. Pepperell, M. Ellis (Gladstone Ports Corporation), D. Blyde (Sea World), S. Gilchrist (Sydney Aquarium), K. Rose (Taronga Wildlife Hospital), J. Roberts, N. McFarlane (IDEXX Laboratories, Brisbane) helped with aspects of this research. M. Booth (NSW DPI) is thanked for the constructive review of the manuscript. R. West and other NSW DPI library staff provided substantial assistance in accessing some of the lit- erature. This study forms part of an ongoing shark research program with grant funding provided by the NSW and Commonwealth governments.

DISCLOSURE
The author has indicated that he has no affiliations or financial in- volvement with any organization or entity with a financial interest in, or in financial competition with, the subject matter or materials discussed in this article.

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How to cite this article: Otway NM. Capture-induced exertional rhabdomyolysis in the Shortfin Mako Shark, Isurus oxyrinchus. Vet Clin Pathol. 2020;00:1–19. https://doi. org/10.1111/vcp.12824
1. Harter TS, Morrison PR, Mandelman JW, et al. Validation of the i-STAT system for the analysis of blood gases and acid-base status in juvenile sandbar shark (Carcharhinus plumbeus). Conserv Physiol. 2015;3. https://doi.org/10.1093/conphys/cov002