The understanding of stress and its impact on elasmobranch health and medicine is an important topic of ongoing investigation. During stressful events such as physical handling and exposure to air, fish have a wide range of physiologic responses that can affect their health and are cause for concern during medical examinations, capture via fishing, and transportation.1–4 In particular, transportation of pelagic sharks in the past has resulted in high rates of mortality and poor long-term survival rate. In fact, mortality rates of some species of wild fish after capture and release events can be as high as 94%.5–7 Respiratory or metabolic (more specifically, lactic) acidosis is an example of a potentially harmful result of such physiologic responses.5,6,8–12
For fish, motion is fueled by either aerobic (oxygen-dependent) processes, as occurs with nonexhaustive swimming, or anaerobic (glycogen-dependent) processes, as occurs with burst movement, excitation, or exhaustive exercise. With stress or exhaustive exercise, large disturbances of ionic, osmotic, and fluid volume homeostasis develop.13,14 If left unrecognized, the results are irreversible and life-threatening. Once glycogen breaks down, lactic acid accumulates and quickly dissociates into lactate by the release of a proton. Exhaustive exercise studied in dogfish results in depletion of arterial HCO3− immediately after exercise and during the first 8 hours of recovery.15 In addition, increased lactate and CO2 concentrations and decreased bicarbonate concentrations cause blood pH to decrease16 and serum electrolytes and osmolality to increase.1,10,16–18 Also, if a fish struggles on a hook, in a net, or during transport for a prolonged period, respiratory inadequacy can result, causing respiratory acidosis. Measurement of blood analytes is a sensible, rapid method that can aid in the assessment of physiologic status and has been accomplished in earlier studies5,7,18 in fish by use of a POC blood analyzera that measures blood gases, Hct, and concentrations of electrolytes, lactate, and glucose.
Analysis of blood analytes linked to acid-base status is a common procedure for evaluating pulmonary performance and immediate metabolic status in mammals. Only during recent years, measurements have been used in aquatic animal medicine for evaluating metabolic rates, energy budgets, and stress.4,5,18–20 Although the primary physiologic responses of fish to stressors involve the immediate release of circulating corticosteroids and catecholamines, these biomarkers are difficult to measure and their use as indicators of a stress response has not yet been validated in shark species. Therefore, secondary stress responses, which include acid-base disturbances, have been evaluated and used in practice.5,6,12,16,21,22 Variations exist among aquatic species in regard to metabolic capacity, means of respiration, and response to stress or low oxygen availability and have recently been associated with recovery from stress.2,5,9,13,23–25 Gill ventilation in most fish is accomplished by actively pumping water through the mouth and over the gills (buccal pumping; typical of benthic species) or by physically forcing water across the gills through swimming (obligate ram ventilation; typical of pelagic species).3,9 Fish that force oxygenated water over their gills and therefore cannot rest by cessation of swimming are more sensitive to oxygen deprivation than are species capable of buccal pumping; during capture and restraint, they are forced into anaerobic metabolism.1,9,16,26 For other aquatic species more tolerant of low-oxygen situations, acid-base disturbances may be less severe because less anaerobic metabolism is necessary. Identification of species-specific values for blood analytes associated with acid-base status during handling can lead to improved evaluation of restraint and capture techniques, anesthesia protocols, and viability of animals for transportation and may potentially curtail the morbidity and mortality rates of elasmobranchs following capture situations.
Factors other than physiologic status, such as venipuncture site, can potentially influence the results of blood sample analysis. Teleost species of fish have an SVS that manages intravascular fluid shifts. The SVS is a tortuous system of anastamoses between arteries running parallel to the primary circulatory system. This system contains a larger volume than does the primary vascular system; blood therein flows slowly, and the vasculature contains endothelial cells that regulate RBC concentration.27,28 These characteristics contribute to differences in blood values among collection sites. Despite the lack of evidence that an SVS exists in sharks, blood passage is reportedly impaired in some portions of the shark circulatory system.27–33 Large veins and venous sinuses have been identified,29,30,33 and for human safety and accessibility reasons, the dorsal sinus immediately caudal to the first (cranial) dorsal fin is a preferred phlebotomy site in large or awake sharks. However, as described for the teleost SVS, a slow fluid turnover rate, low Hct, and lack of connection to the primary arterial circulation influence the results of hematologic and biochemical analysis in samples obtained from this region. In a previous study,34 significant differences were found between Hct values of blood samples obtained from the dorsal sinus and samples collected from the caudal artery in several shark species. A recent study35 corroborated this finding in whale sharks and found similar differences in lactate values between phlebotomy sites.
The purpose of the study reported here was to establish reference ranges for critical care blood analytes in various species of wild and aquarium-housed elasmobranchs by use of a POC blood analyzer and to compare values on the basis of captivity status, species category (pelagic, benthic, or intermediate), and phlebotomy site. We hypothesized that aquarium-housed elasmobranchs acclimated to human activity would have less hematologic evidence of capture stress associated with sample collection, compared with wild elasmobranchs, and that concentrations of blood analytes and blood gas values would differ significantly between samples collected from the caudal tail artery and those collected from the dorsal sinus.
Materials and Methods
Animals—Shark and ray species were classified into 1 of 3 categories on the basis of their method of respiration, tolerance of low-oxygen conditions, and typical dwelling environments. Pelagic species were defined as obligate ram ventilators that are commonly found in open waters and are known to be sensitive to low-oxygen conditions. Intermediate species were defined as ram ventilators that can tolerate low-oxygen conditions more readily than can pelagic species. Benthic species were defined on the basis of their preference for resting on ocean bottoms and typically relying on buccal ventilation. The present study included 2 populations: aquarium-housed (n = 89) and wild (59) elasmobranchs.
Handling and examination of aquarium-housed elasmobranchs—Eighty-nine sharks and rays at the John G. Shedd Aquarium were anesthetized for annual or biannual health screens and were deemed healthy on the basis of typical appetites, gross appearance, and uneventful recovery from anesthesia. Blood samples collected between January 10, 2002, and July 10, 2004 were analyzed for study purposes. Species evaluated included the following: Carcharhinus melanopterus (blacktip reef shark; 15 males and 3 females), Carcharhinus plumbeus (sandbar shark; 2 males and 3 females), Triakis semifasciatum (leopard shark; 1 male and 2 females), Hydrolagus colliei (spotted ratfish; 1 male and 1 female), Aetobatus narinari (spotted eagle ray; 2 males and 2 females), Triaenodon obesus (whitetip reef shark; 6 males and 10 females), Orectolobus japonicus (Japanese wobbegong, 1 males and 2 females) Stegostomafasciatum (zebra shark; 5 males and 4 females), Pristis zijsron (green sawfish; 1 female), Potamotrygon castexi (vermiculate river stingray; 1 female), Potamotrygon menchacai (tiger ray; 4 males and 1 female), Cephaloscyllium ventriosum (swell shark; 1 male and 1 female, Himantura granulata (mangrove whipray; 2 males and 2 females), Chiloscyllium plagiosum (white spotted bamboo shark; 6 males and 7 females), and Heterodontus francisci (hornshark; 1 male, 1 female, and 1 of unknown sex).
Aquarium-housed fish were kept in aquarium settings for ≥ 6 months and were anesthetized at least once prior to inclusion in the present study; most had undergone repeat anesthetic events. Immobilization conditions for sharks involved desensitization in a medical treatment pool and rapid netting and sequential placement into immobilization pools with 2 concentrations of anesthetic. Large sharks (> 45 cm in length) were placed in a round plastic tub containing 870 L of water from the enclosure that was mixed with tricaine methane sulfonateb (125 μg/mL) for anesthetic induction. Once swimming and voluntary movement ceased, they were moved to another tub of water containing tricaine methane sulfonate (100 μg/mL) for the duration of physical examination and sample collection. Small sharks underwent the same anesthetic protocol in 278.5-L tubs. Physical examinations included blood collection, ultrasonographic evaluation of the heart and internal organs, external examination, and morphometric measurements, which included body weight, length, and girth. The duration of anesthetic time was approximately 10 to 15 minutes. A continuous flow of water over the gills was ensured as soon as fish could be safely handled (usually within 5 minutes of induction). As C melanopterus and C plumbeus aged during the years of sample collection and subsequently became larger and more difficult to restrain, midazolam (0.05 to 0.1 mg/kg [0.023 to 0.045 mg/lb], IM, once)c was added to the protocol and administered immediately after netting as a premedication to decrease stress during induction. Valium (0.4 mg/kg [0.18 mg/lb], PO, once)d was added to the anesthetic protocol for 1 sawfish (P zijsron) as a premedication 1 hour prior to any attempted handling because the fish was considered dangerous to handlers. Immobilization for rays involved rapid netting and placement into a 278.5-L tub of water from the enclosure containing tricaine methane sulfonate (125 μg/mL). Anesthetic induction typically occurred in 5 to 10 minutes, and the duration of anesthetic time was < 10 minutes.
Handling of wild elasmobranchs—Sixty-six sharks and rays were caught via hook and line fishing in the coastal waters of Southern Florida several miles from Vaca Key, Marathon, Fla, during a sample collection trip for Dynasty Marine Associatese between April 13, 2004, and July 25, 2009. Species included Ginglymostoma cirratum (nurse shark; 4 males and 2 females), Carcharhinus acronotus (blacknose shark; 12 males, 16 females, and 7 of unknown sex), Rhizoprionodon terraenovae (Atlantic sharpnose shark; 1 male), Carcharhinus limbatus (blacktip shark; 8 males and 10 females), Dasyatis americana (southern stingray; 1 female), and Negaprion brevirostris (lemon shark; 3 males and 2 females).
Animals were immediately brought to boatside, netted, and manually restrained for blood sample collection. Time from initial hooking to phlebotomy was between 1 and 2 minutes. All fish were released after sample collection.
Blood sample collection—Blood was collected from 2 anatomic sites in both populations of fish. Samples were immediately processed as whole blood or were mixed with heparin. In the authors' experience (LMN and NDM), heparin has induced agglutination of cells during blood collection from O japonicus; therefore, the use of heparin was avoided for samples from this species.
The first blood sample from each fish (ie, caudal sample) was collected from tail vasculature by inserting a needle at the ventral midline caudal to the pelvic fins until the vertebral column was contacted. Blood was typically observed in the needle hub; if not, slight movement away from the bone usually resulted in a flash of blood. Samples from this site for all sharks were considered arterial as evidenced by the anatomic location, color of the blood, and active flow Samples from rays were considered to be arterial overall; however, because of some difficulty with sample collection in a few of these fish, some samples may have included mixed arterial and venous blood. These samples could be distinguished from a venous sample by an experienced phlebotomist. Samples were collected from this site with a variety of needle and syringe sizes appropriate for the size and shape of the fish.
The second blood sample (ie, dorsal sample) was collected from the dorsal sinus region (located immediately caudal to the first dorsal fin at the fold where it met the dorsum) in < 3 minutes from aquarium-housed fish and in < 1 minute from wild fish. Samples from this site were collected with a 1-cm3 syringe and a 25-gauge needle.
Volumes of blood in caudal and dorsal samples varied among fish, ranging between 3 to 6 mL and 1 to 3 mL, respectively. A POC blood analyzera was used for blood sample analysis. Samples from wild elasmobranchs were analyzed with 2 of these instruments.a For all fish, the following blood analytes were measured: pH, Pco2, Po2, So2, total CO2, BEecf, Hct, anion gap, and concentrations of HCO3−, lactate, Na+, K+, iCa, Hb, and glucose. Analyzer cartridge models were not consistently available for use throughout the present study These varied according to manufacturer production and the availability or discontinuation of cartridges for the analyzer. However, all cartridge types used (CG4+, EG7+, CG8+, and EC8+) analyzed 6 standard blood gas values (pH, Pco2, Po2, total CO2, HCO3−, and base excess). The CG4+ cartridge also included analysis of lactate and So2; the EG7+ cartridge included analysis of Na+, K+, iCa, Hct, and Hb; the CG8+ cartridge included Na+, K+, iCa, Hct, Hb, and glucose; and the EC8+ cartridge included Na+, K+, Hct, Hb, and anion gap. Occasionally, an aliquot (1 mL) of blood was placed in a small heparinized tubef and run immediately after the caudal sample was collected; 1 agitation was performed to mix the sample with anticoagulant. Only samples analyzed ≤ 3 minutes after collection were included in statistical analyses for the present study because manufacturer instructions indicated that maintaining a completely anaerobic sample was unnecessary for blood tested within this time and that use of heparin does not affect results for such samples.g
Temperature corrections—Because the POC blood analyzers are routinely used to analyze blood samples at 37°C but results of studies36,37 indicate that certain blood gas values should be adjusted according to temperature, temperature-corrected values for pH, Pco2, and Po2 were calculated. All elasmobranchs of the present study were ectothermic species; therefore, blood sample temperature was estimated on the basis of water temperature at the time of collection. For wild elasmobranchs, mean sea surface temperature at all capture locations was 24°C. Water temperature at the John G. Shedd Aquarium was estimated at 26°C (maintained at 25° to 27°C). For comparison, blood pH, Pco2, and Po2 were recalculated for fish from both populations at 24° and 26°C with the following temperature correction formulas5,38:
where T is the estimated sample temperature (24° or 26°C).
Statistical analysis—Samples with analyte values reported as out of range of the POC analyzer were excluded from analysis for that analyte. With the exception of the statistical analysis used to compare variables between blood collection sites, only data from caudal samples were used in statistical analyses. Reference ranges were calculated for several blood variables in 6 species of elasmobranchs; species were included if samples were analyzed from > 5 fish. Data were analyzed for normality with the Shapiro-Wilk test and by examination of skewness and kurtosis. On the basis of analysis for normality, appropriate descriptive statistics for data were calculated for species of fish in aquarium-housed and wild populations. For data that followed a Gaussian distribution, mean, SD, and 95% CIs were calculated. For data that did not follow a Gaussian distribution, median, 25% to 75% quartiles, and minimum and maximum values were determined. Comparison between sexes was not performed because of a preponderance of 1 sex or missing information for some species.
Species of fish were categorized according to their method of respiration, tolerance of low-oxygen conditions, and typical dwelling environment as pelagic, benthic, or intermediate for further analysis of blood sample data. For purposes of analysis, of the 429 samples collected from aquarium-housed elasmobranchs and 168 samples collected from wild elasmobranchs, only 1 set of blood sample data was included from each individual fish in all statistical analyses to correct for repeated measures (ie, if an aquarium-housed fish underwent multiple sample collections during the study, the mean of all results for that fish was calculated). These data were also analyzed for normality with a Shapiro-Wilk test, and skewness and kurtosis were evaluated. A Kruskal-Wallis 1-way ANOVA was used to determine differences among species categories for each variable. Post hoc pairwise comparisons were completed with a Mann-Whitney U test. Appropriate descriptive statistics were calculated when significant differences were found. For data that followed a Gaussian distribution, mean, SD, and 95% CIs were calculated. For data that did not follow a Gaussian distribution, median, 25% to 75% quartiles, and minimum and maximum values were determined. A Mann-Whitney U test was used to compare data between pelagic and benthic species.
To assess agreement between values for caudal and dorsal samples, paired data for both populations were combined. To negate the effects of time, only samples collected and analyzed within 3 minutes of each other were included. Data for aquarium-housed and wild elasmobranchs were combined because of the small number of samples. Data for caudal samples were used as the standard with which dorsal samples were compared. Comparison of data from the 2 sites was conducted as a modification of analysis described elsewhere.39 The Bland-Altman method40 was used to construct difference plots and calculate bias and 95% LOA for each variable. Bias was defined as the mean difference between blood sample values for the 2 sample collection sites, and 95% LOA were calculated as the bias ± (1.96 × SD). Good agreement was defined as a bias and 95% LOA that differed by < 5% of the mean value for the caudal samples.
To further determine the relationship of measured hematologic variables between the 2 sample collection sites, Pearson correlation analysis was performed. Calculated correlation coefficients were interpreted as follows: 0.90 to 1.00, very high correlation; 0.70 to 0.89, high correlation; 0.50 to 0.69, moderate correlation; 0.30 to 0.49, low correlation; and 0 to 0.29, little or no correlation.41 Deming linear regression was performed to detect constant and proportional bias. If the 95% CI for the slope did not include the value of 1, proportional bias was considered to be present. If the 95% CI for the y intercept did not include the value of 0, constant bias was considered to be present. Overall comparison of values between caudal and dorsal blood samples was determined on the basis of Bland-Altman analysis results, correlation, and the presence or absence of constant or proportional bias.
Statistical significance was set at P ≤ 0.05. Statistical analysis was performed with commercially available statistical software packages.h,i
Results
All 89 aquarium-housed elasmobranchs were included in statistical analysis comparing specific blood analytes among the 3 species categories (pelagic, benthic, and intermediate). Samples from 56 of these fish were used to determine species-specific reference values (species with a sample size > 5). Of the 66 wild-caught elasmobranchs, 59 (55 of pelagic species and 4 of benthic species) were included in the statistical analysis comparing values among species categories; 7 individual samples analyzed with the POC analyzer > 3 minutes after collection were excluded from analysis.
Paired samples from 102 (44 aquarium-housed and 58 wild) elasmobranchs were used for comparisons between samples collected from the dorsal sinus region (dorsal samples) and tail vasculature (caudal samples). Caudal samples for sharks were considered arterial; in rays, these samples were considered to be arterial, although some samples may have included mixed arterial and venous blood. Dorsal samples were not collected from rays (n = 15) because no dorsal fin was present; dorsal samples were omitted or sample collection was unsuccessful in 31 aquarium-housed sharks, and samples from the 7 sharks that exceeded the 3-minute analysis time were excluded.
Reference ranges (Table 1) and mean or median values (Table 2) of 8 blood analytes (pH, Pco2, Po2, So2, BEecf, and concentrations of lactate, HCO3−, and glucose) in caudal samples were summarized for those species from which > 5 samples were obtained (aquarium-housed C melanopterus, T obesus, S fasciatum, C plagiosum, and wild C limbatus and C acronotus). Other blood analytes evaluated included iCa and Na+; these analytes had little or no apparent variability among individual fish or among species (iCa, 2.35 to > 2.5 mmol/L; Na+ > 180). Values for K+ concentration and Hct were out of range for the analyzer to calculate. Few fish of either study population had pH values < 7.2, which are considered by mammalian standards to be associated with acidosis. In the present study, the highest blood lactate concentration was 10.61 mmol/L, and samples from only 4 elasmobranchs (3 aquarium-housed and 1 wild) had concentrations > 5 mmol/L for this analyte.
Reference ranges for selected analytes in blood samples collected from aquarium-housed and wild sharks and measured by use of a POC blood analyzer at 37°C.
| Variable | Aquarium-housed sharks | Wild sharks | ||||
|---|---|---|---|---|---|---|
| Carcharhinus melanopterus (n = 18) | Triaenodon obesus (n = 16) | Stegostoma fasciatum (n = 9) | Chiloscyllium plagiosum (n = 13) | Carcharhinus limbatus (n = 16) | Carcharhinus acronotus (n = 33) | |
| pH | 7.127 to 7.275 | 7.19 to 7.294 | 7.253 to 7.455 | 7.186 to 7.354 | 7.170 to 7.322 | 7.153 to 7.316 |
| Total CO2 (mmol/L) | 3.57 to 4.95 | 4.8 to 5.7 | 3.3 to 5, 3 to 12* | 3.86 to 4.3 | 4.25 to 5.75 | 5 to 6, 5 to 7* |
| Total CO2 (mm Hg) | 7.98 to 12.46 | 9.55 to 13.87 | 6.2 to 7.2 | 7.35 to 10.74 | 8.56 to 12.86 | 10.3 to 15.5 |
| Po2 (mm Hg) | 23.75 to 78.03 | 49.73 to 69.15 | 51.15 to 97.13 | 41.92 to 71.33 | 16.37 to 30.57 | 15.5 to 29.75 |
| So2 (%) | 38 to 80.3 | 69.28 to 87.02 | 85.01 to 97.03 | 57.85 to 92.61 | 20.07 to 49.39 | 17 to 44, 7 to 98* |
| HCO3− (mmol/L) | 3.34 to 4.4 | 4.52 to 5.52 | 3 to 4.75 | 3.74 to 4.26 | 4.01 to 5.13 | 4.62 to 5.78 |
| BEecf (mmol/L) | −25.49 to 22.65 | −23.23 to 21.75 | −23.5 to 18.8 | −24.4 to 21.4 | −23.8 to 21.3 | −24 to 21 |
| Lactate (mmol/L) | 2.92 to 5.75 | 1.04 to 2.2 | 0.39 to 0.89 | 0.68 to 1.46 | 0.56 to 1.38 | 0.72 to 2.61 |
| Glucose (mg/dL) | 57.08 to 80.42 | 49.5 to 57.63 | 20 to 34.5 | 20.69 to 28.21 | 25.52 to 39.48 | 55.25 to 71.75 |
Blood samples were collected from the tail vasculature (caudal samples) of elasmobranchs; values were calculated for species with a sample size > 5. Caudal samples for all sharks were considered arterial; for rays, these samples were considered to be arterial overall, although some samples may have included mixed arterial and venous blood. Reference ranges were determined on the basis of measures of centrality (mean and median values) and dispersion (SD and 25% to 75% quartiles or 95% CI).
Multiple reference intervals indicate results when dispersions of 25% to 75% quartiles and 95% CI, respectively, were used in statistical analysis.
Mean or median values (with applicable temperature corrections) for analytes in the same 105 blood samples in Table 1.
| Variable | C melanopterus (n = 18) | Tobesus (n = 16) | S fasciatum (n = 9) | Cplagiosum (n = 13) | C limbatus (n = 16) | C acronotus (n = 33) | ||||||||||||
|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|
| M37 | M26 | M24 | M37 | M26 | M24 | M37 | M26 | M24 | M37 | M26 | M24 | M37 | M26 | M24 | M37 | M26 | M24 | |
| pH | 7.20 | 7.365 | 7.395 | 7.24 | 7.405 | 7.435 | 7.36 | 7.525 | 7.555 | 7.27 | 7.435 | 7.465 | 7.25 | 7.415 | 7.445 | 7.21* | 7.375* | 7.405* |
| Tco2 (mmol/L) | 4.26 | NA | NA | 5.25 | NA | NA | 4.0* | NA | NA | 4.08 | NA | NA | 5.0* | NA | NA | 6.0* | NA | NA |
| Pco2 (mm Hg) | 10.22 | 6.32 | 5.78 | 11.71 | 7.24 | 6.63 | 6.6* | 4.08* | 3.74* | 8.35* | 5.16* | 4.73* | 10.71 | 6.62 | 6.06 | 13.1* | 8.1* | 7.41* |
| Po2 (mm Hg) | 50.89 | 43.92 | 42.80 | 54.74* | 47.24* | 46.04* | 74.14 | 63.98 | 62.35 | 47.67* | 41.14* | 40.09* | 23.47 | 20.25 | 19.74 | 22.5* | 19.42* | 18.92* |
| So2 (%) | 59.15 | NA | NA | 78.15 | NA | NA | 91.02 | NA | NA | 75.23 | NA | NA | 34.73 | NA | NA | 30.0* | NA | NA |
| HCO3− (mmol/L) | 3.87 | NA | NA | 5.02 | NA | NA | 4.0* | NA | NA | 4.0 | NA | NA | 4.57 | NA | NA | 5.20 | NA | NA |
| BEecf (mmol/L) | −24.07 | NA | NA | −22.49 | NA | NA | −22.6* | NA | NA | −22.91 | NA | NA | −22.56 | NA | NA | −22.0* | NA | NA |
| Lactate (mmol/L) | 4.03* | NA | NA | 1.62 | NA | NA | 0.64 | NA | NA | 1.07 | NA | NA | 0.97 | NA | NA | 1.2* | NA | NA |
| Glucose (mg/dL) | 68.75 | NA | NA | 52.67* | NA | NA | 20* | NA | NA | 24.45 | NA | NA | 34.0 | NA | NA | 63.5* | NA | NA |
Values without an asterisk are mean values.
Median value.
M24 = Mean or median value at 24°C. M26 = Mean or median value at 26°C. M37 = Mean or median value at 37°C. Tco2 = Total CO2. NA = Not applicable (ie, temperature is not relevant to calculations for the given analyte).
Among aquarium-housed elasmobranchs of different species categories (pelagic, benthic, and intermediate), the Kruskal-Wallis 1-way ANOVA revealed differences in blood So2 (P = 0.016), lactate (P < 0.001), and glucose (P < 0.001) concentrations in caudal samples. Post hoc pairwise analysis determined differences between pelagic, benthic, and intermediate species for each of these analytes (Table 3). Among wild elasmobranchs of different species categories, the Mann-Whitney U test revealed a significant (P = 0.004) difference between pelagic and benthic species only for blood lactate concentration. Although not all comparisons among species categories were significantly different, mean lactate concentrations for aquarium-housed and wild fish were highest in pelagic species, mean So2 was highest in benthic species, and mean glucose concentration was highest in intermediate species.
Median (25% to 75% quartile) values for analytes in caudal samples of aquarium-housed (n = 89) and wild (59) elasmobranchs that were significantly different among species categories within a population.
| Population and variable | Species category | ||
|---|---|---|---|
| Pelagic | Intermediate | Benthic | |
| Aquarium-housed fish | |||
| So2 (%) | 68.5 (45.0–74.0)a | 80.0 (28.00–97.00)a,b | 81.50 (68.58–92.75)b |
| Lactate (mmol/L) | 3.21 (2.29–4.59)a | 2.05 (1.27–2.41)b | 0.95 (0.50–1.26)c |
| Glucose (mg/dL) | 59.50 (54.25–70.13)a | 63.0 (59.00–71.50)a | 29.00 (21.00–51.00)b |
| Wild fish | |||
| Lactate (mmol/L) | 1.11 (0.75–1.67)a | NA | 0.49 (0.38–0.59)b |
Shark and ray species were categorized as pelagic, intermediate, or benthic on the basis of their method of respiration, tolerance of low-oxygen conditions, and typical dwelling environments. Data from 7 of 66 wild elasmobranchs were excluded from analysis because the time from sample collection to analysis was > 3 minutes.
NA = Notapplicable.
Within a row, different superscript letters indicate significant (P ≤ 0.05) differences.
Overall analysis of analyte data from the caudal and dorsal blood sample collection sites revealed that values for dorsal blood samples were not comparable with those of caudal samples (Table 4). Visual inspection of the Bland-Altman difference plots and bias and 95% LOA data indicated substantial variation among values between the 2 sample collection sites for nearly all analytes (Figure 1). Good agreement between sites was found for BEecf only. Correlation between sites was high for pH, BEecf, and lactate concentration. The remaining analytes had moderate (HCO3− concentration), low (Pco2 or Po2), or little (So2) correlation. Also, Deming linear regression analysis revealed constant and proportional bias for So2. On the basis of results of Bland-Altman analyses, no dorsal samples were included in any of the other described statistical analyses. Because several factors differed between aquarium-housed and wild elasmobranchs (eg, anesthesia use), direct comparisons and statistical analyses between the 2 populations were not performed.
Results of Bland-Altman, Pearson correlation, and Deming linear regression analyses comparing analytes in 102 paired caudal and dorsal blood samples from aquarium-housed (n = 44) and wild (58) elasmobranchs.
| Variable | No. of samples | Bias | 95% LOA | Correlation coefficient (r) | Correlation P value | Linear regression (95% CI) | |
|---|---|---|---|---|---|---|---|
| Slope | y-intercept | ||||||
| pH | 25 | −0.066 | −0.22 to 0.091 | 0.70 | < 0.001 | 0.59 to 1.52 | −3.66 to 3.01 |
| Pco2 (mm Hg) | 25 | 2.95 | −2.90 to 8.80 | 0.30 | 0.143 | −0.37 to 2.40 | −20.16 to 13.88 |
| Po2 (mm Hg) | 23 | −25.91 | −111.30 to 59.44 | 0.43 | 0.04 | 0.14 to 5.64 | −175.3 to 83.11 |
| So2 (%) | 21 | −21.90 | −94.52 to 50.71 | 0.21 | 0.372 | −0.36 to 0.92* | 19.13 to 91.39† |
| HCO3− (mmol/L) | 24 | 0.60 | −0.98 to 2.19 | 0.57 | 0.004 | 0.34 to 1.55 | −3.31 to 2.61 |
| BEecf (mmol/L) | 25 | −0.40 | −3.29 to 2.49 | 0.78 | < 0.001 | 0.65 to 1.33 | −7.65 to 8.06 |
| Lactate (mmol/L) | 7 | 1.56 | −1.13 to 4.26 | 0.89 | < 0.001 | 0.66 to 1.12 | −2.76 to 0.86 |
Paired samples from each fish were collected 1 to 3 minutes apart and were analyzed < 3 minutes after sample collection by use of a POC blood analyzer. Data from aquarium-housed and wild elasmobranchs of various pelagic, benthic, and intermediate species were included in the analysis. Dorsal samples were collected from the dorsal sinus region just caudal to the first (cranial) dorsal fin; dorsal samples were not collected from rays (n = 15 [14 aquarium-housed and 1 wild]) because no dorsal fin was present; dorsal samples were omitted or sample collection was unsuccessful in 31 aquarium-housed sharks, and samples from 7 wild sharks that required > 3 minutes between collection and analysis were excluded.
Indicates proportional bias between values for the 2 collection sites.
Indicates constant bias between values for the 2 collection sites.
Bland-Altman plots of differences in pH (A) Pco2 (B), Po2 (C), So2 (D), HCO3− concentration (E), Beecf (F), and lactate concentration (G) in paired blood samples collected from 102 (44 aquarium-housed and 58 wild) of 155 elasmobranchs of various species categories (ie, pelagic, benthic, and intermediate). Blood samples were collected from the dorsal sinus region (dorsal samples) and caudal tail vasculature (caudal samples). Caudal samples for all sharks were considered arterial; in rays, these samples were considered to be arterial overall, although some samples may have included mixed arterial and venous blood. Paired samples from each fish were collected 1 to 3 minutes apart and were analyzed with a POC blood analyzer < 3 minutes after sample collection. Dorsal samples were not collected from rays (n = 15) because no dorsal fin was present; dorsal samples were omitted or sample collection was unsuccessful in 31 aquarium-housed sharks, and samples from 7 wild sharks that required > 3 minutes between collection and analysis were excluded. Solid lines indicate bias; dashed lines indicate 95% LOA.
Citation: Journal of the American Veterinary Medical Association 241, 1; 10.2460/javma.241.1.117
Bland-Altman plots of differences in pH (A) Pco2 (B), Po2 (C), So2 (D), HCO3− concentration (E), Beecf (F), and lactate concentration (G) in paired blood samples collected from 102 (44 aquarium-housed and 58 wild) of 155 elasmobranchs of various species categories (ie, pelagic, benthic, and intermediate). Blood samples were collected from the dorsal sinus region (dorsal samples) and caudal tail vasculature (caudal samples). Caudal samples for all sharks were considered arterial; in rays, these samples were considered to be arterial overall, although some samples may have included mixed arterial and venous blood. Paired samples from each fish were collected 1 to 3 minutes apart and were analyzed with a POC blood analyzer < 3 minutes after sample collection. Dorsal samples were not collected from rays (n = 15) because no dorsal fin was present; dorsal samples were omitted or sample collection was unsuccessful in 31 aquarium-housed sharks, and samples from 7 wild sharks that required > 3 minutes between collection and analysis were excluded. Solid lines indicate bias; dashed lines indicate 95% LOA.
Citation: Journal of the American Veterinary Medical Association 241, 1; 10.2460/javma.241.1.117
Bland-Altman plots of differences in pH (A) Pco2 (B), Po2 (C), So2 (D), HCO3− concentration (E), Beecf (F), and lactate concentration (G) in paired blood samples collected from 102 (44 aquarium-housed and 58 wild) of 155 elasmobranchs of various species categories (ie, pelagic, benthic, and intermediate). Blood samples were collected from the dorsal sinus region (dorsal samples) and caudal tail vasculature (caudal samples). Caudal samples for all sharks were considered arterial; in rays, these samples were considered to be arterial overall, although some samples may have included mixed arterial and venous blood. Paired samples from each fish were collected 1 to 3 minutes apart and were analyzed with a POC blood analyzer < 3 minutes after sample collection. Dorsal samples were not collected from rays (n = 15) because no dorsal fin was present; dorsal samples were omitted or sample collection was unsuccessful in 31 aquarium-housed sharks, and samples from 7 wild sharks that required > 3 minutes between collection and analysis were excluded. Solid lines indicate bias; dashed lines indicate 95% LOA.
Citation: Journal of the American Veterinary Medical Association 241, 1; 10.2460/javma.241.1.117
Discussion
In the study reported here, a POC blood analyzer was used to measure analytes for assessment of acid-base status in a variety of aquarium-housed and wild elasmobranch species. Blood samples were collected from 2 phlebotomy sites (the dorsal sinus region [dorsal samples] and tail vasculature [caudal samples]). Caudal samples for all sharks were considered arterial; in rays, these samples were considered to be arterial overall, although some samples may have included mixed arterial and venous blood. Dorsal and caudal samples were used to evaluate differences between phlebotomy sites; only caudal samples were used for all other analyses. The acute physiologic effects of capture-related stress are of concern when managing these species, and because the analyzer used is portable, it can be used to obtain boat-side or tank-side results in minutes. Species-specific reference values reported in the present study expand on the growing pool of vital information in elasmobranch medicine and provide a basis for further research.
Metabolic acidosis, one physiologic effect of stress, is a condition considered to be commonly induced in aquatic animals by capture techniques.1,8,10,11 However, little information has been published regarding teleost or elasmobranch critical care blood analytes.8,9,11,16,20,42 Anecdotally, the authors of the present study have observed acid-base disturbances associated with metabolic acidosis (as with mammals) in ill or fatigued elasmobranchs after prolonged capture procedures. In the present study, minimal variation among blood analyte values indicative of metabolic disturbances or capture stress was detected in caudal samples from aquarium-housed or wild elasmobranchs. The blood gas, electrolyte, and biochemical values for both elasmobranch populations were comparable with those reported in studies9,16 on Atlantic sharpnose and dusky sharks when blood samples were collected within several minutes after the animals were first handled. Although lactic acidosis and high blood lactate concentrations (> 10 to 15 mmol/L) have been reported in elasmobranchs during stressful events,5,9–11,16,17,20,22,26,43 few of the sharks and rays of the present study had blood lactate concentrations > 5 mmol/L. Explanations for a lack of extreme acid-base fluctuations include reduced or slow secondary physiologic responses to stress. For example, peak values for circulating lactate concentrations have been found to slowly develop in some teleost and elasmobranch species, sometimes > 1 hour after a stressful event.9,12,16,26,42 Furthermore, in an aquarium environment, aquatic animals are frequently accustomed to human activity and numerous handling efforts and become conditioned or trained to handling. For these reasons, the authors believe that the information obtained in the present study provides adequate baseline values and therefore a point of reference for assessment of acid-base status in these and similar elasmobranch species.
In previous studies1,2,4–6,8–25,43 of aquatic animals during stressful events, both the degree of acid-base imbalance and recovery capacity differed among pelagic, intermediate, and benthic species. In the present study, differences in blood analytes were detected among these 3 species categories regardless of captivity status; for the aquarium-housed population, analysis revealed significant differences in So2, lactate concentration, and glucose concentration. For the wild population, a significant difference was detected in lactate concentrations among the 3 categories. Although not all comparisons among species categories were significantly different, mean lactate concentration was highest in pelagic species, which are most likely to rely on anaerobic metabolism during times of stress.1,9 Mean blood So2 was highest in benthic species, which may be expected on the basis of their multiple modes of respiration regulation. Mean glucose concentration was highest for intermediate species.
In canids and ferrets, hypoglycemia is defined as a serum glucose concentration < 40 and < 60 mg/dL, respectively.44 However, in elasmobranchs, values of 40 to 60 mg/dL are considered normal or slightly high. Some studies6,16 of elasmobranchs during stress have revealed a wide range of circulating glucose concentrations, with values well above or below the established normal ranges for mammals, and reported hyperglycemia (values > 70 mg/dL for some elasmobranch species) was a poor prognostic indicator for recovery. The relationship between stress and serum glucose concentrations is not yet completely clear in elasmobranch medicine.1,8,10,11 Although increased circulating concentrations of iCa, K+, and chloride have also been associated with stress in teleosts and elasmobranchs,10,16 these values were not statistically assessed in the present study because of the limited range of the POC blood analyzer used to assess those analytes. In the authors' opinions, a blood iCa concentration > 2.5 mg/dL as measured with the POC analyzer (the upper measurement capacity limit of the POC analyzer) should be expected in healthy elasmobranchs, and concentrations below this value appear to be indicative of a metabolic disturbance. In the present study, the small sample sizes for specific groups such as aquarium-housed intermediate species and wild benthic species likely reduced our ability to find potential differences in analyte values. Continued comparative studies are needed to validate the reference ranges in the present study as well as the differences detected among pelagic, intermediate, and benthic species.
In addition to determining reference values, differences in values for dorsal and caudal blood samples were examined in the study reported here. Overall analysis of the data revealed substantial differences between blood analyte values for the 2 sites, with higher values for most analytes in the caudal samples. These differences may be attributable to the unique elasmobranch circulatory anatomy as described elsewhere.27–35,45 Identifying the phlebotomy site that provides a more accurate depiction of acid-base status in elasmobranchs was beyond the scope of the present study, but given the mean values of So2 and the determination that samples collected from the tail vasculature were arterial rather than venous, it seems likely that the caudal artery would be a preferred, more accurate site for collecting samples for blood gas and metabolic status analysis. Regardless, significant differences in blood values between collection sites were identified and should be considered when analyzing blood from elasmobranchs. Continued studies identifying blood gas, electrolyte, lactate, and glucose values specific to pelagic, intermediate, and benthic species categories or individual elasmobranch species will be invaluable to providing a more comprehensive database for medical assessment of these species.
ABBREVIATIONS
| BEecf | Base excess in extracellular fluid |
| CI | Confidence interval |
| Hb | Hemoglobin |
| iCa | Ionized calcium |
| LOA | Limits of agreement |
| POC | Point-of-care |
| So2 | Oxygen saturation |
| SVS | Secondary vascular system |
i-Stat, Heska Corp, Fort Collins, Colo.
MS-222, tricaine methanesulfonate, Argent Chemical Laboratories Inc, Redmond, Wash.
Midazolam, 5 mg/mL, Hospira Inc, Lake Forrest, Ill.
Valium, 10-mg tablets, Watson Pharmaceuticals Inc, Corona, Calif.
Dynasty Marine Assoc Inc, Gulf, Marathon, Fla.
Lithium heparin blood collection tubes, BD, Franklin Lakes, NJ.
Heska Corp. i-Stat analyzer cartridge specifications. Available at: www.i-stat.com/products/ctisheets/714285–01L.pdf. Accessed Oct 1, 2010.
SPSS, SPSS Inc, Chicago, Ill.
Prism, version 5, GraphPad Software Inc, La Jolla, Calif.
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