Connecting post-release mortality to the physiological stress response of large coastal sharks in a commercial longline fishery.

Bycatch mortality is a major factor contributing to shark population declines. Post-release mortality (PRM) is particularly difficult to quantify, limiting the accuracy of stock assessments. We paired blood-stress physiology with animal-borne accelerometers to quantify PRM rates of sharks caught in a commercial bottom longline fishery. Blood was sampled from the same individuals that were tagged, providing direct correlation between stress physiology and animal fate for sandbar (Carcharhinus plumbeus, N = 130), blacktip (C. limbatus, N = 105), tiger (Galeocerdo cuvier, N = 52), spinner (C. brevipinna, N = 14), and bull sharks (C. leucas, N = 14). PRM rates ranged from 2% and 3% PRM in tiger and sandbar sharks to 42% and 71% PRM in blacktip and spinner sharks, respectively. Decision trees based on blood values predicted mortality with >67% accuracy in blacktip and spinner sharks, and >99% accuracy in sandbar sharks. Ninety percent of PRM occurred within 5 h after release and 59% within 2 h. Blood physiology indicated that PRM was primarily associated with acidosis and increases in plasma potassium levels. Total fishing mortality reached 62% for blacktip and 89% for spinner sharks, which may be under-estimates given that some soak times were shortened to focus on PRM. Our findings suggest that no-take regulations may be beneficial for sandbar, tiger, and bull sharks, but less effective for more susceptible species such as blacktip and spinner sharks.

Variation of body condition and plasma energy substrates with life stage, sex, and season in wild-sampled nurse sharks Ginglymostoma cirratum.

Reported here are the relationships among morphological (i.e., body condition) and biochemical (i.e., plasma concentrations of triglycerides, cholesterol, free fatty acids, and ketone bodies and ketone body ratios) parameters related to energy storage and use, as well as the variation of such parameters, for 107 free-ranging nurse sharks Ginglymostoma cirratum sampled off South Florida. Immature G. cirratum exhibited a higher variance in body condition, plasma free fatty acid concentrations and ketone body ratios compared to adults. Mature female G. cirratum had significantly higher body condition than mature males, driven by a seasonal increase in mature female body condition during the wet season. Mature male G. cirratum showed a decrease in the ketone body ß-hydroxybutyric acid during the dry season. Taken together, this study provides a baseline assessment of body condition and internal physiological state for a data-poor marine species and demonstrates significant ontogenetic, sexual and seasonal variation in G. cirratum energetic state. As concluded by other studies of energy metabolism in free-ranging sharks, this research highlights the importance of considering intraspecific patterns and sampling context for inferring the drivers of variation.

The power struggle: assessing interacting global change stressors via experimental studies on sharks.

Ocean warming and acidification act concurrently on marine ectotherms with the potential for detrimental, synergistic effects; yet, effects of these stressors remain understudied in large predatory fishes, including sharks. We tested for behavioural and physiological responses of blacktip reef shark (Carcharhinus melanopterus) neonates to climate change relevant changes in temperature (28 and 31 °C) and carbon dioxide partial pressures (pCO2; 650 and 1050 µatm) using a fully factorial design. Behavioural assays (lateralisation, activity level) were conducted upon 7-13 days of acclimation, and physiological assays (hypoxia tolerance, oxygen uptake rates, acid-base and haematological status) were conducted upon 14-17 days of acclimation. Haematocrit was higher in sharks acclimated to 31 °C than to 28 °C. Significant treatment effects were also detected for blood lactate and minimum oxygen uptake rate; although, these observations were not supported by adequate statistical power. Inter-individual variability was considerable for all measured traits, except for haematocrit. Moving forward, studies on similarly 'hard-to-study' species may account for large inter-individual variability by increasing replication, testing larger, yet ecologically relevant, differences in temperature and pCO2, and reducing measurement error. Robust experimental studies on elasmobranchs are critical to meaningfully assess the threat of global change stressors in these data-deficient species.

Non-lethal assessment of reproductive stage for female blue sharks Prionace glauca using sex steroid hormones.

Plasma concentrations of progesterone (P4 ) and 17ß-oestradiol (E2 ) in juvenile, pre-ovulatory, early, mid- or late pregnancy stages of female blue sharks Prionace glauca were analysed. Concentrations of P4 were significantly higher in pregnant than in non-pregnant individuals, whereas E2 concentrations increased with embryonic and follicular development. A highly accurate (86.1%) random forest classification model was developed to predict shark pregnancy. It is proposed that hormone concentrations could be used for the subsequent non-lethal determination of female P. glauca reproductive state.

Serum Protein Analysis of Nurse Sharks.

Serum protein electrophoresis (EPH) is used to assess relative concentrations of blood proteins in clinical and biological studies. Serum EPH fractions have been determined for elasmobranchs using mammalian albumin, alpha 1-, alpha 2-, beta-, and gamma-globulin fractions, and have been deemed fractions 1 through 5, respectively. However, serum EPH fraction concentration reference intervals (RIs) have not been widely established for different elasmobranch species. In this study, RIs for fractions 1 through 5 were determined from 45 wild-caught Nurse Sharks Ginglymostoma cirratum (27 females and 23 males) in South Florida. Serum samples were isolated from whole blood following caudal venipuncture. Body condition was also measured in the field to assess the relative health of the individuals sampled. There was no relationship between body condition and serum EPH fraction concentrations. In addition, there was no difference in body condition or serum EPH fraction concentrations between females and males. Total solids and total protein values were significantly different (P < 0.001). Nurse Shark serum EPH fraction 1 was found within the mammalian albumin migrating band distance and was negligible. Fraction 2 showed no peak in the mammalian alpha 1-globulin range. A thin, medium peak in the mammalian alpha 2-globulin range represented fraction 3. In the mammalian beta-globulin range, fraction 4 consisted of the majority of protein observed. It was represented by a smooth, broad peak. A short, medium broad peak in the mammalian gamma-globulin range represented fraction 5. The Nurse Shark serum EPH fraction RIs provided in this study may be utilized to clinically evaluate the health of Nurse Sharks in captivity and in the wild, and to compare the health of their populations around the world experiencing various anthropogenic stressors and other environmental impacts.

Effects of elevated carbon dioxide on the hematological parameters of a temperate catshark.

Atmospheric CO2 levels have been rising due to an increase in anthropic activities and its implications over marine ecosystems are unprecedented. The present study focused on the effects of ocean acidification (OA) on key hematological parameters of the juvenile small-spotted catsharks (Scyliorhinus canicula). Eggs were reared throughout the entire embryogenesis (~4 months) plus 5 additional months, in two experimental treatments (control: pCO2 ~ 400 µatm; and high CO2 : pCO2 ~ 900 µatm, Δ -0.3 pH units). After blood collection, the following hematological parameters were evaluated: (a) normal blood cells count (erythrocytes, leukocytes, and thrombocytes), (b) presence of erythrocytes with nuclear abnormalities, and (c) erythrocyte nucleus to cytoplasmic ratio. Concomitantly, to determine the cardiac and hematopoietic conditions, the spleen and heart to body ratios were also assessed. The present findings indicate that the measured variables may not be affected by elevated pCO2 in this temperate species, as no significant differences were observed between treatments across all the endpoints tested. Nonetheless, it is worth mentioning a decreasing trend observed in a number of thrombocytes associated with OA, which should foster further investigation, regarding other aspects of their coagulation response. Along with OA, other stressors are expected to impact marine life, such as warming and hypoxia. Thus, future research should aim to investigate the cumulative effect of these stressors on hematological parameters in sharks.

Isotopic variation in blood components based on their biochemistry and physiology: A comparison between sharks and fur seals.

Research using stable isotopes analysis (SIA) of carbon (δ13 C) and nitrogen (δ15 N) in blood components is lacking, because of the challenge of sample collection, processing, and storage in remote areas. There also is a paucity of information regarding the effect of tissue biochemical composition on isotopic ratios with few comparisons among taxa. We collected blood samples from shortfin mako sharks (n = 70; 2016) and Guadalupe fur seals (n = 25; 2017). All samples were centrifuged to obtain plasma from sharks and serum from the Guadalupe fur seals, and all the samples were prepared for SIA and analyzed using a Costech 4010 elemental analyzer interfaced with a Delta V Plus isotope ratio mass spectrometer. We found significant differences between plasma δ13 C values of shortfin mako sharks (-17.6 ± 0.9‰) and serum of Guadalupe fur seals (-20.3 ± 1.2‰), but we did not find any differences for δ15 N values between the two species. The differences in δ13 C values between species are probably due to the specific blood composition and to the different biochemical characteristics and different adaptations within taxa. These findings highlight the importance of further research on the influence of biochemistry features on isotopic results, in this way a more accurate assessment will be possible for this factor, separating it from the dietary influences on stable isotopic values.

Effects of anticoagulants on stable-isotope values (δ13 C and δ15 N) of shark blood components.

The effects of anticoagulant EDTA and sodium heparin (SH) on stable carbon δ13 C and nitrogen δ15 N isotopic values of red blood cells (RBC) and blood plasma in juvenile blacktip reef sharks Carcharhinus melanopterus were analysed. Plasma preserved with anticoagulants was not isotopically distinct from plasma stored in no-additive control tubes but RBC δ15 N values exhibited small enrichments when preserved with EDTA and SH. Results suggest EDTA and SH are viable anticoagulants for stable isotopic analyses of blood fractions but further studies are advised to validate results.

Effects of temperature on the reproductive physiology of female elasmobranchs: The case of the narrownose smooth-hound shark (Mustelus schmitti).

The knowledge of how temperature influences elasmobranchs reproductive physiology allows a better understanding of their reproductive patterns. This study describes the relationship between temperature fluctuations and the plasmatic changes of the sex steroids related to reproduction: testosterone (T), estradiol (E2) and progesterone (P4), throughout the female reproductive cycle of the shark Mustelus schmitti. A total of 123 adult females were bi-monthly sampled in Buenos Aires, Argentina, coastal waters. Bottom temperatures were recorded at each sampling point and blood samples were taken from each female for plasma sex steroids measurement. Sex steroid plasma levels were analyzed in relation with maximum follicular diameter (MFD), uterosomatic index (USI, as indicator of pregnancy) and temperature using Generalized Additive Models. Plasmatic E2 and T increased during follicular growth until MFD reached 1.34 and 1.46 cm, respectively. Peak of T occurred at the follicular stage associated with parturition (MFD, 1.4-1.6 cm), just prior to final maturation and ovulation (MFD, 1.6-2.0 cm). Progesterone significantly increased at this last ovarian phase, while T and E2 decreased. The increase of USI with pregnancy was associated to a decrease in T and mainly E2 levels, while P4 remained unaffected. Prior to ovulation, T plasma levels decreased with temperature below to 13 °C and then increased progressively with a pronounced elevation above 17 °C, while E2 presented an opposite pattern. Progesterone plasma levels changed with temperature showing a similar pattern to that observed for T. Using M. schmitti shark as model species, this study shows a clear picture of how seawater temperature variations can affect the reproductive physiology in elasmobranch females. A hypothetical mechanism (based on T elevation driven by temperature increase and its connection by feedback with a P4 rise and parturition/ovulation induction) is proposed as evidence to support that the increase in temperature can trigger reproductive events in elasmobranchs. In addition to its ecological scope, this work contributes to reinforce the relatively scarce general knowledge of elasmobranchs reproductive physiology.

NAÏVE-POOLED PHARMACOKINETICS OF CEFTIOFUR CRYSTALLINE FREE ACID AFTER SINGLE INTRAMUSCULAR ADMINISTRATION IN SMOOTH DOGFISH (MUSTELUS CANIS).

Pharmacokinetics study of ceftiofur crystalline free acid (CCFA) was conducted in 14 adult captive smooth dogfish (Mustelus canis). A single dose of CCFA at 6.6 mg/kg was administered intramuscularly. Blood samples were collected prior to treatment and at 1, 2, 6, 12, 24, 32, 48, 72, 96, 120, 144, and 168 hr posttreatment. Naïve pooling of data from four sharks was used to generate the average plasma drug concentration at each time point. After concluding the study, additional blood samples were opportunistically collected from five randomly selected sharks at 1,920 hr. Plasma ceftiofur and desfuroylceftiofur metabolite concentrations were determined using reversed-phase high performance liquid chromatography (HPLC). Pharmacokinetic analysis was performed using a noncompartmental technique. Peak plasma concentration (Cmax) was 3.75 µg/ml with a time to Cmax (Tmax) of 96 hr. Ceftiofur plasma concentrations were maintained above 2 µg/ml for at least 168 hr and were still quantifiable at 1,920 hr.

Blood plasma levels of heavy metals and trace elements in white sharks (Carcharodon carcharias) and potential health consequences.

Heavy metals may adversely affect health in marine organisms. As top predators, sharks may be especially vulnerable to exposure over long lifespans. Here we evaluate plasma levels of 14 heavy metals and 12 trace elements in white sharks, Carcharodon carcharias, in South Africa to determine whether they are related to sex, body size, and/or body condition and other health parameters. High levels of mercury and arsenic were found in shark blood at levels considered toxic in other vertebrates. Heavy metal concentrations were not related to body size or sex. Metal concentrations were not related to body condition with exception of copper, which was positively correlated. Protective effects of elements such as selenium, zinc, and iron were not detected. No negative effects on health parameters, such as total leukocytes or granulocyte to lymphocyte ratios were observed. Results suggest that sharks may have protective mechanisms that mitigate harmful effects of heavy metal exposure, providing new opportunities for future studies.

EFFECT OF A NUTRIENT ENEMA ON SERUM NUTRIENT CONCENTRATIONS IN WHITE-SPOTTED BAMBOO SHARKS (CHILOSCYLLIUM PLAGIOSUM).

Ill and anorectic captive sharks present a unique challenge for husbandry and veterinary staff. Providing adequate fluid and nutritional support to sharks while minimizing handling remains difficult. This study aimed to evaluate the ability of a nutrient enema to alter blood analyte concentrations. Thirty-six healthy, fasted white-spotted bamboo sharks (Chiloscyllium plagiosum) were enrolled in the study with 18 sharks receiving a nutrient enema and 18 sharks receiving a non-nutrient saline enema. The metabolic state of sharks was evaluated via measurement of blood glucose, blood urea nitrogen, and ß-hydroxybutyrate as well as other serum biochemistry parameters. Changes in sodium, chloride, calcium, ß-hydroxybutyrate, glucose, total protein, and triglyceride concentrations were seen across time in both groups. Blood glucose absolute concentrations and changes over time differed between the nutrient and nonnutrient groups. This pilot study indicates that it is possible to influence the glucose metabolism of healthy sharks via nutrient enema. Further study is needed to better understand potential therapeutics for ill and anorectic sharks.

Seasonality of Immunological and Health-State Parameters of Wild Broadnose Sevengill Shark, Notorynchus cepedianus.

Seasonal fluctuation in environmental parameters can influence immune responses of vertebrates and consequently influence their health and disease resistance. Although seasonality of immune function is well documented in a broad range of vertebrate taxa, this information remains virtually unexplored in cartilaginous fish. Here we examine seasonal variation in immune and general-health parameters of free-living adult broadnose sevengill sharks, Notorynchus cepedianus, along an annual cycle. We sampled sharks during autumn/winter (i.e., coolest temperatures and nonreproductive period) and spring/summer (i.e., warmest temperatures and active reproductive period) and assessed aspects of immunity, general condition, and reproductive hormone levels. A seasonal influence was observed in some, but not all, parameters evaluated. Lower lymphocyte counts and higher heterophil counts and granulocyte to lymphocyte (G∶L) ratios were observed in sharks sampled during autumn/winter than in those sampled during spring/summer. On the other hand, total leukocyte counts, eosinophil counts, bacterial agglutination mediated by natural antibodies, and hematocrit did not vary seasonally. The observed seasonal patterns could be explained as (1) greater levels of stress based on the G∶L ratio, (2) a sign of immunosuppression or depressed immune investment based on the low lymphocyte counts, and/or (3) a sign of ongoing infection based on the higher heterophil counts in the colder seasons with respect to the warmer ones. In addition, the pattern is in line with the notion that while acquired components are usually depressed by lower temperatures, some innate components might increase to offset that reduction. Immune and health-state parameters were mostly independent of reproductive hormone levels, providing little support for a trade-off with reproduction. Overall, the observed seasonal pattern in immunity of broadnose sevengill sharks could be related to changes in abiotic environmental condition, such as water temperature and photoperiod, although other factors such as availability of high-quality food may play a part.

Changes in sex steroid hormone levels reflect the reproductive status of captive female zebra sharks (Stegostoma fasciatum).

Captive breeding in aquaria is a useful means for ex situ preservation of threatened elasmobranch species. To promote captive breeding, it is important to determine the female reproductive status. However, information regarding reproductive status in female elasmobranchs is limited. Here, we used zebra sharks, Stegostoma fasciatum, as a model for elasmobranch reproduction in captivity. We investigated the relationships among changes in the sex steroid hormone levels, follicle size, and egg-laying period to develop indicators for the female reproductive status. We confirmed that mature female zebra sharks undergo an annual reproductive cycle. Additionally, we showed that the variations in sex steroid hormone levels correlated with reproductive status in mature female zebra sharks. Plasma estradiol-17ß (E2) concentrations increased two months before ovarian follicle development and decreased along with follicle regression. Interestingly, E2 levels were inversely correlated with water temperature (R = -0.901). Moreover, high levels of testosterone (T) correlated well with the laying period. These results strongly suggest that E2 is an indicator for ovarian follicle development, and that T is a useful indicator for both the onset and end of the egg-laying period in captive zebra sharks.

Temperature effects on the blood oxygen affinity in sharks.

In fish, regional endothermy (i.e., the capacity to significantly elevate tissue temperatures above ambient via vascular heat exchangers) in the red swimming muscles (RM) has evolved only in a few marine groups (e.g., sharks: Lamnidae, Alopiidae, and teleosts Scombridae). Within these taxa, several species have also been shown to share similar physiological adaptations to enhance oxygen delivery to the working tissues. Although the hemoglobin (Hb) of most fish has a decreased affinity for oxygen with an increase in temperature, some regionally endothermic teleosts (e.g., tunas) have evolved Hbs that have a very low or even an increased affinity for oxygen with an increase in temperature. For sharks, however, blood oxygen affinities remain largely unknown. We examined the effects of temperature on the blood oxygen affinity in two pelagic species (the regionally endothermic shortfin mako shark and the ectothermic blue shark) at 15, 20, and 25 °C, and two coastal ectothermic species (the leopard shark and brown smooth-hound shark) at 10, 15, and 20 °C. Relative to the effects of temperature on the blood oxygen affinity of ectothermic sharks (e.g., blue shark), shortfin mako shark blood was less affected by an increase in temperature, a scenario similar to that documented in some of the tunas. In the shortfin mako shark, this may act to prevent premature oxygen dissociation from Hb as the blood is warmed during its passage through vascular heat exchangers. Even though the shortfin mako shark and blue shark occupy a similar niche, the effects of temperature on blood oxygen affinity in the latter more closely resembled that of the blood in the two coastal shark species examined in this study. The only exception was a small, reverse temperature effect (an increase in blood oxygen affinity with temperature) observed during the warming of the leopard shark blood under simulated arterial conditions, a finding that is likely related to the estuarine ecology of this species. Taken together, we found species-specific differences in how temperature affects blood oxygen affinity in sharks, with some similarities between the regionally endothermic sharks and several regionally endothermic teleost fishes.

Variability in hematology of white-spotted bamboo sharks (Chiloscyllium plagiosum) in different living environments.

Elasmobranch hematology continues to reveal new peculiarities within this specialized field. This report compares total hematologic values from the same white-spotted bamboo sharks (Chiloscyllium plagiosum) housed in different environments. We compared the hemograms one year apart, using a standardized Natt-Herrick's technique. The total white blood cell (WBC) counts of the sharks were statistically different between the two time points (initial median total WBC count = 18,920 leukocytes/µl, SD = 8,108; 1 year later total WBC count = 1,815 leukocytes/µl, SD = 1,309). The packed cell volumes were additionally found to be statistically different (19%, SD = 2.9 vs. 22%, SD = 2.0). Analysis revealed the only differences between the time points were the temperature and stocking densities at which these sharks were housed. This report emphasizes the need for a thorough understanding of the husbandry of an elasmobranch prior to interpretation of a hemogram and suggests that reference intervals should be created for each environment.

The Greenland shark: A new challenge for the oxidative stress theory of ageing?

The free radical theory of ageing predicts that long-lived species should be more resistant to oxidative damage than short-lived species. Although many studies support this theory, recent studies found notable exceptions that challenge the generality of this theory. In this study, we have analysed the oxidative status of the Greenland shark (Somniosus microcephalus), which has recently been found as the longest living vertebrate animal known to science with a lifespan of at least 272years. As compared to other species, the Greenland shark had body mass-corrected values of muscle glutathione peroxidase and red blood cells protein carbonyls (metric of protein oxidative damage) above 75 percentile and below 25 percentile, respectively. None of the biochemical metrics of oxidative status measured in either skeletal muscle or red blood cells were correlated with maximum lifespan of species. We propose that the values of metrics of oxidative status we measured might be linked to ecological features (e.g., adaptation to cold waters and deep dives) of this shark species rather to its lifespan.

Talking to the dead: using Post-mortem data in the assessment of stress in tiger sharks (Galeocerdo cuvier) (Péron and Lesueur, 1822).

Sharks are very sensitive to stress and prone to a high mortality rate after capture. Since approximately 50 million of sharks are caught as bycatch every year, and current recommendations to reduce the impact of commercial fishing strongly support immediate release, it is imperative to better understand post-release mortality caused by the stress of capture and handling. Blood samples allow the assessment of stress levels which are valuable tools to reduce mortality in commercial, recreational and scientific fishing, being essential for the improvement in those conservation measures. Biochemical analyses are widely used for sharks as stress indicators, with secondary plasma parameters (lactate, glucose and ions) being the most often employed assays. However, it is virtually impossible to determine baseline plasma parameters in free-ranging sharks, since blood withdrawal involves animal capture and restrain, which are stressful procedures. This study aims at analyzing secondary parameters of five healthy tiger sharks captured with circular hooks and handlines in Fernando de Noronha (Northeastern Brazil) and comparing them with secondary parameters of three dead tiger sharks caught off Recife (also Northeastern Brazil). The results showed that the analysis of some plasma constituents in dead animals may be an efficient tool to assess stress and lethality. However, traditional parameters such as glucose and calcium, need to be used with caution. The results also demonstrated the extreme importance of urea and phosphorus for assessing stress response and mortality in tiger sharks, both parameters frequently neglected and of utmost importance for shark's homeostasis.

Temperature Insensitivity and Behavioural Reduction of the Physiological Stress Response to Longline Capture by the Gummy Shark, Mustelus antarcticus.

Many factors influence the physiological stress response to fisheries capture in elasmobranchs. However, the influence of sea surface temperatures (SST) and behaviour are unknown and crucial considering global fishing pressures. We investigated the effect of SST and behaviour on the physiological stress response to capture of the gummy shark, Mustelus antarcticus, and compared our results to a laboratory study using similar conditions to test whether stress responses of in situ capture are consistent with those from laboratory simulations. Capture time for 23 M. antarcticus ranged 32-241 min as measured by hook timers or time depth recorders (TDR) in SSTs ranging 12-20°C. TDR data from 13 M. antarcticus were analysed to quantify capture behaviour as the percentage of time spent moving during capture. Several physiological variables measured from blood samples obtained immediately upon the animals' landing indicated that although warmer SSTs increased metabolic rate, the stress response to capture was not exacerbated by capture duration. During capture movement occurred for an average of 10% of the time and since M. antarcticus can respire whilst stationary, restricted movement probably mitigated potential influences of increased SSTs and capture duration on the stress response. Previous laboratory findings were also shown to be indicative of in situ conditions and we thus advise that studies control for water temperature given the influence it has on variables (e.g. lactate) used to measure capture stress in elasmobranchs. We highlight the importance of seasonal water temperatures and capture behaviour when assessing the resilience to fisheries capture and the implementation of appropriate fisheries management strategies.

PLASMA PROTEIN ELECTROPHORESIS AND SELECT ACUTE PHASE PROTEINS IN HEALTHY BONNETHEAD SHARKS (SPHYRNA TIBURO) UNDER MANAGED CARE.

Preventative health care of elasmobranchs is an important but understudied field of aquatic veterinary medicine. Evaluation of inflammation through the acute phase response is a valuable tool in health assessments. To better assess the health of bonnethead sharks ( Sphyrna tiburo ) under managed care, normal reference intervals of protein electrophoresis (EPH) and the acute phase proteins, C-reactive protein (CRP) and haptoglobin (HP), were established. Blood was collected from wild caught, captive raised bonnethead sharks housed at public aquaria. Lithium heparinized plasma was either submitted fresh or stored at -80°C prior to submission. Electrophoresis identified protein fractions with migration characteristics similar to other animals with albumin, α-1 globulin, α-2 globulin, ß globulin, and γ globulin. These fractions were classified as fractions 1-5 as fractional contents are unknown in this species. Commercial reagents for CRP and HP were validated for use in bonnethead sharks. Reference intervals were established using the robust method recommended by the American Society for Veterinary Clinical Pathology for the calculation of 90% reference intervals. Once established, the diagnostic and clinical applicability of these reference intervals was used to assess blood from individuals with known infectious diseases that resulted in systemic inflammation and eventual death. Unhealthy bonnethead sharks had significantly decreased fraction 2, fraction 3, and fraction 3:4 ratio and significantly increased fraction 5, CRP, and HP. These findings advance our understanding of elasmobranch acute phase inflammatory response and health and aid clinicians in the diagnosis of inflammatory disease in bonnethead sharks.