Hematological and Plasma Biochemical Pro�le of Two Species of Freshwater Stingrays From the Amazon

The demand for �sh by-products and the need for nutritionally adequate and less expensive The evaluation of hematological and plasma biochemical parameters and the subsequent establishment of reference intervals can facilitate the diagnosis of the health status of animals and contribute to animal production, management, and conservation of natural populations. This work aimed to determine the hematological parameters of wild specimens of the stingrays Potamotrygon motoro and Potamotrygon orbignyi from the lower Solimões River region, Amazonas, Brazil, establishing reference blood values. One hundred forty-one stingrays were captured, 92 specimens of P. motoro and 49 of P. orbignyi, of both sexes and at different stages of development (neonates and juveniles). Hematological and plasma biochemical parameters were determined according to the methodology recommended in the literature. No effect of sex was observed on the hematological parameters of juvenile animals for both species. P. motoro neonates presented a distinct hematological pro�le, with signi�cantly lower hematocrit values, hemoglobin, number of erythrocytes, mean corpuscular hemoglobin concentration, monocytes, plasma glucose, total proteins, albumin, and globulin. On the other hand, total cholesterol and urea levels were signi�cantly higher in this same group compared to juveniles of the same species. Comparison between species revealed lower values of triglycerides and total cholesterol in P. orbignyi of both sexes. Due to the lack of sex effect, data from males and females of each species were grouped to establish more robust hematological reference intervals. The results obtained are pioneering for these Amazonian species in whitewater environments and will serve as a basis for evaluating the health status of wild stingrays. They can identify hematological disorders and monitor the animals' physiological adaptations to environmental changes.


Introduction
Stingrays of the subfamily Potamotrygoninae represent the only taxon within the Class Chondrichthyes, composed of exclusively freshwater elasmobranchs and restricted to the continental waters of South America (Carvalho 2016;Carvalho et al. 2016).Due to their high biodiversity and complex polychromatism patterns, these organisms have shown high potential for the aquarium hobby, which is highly coveted in countries such as the United States, Japan, Taiwan, and Germany (Lasso et al. 2016).Furthermore, stingrays are also being used as a food source (Duncan et al. 2010), and investigations have been carried out on consumption risk analysis in the assessment of mercury (Oliveira et al. 2023) and the detection of fraud in the marketing of freshwater stingrays, which are traditionally marketed as being of the genus Potamotrygon, but are Paratrygon aiereba (Andrade et al. 2023), which is a critically endangered species (MMA 2022).
Among the species of freshwater stingrays, Potamotrygon motoro (Müller and Henle 1841) and Potamotrygon orbignyi (Castelnau 1855) stand out from their congeners for having a wide distribution, occurring from the Orinoco river basin, in Venezuela, through the Amazon, in Brazil, to the Prata basin, in Argentina/Uruguay (Lasso et al. 2016).The smaller individuals (disc width < 30 cm) of both species have a high ornamental interest and can be exported by Brazil for aquarium purposes (IBAMA 2008).At the same time, the larger animals are used for food consumption ( Despite being an essential component of Amazonian biodiversity and economic relevance for trade, freshwater stingrays are still little known from a biological perspective, with studies that strengthen the understanding of their physiological aspects in their natural habitat being scarce (Oliveira et al. 2015(Oliveira et al. , 2016(Oliveira et al. , 2017)).Factors such as the di culty of capture and the presence of a stinger in the tail, which poses a risk of accidents when handling the animal, contribute to the scarcity of studies on the physiology of these organisms, especially about hematological parameters, which are very low in comparison with marine elasmobranchs and, mainly, to teleosts (Oliveira et  Hematology is a tool traditionally used to monitor the health of an organism individually.However, assessing the health status of sh and elasmobranch populations can also be helpful, as environmental modi cations generally result in detectable blood changes (Oliveira et al. 2016; Santos et al. 2020; Semeniuk et al. 2009).The characterization of the health status is often based on the assessment of blood constituents, using the reference values of the blood count, leukogram and plasma biochemistry.However, there are several species of stingrays for which these reference ranges are scarce or unknown (Pronina et al. 2022).Several factors can interfere with hematological parameters, making establishing the reference blood range for a given species di cult.In addition to endogenous factors, such as sex, size and stage of development, exogenous factors, such as the physicochemical properties of water, habitat and dietary habits, can also in uence the hematological properties of aquatic organisms (Oliveira et al. 2015(Oliveira et al. , 2016(Oliveira et al. , 2017;;Pérez-Rojas et al. 2022).The signi cant intra-and interspeci c variation and the interference of environmental variables on the hematological parameters of stingrays make it impossible to extrapolate the data present in the literature to other species of the Potamotrygoninae subfamily (Oliveira et al. 2015;Brito et al. 2015).
Furthermore, the work present in the literature with freshwater stingrays in natural environments was carried out mainly in black water environments in the Negro River basin, Amazonas (Brito et Wood et al. 2002), and to date, there are no studies on the determination of hematological reference intervals for stingrays from white water environments in the Amazon hydrographic basin.
To increase knowledge about the biology of the Potamotrygoninae subfamily, this work aimed to compare the hematological parameters of wild specimens of P. motoro and P. orbignyi of both sexes and different stages of development, as well as to establish hematological reference ranges for both.Species from white water environments in the lower Solimões River region, Amazonas.

Animal ethics
The stingray species used in this study are not listed in the Red Book of Brazilian Fauna Threatened with Extinction of the Chico Mendes Institute for Biodiversity Conservation (ICMBio), Ministry of the Environment and Climate Change of Brazil.The animals were collected under a license to capture wild animals for activities with scienti c purposes issued by ICMBio (SISBIO/ICMBio Authorization: 41350-6).All procedures were approved by the Ethics Committee on the Use of Animals (CEUA) of the Federal Institute of Education, Science and Technology of Amazonas (2019/010.02.0905) following the guidelines of the National Council for the Control of Animal Experimentation (CONCEA).

Study area and capture of animals
Field collections occurred from May/2022 to February/2023 in white water environments in the lower Solimões River region, around Manaquiri, Manacapuru and Iranduba, in Amazonas, Brazil (Fig. 1).One hundred forty-one stingrays were captured, including 92 specimens of Potamotrygon motoro and 49 specimens of Potamotrygon orbignyi, of both sexes and at different stages of development.Both species were found sharing the same habitat.Juveniles were captured at night using a trawl net, while neonates were captured using a hand net (rapiché).Immediately after capture, the rays were anesthetized with a benzocaine solution (1:20,000) to obtain blood samples.The captured specimens were identi ed according to the recommendations of Lasso et al. (2016) and Rosa and Carvalho (2016).

Blood collection, biometrics and classi cation of animals
Blood handling and collection procedures followed the recommendations of Oliveira et al. (2012), with the blood sample obtained through puncture of the branchial artery using syringes with 10% EDTA anticoagulant.The erythrogram and blood smears were performed in the eld.At the same time, the plasma was stored in liquid nitrogen and, subsequently, in a -80ºC freezer until the biochemical constituents were determined in the laboratory.
After blood collection, biometrics were performed on all animals, with total length (cm), disc width (cm) and weight (g) measured.The developmental stage was determined from disc width (DW) measurements.For P. motoro (newborns, DW ≤ 14.0 cm; juveniles, 14.1 < DW ≤ 35.0 cm; subadults, 35 < DW ≤ 40; and adults, DW > 40.0 cm), the classi cation was determined according to Araújo (1999).For P. orbignyi (newborns, DW ≤ 13.9 cm; juveniles, 14.0 < DW ≤ 26.0 cm; and adults, DW > 26.1 cm), the classi cation followed the recommendations of Lasso et al. (2013).Sexual determination was carried out by external macroscopic observation of the clasper, present in males and absent in females.After recovering from anesthesia, the animals were marked and returned to their respective capture locations.

Hematological analyzes
The hematocrit (Ht, %) was determined by the microhematocrit method, the hemoglobin concentration (Hb, g.dL − 1 ) was determined by the cyanmethemoglobin method and the erythrocyte count (RBC, million.µL− 1 ) was performed in a Neubauer chamber, after diluting the blood in formaldehyde-citrate solution.From the Ht, Hb and RBC values of each individual, hematimetric indices were calculated: mean corpuscular volume (MCV, fL), mean corpuscular hemoglobin (MCH, pg), and mean corpuscular hemoglobin concentration (MCHC, g.dL − 1 ).
Total leukocyte and thrombocyte counts (cells.µL− 1 ), as well as differential leukocyte counts (cells.µL− 1 and %), were performed on blood samples stained with a combination of May Grunwald-Giemsa-Wright following recommendations by Tavares-Dias and Moraes (2006).To identify cell types, the vocabularies used in previous studies with potamotrigonines were assigned (Brito et  Plasma metabolites were determined using speci c commercial kits for each constituent (Labtest Diagnóstica S.A., Brazil).

Water quality monitoring
During the eld collections, the physical and chemical parameters of the water at the stingray capture sites were measured, measuring the levels of dissolved oxygen (mg.L − 1 ), temperature (°C), electrical conductivity (µS.cm − 1 ) and hydrogen potential, with the aid of a multiparameter meter (Thermo Fisher Scienti c Inc., Orion 5-Star Plus, USA).The concentrations of total ammonia (mg.L − 1 ) and nitrite (mg.L − 1 ) were determined by the spectrophotometric method according to Verdouw et al. (1978) and Boyd and Tucker (1992), respectively.Alkalinity (mg.L − 1 ) and total hardness (mg.L − 1 ) were determined by the titrimetric method, according to Boyd and Tucker (1992).

Statistical analyzes
All quantitative results were initially analyzed according to the assumptions of homoscedasticity of variances using the Levene test and normality of residuals using the Shapiro-Wilk test.The Grubbs test was used to identify and remove outliers.
Blood parameters were subjected to descriptive statistics to obtain the mean, standard deviation, median, minimum and maximum.The reference hematological pro le was presented in 25-75 percentiles and a 90% con dence interval (Friedrichs et al. 2012).
For variables with normal and homoscedastic distribution, the Student's t-test was used to compare male and female juveniles of P. orbignyi, and the Analysis of Variance (ANOVA) test was used to compare neonates, male juveniles and female juveniles of P. orbignyi.P. motoro, followed by Tukey's a posteriori test, assuming a 95% con dence interval.The ANOVA test was also used to compare the water parameters of the three locations, the biometric data of the stingrays and to compare the male and female juveniles between the two species analyzed.
For non-parametric variables, the Mann-Whitney U test was used for the comparison between male and female juveniles of P. orbignyi, and the Kruskal-Wallis test for the comparison between neonates, male juveniles and female juveniles of P. motoro, followed by Dunn's a posteriori test.All analyses were performed at 5% signi cance using Statistica software, version 10.0 (StatSoft, Inc., USA).The relationships between hematological variables were evaluated using a Spearman correlation matrix with the aid of OriginPro Software, version 2023b (OriginLab Corporation, USA).

Results
The physical and chemical parameters of the water from the P. motoro and P. orbignyi stingray capture sites are presented in Table 1.No signi cant differences were observed in the water quality parameters between the three collection points (Manaquiri, Manacapuru and Iranduba).The sample number and biometric data of the stingrays studied are presented in Table 2. None of the stingrays captured showed physical evidence of health problems, such as deformities, lacerations, discolorations and mutilation.There were no signi cant biometric differences between juveniles of both sexes and between the species studied, with only the neonates being signi cantly smaller (p < 0.05) in all measured parameters compared to the other groups.The hematological parameters of neonates and male and female juveniles of P. motoro are shown in Table 3.No signi cant differences existed between male and female juveniles of the species for any of the parameters analyzed.However, neonates had signi cantly lower values (p < 0.05) of Ht, Hb, RBC, MCHC and monocytes (µL), as well as plasma glucose, total proteins, albumin and globulin concentrations.On the other hand, plasma levels of total cholesterol and urea were signi cantly higher (p < 0.05) in this same group compared to the others.About P. orbignyi, no effect of sex was observed on the hematological parameters of juveniles of the species (Table 4).Comparison between male and female specimens of the two species of rays revealed signi cant differences (p < 0.05) only in the levels of triglycerides and total cholesterol, which were lower in P. orbignyi of both sexes (Fig. 2).
Table 3 Comparative hematology between male and female neonates and juveniles of Potamotrygon motoro from the lower Solimões River region, Amazonas, Brazil.Different letters on the same line indicate signi cant differences by ANOVA test (p<0.05).* Parameters did not meet the parametric premise and were compared using the Mann-Whitney U test (p < 0.05).

Hematological parameters
The correlation matrices between the hematological parameters of P. motoro and P. orbignyi are shown in Fig. 3. Despite some signi cant relationships (p < 0.05), only the correlations between Ht-Hb, Ht-RBC, Hb-RBC, total leukocytes-lymphocytes (µL), total leukocytes-monocytes (µL), total leukocytes-heterophils (µL), protein-albumin, protein-globulin, albumin-albumin: globulin relation showed a strong positive correlation (P ≥ 0.7) for both species.The alkaline phosphatase-creatine kinase ratio showed a strong positive correlation (P ≥ 0.7) only for P. orbignyi (Fig. 3B).The results of this work culminated in the establishment of hematological reference intervals for P. motoro (Table 5) and P. orbignyi (Table 6), this being the rst hematological characterization study of both species of freshwater stingray in the region of lower Solimões River, Amazonas.

Discussion
Although there has been an intensi cation in the amount of research evaluating the hematological properties of elasmobranchs in recent years, this information is still developing for several species of freshwater stingrays, especially when considering the biodiversity of the Potamotrygoninae subfamily, which has 38 species distributed across four genera: Paratrygon, Heliotrygon, Plesiotrygon and Potamotrygon (Fontenelle et al. 2021), and the diversity of Amazonian river systems, with their distinct limnological characteristics (Sioli et al. 1984).This is the rst study providing reference values for the hematological and plasma biochemical parameters of P. motoro and P. orbignyi from whitewater environments in the Amazon basin.
The rivers of the Amazon are pretty heterogeneous, presenting different colors and chemical characteristics, being classi ed into three categories: black waters (brown color, rich in dissolved organic matter, pH between 3.8 to 4.9 and poor in electrolytes), clear waters (transparent, little suspended material, pH between 4.5 to 7.0 and de cient in electrolytes) and white waters (turbid, rich in suspended material, pH between 6.2 to 7.2, high concentration of nutrients and electrolytes) (Sioli et al. 1984).As a consequence of these differences, species with a wide geographic distribution, such as those in the present research, can present high physiological plasticity (Duncan et al. 2010), resulting in distinct hematological properties due to the adaptive mechanisms induced by the characteristics of the water.In the present study, the physical-chemical properties of the water from the three capture sites presented values within those expected for white waters in the Amazon region (Duncan et al. 2021;Queiroz et al. 2008).In addition, the absence of signi cant differences between the sampling points demonstrates that water was not a factor that triggered different hematological adaptations between the two species of stingrays captured.
The lack of capture of subadult and adult individuals of both species investigated in the present study is related to stingrays' occupation of different habitats throughout their lives and the shing gear used.Smaller organisms are associated with riverbanks and lakes, enabling capture using trawl nets, while larger animals inhabit river channels, being captured using longlines (Lasso et al. 2013;Shibuya 2022).In hematological studies, capture time is an important factor to consider since the time interval between the capture of the organism and the time of blood collection can trigger primary and secondary physiological stress responses, signi cantly altering the blood count and biochemistry.plasma (Brinn et  this research.Furthermore, Brazilian legislation allows, for ornamental and aquarium purposes, the use of specimens of P. motoro and P. orbignyi with a disk width of up to 30 cm, with the exploitation of adult organisms is prohibited (IBAMA 2008).
The presence of neonatal individuals of P. motoro, as well as the predominance of the species in one of the study locations (Lake Andiroba, Manaquiri, Amazonas), suggests that this is a nursery/growth area.Freshwater stingrays preferably use shallow regions of the river with high food availability to give birth, providing more excellent protection against predators and causing a greater density of newborns and smaller juveniles in these regions (Araujo 1999).This reproductive strategy of giving birth in shallow areas is also observed in marine stingrays of the genus Dasyatis, such as D. crysonota, D. americana and D. marianae (Yokota andLessa 2006, 2007).More studies are needed to con rm the use of the location as a birthing/nursery area by P. motoro and, consequently, to classify it as a priority area for the conservation of the species.
In the present study, no differences were observed in hematology and plasma biochemistry between males and females of the two species analyzed, similar to what was observed in the freshwater stingrays P. wallacei, P. motoro and P. aiereba ) demonstrating that sex is not a determining factor in modifying the hematological pro le of stingrays.Although the present study only analyzed sexually immature individuals, the absence of an effect of sex on hematology has also been reported among adult males and females, as well as pregnant females, of three species of the subfamily Potamotrygoninae (Oliveira et al. 2016).
On the other hand, the size/stage of development strongly in uenced the blood of P. motoro, with lower values observed in the erythrogram of neonates, similar to that observed in P. wallacei (Oliveira et al. 2021).In contrast, no erythrocyte differences were found between neonates and juveniles of P. motoro and P. aiereba from the middle Rio Negro basin (Oliveira et al. 2016), which was attributed to the low sample number of P. motoro (n = 3) and the larger size of P. aiereba neonates (19.2 ± cm LD).Younger sh are generally more sedentary than older organisms.However, as the organism grows, metabolic activity increases, resulting in increased capacity for oxygen carriers and, consequently, modi cation of the animal's erythrocyte pro le (Jawad et al. 2004).
The sedentary lifestyle of neonates may also justify the low concentrations of plasma glucose, total proteins, albumin and globulin compared to P. motoro juveniles in this study since the slower the metabolism, the lower the mobilization of energy substrates through activation of catabolic pathways (Wash and Luer 2004).On the other hand, total cholesterol levels were higher in the neonates in the present study, as observed in P. wallacei, which was attributed to the in uence of the calf present at this stage of development (Oliveira et al. 2016).Urea was also higher in neonates in the present study, as well as in neonates of P. motoro from the Rio Negro (Oliveira et al. 2016), demonstrating that it is an ontogenetic characteristic of the species.Considering the atrophy of the rectal gland in potamotrygonines, which resulted in the inability to accumulate urea in the blood (Thorson et al. 1967), the increase in this component may be related to the composition of the diet (Oliveira et al. 2016), as the Consumption of foods rich in protein can increase nitrogen excretion rates and cause a temporary increase in urea levels (Kajimura et al. 2006; Pérez-Rojas et al. 2022).P. motoro neonates in the present study had a lower quantity of monocytes, as observed in P. wallacei (Oliveira et al. 2016).However, this reduction does not denote immunosuppression, as the values were similar to those observed in other species of freshwater stingrays (Oliveira et al. 2015(Oliveira et al. , 2016)).
Considering the standardization in animal capture and the fact that juvenile stingrays did not show biometric differences, we can infer that the lower values of plasma triglyceride and total cholesterol levels observed in P. orbignyi, compared to P. motoro, are not indicators of stress or ontogenetic effect, probably related to the different eating habits of the species.While P. motoro has a diet rich in fat, feeding on shrimp and crabs, P. orbignyi has an insectivorous diet, with a predominance of Gomphidae dragon y larvae (Boyd and Tucker, 1992;Shibuya et al. 2009).
By comparing the results obtained in the present research with the information available in the literature, it was possible to verify that the erythrogram of the stingrays analyzed presented values within the ranges reported for potamotrygonines in previous studies (Brinn et  Although elasmobranchs have fewer erythrocytes, the cell size is two to three times larger than that observed in teleost sh, resulting in low-viscosity blood, typical of sedentary animals (Wilhelm Filho et al. 1992).Contrary results were observed in P. falkineri and P. motoro from the Paraná River-Paraná, Brazil, and in P. orbignyi and P. scobina from the Piririm River-AP, which presented RBC two to three times higher.In contrast, VCM showed half of the values reported for the taxon (Brito et al. 2015), indicating the triggering of secondary physiological stress responses (Semeniuk et al. 2009; Skomal and Mandelman 2012), probably due to using longlines as shing equipment in that study.
The quantities of total leukocytes and thrombocytes found in the present study and their high intraspeci c variation were similar to those reported in the literature for potamotrigonines (Oliveira et al. 2015(Oliveira et al. , 2016(Oliveira et al. , 2017)).The differential leukocyte count showed the predominance of lymphocytes, followed by monocytes and heterophils, indicating the importance of these three leukocyte types for the immune system of the stingrays analyzed, which was corroborated by the enormously positive correlation found between total leukocytes and the three leukocytes above in the present study.The high expressivity of lymphocytes, monocytes and heterophils was also reported in P. wallacei, P. motoro, P. aiereba, P. orbignyi and P. schroederi from the Rio Negro basin (Oliveira et al. 2015(Oliveira et al. , 2016)).Furthermore, cytochemical characterization demonstrated that heterophils are the essential granulocyte leukocytes in the immune defense of Amazonian potamotrigonines ( In elasmobranchs, little research is aimed at establishing hematological reference values (Harms et al. 2002).Considering the di culties of capture and the risks involved when handling elasmobranchs, reference ranges for sharks and rays are often calculated with a relatively low number of individuals (< 30) (Cain et al. 2004).The present study established reference intervals based on a sampling of 86 individuals of P. motoro and 49 of P. orbignyi, generating robust reference data that can be applied to the health assessment, clinical diagnosis and treatment of stingrays freshwater in a natural environment or under human care.
The information generated in this research adds to the limited hematological and plasma biochemical data available in the literature for elasmobranchs.Considering the intra-and interspeci c changes observed in the taxon, future studies should be carried out aiming at the blood characterization of more giant stingrays in the region of the middle Solimões River, Amazonas, as well as carrying out research with different species of potamotrigonines from Amazonian river systems still not explored.
phase, N: Sample number, ND: Not determined.

Table 1
Mean values ± standard deviation of the physical and chemical parameters of the water from the capture sites of the stingrays Potamotrygon motoro and Potamotrygon orbignyi from the lower Solimões River region, Amazonas, Brazil.The ANOVA test detected No signi cant differences between municipalities (p < 0.05).

Table 2
Mean values ± standard deviation of biometric parameters of Potamotrygon motoro and Potamotrygon orbignyi among neonatal and juvenile specimens (males and females) from the lower Solimões River region, Amazonas, Brazil.Different letters in the same column indicate signi cant differences between groups of freshwater stingrays by ANOVA test (p < 0.05).

Table 4
Comparative hematology between male and female juveniles of Potamotrygon orbignyi from the lower Solimões River region, Amazonas, Brazil.No signi cant differences were detected between the sexes by Student's t-test (p < 0.05).

Table 5
Hematological reference intervals for Potamotrygon motoro from the lower Solimões River region, Amazonas, Brazil.Hematological reference intervals for Potamotrygon orbignyi from the lower Solimões River region, Amazonas, Brazil.
(Cusack et al. 2016)oninaet al. 2022erreira et al. 2010o, P. schroederi, P. orbignyi and P. aiereba from the middle Rio Negro region(Oliveira et al. 2015(Oliveira et al. , 2016)).These contrary results present in the literature about granulocyte leukocytes occur due to the lack of standardization in the identi cation and nomenclature of cell types for elasmobranchs, being an obstacle to a better understanding of the immunological particularities of this taxon(Arnold 2005).Unlike what is observed in marine stingrays (Morón-Elorza 2022a), freshwater species present a wide variation in their plasma biochemical pro le, which may re ect the in uence of exogenous factors, such as the diversity of habitats, eating habits and the physical-chemical properties of water(Brito et al. 2015;Oliveira et al. 2017).Cholesterol and triglyceride levels in potamotrigonines are strongly in uenced by diet, which was also observed in the present study, being higher in species with a high-calorie diet(Pérez-Rojas et al. 2022).Low glucose values are commonly observed in healthy rays and sharks (Morón-Elorza et al. 2022a; Morón-Elorza et al. 2022b), with an increase in this component being an indicator of physiological stress (Grant and Campbell 2020; Semeniuk et al. 2009; Skomal and Mandelman 2012).However, it is crucial to consider that the present study, as well as others, carried out with freshwater stingrays (Brinn et al. 2012; Brito et al. 2015; Oliveira et al. 2015, 2016; Pérez-Rojas et al. 2022), did not use sodium uoride to inhibit the glycolytic pathway, which may have contributed to the low glucose values reported for the taxon.Amazonian potamotrigonines showed a conservative pattern for total proteins, albumin, globulin and uric acid (Brito et al. 2015; Oliveira et al. 2015, 2016).The chloride, potassium, magnesium and phosphorus concentrations in the present study were similar to those reported for several species of freshwater stingrays from different Amazonian river systems (Brito et al. 2015; Duncan et al. 2021; Oliveira et al. 2016).On the other hand, plasma urea and sodium levels were higher than those reported for stingrays from the Negro River(Oliveira et al. 2015(Oliveira et al. , 2016)), presenting values similar to those observed in Heliotrygon gomesi, Plesiotrygon iwamae and P. motoro captured in the environment of white water from the Solimões River (Duncan et al. 2021).P. aiereba stingrays from white water environments also showed higher sodium and urea concentrations than black water samples(Duncan et al. 2009).The osmoregulatory patterns of potamotrigonines are linked to environmental variables(Duncan et al. 2021) in a similar way to that observed in freshwater teleost sh(Duncan et al. 2009).Unlike several species of marine elasmobranchs, freshwater stingrays remain hyperosmotic about the environment.In this sense, rivers with high concentrations of electrolytes, such as the white water ones in the present study, induce more signi cant sodium transport against a gradient chemical by the enzyme Na + /K + -ATPase, driving ionic uptake by the gills and the reabsorption of ions in the distal renal tubules, maintaining osmoregulatory balance(Duncan et al. 2021).The assessment of ALT, AST, ALP and CK has little been explored in research with stingrays(Cain et al. 2004;Cusack et al. 2016;Ferreira et al. 2010; Móron-Elorza et al. 2022a), and even less with freshwater stingrays(Brito et al. 2015;Pronina et al. 2022).These enzymes are intracellular.Therefore, the signi cant increase in plasma components may indicate cellular rupture and potential necrosis(Renshaw et al. 2012).In this work, the values of these enzymes were closer to those observed in the stingrays R. undulata (Móron-Elorza et al. 2022a) and R. bonasus(Ferreira et al. 2010) kept in marine aquariums than those reported for the freshwater stingrays P. falkineri, P. motoro, P. scobina and P. orbignyi captured in the natural environment(Brito et al. 2015).Although different factors can cause changes in enzyme concentrations, such as species, diet, growth phase and physiological condition(Otway 2015), the signi cantly high values observed in potamotrigonines(Brito et al. 2015)were probably a consequence of the use of shing used in that work (longline), since the longer the capture procedure, the greater the level of stress and, consequently, the tissue damage generated to the organism(Cusack et al. 2016).The CK values reported in the present study, as well as the high intraspeci c variation, were higher than those observed in sharks(Dove et al. 2010; Harms et al. 2002; Shibuya et al. 2009) but similar to those surveyed in stingrays P. motoro (Pronina et al. 2022)d undulata kept in an aquarium.The high CK values and the enormously positive correlation observed between CK-ALP of P. orbignyi may be related to increased muscle fatigue due to the organism's attempt to escape at the time of capture with trawl nets.Low plasma AST, ALT and ALP values, as observed in the present study, indicate cell membrane strength and stability.
Oliveira et al. 201621).Basophils are described as rare in elasmobranchs, and when present, they are found in low quantities(Brito etal.2015;Cusacket al. 2016; Dove et al. 2010; Grant and Campbell et al. 2020; MMA 2022;Oliveira et al. 2016).In the present study, the presence of eosinophils and the absence of neutrophils in the blood of P. motoro and P. orbignyi were recorded; however, the presence of these cell types in freshwater stingrays is controversial, as both are found in P. motoro, P. orbignyi and P. falkineri (