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Anticoagulants and their effects on the hematological and biochemical parameters of yellow-spotted amazon river turtle

Anticoagulantes e seus efeitos em parâmetros hematológicos e bioquímicos de tracajá

ABSTRACT:

Knowledge of suitable methods and reagents for assessing the health condition of specimens of a given species is essential. The present study evaluated the efficacy of the heparin anticoagulants 5,000 I.U. mL-1, Na2EDTA, and K3EDTA on the blood parameters of yellow-spotted amazon river turtle Podocnemis unifilis, employing different solutions for red blood cells count. The use of the various anticoagulants evaluated after 10 hours of storage efficiently inhibited coagulation in blood samples from P. unifilis. An increased number of erythrocytes was observed with the use of K3EDTA 5% when compared with heparin. Statistically significant changes in the erythrocyte number were observed with the use of the different solutions. Solutions which featured sodium citrate and formaldehyde in their composition, allowed erythrocytes counting up to 120 hours after blood collection, without a change in values. The use of the heparin anticoagulants 5,000 I.U. mL-1, Na2EDTA 3%, Na2EDTA 5%, K3EDTA 3% was recommended in the hematological analysis of P. unifilis. Also recommended was the use of the formaldehyde-citrate solution containing 1.9 g of sodium citrate and 1.0 mL of formaldehyde (in 50 mL of distilled water) to perform red blood cells counts in yellow-spotted amazon river turtle.

Key words:
blood; Chelonia; erythrocytes; Podocnemis unifilis; tracajá

RESUMO:

O conhecimento sobre métodos e reagentes apropriados para as avaliações da condição de saúde de exemplares de determinada espécie é fundamental. Este estudo avaliou a eficácia dos anticoagulantes heparina 5.000 U.I. mL-1, Na2EDTA (3% e 5%), e K3EDTA (3 e 5%) em parâmetros sanguíneos de tracajá (Podocnemis unifilis), bem como diferentes soluções para contagem de eritrócitos totais. A coagulação foi eficientemente inibida nas amostras de sangue de P. unifilis com o uso dos diferentes anticoagulantes avaliados após 10 horas de armazenamento. Maior número de eritrócitos com o uso de K3EDTA 5% foi observado quando comparado com a coleta de sangue realizada com heparina. Diferenças, estatisticamente significativas, entre as contagens de eritrócitos com o uso das diferentes soluções de reagentes avaliadas foram verificadas. As soluções contendo citrato de sódio e formol na composição possibilitaram contagens de eritrócitos até 120 horas após a coleta, sem alteração em seus valores. Recomenda-se o uso dos anticoagulantes heparina 5000 U.I. mL-¹, Na2EDTA 3%, Na2EDTA 5%, K3EDTA 3% nas análises hematológicas de tracajá. Assim como o uso da solução de formol-citrato contendo 1,9 g de citrato de sódio e 1,0 mL de formol (em 50 mL de água destilada) para realização das contagens de eritrócitos totais em tracajá.

Palavras-chave:
sangue; Chelonia; eritrócitos; Podocnemis unifilis; tracajá

INTRODUCTION:

Turtles are reptiles of the Chelonia order, with both sea and freshwater representatives. They are ectothermic, maintaining the balance of their body temperature by exchanging thermal energy with the environment (POUGH et al., 2008POUGH, F. H. et al. A vida dos vertebrados. 4. ed. São Paulo: Editora Atheneu. 2008. 750p.). They are of great medicinal, economic, and subsistence-related importance for the human populations of the Amazon. Among the most notable genus are Podocnemis and Kinosternon (ALHO, 1985ALHO, C. J. R. Conservation and management strategies for commonly exploited Amazonian turtles. Biological Conservation, 1985. v.32, n.4, p.291-298. Available from: <Available from: https://doi.org/10.1016/0006-3207(85)90019-9 >. Accessed: Apr. 20, 2021. doi: 10.1016/0006-3207(85)90019-9.
https://doi.org/10.1016/0006-3207(85)900...
). From Podocnemididae family, Podocnemis is the most representative genus, with six living species: Podocnemis erythrocephala Spix, 1824; Podocnemis expansa Schweigger, 1812; Podocnemis lewyana Duméril, 1852; Podocnemis sextuberculata Cornalia, 1849; Podocnemis unifilis Tröschel, 1848, and Podocnemis vogli Muller, 1935. Of these, yellow-spotted amazon river turtle, P. unifilis, is one of the most frequently caught species for human consumption, according to the International Union for the Conservation of Nature (IUCN). Additionally, the species adapts well to management conditions in cultivation (PEZZUTI et al., 2008PEZZUTI, J. C. et al. Ecologia de Quelônios pelomedusídeos na Reserva Biológica do Abufari. In: ANDRADE, P. C. M. Criação e Manejo de Quelônios no Amazonas . 2. ed. Ibama-ProVárzea, 2008, p.127-173.). Podocnemis unifilis has great economic importance in the Brazilian Amazon, and its breeding is authorized through Normative Instruction No. 07/2015 (IBAMA, 2015IBAMA. Instrução Normativa n. 07de 30 de abril de 2015. 2015 Available from: <Available from: https://www.icmbio.gov.br/cepsul/images/stories/legislacao/Instrucao_normativa/2015/in_ibama_07_2015_institui_categorias_uso_manejo_fauna_silvestre_cativeiro.pdf >. Accessed: Oct. 29, 2020.
https://www.icmbio.gov.br/cepsul/images/...
), as it is widely consumed by the populations of the region, and for these reasons, this is one of animal species vulnerable to extinction, in the red list of threatened species announced by the IUCN.

To establish the hematological reference values of each species, it is essential that the most suitable reagents used in evaluations are known, as changes caused by the use of certain anticoagulants have been reported (HATTINGH, 1975HATTINGH, J. Heparin and ethylenediamine tetra-acetate as anticoagulants for fish blood. Pflügers Archiv European Journal of Physiology, 1975. v.355, n.4, p.347-352. Available from: <Available from: https://doi.org/10.1007/BF00579855 >. Accessed: Jun. 22, 2019. doi: 10.1007/BF00579855.
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; MAINWARING & ROWLEY, 1985MAINWARING, G.; ROWLEY, A. F. The effect of anticoagulants on Blennius pholis L. leucocytes. Comparative Biochemistry and Physiology - Part A: Physiology, 1985. v.80, n.1, p.85-91. Available from: <Available from: https://doi.org/10.1016/0300-9629(85)90683-8 >. Accessed: Jan. 10, 2020. doi: 10.1016/0300-9629(85)90683-8.
https://doi.org/10.1016/0300-9629(85)906...
; WALENCIK & WITESKA, 2007WALENCIK, J.; WITESKA, M. The effects of anticoagulants on hematological indices and blood cell morphology of common carp (Cyprinus carpio L.). Comparative Biochemistry and Physiology - C Toxicology and Pharmacology, 2007. v.146, n.3, p.331-335. Available from: <Available from: https://doi.org/10.1016/j.cbpc.2007.04.004 >. Accessed: Sep. 09, 2019. doi: 10.1016/j.cbpc.2007.04.004.
https://doi.org/10.1016/j.cbpc.2007.04.0...
; ISHIKAWA et al., 2010ISHIKAWA, M. M. et al. Heparin and Na2EDTA as anticoagulants for hybrid surubim catfish (Pseudoplatystoma reticulatum x P. corruscans): efficacy and hematological changes. Ciência Rural, 2010. v.40, n.7, p.1557-1561. Available from: <Available from: https://doi.org/10.1590/S0103-84782010005000113 >. Accessed: Jan. 17. 2021. doi: 10.1590/S0103-84782010005000113.
https://doi.org/10.1590/S0103-8478201000...
), including those used to determine erythrocyte parameters. Among the anticoagulants used in clinical hematology, potassium (K3EDTA) and sodium (Na2EDTA) ethylenediaminetetraacetic acids (EDTA) are more commonly used, in addition to sodium heparin (HARR et al., 2005). Heparin is the most commonly used anticoagulant during blood tests in humans, fish, reptiles, and birds (HATTINGH & SMITH, 1976; WALENCIK & WITESKA, 2007, PERPIÑÁN et al. 2010PERPIÑÁN, D. et al. Effect of anticoagulant and venipuncture site on hematology and serum chemistries of the spiny softshell turtle (Apalone spinifera). Journal of Herpetological Medicine and Surgery , 2010. v.20, n.2, p.74. Available from: <Available from: https://doi.org/10.5818/1529-9651-20.2.74 >. Accessed: Apr. 18, 2019. doi: 10.5818/1529-9651-20.2.74.
https://doi.org/10.5818/1529-9651-20.2.7...
; FAZIO, 2019FAZIO, F. Fish hematology analysis as an important tool of aquaculture: A review. Aquaculture, 2019. v.500, p.237-242. Available from: <Available from: https://doi.org/10.1016/j.aquaculture.2018.10.030 >. Accessed: Feb. 25, 2022. doi: 10.1016/j.aquaculture.2018.10.030.
https://doi.org/10.1016/j.aquaculture.20...
). Its anticoagulant activity is caused by the acceleration of antithrombin III activity, which in turn inhibits the action of thrombin and other proteases responsible for the coagulation cascade (HARR et al., 2005). Heparin has been used as an anticoagulant of choice for turtles, due to the observation of hemolysis caused by EDTA (JACOBSON, 1987JACOBSON, E. R. Reptiles. Veterinary Clinics of North America - Small Animal Practice, 1987. v.17, n.5, p.1203-1225. Available from: <Available from: https://doi.org/10.1016/S0195-5616(87)50111-5 >. Accessed: Jun. 07, 2020. doi: 10.1016/S0195-5616(87)50111-5.
https://doi.org/10.1016/S0195-5616(87)50...
; MURO et al., 1998MURO, J. et al. Effects of lithium heparin and tripotassium EDTA on hematologic values of Hermann’ s tortoises (Testudo hermanni). Journal of Zoo and Wildlife Medicine , 1998. v.29, n.1, p.40-44. Available from: <Available from: http://www.jstor.org/stable/20095714 >. Accessed: Feb. 25, 2022.
http://www.jstor.org/stable/20095714...
).

The anticoagulant effect of EDTA is due to its action in the chelation of factor IV (Ca2+) in the coagulation cascade, acting as a mediator, as well as in the cell-to-cell relationship during coagulation reactions (HARR et al., 2005; TAVARES-DIAS & OLIVEIRA, 2009TAVARES-DIAS, M. et al. Comparative hematological and biochemical analysis of giant turtles from the amazon farmed in poor and normal nutritional conditions. Veterinarski Arhiv , 2009. v.79, n.6, p.601-610. Available from: <Available from: https://hrcak.srce.hr/53089 >. Accessed: Jan. 29, 2019.
https://hrcak.srce.hr/53089...
). However, EDTA is recommended as an anticoagulant, preferably for the total and differential counting of chelonian leukocytes, due to the preservation properties of leukocyte blood cells (NCCLS, 1990; OVIEDO & RODRÍGUEZ, 2019; BURTIS & BURNS, 2016BURTIS, C. A.; BRUNS, D. E. Tietz - Fundamentos de Química Clínica e Diagnóstico Molecular. Rio de Janeiro: Elsevier. 2016. 1106p.). Nevertheless, these anticoagulants have not been evaluated for blood collection specifically in P. unifilis. The present study compared the most suitable methods and reagents for assessing the qualitative and quantitative characteristics of the blood cells of yellow-spotted amazon river turtle, using sodium heparin, sodium ethylenediaminetetraacetic acid (Na2EDTA 3 and 5%) and potassium ethylenediaminetetraacetic acid (K3EDTA 3 and 5%) as anticoagulants, in addition to different solutions for total erythrocyte counting.

MATERIALS AND METHODS:

Anticoagulant evaluation based on hematological analysis

Podocnemis unifilis (n=10), belonging to the vivarium of Embrapa Amapá, captured with the aid of a dip net and transported to the Laboratory of Nutrition of Aquatic Organisms, Embrapa Amapá, Macapá, AP, were individually weighed (2.1 ± 0.3 kg), measured (carapace length, 25.2 ± 1.2 cm) and identified. The animals were manually restrained with the aid of a damp cloth and the use of rubber gloves for safe handling, and blood samples were collected by puncturing the caudal vessel.

Disposable syringes (capacity of 3.0 mL) and hypodermic needles (25 x 7 mm), without the use of anticoagulants, were used to collect blood samples for the evaluation of anticoagulants. The blood sample ( 2.5 mL) was quickly distributed into five polyethylene microtubes (capacity of 1.5 mL), 500 µL in each, to which 12.5 μL of anticoagulant was added as follows: in microtube 1, sodium heparin 5,000 I.U. mL-1; in microtube 2, Na2EDTA 3%; in microtube 3, Na2EDTA 5%; in microtube 4, K3 EDTA 3%; and, in microtube 5, K3EDTA 5%; all homogenized by inversion. After aliquoting the blood in polyethylene tubes, 10 μL of each aliquot was placed in polyethylene microtubes (capacity of 1.5 mL), kept under refrigeration for a period of 10 hours, and visually (with naked eyes) evaluated every 30 minutes for the occurrence of coagulation and/or hemolysis (HATTINGH & SMITH, 1976HATTINGH, J.; SMITH, M. Anticoagulants for avian and reptilian blood: heparin and EDTA. Pflfigers Archiv European Journal of Physiology, 1976. v.269, p.267-269. Available from: <Available from: https://doi.org/10.1007/BF00594613 >. Accessed: Sep. 26, 2021. doi: 10.1007/BF00594613.
https://doi.org/10.1007/BF00594613...
).

Right after blood samples were mixed with anticoagulant, hematocrit (Ht) was determined using the microhematocrit technique (GOLDENFARB et al., 1971GOLDENFARB, P. B. et al. Reproducibility in the hematology laboratory: the microhematocrit determination. American Journal of Clinical Pathology, 1971. v.56, n.1, p.35-39. Available from: <Available from: https://doi.org/10.1093/ajcp/56.1.35 >. Accessed: Jan. 18, 2021. doi: 10.1093/ajcp/56.1.35.
https://doi.org/10.1093/ajcp/56.1.35...
), with a reading of the red cell percentage on standardized cards. The hemoglobin concentration (Hb) was determined by the cyanmethemoglobin method (COLLIER, 1944COLLIER, H. B. Standardization of blood haemoglobin determinations. Canadian Medical Association Journal, 1944. v.50, n.6, p.550-2. Available from: <Available from: https://www.ncbi.nlm.nih.gov/pmc/articles/PMC1581573/ >. Accessed: Aug. 12, 2020.
https://www.ncbi.nlm.nih.gov/pmc/article...
), with the absorbance reading taken in a spectrophotometer (Biospectro, SP-220, Curitiba, PR, Brazil) at 540 nm. The red blood cells count (RBC) was performed using formaldehyde-citrate solution and a Neubauer chamber (RANZANI-PAIVA et al. 2013RANZANI-PAIVA, M. J. T. et al. Métodos para análise hematológica em peixes. Maringá: EDUEM. 2013. 166p.), under a light microscope (Boeco, model BOE-01, Germany). These data (Ht, Hb and RBC) were used to determine the hematimetric indices (WINTROBE, 1934WINTROBE, M. Variations in the size and hemoglobin content of erythrocytes in the blood of various vertebrates. Folia Haematol, 1934. v. 51, n. 32, p. 32-49. Available from: <Available from: https://www.scirp.org/(S(351jmbntvnsjt1aadkposzje))/reference/ReferencesPapers.aspx?ReferenceID=1246452 >. Accessed: Nov. 09, 2019.
https://www.scirp.org/(S(351jmbntvnsjt1a...
): mean corpuscular volume (MCV) and mean corpuscular hemoglobin concentration (MCHC). The remaining blood was centrifuged at 75 G (Centrifuge, model MCD-2000, MRC Lab, United Kingdom), during 10 min to obtain plasma. The plasma samples were kept at -18ºC until the analysis to determine glucose, total protein and albumin levels using specific colorimetric kits (Ebram®, São Paulo, SP, Brazil), for each metabolite, with absorbance readings from a spectrophotometer (Biospectro, SP-220, Curitiba, PR, Brazil).

Blood evaluation based on different solutions used to total erythrocyte count

The following solutions were evaluated for the total erythrocyte count (red blood cells count, RBC), for which a total volume of 50 mL of each solution was prepared, made up with distilled water: Solution A: Sodium chloride solution 0.9 %; Solution B: 1.45 g sodium citrate and 1.5 ml formaldehyde; Solution C: 1.9 g sodium citrate, 1.0 ml formalin and 0.01 g toluidine blue; Solution D: 1.9 g sodium citrate and 1.0 mL formaldehyde; Solution E: 1.9 g sodium citrate and 0.01 g toluidine blue; Solution F: 1.0 mL formaldehyde and 0.01 g toluidine blue. Blood samples in a sodium chloride solution (Solution A) were kept in the refrigerator, as this solution does not include any preservative-containing reagents.

Another P. unifilis (n=6) were captured with a dip net and transported to the Laboratory of Nutrition of Aquatic Organisms, Embrapa Amapá (Macapá, AP) and individually weighed (2.2 ± 0.1 kg) and measured (carapace length, 25.6 ± 1.0 cm). Blood samples were taken from those animals manually restrained by puncturing the caudal vessel. Disposable syringes (capacity of 3.0 mL) and hypodermic needles (25 x 7 mm) with sodium heparin as anticoagulant, were used to collect blood samples for the evaluation of solutions for erythrocyte counting. and a Neubauer chamber (RANZANI-PAIVA et al. 2013RANZANI-PAIVA, M. J. T. et al. Métodos para análise hematológica em peixes. Maringá: EDUEM. 2013. 166p.), under a light microscope (Boeco, model BOE-01, Germany).

The red blood cells counts (RBC) were performed in each of six different solutions (A to F) in a Neubauer chamber (RANZANI-PAIVA et al. 2013RANZANI-PAIVA, M. J. T. et al. Métodos para análise hematológica em peixes. Maringá: EDUEM. 2013. 166p.) under a light microscope (Boeco, model BOE-01, Germany), at the moment of blood samples collection (0h) until 120h, every 24h, as indicated: the first, at the time of blood collection (0h); the second, 24 hours (1 day) after blood collection; the third, 48 hours (2 days) after collection; the fourth, 72 hours (3 days) after collection; the fifth, 96 hours (4 days) after collection and the sixth, 120 hours (5 days) after collection.

Data analysis

After tests of normality (Shapiro-Wilk) and homogeneity (Levene), the data were subjected to one-way analysis (ANOVA) of variance with the use of parametric and nonparametric multiple comparison tests (ZAR, 2010ZAR, J. H. Biostatistical Analysis. 5. ed. Prentice Hall, 2010. 944p.). The analyses were performed using the GraphPad Instat® Statistical Program, version 3.01 (GraphPad Software, San Diego, CA, USA).

RESULTS AND DISCUSSION:

Different anticoagulants effects on P. unifilis blood sample

This is the first study about the effects of heparin sodium and EDTA (sodium and potassium) as anticoagulants on the hematological parameters of P. unifilis. The use of sodium heparin in the hematological evaluation of turtles and other reptiles has been widely described in several studies (AGUIRRE et al., 1995AGUIRRE, A. A. et al. Adrenal and hematological responses to stress in juvenile green turtles (Chelonia mydas) with and without fibropapillomas. Physiological Zoology, 1995. v.68, n.5, p.831-854. Available from: <Available from: https://doi.org/10.1086/physzool.68.5.30163934 >. Accessed: Feb. 25, 2022. doi: 10.1086/physzool.68.5.30163934.
https://doi.org/10.1086/physzool.68.5.30...
; MAFUVADZE & ERLWANGER, 2007MAFUVADZE, B.; ERLWANGER, K. H. The effect of EDTA, heparin and storage on the erythrocyte osmotic fragility, plasma osmolality and haematocrit of adult ostriches (Struthio camelus). Veterinarski Arhiv, 2007. v.77, n.5, p.427-434. Available from: <Available from: https://hrcak.srce.hr/24763 >. Accessed: Mar. 22, 2020.
https://hrcak.srce.hr/24763...
; SANTOS et al., 2009SANTOS, M. R. D. et al. Valores hematológicos de tartarugas marinhas Chelonia mydas (Linaeus, 1758) juvenis selvagens do Arquipélago de Fernando de Noronha, Pernambuco, Brasil. Brazilian Journal of Veterinary Research and Animal Science, 2009. v.46, n.6, p.491. Available from: <Available from: https://www.revistas.usp.br/bjvras/article/download/26800/28583/31071 >. Accessed: Aug. 11, 2020.
https://www.revistas.usp.br/bjvras/artic...
; SABINO et al., 2010SABINO, A. J. et al. Effect of ethylenediaminetetraacetic acid (EDTA) and heparin on erythrocytes of ostriches (Struthio camelus L.). Revista Ceres, 2010. v.57, n.3, p.338-342. Available from: <Available from: https://doi.org/10.1590/S0034-737X2010000300008 >. Accessed: Oct. 22, 2019. doi: 10.1590/S0034-737X2010000300008.
https://doi.org/10.1590/S0034-737X201000...
, ISHIKAWA et al., 2010ISHIKAWA, M. M. et al. Heparin and Na2EDTA as anticoagulants for hybrid surubim catfish (Pseudoplatystoma reticulatum x P. corruscans): efficacy and hematological changes. Ciência Rural, 2010. v.40, n.7, p.1557-1561. Available from: <Available from: https://doi.org/10.1590/S0103-84782010005000113 >. Accessed: Jan. 17. 2021. doi: 10.1590/S0103-84782010005000113.
https://doi.org/10.1590/S0103-8478201000...
, COSTA et al., 2020COSTA, M. B. F. et al. Plasma proteins and leukocyte kinetics of turtles (Podocnemis unifilis (Tröschel, 1848)) inoculated with inactivated Escherichia coli. Comparative Clinical Pathology, 2020. v.29, n.1, p.305-310. Available from: <Available from: https://doi.org/10.1007/s00580-019-03059-x >. Accessed: Jan. 29, 2021. doi: 10.1007/s00580-019-03059-x.
https://doi.org/10.1007/s00580-019-03059...
), and has been found to be an effective anticoagulant. Blood clotting inhibition was efficient in all the tests with the anticoagulants evaluated. The hematological and biochemical values of apparently healthy P. unifilis using different anticoagulants are shown in table 1. However, in the samples collected with heparin, the mean RBC was lower (P < 0.05) than with the use of K3EDTA 5%, showing that hemolysis or blood clot could be happened with that anticoagulant. A significantly lower MCV was observed with the use of the Na2EDTA 3% anticoagulant than with the use of sodium heparin, Na2EDTA 5% and K3EDTA 5%. Differences were not observed (P > 0.05) in glucose, total protein, and albumin plasma levels, or in Ht, Hb, and MCHC, with the use of the different anticoagulants.

Table 1
Hematological variables and plasma concentrations (glucose, total proteins and albumin) of Podocnemis unifilis by the use of different anticoagulants: sodium heparin (5000 U.I. mL-1), Na2EDTA (3 and 5%) and K3EDTA (3 and 5%).

Some studies have reported that heparin, despite being considered a natural anticoagulant, can interfere with the staining of leukocyte blood cells, in addition to being expensive in comparison with other anticoagulants (GILOR & GILOR, 2011GILOR, S.; GILOR, C. Common laboratory artifacts caused by inappropriate sample collection and transport: How to get the most out of a sample. Topics in Companion Animal Medicine, 2011. v.26, n.2, p.109-118. Available from: <Available from: https://doi.org/10.1053/j.tcam.2011.02.003 >. Accessed: Jan. 27, 2021. doi: 10.1053/j.tcam.2011.02.003.
https://doi.org/10.1053/j.tcam.2011.02.0...
). Whereas EDTA is a more widely used anticoagulant than heparin, as it reduces the agglutination processes, preserving blood cell morphology and staining (GILOR & GILOR, 2011). Nevertheless, several studies have reported that EDTA, when used as an anticoagulant in chelonian, can cause hemolysis (MURO et al., 1998MURO, J. et al. Effects of lithium heparin and tripotassium EDTA on hematologic values of Hermann’ s tortoises (Testudo hermanni). Journal of Zoo and Wildlife Medicine , 1998. v.29, n.1, p.40-44. Available from: <Available from: http://www.jstor.org/stable/20095714 >. Accessed: Feb. 25, 2022.
http://www.jstor.org/stable/20095714...
, PERPIÑÁN et al., 2008PERPIÑÁN, D. et al. Hematology of the pascagoula map turtle (Graptemys gibbonsi) and the southeast asian box turtle (Cuora amboinensis). Journal of Zoo and Wildlife Medicine , 2008. v.39, n.3, p.460-463. Available from: <Available from: https://doi.org/10.1638/2007-0044.1 >. Accessed: Jun. 27, 2020. doi: 10.1638/2007-0044.1.
https://doi.org/10.1638/2007-0044.1...
; OLIVEIRA-JUNIOR et al. 2009OLIVEIRA-JÚNIOR, A. A. et al. Biochemical and hematological reference ranges for Amazon freshwater turtle, Podocnemis expansa (Reptilia: Pelomedusidae), with morphologic assessment of blood cells. Research in Veterinary Science, 2009. v.86, n.1, p.146-151. Available from: <Available from: https://doi.org/10.1016/j.rvsc.2008.05.015 >. Accessed: Jun. 13, 2019. doi: 10.1016/j.rvsc.2008.05.015.
https://doi.org/10.1016/j.rvsc.2008.05.0...
; TAVARES-DIAS et al., 2009TAVARES-DIAS, M.; OLIVEIRA, S. R. A review of the blood coagulation system of fish. Revista Brasileira de Biociências, 2009. v.4849, n.2, p.205-224. Available from: <Available from: http://www.ufrgs.br/seerbio/ojs/index.php/rbb/article/view/1144 >. Accessed: Sep. 18, 2020.
http://www.ufrgs.br/seerbio/ojs/index.ph...
; PERPIÑÁN et al., 2010).

There was a trend towards higher hematocrit values with the use of EDTA in relation to heparin, indicating a desired cell preservation. It was reported that the use of different anticoagulants did not alter most blood parameters. The hematocrit values of P. unifilis in the present study using sodium and potassium EDTA were similar to those reported by BOGAN et al. (2020BOGAN, J. E. et al. Comparison of the effects of three anticoagulants on hematological analytes in the Eastern Indigo snake (Drymarchon couperi). Journal of Herpetological Medicine and Surgery, 2020. v.30, n.2, p.96. Available from: <Available from: https://doi.org/10.5818/17-07-118.2 >. Accessed: Jan. 09, 2021. doi: 10.5818/17-07-118.2.
https://doi.org/10.5818/17-07-118.2...
) for the eastern indigo snake Drymarchon couperi, using K3EDTA. However, it was lower than that reported by ANDRADE (2008ANDRADE, P. C. M. Criação e Manejo de Quelônios no Amazonas. Manaus: Pró-Várzea, 2008.) for P. unifilis in captivity, and by TAVARES-DIAS et al. (2012TAVARES-DIAS, M. et al. Propriedades do sangue de três espécies de quelônios do gênero Podocnemis de vida livre da Reserva Biológica do Abufari, baixo rio Purus, estado do Amazonas, Brasil. Patologia e sanidade de organismos aquáticos. 1. ed. Maringá: Massoni, 2012, p.195-220.) for P. unifilis from the Abufari biological reserve (Amazon State, Brazil). In addition, both studies used heparin as an anticoagulant.

The hemoglobin concentration of P. unifilis did not differ with the use of different anticoagulants in the present study, corroborating previous studies with animals of the same species kept in captivity and under adequate cultivation conditions (TAVARES-DIAS et al, 2009TAVARES-DIAS, M.; OLIVEIRA, S. R. A review of the blood coagulation system of fish. Revista Brasileira de Biociências, 2009. v.4849, n.2, p.205-224. Available from: <Available from: http://www.ufrgs.br/seerbio/ojs/index.php/rbb/article/view/1144 >. Accessed: Sep. 18, 2020.
http://www.ufrgs.br/seerbio/ojs/index.ph...
). However, the number of total erythrocytes was lower with the use of heparin than with the use of K3EDTA 5%. In addition, MURO et al. (1998MURO, J. et al. Effects of lithium heparin and tripotassium EDTA on hematologic values of Hermann’ s tortoises (Testudo hermanni). Journal of Zoo and Wildlife Medicine , 1998. v.29, n.1, p.40-44. Available from: <Available from: http://www.jstor.org/stable/20095714 >. Accessed: Feb. 25, 2022.
http://www.jstor.org/stable/20095714...
) observed a reduction in the number of erythrocytes in Testudo hermanni, also with the use of K3EDTA 5%, in comparison with the use of lithium heparin. In contrast, such differences were not observed in studies with macaws and pythons (HARR et al., 2005). ISHIKAWA et al. (2010ISHIKAWA, M. M. et al. Heparin and Na2EDTA as anticoagulants for hybrid surubim catfish (Pseudoplatystoma reticulatum x P. corruscans): efficacy and hematological changes. Ciência Rural, 2010. v.40, n.7, p.1557-1561. Available from: <Available from: https://doi.org/10.1590/S0103-84782010005000113 >. Accessed: Jan. 17. 2021. doi: 10.1590/S0103-84782010005000113.
https://doi.org/10.1590/S0103-8478201000...
) showed the efficiency of the anticoagulant Na2EDTA 3% in hybrid catfish, known as surubim (P. reticulatum x P. corruscans). Thus, such results demonstrated the importance of knowledge about the most suitable anticoagulant for use in each species, due to their peculiarities and specificities.

The hematological and biochemical parameters of P. unifilis revealed few variations, with mainly minimal differences observed in the blood count. The samples collected with K3EDTA 5% exhibited higher number of RBC, while the MCV differed between the samples with the anticoagulants evaluated. In P. unifilis, MCV was lower with the use of Na2EDTA 3% than with the use of heparin, Na2EDTA 5% and K3EDTA 3%. However, the MCV values of this study, with these different anticoagulants, were lower than those reported for wild yellow spotted amazon river turtle obtained with heparin (OLIVEIRA-JÚNIOR et al., 2009OLIVEIRA-JÚNIOR, A. A. et al. Biochemical and hematological reference ranges for Amazon freshwater turtle, Podocnemis expansa (Reptilia: Pelomedusidae), with morphologic assessment of blood cells. Research in Veterinary Science, 2009. v.86, n.1, p.146-151. Available from: <Available from: https://doi.org/10.1016/j.rvsc.2008.05.015 >. Accessed: Jun. 13, 2019. doi: 10.1016/j.rvsc.2008.05.015.
https://doi.org/10.1016/j.rvsc.2008.05.0...
; TAVARES-DIAS et al., 2012TAVARES-DIAS, M. et al. Propriedades do sangue de três espécies de quelônios do gênero Podocnemis de vida livre da Reserva Biológica do Abufari, baixo rio Purus, estado do Amazonas, Brasil. Patologia e sanidade de organismos aquáticos. 1. ed. Maringá: Massoni, 2012, p.195-220.). This reduction in MCV may be due to osmotic crenation in erythrocytes when blood is collected with high concentrations (7.2 mg mL-1 e 14.4 mg mL-1) of EDTA (OLIVEIRA et al., 2010OLIVEIRA, A. C. De et al. Anticoagulant concentration, time and storage temperature on hematological parameters in automated blood count. Ciência Rural , 2010. v.40, n.12, p.2521-2526. Available from: <Available from: https://doi.org/10.1590/S0103-84782010001200014 >. Accessed: May, 27, 2019. doi: 10.1590/S0103-84782010001200014.
https://doi.org/10.1590/S0103-8478201000...
), even without Ht alterations observed.

Total plasma protein concentrations were not influenced using different anticoagulants in P. unifilis. Similarly, the levels of total plasma proteins of hybrid surubim were not influenced by heparin or Na2EDTA, even in different concentrations of 3%, 5% and 10% (ISHIKAWA et al., 2010ISHIKAWA, M. M. et al. Heparin and Na2EDTA as anticoagulants for hybrid surubim catfish (Pseudoplatystoma reticulatum x P. corruscans): efficacy and hematological changes. Ciência Rural, 2010. v.40, n.7, p.1557-1561. Available from: <Available from: https://doi.org/10.1590/S0103-84782010005000113 >. Accessed: Jan. 17. 2021. doi: 10.1590/S0103-84782010005000113.
https://doi.org/10.1590/S0103-8478201000...
). However, OLIVEIRA et al. (2015OLIVEIRA, A. T. et al. Comparison of the effects of anticoagulants used in blood collection to determine blood parameters of free-living stingrays from the Potamotrygon genus (Elasmobranchii: Potamotrygonidae). Biota Amazônia, 2015. v.5, n.3, p.55-58. Available from: <Available from: http://dx.doi.org/10.18561/2179-5746/biotaamazonia.v5n3p55-58 >. Accessed: Dec. 11, 2020. doi: 10.18561/2179-5746/biotaamazonia.v5n3p55-58.
http://dx.doi.org/10.18561/2179-5746/bio...
) reported that EDTA 5% and 10% were not efficient, and caused a reduction in total plasma proteins, as the coagulation process includes protein retention, reducing its amount in plasma. The use of EDTA, in the concentrations assessed in the present study, allowed hematological and biochemical values to be obtained, corroborating the results of studies carried out in yellow-blotched map turtles (Graptemys flavimaculata) (MARTINEZ-JIMENEZ et al., 2007MARTINEZ-JIMENEZ, D. et al. Comparison of the effect of dipotassium ethylenediaminetetraacetic acid and lithium heparin on hematologic values in the green iguana (Iguana iguana). Journal of Zoo and Wildlife Medicine , 2007. v.35, n.3, p.328-332. Available from: <Available from: https://doi.org/10.1638/03-057 >. Accessed: Jan. 28, 2019. doi: 10.1638/03-057.
https://doi.org/10.1638/03-057...
). Other studies have reinforced the applicability of the use of EDTA for several species, in particular reptiles (HATTINGH, 1975HATTINGH, J. Heparin and ethylenediamine tetra-acetate as anticoagulants for fish blood. Pflügers Archiv European Journal of Physiology, 1975. v.355, n.4, p.347-352. Available from: <Available from: https://doi.org/10.1007/BF00579855 >. Accessed: Jun. 22, 2019. doi: 10.1007/BF00579855.
https://doi.org/10.1007/BF00579855...
; MARKS & CITINO, 1990MARKS, S. K.; CITINO, S. B. Hematology and serum chemistry of the radiated tortoise (Testudo radiata). Journal of Zoo and Wildlife Medicine, 1990. v.21, n.3, p.342-344. Available from: <Available from: https://www.jstor.org/stable/20095073 >. Accessed: Apr. 18, 2019.
https://www.jstor.org/stable/20095073...
; SALAKIJ et al., 2002SALAKIJ, C. et al. Hematology, morphology, cytochemical staining, and ultrastructural characteristics of blood cells in king cobras (Ophiophagus hannah). Veterinary Clinical Pathology, 2002. v.31, n.3, p.116-126. Available from: <Available from: https://doi.org/10.1590/S0034-737X2010000300008 >. Accessed: Sep. 04, 2019. doi: 10.1590/S0034-737X2010000300008.
https://doi.org/10.1590/S0034-737X201000...
; BOGAN et al., 2020BOGAN, J. E. et al. Comparison of the effects of three anticoagulants on hematological analytes in the Eastern Indigo snake (Drymarchon couperi). Journal of Herpetological Medicine and Surgery, 2020. v.30, n.2, p.96. Available from: <Available from: https://doi.org/10.5818/17-07-118.2 >. Accessed: Jan. 09, 2021. doi: 10.5818/17-07-118.2.
https://doi.org/10.5818/17-07-118.2...
).

Different solutions effects on P. unifilis total erythrocyte count

Hemolysis was detected with the use of the reagent containing only 0.9% sodium chloride solution, 72 hours after collection. Solutions B and D, both composed of sodium citrate and formaldehyde, but in different concentrations, demonstrated less variation in the erythrocyte count, which could be performed up to 120 hours after the collection of the blood samples. Solution C, with sodium citrate and toluidine blue dye, revealed a lower number of erythrocytes, demonstrating problems with carrying out the count, and allowing cell visualization up to 72 hours after the collection of blood samples. Differences in the total erythrocyte counts of solution E, with sodium citrate and toluidine blue dye, and solution F, with formaldehyde and the addition of toluidine blue dye, were observed at the time of collection, meaning that the blood cell preservation potential of the use of these solutions was not observed (Table 2). All the reagent solutions evaluated exhibited statistically significant differences in the total erythrocyte count.

Table 2
Different solutions evaluation to red blood cells counts (x 103 μL-1) in Podocnemis unifilis blood samples, performed at the time of blood collection and after 24, 48, 72, 96 and 120 hours.

The use of solution A (sodium chloride), with samples preserved under refrigeration, revealed that the P. unifilis erythrocytes can be counted up to a maximum of 48 hours without significant changes in numbers, and after this period there is a considerable reduction, with secure determination not guaranteed. RADISIC et al. (2020RADISIC, R. et al. Red blood cell osmotic fragility in healthy loggerhead and green sea turtles. Journal of Veterinary Diagnostic Investigation, 2020. v.32, n.6, p.908-911. Available from: <Available from: https://doi.org/10.1177/1040638720957117 >. Accessed: Feb. 13, 2021. doi: 10.1177/1040638720957117.
https://doi.org/10.1177/1040638720957117...
) observed greater osmotic fragility in sodium chloride concentrates of under 0.38%, causing hemolysis in 50% of samples from sea turtles, Caretta caretta e Chelonia mydas. The use of the crystal violet method for counting erythrocytes with dilution in 0.45% sodium chloride solution enabled the erythrocyte nuclei of slider turtles Trachemys scripta to be stained, allowing the differentiation and proper distinguishing of erythrocytes from other cells, such as lymphocytes, thrombocytes, and granulocytes (TSAI et al., 2014TSAI, C. Y. et al. An alternative staining method for counting red-eared slider turtle (Trachemys scripta) blood cells using crystal violet in cells diluted with 0.45% sodium chloride. Journal of Veterinary Diagnostic Investigation , 2014. v.26, n.5, p.610-615. Available from: <Available from: https://doi.org/10.1177/1040638714542868 >. Accessed: Jan. 29, 2019. doi: 10.1177/1040638714542868.
https://doi.org/10.1177/1040638714542868...
).

Solution C revealed problems in the counts performed after 96 hours of collection, although with less damage than was observed with the use of solutions E and F. Thus, solutions B and D proved more suitable for counting the erythrocytes of P. unifilis. It is notable that both solutions B and D were prepared without the use of toluidine blue dye. With solutions E and F it was only possible to visualize the erythrocytes at the time of blood collection, and they could not be seen 24 hours after blood collection due to the large number of lysed cells. Of these two solutions, solution F exhibited a very low erythrocyte count number, the lowest among all the solutions evaluated. The possible cause of the hemolysis that occurred in a short period of time with the use of solution F is presumed to be due to the absence of sodium citrate in the composition, even considering the fact that citrate is widely used in blood clotting time tests. When comparing the counts using evaluated solutions, it is possible to observe that the counts performed with solution D had a lower coefficient of variation and can be recommended as the first choice in hematological studies of this species of chelonian, P. unifilis. This solution was prepared only with sodium citrate and formaldehyde, without toluidine blue dye.

The total erythrocyte count in P. unifilis, using methodologies applied to fish (TAVARES-DIAS et al., 2002TAVARES-DIAS, M. et al. Haematological characteristics of Brazilian teleosts. VI. Parameters of jundiá Rhamdia quelen (Pimelodidae). Ciência Rural , 2002. v.32, n.4, p.693-698. Available from: <Available from: https://doi.org/10.1590/S0103-84782002000400024 >. Accessed: Jun. 21, 2020. doi: 10.1590/S0103-84782002000400024.
https://doi.org/10.1590/S0103-8478200200...
; WALENCIK & WITESKA, 2007WALENCIK, J.; WITESKA, M. The effects of anticoagulants on hematological indices and blood cell morphology of common carp (Cyprinus carpio L.). Comparative Biochemistry and Physiology - C Toxicology and Pharmacology, 2007. v.146, n.3, p.331-335. Available from: <Available from: https://doi.org/10.1016/j.cbpc.2007.04.004 >. Accessed: Sep. 09, 2019. doi: 10.1016/j.cbpc.2007.04.004.
https://doi.org/10.1016/j.cbpc.2007.04.0...
; RANZANI-PAIVA et al., 2013RANZANI-PAIVA, M. J. T. et al. Métodos para análise hematológica em peixes. Maringá: EDUEM. 2013. 166p.), makes the determination of this parameter for this species of chelonian imprecise. However, such evaluations are necessary to investigate whether the use of the formaldehyde-citrate reagent with toluidine blue dye could be the cause of the considerable reduction in these cells number during counting. We sought to evaluate the disassociation of some reagents from the complete solution, as a means of identifying which component was causing damage to the cells and making them difficult to determine. The lack of information on this theme makes the present study necessary, as it elucidated which methodology and reactive solutions are suitable for the yellow-spotted amazon river turtle.

CONCLUSION:

Blood samples from P. unifilis using EDTA (Na2EDTA 3%, Na2EDTA 5%, K3EDTA 3%, K3EDTA 5%) and sodium heparin (5000 U.I. mL-¹) prevented clotting within 10 hours of storage. However, a higher number of erythrocytes was observed with the use of K3EDTA 5%. Therefore, we recommend that the heparin anticoagulants 5,000 I.U. mL-¹, Na2EDTA 3%, Na2EDTA 5%, K3EDTA 3% are used in these hematological analyzes. We also recommend the use of a formaldehyde-citrate solution composed of 1.9 g of sodium citrate and 1.0 mL of formaldehyde in 50 mL of distilled water, when performing total erythrocyte counts in P. unifilis.

ACKNOWLEDGEMENTS

The authors thank to Empresa Brasileira de Pesquisa Agropecuária (Embrapa Amapá) and the Amapá State Research Support Foundation (FAPEAP). We thank to Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES) for the Y.I.C. Furtado scholarship (grant #88887.497689/2020-00).

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  • CR-2021-0489.R2

BIOETHICS AND BIOSSECURITY COMMITTEE APPROVAL

  • BIOETHICS AND BIOSSECURITY COMMITTEE APPROVAL

    This experiment is authorized by Ethics Committee for Animal Use (CEUA) of Embrapa Amapá, under procedural nr 010/2017-CEUA/CPAFAP.

Edited by

Editors: Rudi Weiblen (0000-0002-1737-9817)
Felisbina Luisa Pereira Guedes Queiroga (0000-0001-6130-8381)

Publication Dates

  • Publication in this collection
    23 May 2022
  • Date of issue
    2022

History

  • Received
    24 June 2021
  • Accepted
    29 Dec 2021
  • Reviewed
    22 Mar 2022
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