Journal of Comparative Physiology B

, Volume 186, Issue 6, pp 787–800 | Cite as

Blood mixtures: impact of puncture site on blood parameters

  • X. Bonnet
  • M. S. El Hassani
  • S. Lecq
  • C. L. Michel
  • E. H. El Mouden
  • B. Michaud
  • T. Slimani
Original Paper


Various puncture routes, veins, arteries, heart, are used to take blood in animals. For anatomical reasons, differences in blood composition are expected among puncture sites. However, this issue has been rarely assessed and contrasted results have been reported: strong effects of puncture site versus a lack of effect. We captured free-ranging freshwater turtles from different locations to compare the mean concentrations of 12 blood parameters (metabolites, hormone, ions, and enzyme) among three puncture sites: (1) a lateral branch of the jugular vein, (2) a dorsal subcarapacial cervical plexus (sometimes incorrectly referred as the ‘cervical sinus’ in the literature), and (3) a caudal plexus site (sometimes incorrectly referred as the ‘caudal sinus’). Because we used very small syringes (27–30G), we were able to separate lymph, blood, or blood–lymph mixtures. Our results show very strong effects of puncture site and of mixture level (mean maximal difference between sites was 250 %). We also found strong sex and geographical effects. Typically, there were differences in concentrations of blood solutes sampled from the lateral jugular vein and subcarapacial plexus, mainly due to sampling a mixture of blood and lymph from the ‘blood’ at the subcarapacial site and pure blood from the lateral jugular site, and likewise, samples from the caudal site were highly variable due to often sampling a mixture of blood and lymph. These results have technical and fundamental implications, especially when performing comparative analyses. Further, by selecting precise puncture sites, physiological differences between lymph and blood compartments could be investigated.


Corticosterone Lymph Plasma metabolites Hemodilution Turtle 



We thank Tony Chevalier, François Brischoux, and Mohamed Radi for their help during field work. Two reviewers provided abundant and constructive comments.

Compliance with ethical standards

Conflict of interest

The authors declare that no competing interest.


This work was supported by the Hassan II Academy of Sciences and Technics (ICGVSA project).

Data availability

The dataset used in this study would be deposited in the dryad platform (


  1. Aasland KE, Skjerve E, Smith AJ (2010) Quality of blood samples from the saphenous vein compared with the tail vein during multiple blood sampling of mice. Lab Anim 44:25–29CrossRefPubMedGoogle Scholar
  2. Aguirre AA, Balazs GH (2000) Blood biochemistry values of green turtles, Chelonia mydas, with and without fibropapillomatosis. Comp Haematol Int 10:132–137CrossRefGoogle Scholar
  3. Arnold JM, Oswald SA, Voigt CC, Palme R, Braasch A, Bauch C, Becker PH (2008) Taking the stress out of blood collection: comparison of field blood-sampling techniques for analysis of baseline corticosterone. J Avian Biol 39:588–592CrossRefGoogle Scholar
  4. Arora KL (2010) Differences in hemoglobin and packed cell volume in blood collected from different sites in Japanese quail (Coturnix japonica). Int J Poult Sci 9:828–830CrossRefGoogle Scholar
  5. Bernardi C, Monetal D, Brughera M, Di Salvo M, Lamparelli D, Mazue G, Iatropoulos MJ (1996) Haematology and clinical chemistry in rats: comparison of different blood collection sites. Comp Haematol Int 6:160–166CrossRefGoogle Scholar
  6. Bonnet X, Naulleau G, Mauget R (1994) The influence of body condition on 17-β Estradiol levels in relation to vitellogenesis in female Vipera aspis (Reptilia viperidae). Gen Comp Endocrinol 93:424–437CrossRefPubMedGoogle Scholar
  7. Bonnet X, Naulleau G, Bradshaw D, Shine R (2001) Changes in plasma progesterone in relation to vitellogenesis and gestation in the viviparous snake, Vipera aspis. Gen Comp Endocrinol 121:84–94CrossRefPubMedGoogle Scholar
  8. Bonnet X, Delmas V, El-Mouden H, Slimani T, Sterijovski B, Kuchling G (2010) Is sexual body shape dimorphism consistent in aquatic and terrestrial chelonians? Zoology 113:213–220CrossRefPubMedGoogle Scholar
  9. Bonnet X, Fizesan A, Michel CL (2013) Shelter availability, stress level, and digestive performance in the aspic viper. J Exp Biol 216:815–822CrossRefPubMedGoogle Scholar
  10. Brüssow KP, Schneider F, Tuchscherer A, Egerszegi I, Ratky J (2008) Comparison of luteinizing hormone, leptin and progesterone levels in the systemic circulation (Vena jugularis) and near the ovarian circulation (Vena cava caudalis) during the oestrous cycle in Mangalica and Landrace gilts. J Reprod Dev 54:431–438CrossRefPubMedGoogle Scholar
  11. Bulté G, Verly C, Blouin-Demers G (2006) An improved blood sampling technique for hatchling emydid turtles. Herpetol Rev 37:318–319Google Scholar
  12. Chan YK, Davis PF, Poppitt SD, Sun X, Greenhill NS, Krishnamurthi R, Przepiorski A, McGi AT, Krissansen GW (2012) Influence of tail versus cardiac sampling on blood glucose and lipid profiles in mice. Lab Anim 46:142–147CrossRefPubMedGoogle Scholar
  13. Christensen SD, Mikkelsen LF, Fels JJ, Bodvarsdottir TB, Hansen AK (2009) Quality of plasma sampled by different methods for multiple blood sampling in mice. Lab Anim 43:65–71CrossRefPubMedGoogle Scholar
  14. Christopher MM, Berry KH, Wallis IR, Nagy KA, Henen BT, Peterson CC (1999) Reference intervals and physiologic alterations in hematologic and biochemical values of free-ranging desert tortoises in the Mojave Desert. J Wildl Dis 35:212–238CrossRefPubMedGoogle Scholar
  15. Clarke IJ, Cummins JT (1982) The temporal relationship between gonadotropin releasing hormone (GnRH) and luteinizing hormone (LH) secretion in ovariectomized ewes. Endocrinology 111:1737–1739CrossRefPubMedGoogle Scholar
  16. Costa DP, Sinervo B (2004) Field physiology: physiological insights from animals in nature. Annu Rev Physiol 66:209–238CrossRefPubMedGoogle Scholar
  17. Crait JR, Prange HD, Marshall NA, Harlow HJ, Cotton CJ, Ben-David M (2012) High-altitude diving in river otters: coping with combined hypoxic stresses. J Exp Biol 215:256–263CrossRefPubMedPubMedCentralGoogle Scholar
  18. Cuadrado M, Molina-Prescott I, Flores L (2003) Comparison between tail and jugular venipuncture techniques for blood sample collection in common chameleons (Chamaeleo chamaeleon). Vet J 166:93–97CrossRefPubMedGoogle Scholar
  19. Dahan R, Sutton GA, Oreff GL, Kelmer G (2015) Agreement among three different equine venipuncture sites with regard to measurement of packed cell volume and total solids. Aust Vet J 93:109–111CrossRefPubMedGoogle Scholar
  20. Drake KK, Nussear KE, Esque TC, Barber AM, Vittum KM, Medica PA, Tracy CR, Hunter KW (2012) Does translocation influence physiological stress in the desert tortoise? Anim Conserv 15:560–570CrossRefGoogle Scholar
  21. Dupoué A, Angelier F, Lourdais O, Bonnet X, Brischoux F (2014) Effect of water deprivation on baseline and stress-induced corticosterone levels in the Children’s python (Antaresia childreni). Comp Biochem Physiol 168:11–16CrossRefGoogle Scholar
  22. Dyer SM, Cervasio EL (2008) An overview of restraint and blood collection techniques in exotic pet practice. Vet Clin N Am Exot Anim Pract 11:423–443CrossRefGoogle Scholar
  23. Evron S, Tress V, Ezri T, Szmuk P, Landau O, Hendel D, Schechter P, Medalion B (2007) The importance of blood sampling site for determination of hemoglobin and biochemistry values in major abdominal and orthopedic surgery. J Clin Anesth 19:92–96CrossRefPubMedGoogle Scholar
  24. Fauvel T, Brischoux F, Briand MJ, Bonnet X (2012) Do researchers impact their study populations? Assessing the effect of field procedures in a long term population monitoring of sea kraits. Amphib Reptil 33:365–372CrossRefGoogle Scholar
  25. Fazio E, Liotta A, Medica P, Giacoppo E, Ferlazzo A (2012) Effects of different health status on blood haematochemical values of loggerhead sea turtles (Caretta caretta). Comp Clin Pathol 21:105–109CrossRefGoogle Scholar
  26. Feder ME (1987) New directions in ecological physiology: conclusion. In: Feder ME, Bennett AF, Burggren WW, Huey RB (eds) New directions in ecological physiology. Cambridge University Press, Cambridge, pp 347–351Google Scholar
  27. Feder ME, Block BA (1991) On the future of animal physiological ecology. Funct Ecol 5:136–144CrossRefGoogle Scholar
  28. Fernández I, Peña A, Del Teso N, Pérez V, Rodríguez-Cuesta J (2010) Clinical biochemistry parameters in C57BL/6J mice after blood collection from the submandibular vein and retroorbital plexus. J Am Assoc Lab Anim Sci 49:202–206PubMedPubMedCentralGoogle Scholar
  29. Fluttert M, Dalm S, Oitzl MS (2000) A refined method for sequential blood sampling by tail incision in rats. Lab Anim 34:372–378CrossRefPubMedGoogle Scholar
  30. Fritz U, Barata M, Busack SD, Fritzsch G, Castilho R (2006) Impact of mountain chains, sea straits and peripheral populations on genetic and taxonomic structure of a freshwater turtle, Mauremys leprosa (Reptilia, Testudines, Geoemydidae). Zool Scr 35:97–108CrossRefGoogle Scholar
  31. Gottdenker NL, Jacobson ER (1995) Effect of venipuncture sites on hematologic and clinical biochemical values in desert tortoises (Gopherus agassizii). Am J Vet Res 56:19PubMedGoogle Scholar
  32. Hasbún CR, Lawrence AJ, Naldo J, Samour JH, Al-Ghais SM (1998) Normal blood chemistry of free-living green sea turtles, Chelonia mydas, from the United Arab Emirates. Comp Haematol Int 8:174–177CrossRefGoogle Scholar
  33. Hem A, Smith AJ, Solberg P (1998) Saphenous vein puncture for blood sampling of the mouse, rat, hamster, gerbil, guinea pig, ferret and mink. Lab Anim 32:364–368CrossRefPubMedGoogle Scholar
  34. Hoggatt J, Hoggatt AF, Tate TA, Fortman J, Pelus LM (2015) Bleeding the laboratory mouse: not all methods are equal. Exp Hematol 44:132–137CrossRefPubMedGoogle Scholar
  35. Hunter BG, Schlipf JW, Cebra C (2013) Comparison of transverse facial venous sinus and jugular blood values in healthy and critically ill horses. Equine Vet J 45:15–19CrossRefGoogle Scholar
  36. Jacobson ER (2000) Collecting biological samples for clinical evaluation. Concepts of reptile disease and surgical techniques. University of Florida. Accessed 1 Feb 2016
  37. Jensen AL, Wenck A, Koch J, Poulsen JD (1994) Comparison of results of haematological and clinical chemical analyses of blood samples obtained from the cephalic and external jugular veins in dogs. Res Vet Sci 56:24–29CrossRefPubMedGoogle Scholar
  38. Johnson AL (1981) Comparison of three serial blood sampling techniques on plasma hormone concentrations in the laying hen. Poult Sci 60:2322–2327CrossRefPubMedGoogle Scholar
  39. Keller C (1997) Ecología de poblaciones de Mauremys leprosa y Emys orbicularis en el Parque Nacional Doñana. PhD Thesis, Universidad de SevillaGoogle Scholar
  40. Kurle CM (2002) Stable-isotope ratios of blood components from captive northern fur seals (Callorhinus ursinus) and their diet: applications for studying the foraging ecology of wild otariids. Can J Zool 80:902–909CrossRefGoogle Scholar
  41. Lagarde F, Bonnet X, Henen B, Nagy K, Corbin J, Lacroix A, Trouvé C (2003) Plasma steroid and nutrient levels during the active season in wild Testudo horsfieldi. Gen Comp Endocrinol 134:139–146CrossRefPubMedGoogle Scholar
  42. Lefevre A, Ballesta S, Pozzobon M, Charieau JL, Duperrier S, Sirigu A, Duhamel JR (2015) Blood microsampling from the ear capillary in non-human primates. Lab Anim 49:349–352CrossRefPubMedGoogle Scholar
  43. Lindman HR (1974) Analysis of variance in complex experimental designs. WH Freeman and Co, New YorkGoogle Scholar
  44. López-Olvera JR, Montané J, Marco I, Martínez-Silvestre A, Soler J, Lavín S (2003) Effect of venipuncture site on hematologic and serum biochemical parameters in marginated tortoise (Testudo marginata). J Wildl Dis 39:830–836CrossRefPubMedGoogle Scholar
  45. Mashhadi VN, Mishmast Z, Mohri M, Seifi HA (2009) Variation of serum calcium, phosphorus and magnesium concentrations due to venipuncture site in Holstein dairy cows. Comp Clin Pathol 18:149–152CrossRefGoogle Scholar
  46. Medeiros NC, Locatelli-Dittrich R, Schmidt EM, Alvares AA, Patrício LL, Lange RR, de Souza RA (2012) Efeito do sítio de venopunção nos parâmetros hematológicos em tigre-d’água-americano, Trachemys scripta elegans. Pesquisa Veterinária Brasileira 32:37–40CrossRefGoogle Scholar
  47. Meir JU, Milsom WK (2013) High thermal sensitivity of blood enhances oxygen delivery in the high-flying bar-headed goose. J Exp Biol 216:2172–2175CrossRefPubMedGoogle Scholar
  48. Mella JR, Chiswick EL, King E, Remick DG (2014) Location, location, location: cytokine concentrations are dependent on blood sampling site. Shock 42:337–342CrossRefPubMedPubMedCentralGoogle Scholar
  49. Michel CL, Bonnet X (2014) Effect of a brief stress on progesterone plasma levels in pregnant and non-pregnant guinea pigs. Anim Biol 64:19–29CrossRefGoogle Scholar
  50. Miller M (2003) Effect of venipuncture site and anticoagulant on selected hematologic values in black rhinoceros (Diceros bicornis). J Zoo Wildl Med 34:59–64CrossRefPubMedGoogle Scholar
  51. Moniello G, Bovera F, Solinas IL, Piccolo G, Pinna W, Nizza A (2005) Effect of age and blood collection site on the metabolic profile of ostriches. South Afr J Anim Sci 35:268–272Google Scholar
  52. Muñoz FJ, Galván A, Lerma M, De la Fuente M (2000) Seasonal changes in peripheral blood leukocyte functions of the turtle Mauremys caspica and their relationship with corticosterone, 17-β-estradiol and testosterone serum levels. Vet Immunol Immunopathol 77:27–42CrossRefPubMedGoogle Scholar
  53. Mylniczenko ND, Curtis EW, Wilborn RE, Young FA (2006) Differences in hematocrit of blood samples obtained from two venipuncture sites in sharks. Am J Vet Res 67:1861–1864CrossRefPubMedGoogle Scholar
  54. Nemzek JA, Bolgos GL, Williams BA, Remick DG (2001) Differences in normal values for murine white blood cell counts and other hematological parameters based on sampling site. Inflamm Res 50:523–527CrossRefPubMedGoogle Scholar
  55. Oliver G, Detmar M (2002) The rediscovery of the lymphatic system: old and new insights into the development and biological function of the lymphatic vasculature. Genes Dev 16:773–783CrossRefPubMedGoogle Scholar
  56. Ottaviani G, Tazzi A (1977) The lymphatic system. In: Gans C, Parsons TS (eds) Biology of the reptilia, vol 6. Academic Press, New York, pp 315–462Google Scholar
  57. Perpiñán D, Armstrong DL, Dórea F (2010) Effect of anticoagulant and venipuncture site on hematology and serum chemistries of the spiny softshell turtle (Apalone spinifera). J Herpetol Med Surg 20:74–78CrossRefGoogle Scholar
  58. Promislow DE (1991) The evolution of mammalian blood parameters: patterns and their interpretation. Physiol Zool 64:393–431CrossRefGoogle Scholar
  59. Reynolds SJ, Christian KA, Tracy CR (2009) Application of a method for obtaining lymph from anuran amphibians. J Herpetol 43:148–153CrossRefGoogle Scholar
  60. Rice WR (1989) Analyzing tables of statistical tests. Evolution 43:223–225CrossRefGoogle Scholar
  61. Rogers IT, Holder DJ, Mcpherson HE, Acker WR, Brown EG, Washington MV, Motzel SL, Klein HJ (1999) Influence of blood collection sites on plasma glucose and insulin concentration in conscious C57BL/6 mice. J Am Assoc Lab Anim Sci 38:25–28Google Scholar
  62. Salemink PJM, Korsten J, De Leeuw P (1994) Prothrombin times and activated partial thromboplastin times in toxicology: a comparison of different blood withdrawal sites for Wistar rats. Comp Haematol Int 4:173–176CrossRefGoogle Scholar
  63. Schwabenbauer C (1991) Influence of the blood sampling site on some haematological and clinical-chemical parameters in sprague-dawley rats. Comp Haematol Int 1:112–116CrossRefGoogle Scholar
  64. Seymour RS (1979) Blood lactate in free-diving sea snakes. Copeia 1979:494–497CrossRefGoogle Scholar
  65. Stewart K, Mitchell MA, Norton T, Krecek RC (2012) Measuring the level of agreement in hematologic and biochemical values between blood sampling sites in leatherback sea turtles (Dermochelys coriacea). J Zoo Wildl Med 43:719–725CrossRefPubMedGoogle Scholar
  66. Stoot LJ, Cairns NA, Cull F, Taylor JJ, Jeffrey JD, Morin F, Mandelman JW, Clark TD, Cooke SJ (2014) Use of portable blood physiology point-of-care devices for basic and applied research on vertebrates: a review. Conserv Physiol. doi: 10.1093/conphys/cou011 PubMedPubMedCentralGoogle Scholar
  67. Tietz NW, Rinker D, Shaw LM (1983) IFCC method for alkaline phosphatase. J Clin Chem Clin Biochem 21:731–748PubMedGoogle Scholar
  68. Tretbar LL (2008) Structure and Function of the Lymphatic System. In: Tretbar LL, Morgan CL, Lee BB, Simonian SJ, Blondeau B (eds) Lymphedema: diagnosis and treatment. Springer, London, pp 1–11CrossRefGoogle Scholar
  69. Van Herck H, Baumans V, Brandt CJWM, Hesp APM, Sturkenboom JH, Van Lith HA, Beynen AC (1998) Orbital sinus blood sampling in rats as performed by different animal technicians: the influence of technique and expertise. Lab Anim 32:377–386CrossRefPubMedGoogle Scholar
  70. Virolainen JV, Love RJ, Tast A, Peltoniemi OA (2005) Plasma progesterone concentration depends on sampling site in pigs. Anim Reprod Sci 86:305–316CrossRefPubMedGoogle Scholar
  71. Voss M, Shutler D, Werner J (2010) A hard look at blood sampling of birds. Auk 127:704–708CrossRefGoogle Scholar
  72. Walker AM (1933) Comparison of the chemical composition of aqueous humor, cerebrospinal fluid, lymph, and blood from frogs, higher animals and man. J Biol Chem 101:269–287Google Scholar
  73. Warren MF (1940) The lymphatic system. Annu Rev Physiol 2:109–124CrossRefGoogle Scholar
  74. Weitten M, Robin JP, Oudart H, Pévet P, Habold C (2013) Hormonal changes and energy substrate availability during the hibernation cycle of Syrian hamsters. Hormon Behav 64:611–617CrossRefGoogle Scholar
  75. Wells RMG, Tetens V, Devries AL (1984) Recovery from stress following capture and anaesthesia of antarctic fish: haematology and blood chemistry. J Fish Biol 25:567–576CrossRefGoogle Scholar
  76. Wess G, Reusch C (2000) Capillary blood sampling from the ear of dogs and cats and use of portable meters to measure glucose concentration. J Small Anim Pract 41:60–66CrossRefPubMedGoogle Scholar
  77. Wiedmeyer CE, Johnson PJ, Cohn LA, Meadows RL (2003) Evaluation of a continuous glucose monitoring system for use in dogs, cats, and horses. J Am Vet Med Assoc 223:987–992CrossRefPubMedGoogle Scholar
  78. Zawieja D (2005) Lymphatic biology and the microcirculation: past, present and future. Microcirculation 12:141–150CrossRefPubMedGoogle Scholar
  79. Zera AJ, Harshman LG (2001) The physiology of life history trade-offs in animals. Annu Rev Ecol Syst 32:95–126CrossRefGoogle Scholar

Copyright information

© Springer-Verlag Berlin Heidelberg 2016

Authors and Affiliations

  • X. Bonnet
    • 1
  • M. S. El Hassani
    • 2
  • S. Lecq
    • 1
    • 3
  • C. L. Michel
    • 1
    • 4
  • E. H. El Mouden
    • 2
  • B. Michaud
    • 1
  • T. Slimani
    • 2
  1. 1.Centre d’Etude Biologique de ChizéUMR 7372 CNRS-ULRVilliers en BoisFrance
  2. 2.Laboratoire Biodiversité et Dynamique des Ecosystèmes, Faculté des Sciences SemlaliaUniversité Cadi AyyadMarrakechMorocco
  3. 3.Saint NazaireFrance
  4. 4.CarnoulesFrance

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