Journal of Oceanology and Limnology

, Volume 37, Issue 2, pp 767–776 | Cite as

Physiological responses in vitamin C system during hibernation in juvenile Chinese soft-shelled turtle Pelodiscus sinensis

  • Bojian Chen
  • Cuijuan NiuEmail author
  • Lin Yuan
  • Wenyi Zhang
Aquaculture and Fisheries


Vitamin C (Vc) is an important antioxidant that helps turtles tolerating stressful environment. This work quantified changes in tissue Vc levels during winter hibernation in the soft-shelled turtle Pelodiscus sinensis, to reveal the stress response pattern of tissue Vc during hibernation and contribute basic data for turtle culture. We sampled juvenile soft-shelled turtles at pre-hibernation (17.0°C mud temperature; MT), during hibernation (5.8°C MT) and after arousal (20.1°C MT) in the field. The transcript levels of the gene encoding L-gulonolactone oxidase (GLO), the key enzyme for Vc synthesis, decreased significantly during hibernation. However, GLO activity did not match the GLO transcription patterns and remained stable during hibernation, and showed temperature-dependent kinetic characteristics. Vitamin C levels in the brain, liver, kidney, and spleen (but not muscle) all decreased significantly during hibernation, but recovered to pre-hibernation levels or even higher levels after arousal. The soft-shelled turtle endured 5 months of hibernation with no significant oxidative damage in most tissues, except in the spleen. Splenic Vc was nearly exhausted during hibernation, accompanied by a significantly elevated malonaldehyde (MDA) level. Although the high level of oxidative damage quickly decreased after arousal, the potential tissue damage in the spleen during hibernation might account for the weakened immune capacity of turtles after hibernation.


Chinese soft-shelled turtle hibernation reactive oxygen species (ROS) L-gulonolactone oxidase oxidative stress 


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This study was approved by the Ethic and Animal Welfare Committee (EAWC) of Beijing Normal University. We are very grateful to Mr. CAO Zhendong, Dr. LIU Kai and Dr. ZHANG Zuobing for their help (technical assistance) in this study.


  1. Baker P J, Costanzo J P, Lee Jr R E. 2007. Oxidative stress and antioxidant capacity of a terrestrially hibernating hatchling turtle. J. Comp. Physiol. B, 177 (8): 875–883, https://doi. org/10.1007/s00360–007–0185–0.CrossRefGoogle Scholar
  2. Bickler P E, Buck L T. 2007. Hypoxia tolerance in reptiles, amphibians, and fishes: life with variable oxygen availability. Ann. Rev. Physiol., 69 (1): 145–170, Scholar
  3. Bradford M M. 1976. A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein–dye binding. Anal. Biochem., 72 (1–2): 248–254,–2697(76)90527–3.CrossRefGoogle Scholar
  4. Buckley R. 2006. Choosing and using statistics: a biologist's guide, 2nd edition. Austral Ecol., 31 (3): 425, https://doi. org/10.1111/j.1442–9993.2006.01623.x.CrossRefGoogle Scholar
  5. Chen B J, Niu C J, Yuan L. 2015. Ascorbic acid regulation in stress responses during acute cold exposure and following recovery in juvenile Chinese soft–shelled turtle ( Pelodiscus sinensis ). Comp. Biochem. Phys. A, 184: 20–26, Scholar
  6. Ching B Y, Ong J L, Chng Y R, Chen X L, Wong W P, Chew S F, Ip Y K. 2014. l–gulono–γ–lactone oxidase expression and vitamin C synthesis in the brain and kidney of the African lungfish, Protopterus annectens. FASEB J., 28 (8): 3 506–3 517,–249508.CrossRefGoogle Scholar
  7. Ching S, Mahan D C, Dabrowski K. 2001. Liver L–gulonolactone oxidase activity and tissue ascorbic acid concentrations in nursing pigs and the effect of various weaning ages. J. Nutr., 131 (7): 2 002–2 006, https://doi. org/10.1093/jn/131.7.2002.CrossRefGoogle Scholar
  8. Dabrowski K. 1990. Gulonolactone oxidase is missing in teleost fish. The direct spectrophotometric assay. Biol. Chem. Hoppe–Seyler., 371 (1): 207–214, https://doi. org/10.1515/bchm3.1990.371.1.207.Google Scholar
  9. Davies K J. 2000. Oxidative stress, antioxidant defenses, and damage removal, repair, and replacement systems. IUBMB life, 50 (4): 279–289, Scholar
  10. Drew K L, Tøien Ø, Rivera P M, Smith M A, Perry G, Rice M E. 2002. Role of the antioxidant ascorbate in hibernation and warming from hibernation. Comp. Biochem. Phys. C, 133 (4): 483–492,–0456(02) 00118–7.Google Scholar
  11. Galli G L J, Richards J G. 2012. The effect of temperature on mitochondrial respiration in permeabilized cardiac fibres from the freshwater turtle, Trachemys scripta. J. Therm. Biol., 37 (3): 195–200, 2011.12.012.CrossRefGoogle Scholar
  12. Garbarino V R, Orr M E, Rodriguez K A, Buffenstein R. 2015. Mechanisms of oxidative stress resistance in the brain: Lessons learned from hypoxia tolerant extremophilic vertebrates. Arch. Biochem. Biophys., 576: 8–16, Scholar
  13. Hermes–Lima M, Storey J M, Storey K B. 2001. Antioxidant defenses and animal adaptation to oxygen availability during environmental stress. Cell Mol. Response Stress, 2: 263–287,–1254(01)80022–X.CrossRefGoogle Scholar
  14. Hermes–Lima M, Zenteno–Savín T. 2002. Animal response to drastic changes in oxygen availability and physiological oxidative stress. Comp. Biochem. Phys. C, 133 (4): 537–556,–0456(02)00080–7.CrossRefGoogle Scholar
  15. Hochachka P W, Buck L T, Doll C J, Land S C. 1996. Unifying theory of hypoxia tolerance: molecular/metabolic defense and rescue mechanisms for surviving oxygen lack. Proc. Natl. Acad. Sci. USA, 93 (18): 9 493–9 498, Scholar
  16. Jackson D C, Ultsch G R. 2010. Physiology of hibernation under the ice by turtles and frogs. J. Exp. Zool. A, 313 (6): 311–327, Scholar
  17. Jackson D C. 2000. Living without oxygen: lessons from the freshwater turtle. Comp. Biochem. Phys. A, 125 (3): 299–315,–6433(00)00160–4.CrossRefGoogle Scholar
  18. Jing R Z, Niu C J, Qian M Y, Huang C X, Zhang Z B, Qian Y Q. 2011. Effect of dietary Vitamin C and hibernation on energy metabolism in juvenile soft–shelled turtle ( Pelodiscus sinensis ). J. B eijing Normal Univ. ( Nat. Sci.), 47 (2): 185–191. (in Chinese with English abstract)Google Scholar
  19. Knight J A. 2000. Review: free radicals, antioxidants, and the immune system. Ann. Clin. Lab. Sci., 30 (2): 145–158.Google Scholar
  20. Krivoruchko A, Storey K B. 2010. Activation of antioxidant defenses in response to freezing in freeze–tolerant painted turtle hatchlings. B iochim. B iophys. A cta, 1800 (7): 662–668, Scholar
  21. Leceta J, Zapata A. 1985. Seasonal changes in the thymus and spleen of the turtle, Mauremys caspica. A morphometrical, light microscopical study. Dev. Comp. Immunol., 9 (4): 653–668,–305X(85)90030–8.CrossRefGoogle Scholar
  22. Liu Q, Yang H, Wang S, Ma J, You J. 2004. The prevention and treatment of overwintering death in Chinese soft–shelled turtle Pelodiscus sinensis. Fishery Guide, (22): 53. (in Chinese)Google Scholar
  23. Liu X N, Tan B P. 1997. Preliminary study on prevention methods in overwintering death disease of Chinese softshelled turtle. Reservoir Fisheries, (1): 26–27, 49. (in Chinese)Google Scholar
  24. López–Torres M, Pérez–Campo R, Cadenas S, Rojas C, Barja G. 1993. A comparative study of free radicals in vertebrates—II. Non–enzymatic antioxidants and oxidative stress. Comp. Biochem. Phys. B, 105 (3–4): 757–763,–0491(93)90117–N.Google Scholar
  25. Ma Y L, Rice M E, Chao M L, Rivera P M, Zhao H W, Ross A P, Zhu X W, Smith M A, Drew K L. 2004. Ascorbate distribution during hibernation is independent of ascorbate redox state. Free Radical Bio. Med., 37 (4): 511–520, Scholar
  26. Mæland A, Waagbø R. 1998. Examination of the qualitative ability of some cold water marine teleosts to synthesise ascorbic acid. Comp. Biochem. Phys. A, 121 (3): 249–255,–6433(98)10125–3.CrossRefGoogle Scholar
  27. Munang’andu H M, Fredriksen B N, Mutoloki S, Dalmo R A, Evensen Ø. 2013. Antigen dose and humoral immune response correspond with protection for inactivated infectious pancreatic necrosis virus vaccines in Atlantic salmon ( Salmo salar L). Vet. Res., 44: 7,–9716–44–7.CrossRefGoogle Scholar
  28. Munoz F J, de La Fuente M, Beaupre S J. 2004. Seasonal changes in lymphoid distribution of the turtle Mauremys caspica. Copeia, 2004 (1): 178–183, 1643/CP–02–058R2.CrossRefGoogle Scholar
  29. Nandi A, Mukhopadhyay C K, Ghosh M K, Chattopadhyay D J, Chatterjee I B. 1997. Evolutionary significance of vitamin C biosynthesis in terrestrial vertebrates. Free Radical Bio. Med., 22 (6): 1 047–1 054, 1016/S0891–5849(96)00491–1.CrossRefGoogle Scholar
  30. Pérez–Pinzón M A, Rice M E. 1995. Seasonal–and temperaturedependent variation in CNS ascorbate and glutathione levels in anoxia–tolerant turtles. Brain Res., 705 (1–2): 45–52,–8993(95)01136–6.CrossRefGoogle Scholar
  31. Qian M Y, Niu C J, Jing R Z, Qian Y Q, Zhang Z B. 2008. Effect of dietary Vc and hibernation on biosynthesis of Vc and liver Vc concentration in juvenile soft–shelled turtles Pelodiscus sinensis. Acta Zool. Sin., 54 (2): 309–316. (in Chinese with English abstract)Google Scholar
  32. Reese S A, Crocker C E, Carwile M E, Jackson D C, Ultsch G R. 2001. The physiology of hibernation in common map turtles ( Graptemys geographica ). Comp. Biochem. Phys. A, 130 (2): 331–340,–6433(01)00398–1.CrossRefGoogle Scholar
  33. Reese S A, Ultsch G R, Jackson D C. 2004. Lactate accumulation, glycogen depletion, and shell composition of hatchling turtles during simulated aquatic hibernation. J. Exp. Biol., 207 (16): 2 889–2 895, 1242/jeb.01124.CrossRefGoogle Scholar
  34. Rice M E, Forman R E, Chen B T, Avshalumov M, Cragg S J, Drew K L. 2002. Brain antioxidant regulation in mammals and anoxia–tolerant reptiles: balanced for neuroprotection and neuromodulation. Comp. Biochem. Phys. C, 133 (4): 515–525,–0456(02)00116–3.Google Scholar
  35. Rice M E, Lee E J, Choy Y. 1995. High levels of ascorbic acid, not glutathione, in the CNS of anoxia–tolerant reptiles contrasted with levels in anoxia–intolerant species. J. Neurochem., 64 (4): 1 790–1 799,–4159.1995.64041790.x.CrossRefGoogle Scholar
  36. Rice M E. 1999. Ascorbate compartmentalization in the CNS. Neurotox. Res., 1 (2): 81–90, Scholar
  37. Rice M E. 2000. Ascorbate regulation and its neuroprotective role in the brain. Trends Neurosci., 23 (5): 209–216,–2236(99)01543–X.CrossRefGoogle Scholar
  38. Schmittgen T D, Livak K J. 2008. Analyzing real–time PCR data by the comparative CT method. Nat. Protoc., 3 (6): 1 101–1 108, Scholar
  39. Shao Q. 2012. Soft–shelled turtles. 460. In: Lucas J S, Southgate P C eds. Aquaculture (Second edition): Farming Aquatic Animals and Plants. Blackwell Publishing Ltd., West Sussex, UK.Google Scholar
  40. Sizer I W. 2006. Effects of temperature on enzyme kinetics. In: Nord F F, Werkman C H eds. Advances in Enzymology and Related Areas of Molecular Biology. Interscience Publishers, Inc., New York. p.35–62.Google Scholar
  41. Skjærven K H, Penglase S, Olsvik P A, Hamre K. 2013. Redox regulation in Atlantic cod ( Gadus morhua ) embryos developing under normal and heat–stressed conditions. Free Radical Bio l. Med., 57: 29–38, 1016/j.freeradbiomed.2012.11.022.CrossRefGoogle Scholar
  42. Storey K B, Storey J M. 2004. Metabolic rate depression in animals: transcriptional and translational controls. Biol. Rev. Camb. Philos. Soc., 79 (1): 207–233, https://doi. org/10.1017/S1464793103006195.CrossRefGoogle Scholar
  43. Storey K B. 2004. Strategies for exploration of freeze responsive gene expression: advances in vertebrate freeze tolerance. Cryobiology, 48 (2): 134–145, https://doi. org/10.1016/j.cryobiol.2003.10.008.CrossRefGoogle Scholar
  44. Storey K B. 2006. Reptile freeze tolerance: metabolism and gene expression. Cryobiology, 52 (1): 1–16, https://doi. org/10.1016/j.cryobiol.2005.09.005.CrossRefGoogle Scholar
  45. Storey K B. 2007. Anoxia tolerance in turtles: metabolic regulation and gene expression. Comp. Biochem. Phys. A, 147 (2): 263–276, 019.CrossRefGoogle Scholar
  46. Ultsch G R. 1989. Ecology and physiology of hibernation and overwintering among freshwater fishes, turtles, and snakes. Biol. Rev., 64 (4): 435–515,–185X.1989.tb00683.x.CrossRefGoogle Scholar
  47. Venditti P, Di Stefano L, Di Meo S. 2010. Oxidative stress in cold–induced hyperthyroid state. J. Exp. Biol., 213 (17): 2 899–2 911, Scholar
  48. Wang X Z, Wang M B, Sun X Z, Xin X Y. 2008. The investigation on overwintering death in Chinese softshelled turtles and preventive measures. Shandong Fisheries, (11): 40. (in Chinese)Google Scholar
  49. Zhang J, Wang F, Jiang Y L, Hou G J, Cheng Y S, Chen H L, Li X. 2017a. Modern greenhouse culture of juvenile softshelled turtle, Pelodiscus sinensis. Aquacult. Int., 25 (4): 1 607–1 624,–017–0137–y.CrossRefGoogle Scholar
  50. Zhang W Y, Niu C J, Chen B J, Yuan L. 2016. Antioxidant responses in hibernating Chinese soft–shelled turtle Pelodiscus sinensis hatchlings. Comp. Biochem. Phys. A, 204: 9–16, Scholar
  51. Zhang W Y, Niu C J, Liu Y K, Chen B J. 2017b. Glutathione redox balance in hibernating Chinese soft–shelled turtle Pelodiscus sinensis hatchlings. Comp. Biochem. Phys. B, 207: 9–14, 02.003.CrossRefGoogle Scholar
  52. Zimmerman L M, Paitz R T, Vogel L A, Bowden R M. 2010b. Variation in the seasonal patterns of innate and adaptive immunity in the red–eared slider ( Trachemys scripta ). J. Exp. Biol., 213 (9): 1 477–1 483, Scholar
  53. Zimmerman L M, Vogel L A, Bowden R M. 2010a. Understanding the vertebrate immune system: insights from the reptilian perspective. J. Exp. Biol., 213 (5): 661–671, Scholar

Copyright information

© Chinese Society for Oceanology and Limnology, Science Press and Springer-Verlag GmbH Germany, part of Springer Nature 2019

Authors and Affiliations

  • Bojian Chen
    • 1
    • 2
  • Cuijuan Niu
    • 2
    Email author
  • Lin Yuan
    • 2
  • Wenyi Zhang
    • 2
  1. 1.College of Animal Science and TechnologyNorthwest Agriculture and Forestry UniversityYanglingChina
  2. 2.Ministry of Education Key Laboratory for Biodiversity Science and Ecological Engineering, College of Life SciencesBeijing Normal UniversityBeijingChina

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