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Metabolic Switching During the Period of Hibernation in Duttaphrynus Melanostictus (Schneider, 1799) from India

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Metabolic Switching During the Period of Hibernation in Duttaphrynus Melanostictus (Schneider, 1799) from India Pratihar_Duttaphrynus_hibernation_physiology_Pratihar_Duttaphrynus_hibernation_physiology.qxd 01.08.2011 11:34 Seite 1 HERPETOZOA 24 (1/2): 43 - 49 43 Wien, 30. Juli 2011 Metabolic switching during the period of hibernation in Duttaphrynus melanostictus (ScHnEidER , 1799) from india (Anura: Bufonidae) Umstellungen im Stoffwechsel bei indischen Schwarznarbenkröten Duttaphrynus melanostictus (ScHnEidER , 1799) während der Winterruhe (Anura: Bufonidae) SUMAn PRATiHAR KURZFASSUnG Hibernation ist ein winterlicher Ruhezustand. Zwei Formen von Umweltstreß - nahrungsmangel und nie - drige Umgebungstemperaturen - charakterisieren die Bedingungen, mit denen Amphibien zurecht kommen müs - sen, um diese Ruhephase zu überstehen. die vorliegende Untersuchung berichtet über die Wirkungen der Winterruhe auf verschiedene biochemische Parameter des Blutplasmas, und zwar die Plasmaproteinfraktionen SGOT und SGPT, die alkalische Phosphatase, das cholesterol und die Lipase bei der indischen Schwarznarbenkröte Dutta phrynus melanostictus (ScHnEidER , 1799). die SGOT- und SGPT-Werte waren bei hibernierenden Kröten signifikant vermindert und die cholesterol- und Lipasewerte des Blutes erhöht. die während der Winterruhe verstärkte metabolische Abhängigkeit von Fetten ist nicht nur eine Wirkung der niedrigen Temperaturen sondern Teil der während des Jahres ablaufenden homöo - statischen Regulationsvorgänge. ABSTRAcT Hibernation is winter dormancy. Two different environmental stresses, a lack of food and low ambient tem - peratures, characterize the conditions amphibians must contend with in order to survive the winter dormancy. The present investigation reports the effect of hibernation on different blood-plasma biochemical parameters, viz. plas - ma protein’s fraction, SGOT, SGPT, alkaline phosphatase, cholesterol, and lipase in the indian common Toad Duttaphrynus melanostictus (ScHnEidER , 1799). Results clearly indicate that the SGOT, SGPT decreased significantly in hibernating toads with a concomi - tant significant increase in blood cholesterol, and lipase. during hibernation, increased metabolic dependency towards lipids is not only a response to low temperature, but part of circanual homeostatic adjustment. Thus, dur - ing hibernation, metabolic switching appears to be essential for survival. KEY WORdS Amphibia: Anura: Bufonidae, Duttaphrynus melanostictus , hibernation, overwintering, physiology, metab - olism, Midnapur, West Bengal, india inTROdUcTiOn To many amphibians and reptiles the sonal extremes and, second, by significantly ability to avoid seasonal and periodic envi - lowering the ectotherm’s energetic costs ronmental rigors by entering a state of meta - during times that would not be favorable for bolic inactivity is a crucial element in their activity (that is, when food is rare or not survival. Specifically, winter dormancy and available). summer estivation — the usual context in Hibernation is a physiological and which these terms are applied to ectotherms behavioral phenomenon of depressed metab - — permit these animals to survive and olism, which enables different animals flourish, first, by reducing the impact of sea - across phyla, particularly anurans, to survive Pratihar_Duttaphrynus_hibernation_physiology_Pratihar_Duttaphrynus_hibernation_physiology.qxd 01.08.2011 11:34 Seite 2 44 S. P RATiHAR during hostile temperature during winter (cHURcHiLL & S TOREY 1993; E d WARdS et al. season. Metabolic rate is strongly reduced 2004; c OSTAnZO & L EE 2005) blood urea and the hypothalamic set point for core body (cOSTAnZO & L EE 2005) and in SdS-PAGE temperature is lowered ( PRATiHAR & K UndU (= sodium dodecyl sulfate polyacrylamide 2010). increased urea concentration in the gel electrophoresis) pattern ( dAS et al. 2004; blood acts as cryoprotectant agent, which BULBUL & K UTRUP 2007). Serum enzyme helps the hibernating animals to sustain dur - levels play a crucial role during hibernation. ing winter ( PRATiHAR & K UndU 2007). Duttaphrynus melanostictus (ScHnEi- increased metabolic dependency towards dER , 1799) is a species of toad that is com - lipids in hibernating condition is not only mon in South Asia. it inhabits southwestern response to low temperatures but part of the and southern china (including Taiwan and circannual homeostatic adjustment that is at Hainan), and occurs throughout southern least partly regulated by thyroid hormones Asia, from northern Pakistan and nepal (PRATiHAR & K UndU 2009). Available through india to Sri Lanka, Andaman reports indicate that environmental cold islands, Sumatra, Java, Borneo and Bali. in stress in hibernating toads causes significant this study, metabolic switching during changes in plasma-protein and glucose hibernation was investigated. MATERiALS And METHOdS Twenty adult common indian Toads sumption, which is directly proportional to Duttaphrynus melanostictus (ScHnEidER , the ALAT activity in the sample, was meas - 1799 ), each weighing 100-120 g, were col - ured photometrically (340 nm). The photo - lected from a selected site in Midnapur metric determination of as partate transami - (Midnapore), West Bengal, india (22°15’n nase (AST = SGOT = serum glutamic oxalo - 87°39’E) throughout the year as hibernating acetic transaminase) was based on the refer - (January-March, n = 5), in stages of arousal ence method of the in ternational Federation from hibernation (April-May, n = 5), repro - of clinical chemistry (Ecoline, Merck™ ductive (June-August, n = 5) and pre-hiber - diagnostic kit). The rate of nAdH consump - nating phase (September-december, n = 5). tion was measured photometrically (340 nm) Blood samples were drawn via cardiac and directly proportional to the AST activity puncture at the time of euthanasia, using a in the sample. sterile hypodermic syringe, and collected in Serum chol esterol was also analyzed tubes. Animals handling was in accordance by using a standard kit (Merck™-diag - with the ethical guideline laid down by the nostica PdLFT0189). cholesterol re acts committee for the purpose of control & with ferric chloride in the presence of acetic supervision of experimental animals (cPc - acid. The optical density (extinction) of the SEA) constituted by the Animal Welfare red color thus produced was measured col - division of the Government of india on the orimetrically at 560 nm. use of animals in scientific research. The Quantichrom™ Alkaline Plasma protein concentration was esti - Phosphate Assay Kit (dALP-250) was used mated photometrically using a standard to measure alkaline phosphate activity curve against a known protein (BSA, Bovine directly in the biological sample without pre - Serum Albumin) following Lowry’s method treatment. This method utilizes p-nitro - for protein quantitation (L OWRY et al. 1951). phenyl phosphate that is hydrolyzed by alka - For the photometric determination of alanine line phosphate, resulting in a yellow colored aminotransferase (ALAT) (= ALT - Alanine product (maximal absorbance at 405 nm). transaminase = SGPT = serum glutamic pyru - The rate of the reaction is directly propor - vic transaminase) based on the reference tional to the enzyme activity. method of the international Federation of Serum lipase was analyzed by using a clinical chemistry, the rate of nAdH (= Merck™ kit. Plasma protein fractions were nicotinamid-Adenin-dinukleo tid) con - analyzed after SdS-PAGE (sodium dodecyl Pratihar_Duttaphrynus_hibernation_physiology_Pratihar_Duttaphrynus_hibernation_physiology.qxd 01.08.2011 11:34 Seite 3 Metabolic switching during hibernation in Duttaphrynus melanostictus 45 g n i t a n r February e b Februar i H november g n i t a n r e June-July b i Juni-Juli H - n o n Late March Ende März Blood cholesterol level (mg/dl) cholesterol-konzentration im Blut (mg/dl) Fig. 1: Bar diagram showing changes in the blood cholesterol level (mg/dl) of Duttaphrynus melanostictus (ScHnEidER , 1799) from india during different annual activity phases, hibernation included. A significant (* P < 0.05) difference was detected in the blood cholesterol levels of hibernating (February, n = 5) versus non-hibernating ( n = 15) individuals. Abb. 1: Balkendiagramm zur darstellung der Veränderungen im Serum-cholesterolspiegel (mg/dl) bei indischen Duttaphrynus melanostictus (ScHnEidER , 1799) während unterschiedlicher Aktivitätsphasen im Jahresverlauf einschließlich der Winterruhe. der cholesterol-Wert im Blut der überwinternden (Februar, n = 5) individuen war gegenüber dem der nicht überwinternden ( n = 15) individuen signifikant (* P < 0.05) verschieden. ) L / U i ( T O G S m u r e S October- Late december January April-May July-August november Ende dezember Jänner April-Mai Juli-August Fig. 2: Variation of serum SGOT concentration (iU/L) throughout the year in Duttaphrynus melanostictus (ScHnEidER , 1799) from india. A significant (* P < 0.05) difference was detected in the serum SGOT levels of hibernating (January, n = 5) versus non-hibernating ( n = 15) individuals . Abb. 2: Veränderungen im Serum-SGOT-Spiegel (iU/L) bei indischen Duttaphrynus melanostictus (ScHnEidER , 1799) im Jahresverlauf. der Serum SGOT-Wert der überwinternden (Jänner, n = 5) individuen war gegenüber dem der nicht überwinternden ( n = 15) individuen signifikant (* P < 0.05) verschieden. Pratihar_Duttaphrynus_hibernation_physiology_Pratihar_Duttaphrynus_hibernation_physiology.qxd 01.08.2011 11:34 Seite 4 46 S. P RATiHAR ) L / U i ( T P G S m u r e S October- Late december January April-May July-August november Ende dezember Jänner April-Mai Juli-August Fig. 3: Variation of
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