Liudmila A. Kovalchuk, Dr. Sc., Chief Researcher of the Laboratory of Evolutionary Ecology, Institute of Ecology of Plant and Animal, Ural Branch of the RAS (620144, Russia, Yekaterinburg, 8 Marta 202);; e-mail:
Liudmila V. Chernaya
, PhD, Senior Researcher of the Laboratory of Evolutionary Ecology, Institute of Ecology of Plant and Animal, Ural Branch of the RAS (620144, Russia, Yekaterinburg, 8 Marta 202); iD ORCID:; e-mail:
Vladimir A. Mishchenko, Research Engineer of the Laboratory of Evolutionary Ecology, Institute of Ecology of Plant and Animal, Ural Branch of the RAS (620144, Russia, Yekaterinburg, 8 Marta 202); iD ORCID:
Dmitry L. Berzin, PhD, Researcher of the Laboratory of Functional Ecology of Terrestrial Animals, Institute of Ecology of Plant and Animal, Ural Branch of the RAS (620144, Russia, Yekaterinburg, 8 Marta 202); iD ORCID:
Nikolay V. Mikshevich
, PhD, Docent of the Department of Life Safety of the Ural State Pedagogical University (620017, Russia, Yekaterinburg, Pr. Kosmonavtov, 26); iD ORCID:; e-mail:

Reference to article

Kovalchuk L.A., Chernaya L.V., Mishchenko V.A., Berzin D.L., Mikshevich N.V. 2023. Estimation of hematological and biochemical parameters of a representative of the amphibious fauna of the Urals: Salamandrella keyserlingii (Caudata, Amphibia). Nature Conservation Research 8(1): 34–48.

Section Research articles

The paper has presented for the first time the results of a study of hematological and biochemical parameters of a threatened species of the fauna of the Middle Urals, Salamandrella keyserlingii (Caudata, Amphibia). Mature males of S. keyserlingii (n = 30; sampled in spring and summer) living in the Protected Area «Kalinovsky Forest Park» (Sverdlovsk Region, Russia), located in the urban agglomeration of Yekaterinburg city, were used as research objects. The leukocyte composition of S. keyserlingii blood is represented by granulocytes (neutrophils: early, bands, segments; basophils, eosinophils) and agranulocytes (monocytes and lymphocytes). The peripheral blood of individuals was characterised by the predominance of agranulocytes. They provide immune surveillance and selective reactivity of the organism (adaptive immunity) both in spring (55.7%) and summer (60.3%) seasons (p < 0.001). The content of granulocytes, providing non-specific urgent protection of the organism from infections and toxic effects, is ranged as 39.3–44.4% in males. In the spring and summer periods, the leukocyte formula of the peripheral blood of S. keyserlingii has a high lymphoid profile (52.4–56.7%). Seasonal variability of the integral leukocyte index (ISL) of S. keyserlingii was 0.80 in spring and 0.65 in summer. The content of free amino acids in the blood plasma of S. keyserlingii has been assessed for the first time from the standpoint of their participation in the processes of immunomodulation and the formation of adaptive reactions that ensures the survival and eurybiont of the species in a wide temperature range. The amino acid fund of S. keyserlingii blood plasma was represented by 17 amino acids: alanine, serine, threonine, lysine, leucine, valine, glycine, aspartic acid, glutamic acid, arginine, histidine, methionine, proline, phenylalanine, cystine, tyrosine, and isoleucine. The analysis of the metabolic groups of amino acids in the blood plasma of S. keyserlingii shows a high percentage of glycogenic amino acid (GGAA: 82.1%) and essential amino acids (EAA: 53.4%). The high content of amino acids in the blood indicates their relevance in the processes of protein synthesis, active participation in both immune processes and in the processes of detoxification and the formation of adaptive reactions during the summer period of amphibian's growth and development. The obtained results of studies of hematological and biochemical parameters of S. keyserlingii's blood significantly expand the understanding of the adaptation mechanisms of amphibians to conditions of natural and anthropogenic habitat transformations. This is undoubtedly of theoretical interest and applies significance for development of environmental measures in the monitoring system of natural and artificial aquatic ecosystems.


amino acids, caudate amphibians, lymphocytes, peripheral blood, Siberian salamander

Artice information

Received: 26.05.2022. Revised: 30.08.2022. Accepted: 02.09.2022.

The full text of the article

Alfimov A.V., Berman D.I. 2010. Reproduction of the Siberian Salamander, Salamandrella keyserlingii (Amphibia, Caudata, Hynobiidae), in Water Bodies on Permafrost in Northeastern Asia. Biology Bulletin 37(8): 807–822. DOI: 10.1134/S1062359010080054
Alford R.A., Bradfield K.S., Richards S.J. 2007. Global warming and amphibian losses. Nature 447(7144): E3–E4. doi: 10.1038/nature05940
Ananjeva N.B., Uteshev V.K., Orlov N.L. Gakhova E.N. 2015. Strategies for Conservation of Endangered Amphibian and Reptile Species. Biology Bulletin 42(5): 432–439. DOI: 10.1134/S1062359015050027
Berman D.I., Leirikh A.N., Mikhailova E.I. 1984. On the wintering of the Siberian salamander Hynobius keyserhngii in the Upper Kolyma. Journal of Evolutionary Biochemistry and Physiology 3: 323–327. [In Russian]
Berman D.I., Meshcheryakova E.N., Bulakhova N.A. 2016. Extreme negative temperatures and body mass loss in the Siberian salamander (Salamandrella keyserlingii, amphibia, hynobiidae). Doklady Biological Sciences 468(1): 137–141. doi: 10.1134/S001249661603011X
Bolshakov V.N., Vershinin V.L. 2005. Amphibians and reptiles of the Middle Urals. Yekaterinburg: Ural Branch of the RAS. 126 p. [In Russian]
Bosch J., Martínez-Solano I. 2006. Chytrid fungus infection related to unusual mortalities of Salamandra salamandra and Bufo bufo in the Peñalara Natural Park, Spain. Oryx 40(1): 84–89. DOI: 10.1017/S0030605306000093
Bulakhova N.A., Mikhailova E.I., Berman D.I. 2021. Phenology of Siberian salamander (Salamandrella keyserlingii, Caudata, Hynobiidae) in climatically different regions of northeast Asia. Arctic and Subarctic Natural Resources 26(2): 117–135. DOI: 10.31242/2618-9712-2021-26-2-8 [In Russian]
Chernaya L.V., Kovalchuk L.A., Nokhrina E.S. 2016. Role of the tissue free amino acids in adaptation of medicinal leeches Hirudo medicinalis L., 1758 to extreme climatic conditions. Doklady Biological Sciences 466: 42–44. DOI: 10.1134/S00124996616010129
Chessel D., Dufour A.B., Thioulouse J. 2004. The ade4 package–I: One-table methods. R News 4: 5–10.
Coico R., Sunshine G., Benjamini E. 2003. Immunology. A Short Course. New York: Wiley-Liss. 237 p.
Cooper E.L., Klempau A.E., Zapata A.G. 1985. Reptilian immunity. In: C. Gans, F. Billett, P.F.A. Maderson (Eds.): Biology of the Reptilia. Vol. 14: Development A. New York: John Wiley & Sons. P. 601–636.
Council of Europe. 1986. European Convention for the protection of vertebrate animals used for experimental and other scientific purposes (ETS123). Strasbourg: Council of Europe. Available from
Davis A.K., Maney D.L., Maerz J.C. 2008. The use of leukocyte profiles to measure stress in vertebrates: A review for ecologists. Functional Ecology 22(5): 760–772. DOI: 10.1111/j.1365-2435.2008.01467.x
Dray S., Dufour A., Thioulouse J. 2022. ade4: Analysis of Ecological Data: Exploratory and Euclidean Methods in Environmental Sciences. R package .version 1.7-19. Available from
Forman H.J., Zhang H., Rinna A. 2008. Glutathione: Overview of its protective roles, measurement, and biosynthesis. Molecular Aspects of Medicine 30(1–2): 1–12. DOI: 10.1016/j.mam.2008.08.006
Fournier M., Robert J., Salo H.M., Dautremepuits C., Brouseau P. 2005. Immunotoxicology of Amphibians. Applied Herpetology 2(3): 297–309. DOI: 10.1163/1570754054507451
Gallant A.L., Klaver R.W., Casper G.S., Lannoo M.J. 2007. Global Rates of Habitat Loss and Implications for Amphibian Conservation. Copeia 4: 967–979. DOI: 10.1643/0045-8511(2007)7[967:GROHLA]2.0.CO;2
Garaeva S.N., Redkozubova G.V., Postolati G.V. 2009. Amino acids in a living organism. Kishinev: Printing House of the Academy of Sciences of Moldova. 552 p. [In Russian]
Global Amphibian Assessment. 2004. International Union for Conservation of Nature and Natural Resources. Cambridge, UK. Available from
Hussain Q., Pandit A.K. 2012. Global amphibian declines: A review. International Journal of Biodiversity and Conservation 4(10): 348–357. DOI: 10.5897/IJBC12.008
IUCN. 2020. IUCN Red List Quadrennial Report 2017–2020. Available from:
James L.B. 1987. Amino acid analysis: a fall-off in performance. Journal of Chromatography A 408: 291–295. DOI: 10.1016/s0021-9673(01)81812-4
Karanova M.V. 2011. The Effect of Cold Shock on the Free Amino Acid Pool of Rotan Pondfish Perccottus glehni (Eleotridae, Perciformes). Biology Bulletin 38(2): 116–124. DOI: 10.1134/S106235901102004X
Karanova M.V. 2020. Secondary Metabolites and Aspartic Acid in the Brain of the Frog Rana temporaria as Low-Temperature Adaptogens. Journal of Evolutionary Biochemistry and Physiology 56(3): 207–212. doi: 10.31857/S0044452920030043 [In Russian]
Khaitov R.M. 2013. Immunology: structure and function of the immune system. Moscow: GEOTAR-Media 280 p. [In Russian]
Kovalchuk L.A., Mishchenko V.A., Mikshevich N.V., Chernaya L.V., Chibiryak M.V., Yastrebov A.P. 2018a. Free Amino Acid Profile in Blood Plasma of Bats (Myotis dasycneme Boie, 1825) Exposed to Low Positive and Near-Zero Temperatures. Journal of Evolutionary Biochemistry and Physiology 54(4): 281–291. DOI: 10.1134/S002209301804004X
Kovalchuk L.A., Mishchenko V.A., Chernaya L.V., Snitko V.P. 2018b. Species-specific features of blood plasma amino acid spectrum of bats (Mammalia: Chiroptera) in the Urals. Russian Journal of Ecology 49(4): 325–331. DOI: 10.1134/S1067413618040082
Kovalchuk L.A., Chernaya L.V., Mishchenko V.A., Berzin D.L., Bolshakov V.N. 2021a. Amino Acid Spectrum in the Blood of the Endemic and Invasive Amphibian Species in the Fauna of the Ural. Doklady Biochemistry and Biophysics 500: 327–330. DOI: 10.1134/S1607672921050124
Kovalchuk L.A., Mishchenko V.A., Chernaya L.V., Bolshakov V.N. 2021b. Characteristic Immunohematological Parameters of Migratory (Vespertilio murinus Linnaeus, 1758) and Resident (Myotis dasycneme Boie, 1825) Bat Species of the Ural Fauna. Doklady Biological Sciences 501: 210–213. DOI: 10.1134/S001249662106003X
Kovalchuk L.A., Chernaya L.V., Mishchenko V.A., Mikshevich N.V. 2021c. Amino acid spectrum of blood of the lake frog Pelophylax ridibundus introduced into the ponds of the Middle Urals. Hydrobiological Journal 57(3): 80–89. DOI: 10.1615/HYDROBJ.V57.I3.90
Kunitsyn A.A. 2009. Materials on the distribution and ecology of the Siberian salamander (Hynobius keyserlingii, Dybowski, 1870) in the Baikal region. Baikal Zoological Journal 3: 31–34. [In Russian]
Kuranova V.N., Fokina E.V. 2008. Variability in the development and growth of the Siberian salamander Salamandrella keyserlingii (Caudata, Amphibia) In: Issues of Herpetology. St. Petersburg: Zoological Institute RAS. P. 227–233. [In Russian]
Kuzmin S.L. 2012. Amphibians of the former USSR. Moscow Moscow: KMK Scientific Press Ltd. 370 p. [In Russian]
LaFonte B.E., Johnson P.T. 2013. Experimental infection dynamics: using immunosuppression and in vivo parasite tracking to understand host resistance in an amphibian-trematode system. Journal of Experimental Biology 216(19): 3700–3708. DOI: 10.1242/jeb.088104
Li Y., Cohen J.M., Rohr J.R. 2013. Review and synthesis of the effects of climate change on amphibians. Integrative Zoology 8(2): 145–161. DOI: 10.1111/1749-4877.12001
Litvinov N.A., Faizulin A.I., Shurakov A.I., Ganshchuk S.V. 2010. Analysis of clutch status of Siberian newt Salamandrella keyserlingii Dybowski, 1870 (Caudata, Amphibia) near the Urals region. Povolzhsky Journal of Ecology 4: 438–441. [In Russian]
Lyapustin S.N. 2008. Fight against smuggling of objects of fauna and flora in the Russian Far East (late 19th – early 21st centuries). Vladivostok: VF RTA. 252 p. [In Russian]
Lyapustin S.N., Fomenko P.V. 2010. Amphibians of the Far East: a brief guide for customs officials. Vladivostok: VF RTA. 56 p. [In Russian]
Mazo V.K. 1998. Glutathione as a component of the antioxidant system of the gastrointestinal tract. Russian Journal of Gastroenterology, Hepatology, Coloproctology 1: 47–53. [In Russian]
Mineeva O.V., Mineev A.K. 2011. Disorders of blood leukocyte formula in the Lake frog of the Saratov reservoir. Vestnik of Lobachevsky University of Nizhni Novgorod 2(2): 94–97. [In Russian]
Mineeva O.V., Mineev A.K. 2014. Features of haematological parameters of lake frog Rana ridibunda Pallas, 1771 of the Saratov reservoir. Samarskaya Luka: problems of regional and global ecology 23(2): 178–184. [In Russian]
Nikolaev V.Yu., Romanova E.B. 2016. Immunohematological characteristics of amphibians in reservoirs of the Nizhny Novgorod region of different hydrochemical composition. Problems of regional ecology 3: 31–35. [In Russian]
Oksanen J., Simpson G.L., Blanchet F.G., Kindt R., Legendre P., Minchin P.R., O'Hara R.B., Solymos P., Szoecs E., Wagner H., Barbour M., Bedward M., Bolker B., Borcard D., Carvalho G., Chirico M., Caceres M., Durand S., Evangelista H.B.A., FitzJohn R., Friendly M., Furneaux B., Hannigan G., Hill M.O., Lahti L., McGlinn D., Ouellette M.H., Cunha E.R., Smith T., Stier A. et al. 2020. vegan: Community Ecology Package. R package. Version 2.5-7. Available from
Pounds J.A., Bustamante M.R., Coloma L.A., Consuegra J.A., Fogden M.P., Foster P.N., La Marca E., Masters K.L., Merino-Viteri A., Puschendorf R., Ron S.R., Sánchez-Azofeifa G.A., Still C.J., Young B.E. 2006. Widespread amphibian extinctions from epidemic disease driven by global warming. Nature 439(7073): 161–167. doi: 10.1038/nature04246
R Core Team. 2020. R: A language and environment for statistical computing. Vienna: R Foundation for Statistical Computing. Available from
Red Data Book of the Sverdlovsk Region: animals, plants, fungi. Yekaterinburg: Mir, 2018. 450 p. [In Russian]
Rohr J.R., Raffel T.R., Romansic J.M., McCallum H., Hudson P.J. 2008. Evaluating the links between climate, disease spread, and amphibian declines. Proceedings of the National Academy of Sciences of the United States of America 105(45): 17436–17441. DOI: 10.1073/pnas.0806368105
Romanova E.B., Fadeeva G.A., Vershinina K.S., Nikolaev I.Yu. 2013. Leukogram changes in the marsh frog (Pelophylax ridibundus Pallas, 1771) with helminthiases. Vestnik of Lobachevsky University of Nizhni Novgorod 5(1): 141–147. [In Russian]
Romanova E.B., Shapovalova K.V., Ryabinina E.S. 2018. The leukocyte blood composition and micronuclei in the erythrocytes of amphibians in the polluted reservoirs of Nizhni Novgorod Region. Principles of the Ecology 7(2): 125–139. DOI: 10.15393/ [In Russian]
Romanova E.B., Shapovalova K.V., Ryabinina E.S., Gelashvili D.B. 2019. Leukocytic indices and micronucleus in erythrocytes as population markers of the immune status of Pelophylax ridibundus (Pallas, 1771) (Amphibia: Ranidae) living in various biotopic conditions // Biology Bulletin 46(10): 1230–1238. DOI: 10.1134/S1062359019100273
Severin E.S. (Ed.). 2004. Biochemistry. Moscow: GEOTAR-Media. 784 p. [In Russian]
Shitikov V.K., Rosenberg G.S. 2014. Randomization and bootstrap: statistical analysis in biology and ecology using R. Togliatti: Cassandra. 314 p. [In Russian]
Sils E.A. 2008. Comparative analysis of hematological parameters of moor frog (Rana arvalis, Nilsson, 1842) and lake frog (Rana ridibunda, Pallas, 1771) of urban populations. Vestnik of Orenburg State Pedagogical University 10(92): 230–235. [In Russian]
Solomonov N.G., Sedalishchev V.T., Solomonov K.S., Kirillin R.A., Protopopov S.G. 2018. Wintering of Siberian salamander Salamandrella keyserlingii (Dybowski, 1870) in Central Yakutia. Arctic and Subarctic Natural Resources 23(1): 111–116. [In Russian]
Strayer D., Dudgeon D. 2010. Freshwater biodiversity conservation: recent progress and future challenges. Journal of the North American Benthological Society 29(1): 344–358. DOI: 10.1899/08-171.1
Stuart S.N., Chanson J.S., Cox N.A., Young B.E., Rodrigues A.S.L., Fischman D.L., Waller R.W. 2004. Status and trends of amphibian declines and extinctions worldwide. Science 306(5702): 1783–1786. DOI: 10.1126/science.1103538
Vafis A.A., Peskova T.Yu. 2009. Blood Change of Lake Frog (Rana ridibunda) Influenced by Sugar-Beet Mill Sewage. Problems of Contemporary Science and Practice. Vernadsky University 2(16): 8–18. [In Russian]
Vershinin V.L. 2014. Functional specific of amphibian populations in urbanustic gradient. Proceedings of Samara Scientific Center of RAS 16(5–1): 344–348. [In Russian]
Vershinin V.L., Vershinina S.D. 2013. Comparative analysis of hemoglobin content in four species of anurans from the Ural uplands. Doklady Biological Sciences 450(1): 155–157. doi: 10.1134/S0012496613030137
Vershinin V.L., Vershinina S.D., Berzin D.L., Zmeeva D.V., Kinev A.V. 2015. Long-term observation of amphibian populations inhabiting urban and forested areas in Yekaterinburg, Russia. Scientific Data 2: 150018. DOI: 10.1038/sdata.2015.18
Vorobyeva E.I. (Ed.). 1994. Siberian Salamander (Salamandrella keyserlingii Dybowski, 1870): Zoogeography, systematics, morphology. Moscow: Nauka. 367 p. [In Russian]
Whittaker K., Koo M.S., Wake D.B., Vredenburg V.T. 2013. Global Declines of Amphibians. In: Encyclopedia of Biodiversity (Second Edition). Cambridge: Academic Press. P. 691–699. DOI: 10.1016/B978-0-12-384719-5.00266-5
Wu G. 2009. Amino acids: metabolism, functions, and nutrition. Amino Acids 37(1): 1–17. DOI: 10.1007/s00726-009-0269-0
Yarri D. 2005. The Ethics of Animal Experimentation: A Critical Analysis and Constructive Christian Proposal. Oxford: Oxford University Press. 240 p. DOI: 10.1093/0195181794.001.0001
Yartsev V.V., Evseeva S.S. 2018. Histological Characteristics of the Skin of Salamandrella keyserlingii (Caudata, Hynobiidae) Females in Aqutic and Terrestrial Phases of Seasonal Cycle. Current Studies in Herpetology 18(3): 159–167. DOI: 10.18500/1814-6090-2018-18-3-4-159-167 [In Russian]
Zimmerman L.M., Vogel L.A., Bowden R.M. 2010. Understanding the vertebrate immune system: insights from the reptilian perspective. Journal of Experimental Biology 213(5): 661–671. DOI: 10.1242/jeb.038315
Zmeeva D.V. 2009. Population specifics of the reproductive performance of the Siberian salamander in the forest park belt of Yekaterinburg. In: Evolutionary and population ecology (back to the future). Yekaterinburg: Ural Branch of RAS. P. 62. [In Russian]