7 Annales Universitatis Paedagogicae Cracoviensis Studia Naturae, 3 (supplement): 7–16, 2018, ISSN 2543-8832 DOI: 10.24917/25438832.3supp.1 Mateusz Ciepliński1*, Mariusz Kasprzak1, Monika Grandtke1, Aleksandra Steliga2, Piotr Kamiński1,3, Leszek Jerzak1 1Faculty of Biological Sciences, University of Zielona Góra, Prof. Z. Szafrana Str., 65-516 Zielona Góra, Poland, *m.cieplinski@wnb.uz.zgora.pl 2Department of Health Sciences, Pomeranian University of Słupsk, 64 Bohaterów Westerplatte Str., 76-200 Słupsk, Poland 3Department of Medical Biology and Biochemistry, Department of Ecology and Environmental Protection, Faculty of Medicine, Collegium Medicum in Bydgoszcz, Nicolaus Copernicus University in Toruń, 9M. Skłodowskiej-Curie, 85-094 Bydgoszcz, Poland The impact of UDN on selectwed blood parameters of female sea trout Salmo trutta m. trutta L. spawners Introduction Sea trout – Salmo trutta morpha trutta L. (Fig. 1) is an anadromous form of brown trout – Salmo trutta (Bouza et al., 1999). It belongs to Salmonidae family which in- cludes Atlantic salmon (Salmo salar L.), arctic charr (Salvelinus alpinus L.) and rain- bow trout (Oncorhynchus mykiss Walbaum) (Frank-Gopolos et al., 2015). Anadro- mous �sh a�ect many other species in their environment and have a profound e�ect on the stability of aquatic and terrestrial ecosystems in which they occur; therefore, they are considered as a ‘keystone’ species (Willson, Halupka, 1995). Main threats to anadromous �sh populations in Poland are migration barriers, over�shing, poaching, and environmental changes linked with water pollution (HEL- COM, 2015; Kazuń et al., 2011; Radtke et al., 2012; “�e Valley of Słupia”…, 2017). Another important factor limiting sea trout populations in Poland is Ulcerative Der- mal Necrosis (UDN), which is like the disease observed in the Słupia river from 2007 (Grudniewska et al., 2011; Grudniewska et al., 2012; Kazuń et al., 2011). UDN is an infectious skin disease of unknown etiology a�ecting adult, wild, anadromous salmo- nids migrating from open seas to fresh water during the spawning season (Bruno et al., 2013; Harris et al., 2011). First documented mentions of the disease originate from late 19th century Great Britain (Roberts, 1993). In the second half of the XX century, outbreaks were reported in many European countries: Austria, Belgium, France, Luxembourg, Germany, Great Britain, Switzerland, Sweden, and Canada (Grudniewska et al., 2012; Johansson et al., 1982). �e highest mortalities are observed in November and December. UDN M at eu sz C ie pl iń sk i, M ar iu sz K as pr za k, M on ik a G ra nd tk e, A le ks an dr a S te lig a, P io tr K am iń sk i, Le sz ek J er za k 8 Fig. 1. Sea trout with author for scale (Photo. M. Ciepliński) occurrence is linked to low water temperature during these months. Large �sh con- centrations during spawning season create perfect conditions for contagious diseases to spread (Munro, 1970). �e �rst visible signs of UDN are small, grey lesions on the operculum, �ns, and head (Bruno et al., 2013). �ese rapidly ulcerate and frequently become infected with opportunistic pathogens, mainly the oomycete Saprolegnia di- clina Humphrey, which has expansive growth that makes skin damage even greater (Roberts, 1993). Such extensive epidermal damage induces osmotic haemodilution leading to circulatory failure and the death of infected �sh (Bruno et al., 2013). �e Słupia River is one of the �rst rivers where occurrence of sea trout spawners with UDN-like symptoms was reported (Bartel et al., 2009). Since then, the disease oc- currence is observed on a yearly basis (“�e Valley of Słupia”, management personal communication). Tab. 1. Number of healthy, sick and agonal (n) female sea trout Salmo trutta m. trutta L. blood samples used in the present study in each and all years of research Health status Year 2014 2015 2016 2017 2014–2017 Healthy (1) 15 8 1 32 56 Sick (2) 8 31 19 5 63 Agonal (3) 8 6 17 0 31 Total 31 45 37 37 150 9 The im pact of U D N on selectw ed blood param eters of fem ale sea trout Salm o trutta m . trutta L. spaw ners Blood tests are a very valuable tool in human and veterinary medicine. Due to its properties, blood is virtually the easiest tissue to obtain and to test. Blood tests are also one of the most comprehensive ways to assess the status of animal health. Despite rapid development of laboratory techniques, the haematology of �sh is still underes- timated in the assessment of �sh welfare. Many factors in�uence �sh blood results, inter alia, age, sex, reproductive and nutritional status, water temperature, and oxygen concentration (Řehulka, Adamec, 2004; Witeska, 2013). Blood tests reference values found in literature are scarce and o�en vary signi�cantly between reports. In the present study, the authors decided to investigate the impact of UDN on selected haematological (red blood cell count – RBC, haemoglobin concentration – HGB, haematocrit HCT) and biochemical (total protein, albumin and BUN concen- tration) parameters of female sea trout spawners from the Słupia River during four (2014–2017) consecutive spawning seasons. We assume that, along with health dete- rioration, selected blood parameters, i.e. RBC, HGB, HCT, and concentrations of total protein, albumin, and blood urea nitrogen (BUN) will change signi�cantly. Material and methods Fish Mature, spawning sea trout (Salmo trutta m. trutta) females (n = 150) (mean mass 2.02 ± 0.90 kg, mean length 60.01 ± 7.64 cm) were caught during four consecutive spawn- ing seasons in November 2014–2017 (Tab. 1) on a Polish Angling Association trapping point (54°27ʹ37.4ʹʹN; 17°02ʹ21.1ʹʹE) on the Słupia River, Słupsk, northern Poland. Water temperature during catches ranged between 4.6–8.6°C (data provided by “�e Valley of Słupia” Landscape Park). All animals with evident signs of a disease were a bycatch of a targeted trapping of healthy spawners necessary for arti�cial spawning. All specimens were euthanized with blunt force trauma to the cranium region which was followed by pithing by PAA workers. Healthy specimens were euthanized a�er arti�cial spawning. All �sh used in the present research were donated by the PAA. All applicable inter- national and national guidelines for the care and use of animals were followed by the authors. Blood and other tissues for further examination, not presented in this article, were collected. Photographic documentation of �sh skin infection patterns covered both sides of the body. Photographs were made with a Nikon D80 camera. Health status assessment On the basis of photographic documentation, infection schemes were prepared for each UDN a�ected specimen in the GIMP 2.8.18 so�ware (�e GIMP team, www.gimp.org, 1997–2016). All visible areas of damaged skin were mapped on an outline depicting trout contour (Fig. 2A). Salmo trutta m. fario L. illustration (Böhmig, Brauer, 1909) was M at eu sz C ie pl iń sk i, M ar iu sz K as pr za k, M on ik a G ra nd tk e, A le ks an dr a S te lig a, P io tr K am iń sk i, Le sz ek J er za k 10 Fig. 2. Salmo trutta m. trutta L.: A – sample (le� body side) infection scheme made on the basis of pho- tographic documentation; areas of damaged skin were mapped on an outline depicting trout contour, B – Healthy Salmo trutta m. trutta female specimen,; C – sick female specimen, D – agonal female specimen (Photo. M. Ciepliński) 11 chosen due to its simplicity. For each specimen, two schemes were prepared (for both sides of the body). Obtained images were then analysed with ImageJ 1.50i (Rasband, 1997) so�ware to determine the percentage of skin damage. Obtained results for both sides of the body were then averaged, due to high bilateral symmetry of lesions. Skin damage as little as 10% is known to result in nearly 50% mortality (Noga, 2000). �is fact being known, the authors decided to group �sh into three categories (Fig. 2): (2B) healthy specimens with no visible signs of UDN; (2C) sick, where from 0.01% up to 10% skin was damaged (mean = 2.77%); and, (2D) agonal, where lesions covered more than 10% of the body surface (mean = 23.94%). Laboratory analysis Blood for haematological analysis was drawn from caudal vein by means of a 1.2 × 44 mm needle and 5 ml syringe. About 8–10 ml of blood in total was ob- tained from each specimen. We used a minimum two 5 ml syringes, and not one 10 ml, because the high vacuum created by such syringe could damage erythrocytes. Immediately a�er collection, 2 ml of blood was then transferred to standard test tubes containing K2EDTA (1.8 mg/ml K2EDTA for 2ml of blood) (Medlab-Prod- ucts) for basic haematological analysis. �e remaining amount of blood was le� to clot in 7 ml conical centrifuge tubes. Clot was then centrifuged to obtain se- rum for biochemical analysis (total protein, albumin, and BUN concentrations). In 2014, serum samples were not collected. Basic haematological analysis (RBC, HGB, HCT) was performed immediately a�er collection with use of manual methods. Red blood cell count (RBC) was determined with use of Bürker hemocytometer and Natt and Herrick (1952) stain. Drabkin’s (Drabkin, 1945) cyanmethemoglobin method was used to assess haemoglobin concentration (HGB). Hematocrit (HCT) was de- termined according to Turgeon (2012). Biochemical analysis (total protein, albu- min, and blood urea nitrogen concentration (BUN)) was performed with use of an ARCHITECT c 4000 clinical chemistry analyser (Abbott Diagnostics). Unfortu- nately, on a few samples, concentrations of selected biochemical parameters were beyond the measuring range of the analyser. Statistics analysis Statistical analysis was performed in search for di�erences in blood parameters be- tween di�erent health status groups. Due to the lack of normal distribution in all pre- sented parameters, Kruskal-Wallis tests for independent groups and were used. Anal- ysis was made with Statistica 12.5 so�ware (StatSo�, 2006). The im pact of U D N on selectw ed blood param eters of fem ale sea trout Salm o trutta m . trutta L. spaw ners M at eu sz C ie pl iń sk i, M ar iu sz K as pr za k, M on ik a G ra nd tk e, A le ks an dr a S te lig a, P io tr K am iń sk i, Le sz ek J er za k 12 Results A comparison of selected sea trout (Salmo trutta m. trutta, female spawners) hemato- logical parameters in relation to health status is presented in table 2. Signi�cantly lower RBC counts (p < 0.05), HGB (p < 0.01) and HCT (p < 0.01) were observed in agonal �sh than in the remaining two health status groups, be- tween which no statistically signi�cant di�erences were observed. In total protein (p < 0.0001) and albumin (p < 0.0001) concentrations, all groups di�ered signi�cantly. In these parameters, a clear value drop is visible along with health status deterioration. Agonal specimens had signi�cantly higher urea concentrations (p < 0.0001) than the other two groups. Tab. 2. Selected haematological parameters of healthy, sick and agonal sea trout Salmo trutta m. trutta L.; values are mean ± SD; means in parameters marked with di�erent superscript are signi�cantly di�erent (p < 0.05) Health status Parameter RBC [T/l] HGB [g/dl] HCT [%] Total protein [g/dl] Albumin [g/dl] BUN [mg/dl] Healthy (1) 1.16a ± 0.23 n = 56 9.79a ± 1.86 n = 56 46.93a ± 7.52 n = 56 5.34a ± 1.87 n = 40 1.81a ± 0.41 n = 41 4.63a ± 1.48 n = 40 Sick (2) 1.18a ± 0.26 n = 63 10.20a ± 1.64 n = 63 46.28a ± 6.98 n = 63 3.35b ± 1.13 n = 52 1.52b ± 0.45 n = 52 4.74a ± 1.70 n = 63 Agonal (3) 1.02b ± 0.27 n = 31 8.95b ± 3.87 n = 31 38.61b ± 1.87 n = 31 1.31c ± 0.46 n = 17 0.64c ± 0.25 n = 16 6.74b ± 1.65 n = 31 Note: RBC – red blood cell count, HGB – haemoglobin concentration, HCT – haematocrit, BUN – blood urea nitrogen Discussion Skin integrity is vital for the maintenance of �sh homeostasis. In a fresh water envi- ronment, it prevents water intake, which is consistent with concentration gradient. �e damage caused by infection development disturbs this property (Noga, 2000). Uncontrolled water uptake leads to the decrease in hematologic parameters. A similar e�ect is observed in total protein and albumin concentrations, both of which help to maintain proper colloid osmotic pressure (Harr, 2006). Although this parameter was not determined in the current study, globulin concentration, which is a di�erence between total protein and albumin concentrations, would also decrease indicating im- munode�ciency (�rall et al., 2012), and elevated blood urea nitrogen in �sh is a sign of gill dysfunction (Nelson et al., 1999). In the present study, results show that skin damage of more than 10% leads to se- vere hemodilution and a signi�cant drop in investigated haematological parameters 13 (RBC, HGB, HCT). A slight increase in HGB observed in sick �sh, as compared to healthy �sh, may suggest that, during the �rst stages of UDN-like infection, the �sh organism is trying to defend itself from hypoxia caused by developing respiratory fail- ure (A�onso et al., 2002). �e decrease of HTC, in relation to disease development, is the consequence of excessive water uptake via damaged skin and drop in red blood cell count (Weiss, Wardrop, 2010). Even slight, super�cial skin damage (less than 10% of body surface) leads to a signi�cant decrease in total serum protein and albumin concentrations directly caused by water in�ux (Harr, 2006). �is e�ect is even greater in severely diseased �sh. �e a�ermath of gill damage is an increase in BUN concen- tration (Nelson et al., 1999). Due to many factors in�uencing �sh blood results (i.e. age, sex, reproductive and nutritional status, water temperature, oxygen concentration) (Řehulka, Adamec, 2004; Witeska, 2013), normal values that have been presented could serve as reference only for healthy female sea trout during the spawning season. Acknowledgements �e research was entirely funded by Faculty of Biological Sciences, University of Zielona Góra. We would like to thank the management and sta� of “�e Valley of Słupia” Landscape Park for help and support during �eldwork, as well as the Polish Angling Association of the district of Słupsk for providing essential research material. References A�onso, E.G., Polez, V.L.P., Corrêa, C.F., Mazon, A.F., Araújo, M.R.R., Moraes, G., Rantin, F.T. (2002). Blood parameters and metabolites in the teleost �sh Colossoma macropomum exposed to sul�de or hypoxia. Comparative Biochemistry and Physiology Part C: Toxicology & Pharmacology, 133(3), 375–382. 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Erythrocytes in teleost �shes: a review. Zoology and Ecology, 23(4), 275–281. DOI: 10.1080/21658005.2013.846963 15 Abstract Blood tests were performed on 150 female sea trouts Salmo trutta m. trutta L. during four spawning seasons (2014–2017). Fish were caught on a Polish Angling Association trapping point, the Słupia River, Słupsk (northern Poland). �e blood for analysis was drawn from caudal vein of 56 healthy and 94 UDN (Ulcer- ative Dermal Necrosis) infected females. Fish were divided into three groups: (1) healthy, with no visible signs of UDN; (2) sick, with up to 10% skin damage; and, (3) agonal, where more than 10% of body surface was infected. A statistically important decrease in red blood cell count (RBC), haemoglobin concentration (HGB) and haematocrit (HCT) were found between sick and agonal �sh groups. �e concentration of total plasma protein and albumin decreased in relation to �sh health deterioration. Blood urea nitrogen (BUN) had an inverse proportionality to total plasma protein and albumin concentration. Based on the decrease of RBC, HGB, and HCT due to the development of UDN symptoms, a decrease in �sh condition was observed. Signi�cantly higher urea concentrations observed in agonal �sh may indicate respiratory and excretory systems failure. Key words: blood, haematology, sea trout, Ulcerative Dermal Necrosis Received: [2018.06.29] Accepted: [2018.12.10] Wpływ UDN na wybrane parametry krwi samic troci wędrownej Salmo trutta m. trutta L. w czasie tarła Streszczenie W latach 2014 do 2017 w czterech sezonach rozrodczych przeprowadzono badania krwi na 150 samicach troci wędrownej Salmo trutta m. trutta L. Ryby odłowiono na Punkcie Odłowu Troci na rzece Słupi w Słup- sku (północna Polska). Z żyły ogonowej pobrano krew od 56 zdrowych oraz 94 chorych na UDN (Ulce- rative Dermal Necrosis – wrzodziejąca martwica skóry) samic. Badane osobniki podzielono na 3 grupy: (1) zdrowe bez widocznych objawów UDN, (2) chore ze zmianami do 10% powierzchni ciała oraz (3) ago- nalne, gdzie więcej niż 10% powierzchni ciała zostało uszkodzone. Istotne statystycznie zmniejszenie warto- ści liczby krwinek czerwonych (RBC), stężenia hemoglobiny (HGB) oraz hematokrytu (HCT) stwierdzono pomiędzy osobnikami chorymi i  agonalnymi. Stężenie białka całkowitego oraz albumin zmniejszało się istotnie statystycznie wraz z pogorszeniem stanu zdrowia ryb. Stężenie mocznika zmieniało się odwrotnie proporcjonalnie do stężenia białka i albumin. Wraz ze wzrostem intensywności rozwoju UDN stwierdzono osłabienie kondycji wyrażające się zmniejszeniem RBC, HGB jak i  HCT. Znacznie wyższe stężenie BUN u ryb agonalnych może wskazywać na niewydolność układu oddechowego i wydalniczego. Słowa kluczowe: krew, hematologia, troć wędrowna, wrzodziejąca martwica skóry UDN Information on the authors Mateusz Ciepliński http://orcid.org/0000-0002-3386-9744 He is a PhD student and a scienti�c-technical worker on the Faculty of Biological Sciences, University of Zielona Góra. His research interests focuses on vertebrate haematology and its practical application in veterinary medicine. Mariusz Kasprzak http://orcid.org/0000-0001-9088-8098 He is focused on the relationship between the quality of the environment and the condition of the ani- mals, and is an assistant professor in the Department of Zoology at the Faculty of Biological Sciences, University of Zielona Góra. Monika Grandtke http://orcid.org/0000-0001-9472-252X She is a PhD student at the Faculty of Biological Sciences at the University of Zielona Góra. She works at the University of Zielona Góra as a scienti�c and technical assistant. She is interested in the in�uence of the environment with varying degrees of pollution on the white and red blood cell parameters of storks. The im pact of U D N on selectw ed blood param eters of fem ale sea trout Salm o trutta m . trutta L. spaw ners M at eu sz C ie pl iń sk i, M ar iu sz K as pr za k, M on ik a G ra nd tk e, A le ks an dr a S te lig a, P io tr K am iń sk i, Le sz ek J er za k 16 Aleksandra Steliga http://orcid.org/0000-0002-2153-9414 She is a PhD student at Gdański Uniwersytet Medyczny in Gdańsk. She works at the Department of Health Sciences on Pomeranian University in Słupsk and is interested in the haematology of vertebrates and human glial cells. Piotr Kamiński http://orcid.org/0000-0003-1978-6018 He is an assistant professor in the Department of Biotechnology at the Faculty of Biological Sciences, Uni- versity of Zielona Góra and Nicolaus Copernicus University in Toruń, Collegium Medicum in Bydgoszcz. He is fascinated by animal ecophysiology. Leszek Jerzak http://orcid.org/0000-0001-5332-279X He is full professor in nature protection at the University of Zielona Góra, Poland (Faculty of Biological Sciences). He studies the biology and ecology of animals (especially the White Stork and the Magpie) and co-operates with research centres in Germany, USA, Ireland, and Russia.