1. AKNOWLEDGMENTS This study was co-financed by the project “Innovations in finfish aquaculture with special references to reproduction” (acronym: InnovaFish), Operational Programme Sustainable Development of the Fisheries Sector and Coastal Fishing Areas 2007-2013" (OR14-61724-OR1400003/09/10/11). www.eng.InnovaFish.pl Sławomir Krejszeff 1, Daniel Żarski 1, Piotr Gomułka 2, Krzysztof Kupren 1, Katarzyna Palińska-Żarska 1, Joanna Nowosad 1, Katarzyna Stańczak 3 and Dariusz Kucharczyk 1 1 – Department of Lake and River Fisheries, University of Warmia and Mazury, Olsztyn, Poland. 2 – Department of Ichthyology, University of Warmia and Mazury, Olsztyn, Poland. 3 – Department of Fish Biology and Pisciculture, University of Warmia and Mazury, Olsztyn, Poland. Corresponding author: Sławomir Krejszeff, University of Warmia and Mazury, ul. Oczapowskiego 5, 10-719 Olsztyn, Poland, phone: +48 (089) 523 39 69, e-mail: s.krejszeff@wp.pl References: FAO 2013 statistics. http://www.fao.org/fishery/statistics/en García V., J.D. Celada, J.M. Carral, R. González, Á. González, M. Sáez-Royuela. 2011. A comparative study of different preparations of decapsulated Artemia cysts as food for tench (Tinca tinca L.) larvae. Animal Feed Science and Technology 170: 72-77. Gela D., M. Kocour, M. Flajšhans, O. Linhart, M. Rodina. 2010.Comparison of performance of genome manipulated and standard tench, Tinca tinca (L.), groups under pond management conditions. Reviews in Fish Biology and Fisheries 20:301-306. Gilderhus P.A. 1990. Benzocaine as a fish anesthetic: efficacy and safety for spawning-phase salmon. The Progressive Fish- Culturist 52: 189-191. Hamackova J., A. Lepicoa, P. Kozak, Z. Stupka, J. Kouril, P. Lepic. 2004. The efficacy of various anaesthetics in tench (Tinca tinca L.) related to water temperature. Veterinarni Medicina 49: 467-472. Kocour M., D. Gela, M. Rodina, M. Flajšhans. 2010. Performance of different tench, Tinca tinca (L.), groups under semi- intensive pond conditions: it is worth establishing a coordinated breeding program. Reviews in Fish Biology and Fisheries 20: 345-355. Krejszeff S., D. Żarski, D. Kucharczyk, K. Kupren, K. Targońska, A. Mamcarz. 2010. An experimental device for eggs incubation and fish larvae rearing under laboratory conditions. Polish Journal of Natural Sciences25:190-199. Krejszeff S., D. Żarski, K. Palińska, I. Trąbska, K. Kupren, K. Targońska, M. Bowszys, D. Kucharczyk. 2013. Procedure for harmless estimation of fish larvae weight. Italian Journal of Animal Science (in press) Massee K.C., M.B. Rust, R.W. Hardy, R.R. Stickney. 1995. The effectiveness of tricaine, quinaldine sulfate and metomidate as anesthetics for larval fish. Aquaculture 134: 351-359. Myszkowski L., R. Kamiński, J. Wolnicki. 2003. Response of juvenile tench Tinca tinca (L.) to the anaesthetic 2–phenoxyethanol. Journal of Applied Ichthyology 19: 142-145. Podhorec P., M. Socha, M. Sokolowska-Mikolajczyk, B. Drozd, T. Policar, V. Stejskal, J. Kouril. 2011. Effective dose of mGnRHa for induction of ovulation in tench (Tinca tinca L.). Aquaculture 319: 184-187. Steffens W. 1995. The tench (Tinca tinca L.), a neglected pond fish species. Polish Archives of Hydrobiology 42: 161-180. The aquaculture of tench has got long tradition and it began in the Middle Ages (Steffens, 1995). The growth of consumers demand for tench has been observed since the middle 80s since then the global production has been increasing. Although only part of this comprises aquaculture production (FAO 2013). Tench is a species with big potential for aquaculture and is considered as a candidate for diversification of aquaculture production (Gela et al., 2010; García et al., 2011). In order to increase efficiency of tench production the research focused on the following elements: reproduction, larvae and juvenile rearing, market size fish rearing (Kocour et al., 2010; Podhorec et al., 2011). From the mentioned above larvae rearing is the most important bottleneck because much more factors and phenomena affect the survival and growth rate of larvae then in case of juvenile and adults (Krejszeff et al., 2013). Elaborating of methods allowing measuring of body weight and length without mortality should facilitate carrying out experiments on larvae rearing by lowering the number of specimens. It will result in diminishing the size of rearing tanks. Reducing the size of tanks, in turn, allows to increase the number of rearing treatments and repetitions. Non mortal larvae sampling allows also to increase the frequency of getting samples. Anesthesia is one of methods allowing for such manipulations. The aim of this study was to examine the dynamics of efficiency of MS-222 as an anesthetic during larval period of tench. During the experimental period (from 4 to 34 DPH) the efficacy of MS-222 was tested in 10 days intervals. The efficacy of MS-222 as an anesthetic for tench larvae was evaluated using the following guidelines (Gilderhus, 1990): an induction time of approximately 3min (criterion 1) and a recovery time of 10min or less (criterion 2). An added criterion (criterion III) was post-anesthesia survival of 100% of larvae. Each time the test was started with MS-222 concentration which was recognized by the authors as appropriate for the specific DPH. If the tested concentration didn’t met the set criteria the next tested concentration was higher or lower by 20ppm or 10ppm. Efficacy was tested on 7 larvae per concentration. The exposure duration was set for 15min. During the experiment period, larvae were reared in an experimental device for eggs incubation and larvae rearing, described by Krejszeff et al. (2010). The rearing was carried out at 27 °C (±0.1°C). The photoperiod was set at 12 hours of light and 12 hours of darkness and pH ranged between 8.24 and 8.66. The efficient concentration of anaesthetic in juveniles and adults fish depends on many factors, not only the species and temperature but also on the size. The younger fish has got less developed scales cover and smaller ratio between the skin area and body weight. Therefore the amount of the anaesthetic getting through the skin of younger fish in relation to the total amount of absorbed anaesthetic is bigger than in case of older fish (Massee et al., 1995; Myszkowski et al. 2003; Hamačkova et al., 2004). The same correlation has been observed in larvae of tench. During the growth the larvae sensitivity was decreasing. Therefore establishing the effective dose demanded an increase of MS-222 concentration parallel to the growth of fish. During carried-out tests, apart from dynamics of efficacy, the changes in the safety of MS- 222 application was observed. In the 24th DPH and 34th DPH increasing efficient concentration by 11,11% resulted in mortality. While in the 4th DPH and 14th DPH increasing effective concentration by 50% and 25% didn’t have a negative impact on fish. The observed results show high dynamics of MS-222 efficacy change in tench larvae. It results in the conclusion that application of this anesthetic should be preceded by the evaluation of larvae development stage. The additional hindrance is the fact that at the beginning of larval development fish could recover during exposure in the concentration which meets the first criterion. It results in the necessity of use of higher concentrations. During each day of a test, it was possible to determine the concentration meeting the criterion 1. In 4th DPH it was 40ppm, in 14th DPH it was 80ppm, in 24th DPH and in 34th DPH it was 90ppm (Table I). It was also observed that the larvae started to recover during exposure in the 4th DPH in the concentration of 40ppm, in the 14th DPH in the concentration of 60ppm and in the 34th DPH in the concentration of 80ppm. During each day of the test, each tested concentration met criterion 2 except concentration 100ppm in 24th DPH and 34th DPH (Table II). Apart from this the third criterion wasn’t met in case of 24th DPH and 34th DPH in 100ppm concentration. The recorded mortality was 100% in 24th DPH and 86% in 34th DPH. MS-222 application in tench larvae resulted in highly dynamic change of anesthetic efficacy. Therefore usefulness of the drug is limited. The evaluation of larvae development stage is essential before MS-222 anesthesia application for tench larvae. Table I. Time (s) of induction of anaesthesia (mean±SD, n=7) at various concentrationsTable II. Time (s) of recovery from anaesthesia (mean±SD, n=7) at various concentrations Age (DPH)Dose (ppm) 204050608090100 4>900193±20132±2292±19--- 14->900-295±38162±27-68±17 24---->900160±33135±42 34----313±88164±22117±22 Age (DPH)Dose (ppm) 4050608090100 459±2893±15122±37--- 14 -115±16217±93-473±119 24----83±28>600 34---83±2792±8>600