1. COMPARISON OF TEMPERAURE SHOCKS TIMING TO INDUCED ARTIFICIAL MITOTIC GYNOGENESIS AND ANDROGENESIS IN COMMON TENCH Joanna Nowosad*, Dariusz Kucharczyk, Daniel Żarski, Sławomir Krejszeff, Katarzyna Targońska, Roman Kujawa Department of Lake and River Fisheries, University of Warmia and Mazury in Olsztyn, ul. Warszawska 117, pok. 133, 10-719 Olsztyn, Poland * corresponding author e-mail: nowosad.joanna@gmail.com COMPARISON OF TEMPERAURE SHOCKS TIMING TO INDUCED ARTIFICIAL MITOTIC GYNOGENESIS AND ANDROGENESIS IN COMMON TENCH Joanna Nowosad*, Dariusz Kucharczyk, Daniel Żarski, Sławomir Krejszeff, Katarzyna Targońska, Roman Kujawa Department of Lake and River Fisheries, University of Warmia and Mazury in Olsztyn, ul. Warszawska 117, pok. 133, 10-719 Olsztyn, Poland * corresponding author e-mail: nowosad.joanna@gmail.com MATERIAL AND METHODS Breeders and reproduction procedure In the experiment the wild breeders of tench (15 females and 10 males) and yellow-coloured (9 females and 5 males) were used. The selected fish (0.5-1.5 kg of body mass) were transported to the hatchery of Department of Lake and River Fishery where they were placed in 1000 dm 3 containers equipped for thermo-oxygen control (Kujawa et al. 1999). All the manipulations with spawners were carried out after having the fish anaesthetized in solution of 2-phenoxyethanol (Sigma-Aldrich, Germany) at dose 0.5 cm 3 dm -3. The reproduction of tench, common carp and common bream was stimulated by double injections of Ovopel (Unic-trade, Hungary). In each experiment milt was collected from few males. The quality of sperm was expressed as the percentage of motile spermatozoa. Samples of sperm with 70 - 80% (or more) motile spermatozoa were pooled and used for further treatments. Spawning occurred after 8-10 h for tench after resolving injection at water temperature 21-22°C. Genetic inactivation of DNA spermatozoa and oocytes and induction of gynogenesis and androgenesis Tench spermatozoa was diluted in 0.9% NaCl in ratio 1:9 and irradiated. Eggs during irradiation were kept on Petri dishes in artificial ovarian fluid composed for common carp (Bongers et al. 1994). The dishes with diluted sperm or eggs were placed on a rocking table with a cycle of ~ 1 s. During stirring the eggs were able to roll in the fluid. The UV lamp (30 W, 6.4 W m -2 ) was switched on for at least 30 min before the onset of irradiation. Gametes were exposed to the 9 min UV irradiation (dose of UV irradiation 3456 J m -2 ). Inseminated eggs were later exposed to the thermal shock from 20 to 60 minutes after eggs activation (40°C; 2 min duration). It constituted E20, E30, E40, E50 and E60 groups respectively. The whole procedure during both experiments was carried out in darkness to avoid genetic photo-reactivation. Before application of thermal shock eggs were kept at temperature 21°C. Eggs were incubated in a laboratory recirculated system at 21°C. All experimental groups were in triplicates. The success of induction of androgenetic development was determined in two ways: observed haploid syndrome and using colour (wild-dark coloured or yellow) and morphological marker: differences between embryos of pure species and their hybrids [Kucharczyk 2002, Mamcarz et al. 2006]. Differences in hatching success and in survival of tench embryos were analysed using ANOVA and tested by post-hoc Duncan’s multiple range test (P < 0.05). INTRODUCTION Gynogenesis and androgenesis are the processes in which nuclear DNA of solely maternal or paternal origin is transmitted to the offspring. In case of possessing gametes of the same species what must be done before fertilization is genetic inactivation of the spermatozoa or oocyte that means the destruction of its set of chromosomes. This inactivation can be carried out via ion radiation (i.e. UV, gamma, X radiation). The application of gamma or X radiation shows technical difficulties including safety measures (Arai et al., 1992) and residual chromosome fragments in the oocyte (Ocalewicz et al. 2010). The UV irradiation has been used successfully for genetic inactivation of cyprinid spermatozoa and oocytes (Bongers et al., 1994). Later the oocytes are inseminated with spermatozoa and next, in order to duplicate the chromosomes of the zygote, the oocytes are exposed to an environmental shock. In case of mitotic gynogenesis and androgenesis the only possible shock is so called “late” shock, which means disorder of the first mitotic division. The duplication of the paternal chromosome set is made by application of environmental shock to suppress the first cleavage in eggs. Mitotic gynogenesis and androgenesis makes possible to produce groups of fish with defined genetic featuresThe aim of this study is comparison of temperature shock timing applied to disorder of the first mitotic cleavage in artificial gynogenesis and androgenesis of common tench Tinca tinca (L.). DISCUSSION AND CONCLUSIONS Changeable environmental conditions are the cause of the decrease in production of tench and even the possibility of the extinction of some wild populations. Tench is economically important so the fall in its production is a big problem, the main reasons of which are, first of all, growing inter-species competition, pollution of rural and industrial origin or robber exploitation. One of the solutions is applications of methods of modern genome engineering. The low hatching rate of tench embryos from genetically inactivated oocytes (0-2%) has been observed in many other fish species. Such low survival rates were probably connected with synergic effect of few sub- lethal manipulations (irradiation of gametes and temperature shock) as well as inbred increasing. Gynogenetic and androgenetic origin (haploid or diploid embryos) was checked using a recessive colour marker (“blond”) and morphological marker. The survival of larvae of mitotic gynogenetic and androgenetic origin is variable, usually very low and it was dependent from many different factors. The obtained results in present work showed, that survival of larvae from treated groups was between 0 and 2%. It was similar to data published for wild cyprinids (Kucharczyk 2002). The obtained result showed that it is thermal shock applied due to disorder of the first mitotic cleavage in artificial gynogenesis and androgenesis of common tench are different. REFERENCE Arai, K.; Masaoka, T.; Suzuki, R.; 1992: Optimum conditions of UV irradiation for genetic inactivation of loach eggs. Nippon Suisan Gakk. 58: 1197–1201. Bongers, A.B.J.; Veld, E.P.C.; Abo-Hasema, K; Bremmer, I.M.; Eding, E.H.; Komen, J.; Richter, C.J.J.; 1994: Androgenesis in common carp (Cyprinus carpio L.) using UV irradiation in a synthetic ovarian fluid and heat shock. Aquaculture 122: 119–132. Kucharczyk, D.; 2002: Rozród kontrolowany i androgeneza wybranych gatunków ryb karpiowatych. Rozprawy i Monografie 81, Wyd. UWM, Olsztyn, No. 63; 81p (in Polish). Kujawa, R.; Kucharczyk, D.; Mamcarz, A.; 1999: A model system for keeping spawners of wild and domestic fish before artificial spawning. Aquacult. Eng. 20: 85–89. Ocalewicz, K.; Dobosz, S.; Kuzminski, H.; Nowosad, J.; Goryczko, K.; 2010: Chromosome rearrangements and survival of androgenetic rainbow trout (Oncorhynchus mykiss). J Appl Genet 51: 309–317. RESULTS The survival in newly hatched larvae (5 hours after hatching) in all experimental groups in which gynogenesis and androgenesis was involved, it was significantly lower than in control groups. Haploid embryos, yellow-coloured, showed morphological abnormalities. The highest yield of gynogenesis (P<0.05) was noted when eggs were exposed to the shock 40 min after egg activation whereas androgenesis (P<0.05) was noted when eggs were exposed to the shock 30 min after egg activation (Table 1). Haploid larvae were viable for next 3-4 days after hatching. In case of low eggs quality (low survival in groups C), the small number of spontaneous andro- or gyno-genotes was noted. CI20253035405060 Androgenesis 70.25±22.411.78±0.320.18±0.050.34±0.151.74±0.230.66±0.150.23±0.090.11±0.120.03±0.05 Gynogenesis 70.25±0.061.9±0.180.09±0.060.13±0.050.41±0.110.74±0.291.76±0.320.39±0.20.08±0.07 Table 1. The survival to the hatching stage of tench embryos. C – control groups, I – control groups of quality of irradiation, 20 to 60: time (min) of beginning heat shock after gametes activation