Efficient method for cryopreservation of European huchen (Hucho hucho L.) and grayling (Thymallus thymallus L.) semen Joanna Nynca a, ⁎, Grzegorz J. Dietrich a , Joanna Grudniewska b , Stefan Dobosz b , Ewa Liszewska a , Maksymilian Krzyś c , Rafał Różyński b , Andrzej Ciereszko a a Department of Gametes and Embryo Biology, Institute of Animal Reproduction and Food Research, Polish Academy of Sciences, Tuwima 10, 10-748 Olsztyn, Poland b Inland Fisheries Institute, Department of Salmonid Research, Rutki, Żukowo, Poland c Salmonid Fish Breeding and Stocking Station of Polish Anglers Association in Łopuszna, Gorczyńska 102, 34-432 Łopuszna, Poland abstract article info Article history: Received 23 July 2014 Received in revised form 9 September 2014 Accepted 19 September 2014 Available online 28 September 2014 Keywords: Fish Milt Sperm freezing Methanol–glucose extender Fertilization rate The objective of this study was to test the effect of cryopreservation with the use of a methanol–glucose extender on sperm motility parameters and fertilization rates of European huchen (Hucho hucho) and grayling (Thymallus thymallus), belonging to the Salmonidae family of Salmoniformes. Distinct differences between those species in absolute fertilization rates, sperm concentrations and sperm motility parameters were noticed. Post-thaw sperm motility of huchen was low (about 45%) compared to sperm motility of cryopreserved grayling semen (about 68%). Absolute fertilization rates of cryopreserved huchen semen were high: 87–88% of eyed embryos and 75– 80% of hatched larvae. On the other hand, absolute fertilization rates of cryopreserved grayling semen were low (about 50%), but the relative fertilization rates amounted to 100% of control with fresh semen. To the best of our knowledge, this study is the first report to demonstrate that the post-thaw fertilization rate of semen can be comparable to that of fresh semen at a ratio as low as 1.5 × 10 5 and 5 × 10 5 spermatozoa per egg for grayling and huchen, respectively. The described method seems to be universal for the cryopreservation of semen of Salmonidae. In our opinion, it can be a very useful tool for ongoing efforts for conservation of European huchen and grayling in Central Europe, for aquaculture of those species, as well as this procedure can be implemented in hatchery conditions after scaling-up. © 2014 Elsevier B.V. All rights reserved. 1. Introduction The European huchen (Hucho hucho L.) and European grayling (Thymallus thymallus L.) belonging to the Salmonidae family of Salmoniformes (Froese and Pauly, 2014; Nelson, 2006), are of both commercial and recreational importance. The European huchen also known as the Danube salmon or Danube huchen is recognized as one of the most endangered salmonid fish in Europe (Weiss et al., 2011). Ac- cording to IUCN criteria, it is classified as endangered species (EN, Witkowski et al., 2013a). It is estimated that the huchen distribution has been reduced by two-thirds of its original range, mainly due to overfishing, river regulations, hydropower development and pollution related to agricultural and industrial activities (Holčik, 1990). The rarity of the size of the huchen (up to 60–70 kg) makes this species extremely attractive for anglers as well as equally important for the conservation of the running waters (Holčik, 1990; Witkowski et al., 2013a). The European grayling, also commonly referred to as the “Lady of the Stream”, is a non-anadromous freshwater fish (Northcote, 1995). Although less important for commercial fisheries than many other Salmoniformes, T. thymallus is considered culturally important as a tar- get for sport fisheries. Especially due to locally declining population sizes, grayling is listed among the “Threatened fishes of Europe” (Lelek, 1984) and large-scale stocking practices of the species are now commencing. Semen cryopreservation is recognized as a tool with great potential for the preservation of genetic variability of depleted stocks and to be used actively in supportive breeding. As such, semen cryopreservation seems to be suited for the conservation of European huchen and gray- ling, but efficacy of this method is limited. Lahnsteiner et al. (1996) and Lahnsteiner (2000) recorded relative fertilization rates (90–100%) for cryopreserved huchen semen, nonetheless low absolute fertilization rates (20–30%). Moreover, quite high sperm/egg ratios 2.5–5.5 × 10 6 were used in their fertilization experiments (Lahnsteiner, 2000; Lahnsteiner et al., 1996). Glogowski et al. (1997) successfully cryopreserved European huchen semen using the pellet method, but absolute fertilization rates were low (~30%) and quite high sperm/egg ratio — 3 × 10 6 was used. Also the described cryopreservation methods of grayling semen (Lahnsteiner et al., 1992, 1996) secure good relative fertilization rates, but a high sperm-to-egg ratio (1.0–1.5 × 10 6 ) is re- quired for the fertilization. The above ratios are similar to the recom- mended numbers for fresh semen (Billard, 1992). However it should Aquaculture 435 (2015) 146–151 ⁎ Corresponding author. Tel.: +48 89 539 31 34; fax: +48 89 524 01 24. E-mail address: j.nynca@pan.olsztyn.pl (J. Nynca). http://dx.doi.org/10.1016/j.aquaculture.2014.09.031 0044-8486/© 2014 Elsevier B.V. All rights reserved. Contents lists available at ScienceDirect Aquaculture journal homepage: www.elsevier.com/locate/aqua-online