Efficient method for cryopreservation of European huchen (Hucho hucho L.) and grayling (Thymallus thymallus L.) semen
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). According 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 target 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 grayling, 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 × 106 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 × 106 was used. Also the described cryopreservation methods of grayling semen (Lahnsteiner et al., 1992, Lahnsteiner et al., 1996) secure good relative fertilization rates, but a high sperm-to-egg ratio (1.0–1.5 × 106) is required for the fertilization. The above ratios are similar to the recommended numbers for fresh semen (Billard, 1992). However it should be underlined that Billard's recommendations were made for the rainbow trout and not all other salmonids. Improvement of the efficacy of the cryopreservation method for huchen and grayling semen seems to be a prerequisite for the implementation of this method into hatchery practice.
A difficulty faced in semen cryopreservation is that the elaborated freezing–thawing procedure suitable for particular fish species cannot be adapted to the other species. Recently, we have developed an exceptionally efficient method for the cryopreservation of rainbow trout semen, securing post-thaw quality similar to that of fresh semen (Ciereszko et al., 2014). This method is based on the use of a simple methanol–glucose extender and seems to be effective for other species of the Salmonidae family, e.g. brown trout (Nynca et al., 2014). In this study, we sought to determine if methanol–glucose extender is efficient for the cryopreservation of European huchen and grayling semen as well and to test specific characteristics of spermatozoa of both species in response to cryopreservation. The objective of this study was to test the effect of cryopreservation on motility parameters and fertilization rates of European huchen and grayling semen.
Section snippets
Source of milt
European huchen (10 years of age; n = 7) were maintained at the Salmonid Fish Breeding and Stocking Station of the Polish Anglers Association in Łopuszna, in southern Poland (49°48′N, 20°14′E) close to the border with Slovakia. Fish were stocked in concrete tanks and fed raw fish and a commercial diet for spawners. Fish were not stimulated hormonally for spermiation or ovulation. Matured grayling males (2–3 years of age, n = 9) were cultivated at the Rutki Salmonid Research Laboratory at the
Effect of cryopreservation on sperm motility parameters
The percentage motility of fresh huchen semen was relatively low (about 75%), no changes were observed after equilibration but cryopreservation caused a decrease by about 40% (Fig. 1). Among other motility parameters only those related to linearity of movement (LIN, VSL) increased which happened already at equilibration period.
Contrary to European huchen, the percentage of motile sperm of grayling after cryopreservation was exceptionally high (more than 67%), but was significantly lower
Discussion
The results of this study demonstrated the very high usefulness of the cryopreservation method based on the employment of methanol–glucose extender for freezing European huchen and grayling semen. Distinct differences between those species in absolute fertilization rates, sperm concentrations and sperm motility parameters were noticed. Cryopreservation with the use of glucose–methanol extender secured high fertilization rates and low sperm-to-egg ratio, which makes the described method very
Acknowledgments
This work was supported by Iuventus grant IP2011 0390 71 from the Polish Ministry of Higher Education, funds of the National Science Centre, Poland granted on research project nr 2011/01/D/NZ9/03738, and funds appropriated to the Institute of Animal Reproduction and Food Research, Polish Academy of Sciences.
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