Aquaculture 435 (2015) 146–151

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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, Polandb Inland Fisheries Institute, Department of Salmonid Research, Rutki, Żukowo, Polandc Salmonid Fish Breeding and Stocking Station of Polish Anglers Association in Łopuszna, Gorczyńska 102, 34-432 Łopuszna, Poland

⁎ Corresponding author. Tel.: +48 89 539 31 34; fax: +E-mail address: j.nynca@pan.olsztyn.pl (J. Nynca).

http://dx.doi.org/10.1016/j.aquaculture.2014.09.0310044-8486/© 2014 Elsevier B.V. All rights reserved.

a b s t r a c t

a r t i c l e i n f o

Article history:Received 23 July 2014Received in revised form 9 September 2014Accepted 19 September 2014Available online 28 September 2014

Keywords:FishMiltSperm freezingMethanol–glucose extenderFertilization rate

The objective of this studywas to test the effect of cryopreservationwith the use of amethanol–glucose extenderon spermmotility parameters and fertilization rates of European huchen (Hucho hucho) and grayling (Thymallusthymallus), belonging to the Salmonidae family of Salmoniformes. Distinct differences between those species inabsolute fertilization rates, sperm concentrations and spermmotility parameters were noticed. Post-thaw spermmotility of huchen was low (about 45%) compared to sperm motility of cryopreserved grayling semen (about68%). 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 werelow (about 50%), but the relative fertilization rates amounted to 100% of control with fresh semen. To the bestof our knowledge, this study is the first report to demonstrate that the post-thaw fertilization rate of semencan be comparable to that of fresh semen at a ratio as low as 1.5 × 105 and 5 × 105 spermatozoa per egg forgrayling and huchen, respectively. The described method seems to be universal for the cryopreservation ofsemen of Salmonidae. In our opinion, it can be a very useful tool for ongoing efforts for conservation ofEuropean huchen and grayling in Central Europe, for aquaculture of those species, as well as this procedurecan 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 ofSalmoniformes (Froese and Pauly, 2014; Nelson, 2006), are of bothcommercial and recreational importance. The European huchen alsoknown as the Danube salmon or Danube huchen is recognized as oneof themost 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 distributionhas been reduced by two-thirds of its original range, mainly due tooverfishing, river regulations, hydropower development and pollutionrelated to agricultural and industrial activities (Holčik, 1990). The rarityof the size of the huchen (up to 60–70 kg)makes this species extremelyattractive for anglers as well as equally important for the conservationof the running waters (Holčik, 1990; Witkowski et al., 2013a). TheEuropean grayling, also commonly referred to as the “Lady of theStream”, is a non-anadromous freshwater fish (Northcote, 1995).Although less important for commercial fisheries than many other

48 89 524 01 24.

Salmoniformes, T. thymallus is considered culturally important as a tar-get for sport fisheries. Especially due to locally declining populationsizes, grayling is listed among the “Threatened fishes of Europe”(Lelek, 1984) and large-scale stocking practices of the species are nowcommencing.

Semen cryopreservation is recognized as a tool with great potentialfor the preservation of genetic variability of depleted stocks and to beused actively in supportive breeding. As such, semen cryopreservationseems 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 fertilizationrates (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) successfullycryopreserved European huchen semen using the pellet method, butabsolute fertilization rates were low (~30%) and quite high sperm/eggratio— 3 × 106 was used. Also the described cryopreservation methodsof grayling semen (Lahnsteiner et al., 1992, 1996) secure good relativefertilization rates, but a high sperm-to-egg ratio (1.0–1.5 × 106) is re-quired for the fertilization. The above ratios are similar to the recom-mended numbers for fresh semen (Billard, 1992). However it should

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be underlined that Billard's recommendations were made for the rain-bow trout and not all other salmonids. Improvement of the efficacy ofthe cryopreservation method for huchen and grayling semen seems tobe a prerequisite for the implementation of this method into hatcherypractice.

A difficulty faced in semen cryopreservation is that the elaboratedfreezing–thawing procedure suitable for particular fish species cannotbe adapted to the other species. Recently, we have developed an excep-tionally efficient method for the cryopreservation of rainbow troutsemen, securing post-thaw quality similar to that of fresh semen(Ciereszko et al., 2014). This method is based on the use of a simplemethanol–glucose extender and seems to be effective for other speciesof the Salmonidae family, e.g. brown trout (Nynca et al., 2014). In thisstudy, we sought to determine if methanol–glucose extender is efficientfor the cryopreservation of European huchen and grayling semen aswell and to test specific characteristics of spermatozoa of both speciesin response to cryopreservation. The objective of this study was to testthe effect of cryopreservation on motility parameters and fertilizationrates of European huchen and grayling semen.

2. Materials and methods

2.1. Source of milt

European huchen (10 years of age; n = 7) were maintained at theSalmonid Fish Breeding and Stocking Station of the Polish Anglers Asso-ciation in Łopuszna, in southern Poland (49°48′N, 20°14′E) close to theborder with Slovakia. Fish were stocked in concrete tanks and fed rawfish and a commercial diet for spawners. Fish were not stimulatedhormonally for spermiation or ovulation. Matured grayling males (2–3 years of age, n = 9) were cultivated at the Rutki Salmonid ResearchLaboratory at the Institute of Inland Fisheries in Olsztyn, Poland.Grayling males with a mean weight of 263.3 ± 36.6 g were kept in3 m3 concrete ponds supplied with water from the Radunia Riverwith oxygen saturation levels maintained at 85–95% and temperatureof 7–10 °C during spawning. Prior to milt or egg collections, the fishwere anesthetized using Propiscin (1 ppm IFI, Żabieniec, Poland). Miltwas obtained by abdominal massage, with special care to avoid blood,urine or feces contamination.

2.2. Semen cryopreservation

Cryopreservation was performed using the method recently de-scribed (Ciereszko et al., 2014; Dietrich et al., 2014; Nynca et al.,2014). The motility parameters of the fresh sperm and frozen–thawedsperm were examined with Computer Assisted Sperm Analysis(CASA). Semen samples were diluted 1:5 in a 0.18 M glucose and 9%methanol extender (final concentrationwas 0.15Mand 7.5% for glucoseand methanol, respectively). Sperm was loaded into 0.25 ml plasticstraws (IMV Technologies, L'Aigle, France). The straws were placed ona 3 cm adjustable floating rack and equilibrated on ice for 15 min,followed by floating in liquid nitrogen for 5 min in Styrofoam boxwith an isolating Neopor block (Minitüb GmbH, Tiefenbach,Germany). Straws were thawed in a water bath at 40 °C for 5 s.

2.3. Effect of cryopreservation on motility parameters and fertilizationability of semen

The cryopreservation of semen of seven individual males of huchenand nine individuals of grayling was performed as described above.Sperm motility parameters were measured for fresh, fresh-diluted andfrozen–thawed semen. Following thawing, the semen was used forfertilization assays within 1–2 min. Fertility trials were performedwith fresh and cryopreserved sperm on April 16, 2014 and April 23,2014 for grayling and huchen, respectively. The eggs collected fromtwo grayling females were divided into batches of 108 ± 14 eggs

(1.5 g). Then, 5 ml of D532 (20 mM Tris, 30 mM glycine, 125 mMNaCl, pH 9.0; Billard, 1992) was added to the eggs together withcryopreserved sperm. Mean values (±SD) of semen volumes added tothe eggs were 16.0 ± 4.7, 32.1 ± 9.4, and 64.2 ± 18.7 μl at thespermatozoa-to-egg ratios of 150,000; 300,000 and 600,000:1. Eggs ob-tained from two huchen females by abdominal massage were mixedand divided into batches of 3.5 g, 100 ± 9 eggs. Then, 10 ml of D532(20 mM Tris, 30 mM glycine, 125 mM NaCl, pH 9.0; Billard, 1992) wasadded to the eggs together with fresh or cryopreserved sperm. Themean values (±SD) of huchen semen volumes added to the eggswere 295.9 ± 286.5 μl and 147.9 ± 143.3 μl sperm-to-egg ratios of1,000,000:1 and 500,000:1, respectively. These ratios are higherthan that of previously used for rainbow trout (Ciereszko et al.,2014) and brown trout (Nynca et al., 2014) because quality of Dan-ube salmon semen appeared to be lower than those species, whichwas manifested by low sperm concentration and high concentrationrange (see below). Excess fresh semen (50 μl for grayling and 100 μlfor huchen) combined from three males was used at the beginningand at the end of the fertilization trials to test the quality of the eggs.All fertilization trials were done in duplicate. The fertilization rateswere measured at the eyed (April 28, 2014 for grayling and May 01,2014 for huchen) and hatching stages (May 5, 2014 for grayling andJune 1, 2014 for huchen).

2.4. Semen analysis

The motility parameters of sperm were examined with ComputerAssisted Sperm Analysis using the Hobson Sperm Cell Tracker (HobsonVision Ltd, Baslow, UK) as described byDietrich et al. (2005). Spermwasactivated at a dilution ratio of 1:300 with 1 mM CaCl2, 20 mM Tris,30 mMglycine, 125mMNaCl, and pH 9.0 (Billard, 1992) supplementedwith 0.5% bovine serum albumin. The sperm motility parameters: per-centage of motile sperm (MOT), straight line velocity (VSL), curvilinearvelocity (VCL), average path velocity (VAP), linearity (LIN) and ampli-tude of lateral head displacement (ALH) were measured over a 12-second period, between 5 and 17 second post-activation time. Videorecordings (two replicates per sample) were made using an OlympusBX40 microscope (Olympus Optical, Tokyo, Japan) with a 10× negativephase objective and a Sony CCD black andwhite camera (SPT-M108CE).Sperm concentration was measured using NucleoCounter SP-100(Chemometec, Denmark) as described by Nynca and Ciereszko (2009).Briefly, semen was diluted 100 times with a sperm immobilizing solu-tion (100 mM NaCl, 40 mM KCl, 3 mM CaCl2, 1.5 mM MgCl2 and50 mM Tris, pH 8.5; Morisawa and Morisawa, 1988) then 51 timeswith Reagent S100. Osmolality of seminal plasma was measured usinga Vapor Pressure Osmometer 5520 (WESCOR, Logan, USA).

2.5. Statistical analysis

All the results are presented as mean ± SD. All analyses were per-formed at a significance level of 0.05 using GraphPad Prism software(GraphPad Software Inc. San Diego, CA, USA). For statistical proceduresdata percentages were transformed by arcsine square root transforma-tion. Spermmotility datawere subjected to repeatedmeasures one-wayANOVA followed by Tukey's post hoc test. Fertilization rates of cryopre-served semen were subjected to repeated measures two-way analysisof variance (ANOVA) followed by Sidak's post hoc test.

3. Results

3.1. Effect of cryopreservation on sperm motility parameters

The percentage motility of fresh huchen semen was relativelylow (about 75%), no changes were observed after equilibration butcryopreservation caused a decrease by about 40% (Fig. 1). Among

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Fig. 1. Sperm motility of fresh, fresh-diluted and cryopreserved semen of European huchen (n = 7). Results are expressed as mean ± SD. Different superscripts indicate significantdifferences between parameters of fresh, fresh-diluted and cryopreserved semen (P b 0.05).

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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 ofgrayling after cryopreservation was exceptionally high (more than67%), but was significantly lower compared to the fresh semen (by24%). No differences were observed between other sperm motilityparameters (VCL, VSL, VAP, LIN, ALH) of fresh and cryopreservedsemen (Fig. 2). The 15 min equilibration period of semen dilutedwith an extender increased the VSL and LIN of fresh semen. For thepercentage of sperm motility, VCL, VAP and ALH no differences werefound between fresh and equilibrated semen (Fig. 2).

Sperm concentration of European huchen was lower by about 50%compared to grayling. Also European huchen semen was characterizedby higher range of sperm concentration values in comparison tograyling (Table 1). On the other hand, no difference in seminal plasmaosmolality was detected between both species.

3.2. Fertilization rates of cryopreserved semen

Absolute fertilization rates of cryopreserved European huchensemenwere high for both spermatozoa-to-egg ratios (Fig. 3A). Percent-age of eyed eggs amounted 87–88% and 75–80% for hatched larvae andthese values did not differ. Hatching rateswere lower compared to ratesat eyed stage. The fertilization rate of eggs fertilized with an excess offresh semen amounted to 94.8% for eyed embryos and 86.8% for hatchedlarvae.

Absolute fertilization rates of cryopreserved grayling semen weremuch lower compared to European huchen. However, relative fertiliza-tion of eggs with post-thaw grayling semen yielded very high eyedstage (more than 50%) for all sperm-to egg ratios (Fig. 3B). Similar tothe percentage of eyed embryos, no differences were observed amongthe percentage of hatching rates for the sperm-to-egg ratios used forfertilization (Fig. 3B). The hatching rate was still very high for all tested

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Fig. 2. Sperm motility of fresh, fresh-diluted and cryopreserved semen of grayling (n = 9). Results are expressed as mean ± SD. Different superscripts indicate significant differencesbetween parameters of fresh, fresh-diluted and cryopreserved semen (P b 0.05).

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sperm-to-egg ratios, but the decrease in survival from the eyed eggstage to hatched stage, similar to European huchen, was significant(Fig. 3B). The fertilization rates of eggs fertilized with an excess offresh semenwere 51.5 ± 9.7% and 48.1 ± 8.0 for the eyed and hatchingstages, respectively.

4. Discussion

The results of this study demonstrated the very high usefulness ofthe cryopreservation method based on the employment of methanol–glucose extender for freezing European huchen and grayling semen.Distinct differences between those species in absolute fertilizationrates, sperm concentrations and sperm motility parameters were no-ticed. Cryopreservation with the use of glucose–methanol extender

Table 1The average sperm concentration and osmolality of European huchen and grayling semen used

Parameter European huchen

Mean ± SD

Sperm concentration×109 spermatozoa ml−1

3.32 ± 2.16a

OsmolalitymOsmol kg−1

226 ± 35a

Values marked with similar superscripts did not differ significantly from each other at P b 0.05

secured high fertilization rates and low sperm-to-egg ratio, whichmakes the described method very efficient. As such, in our opinion thecryopreservation procedure described herein could be a very usefultool for ongoing efforts for the conservation of huchen and grayling inCentral Europe and can be implemented into hatchery practice.

To the best of our knowledge this study is the first report to demon-strate that the post-thaw fertilization rate of semen can be comparableto that of fresh semen at a ratio as low as 1.5 × 105 and 5 × 105 sperma-tozoa per egg for grayling and huchen, respectively. Lahnsteiner et al.(1996) had obtained similar results of the fertilizing ability of cryopre-served grayling semen. However, a sperm/egg ratio applied by theseauthors was about ten times higher (1.0–1.5 × 106) than those usedin our study. Recently, Horváth et al. (2012) reported very variable(30–90%) fertilization rates for cryopreserved grayling semen for

for cryopreservation.

Grayling

Range Mean ± SD Range

0.65–7.54 6.11 ± 1.92b 4.10–8.77

188–261 239 ± 29a 177–272

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Fig. 3. The sperm fertilization ability of A — European huchen (n = 7) and B— grayling (n = 9) cryopreserved semen in relation to different sperm-to-egg ratios. White bars— the per-centage of eyed embryos; gray bars— the percentage of hatched larvae. Results are expressed as mean SD. Different superscripts indicate significant differences between percentages ofeyed embryos and hatched larvae (P b 0.05).

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different stripping collection periods. However, those authors didnot provide the sperm/egg ratio, so we are unable to compare theirresults with the results of our study. Lahnsteiner et al. (1996)and Lahnsteiner (2000) recorded excellent relative fertilization rates(90–100%) for cryopreserved semen of European huchen, but absolutefertilization rates were low (20–30%). Moreover, quite high sperm/eggratios — 4 × 106 (Lahnsteiner et al., 1996) and 2.5–5.5 × 106 wereused in their studies (Lahnsteiner, 2000). It has to be underlined thatour results clearly suggest that the sperm/egg ratio for fertilizationwith cryopreserved semen can be lower than 1.5 × 105 and 5 × 105

spermatozoa per egg for grayling and huchen, respectively. This sugges-tion should be tested in further experimental studies. Moreover, addi-tional studies are necessary to improve fertilization rates of grayling inhatchery conditions, which is prerequisite for better efficacy for theuse of cryopreserved sperm.

Cryopreservation induces profound changes in sperm motilityparameters, and the most common is a decrease in the percentage ofspermmotility. Grayling sperm seems to be distinguished by exception-ally high post-thaw spermmotility (more than 67%). Those results wereslightly lower than the post-thaw motility of brown trout (about 74%;Nynca et al., 2014) and higher than recorded for rainbow trout semen(about 60%; Ciereszko et al., 2014). In contrast, post-thaw spermmotil-ity of huchen was the lowest among mentioned species, about 45%.Sperm motility parameters measured by CASA appeared to be usefulfor the comparative analysis of changes in trajectory of sperm speedand movement after cryopreservation. In the case of cryopreservedgrayling semen, motility parameters were not affected by the freez-ing–thawing procedures. In cryopreserved huchen semen, only anincrease in LIN was recorded. This is a striking difference compared torainbow and brown trout (Ciereszko et al., 2014; Nynca et al., 2014),because the cryopreservation with the same procedure led to a declinein VSL and VAP of brown trout spermatozoa and in VSL, VAP and ALH ofrainbow trout sperm. Furthermore, European huchen semen andgrayling semen, compared to rainbow trout and brown trout, seem tobe characterized by a significant increase in LIN and VSL just after equil-ibration. It can be suggested that the described differences in the chang-es of sperm motility parameters indicate the species-specific variationsrelated to cryopreservation between properties of grayling, huchen,brown trout and rainbow trout spermatozoa related to cryopreserva-tion. However, it should be stressed out that any suggestions regardingthe specificity of European huchen semen have to be treated with cau-tion at present because the quality of semen used in our experimentmay not be fully representative for this species (see below).

We have observed exceptionally low sperm concentration forEuropean huchen maintained in the Łopuszna hatchery. On theother hand, the sperm concentration values determined for graylingseem to be within normal range. The values for European huchenrecorded in our study are lower (3.32 × 109 spermatozoa ml−1)than the range 4–8 × 109 spermatozoa ml−1 reported by

Lahnsteiner (2000). At the same time average concentration forgrayling (6.11 × 109 spermatozoa ml−1) was within this range. Al-though, no systematic studies on sperm concentration were per-formed, the results of fragmentary observations suggest a steadydecline in sperm concentration in European huchen maintained inthe Łopuszna hatchery. Glogowski et al. (1997) recorded a concen-tration of 7.6 × 109 spermatozoa ml−1. Glogowski et al. (2003) re-ported a concentration of 6.81 × 109 spermatozoa ml−1 in year2000 and 5.19 × 109 spermatozoa ml−1 in year 2003. Formickiet al. (2013) reported 4.87 × 109 spermatozoa ml−1 for 2012 andwe recorded 3.32 × 109 ml−1 spermatozoa in this study. It is possiblethat the low sperm concentration described in our study can be relat-ed to possible inbreeding of European huchen maintained in theŁopuszna hatchery, because this population was created from a re-stricted number of founders (Witkowski et al., 2013b). This sugges-tion is supported by the low values of genetic variability of thispopulation (Fopp-Bayat, unpublished observations). Moreover, thediscrepancy in recorded sperm concentrations might as well be at-tributed to the differences in the concentration assessment methodsused in the experiments, such as flow cytometry, the type of hemo-cytometer or NucleoCounter apparatus. Nevertheless, despite lowsperm concentration, our results demonstrated that sperm can suc-cessfully be cryopreserved, which can be useful for future programsfor increasing the genetic variability of this population.

European huchen is another salmonid species, after rainbow trout(Ciereszko et al., 2014) and brown trout (Nynca et al., 2014) for whichthe same cryopreservation procedure has successfully been applied.Furthermore, for the first time this method was effectively usedfor the semen cryopreservation of grayling also belonging to theSalmonidae family. Recently, Dietrich et al. (2014) have demonstratedthe usefulness of the elaborated procedure for the efficient cryopreser-vation of testicular semen of sex-reversed female rainbow trout. There-fore, it is quite likely that this procedure can be universal for the semencryopreservation of salmonids. Aswe discussed previously (Nynca et al.,2014), perhaps, the salmonid spermatozoa respond in a similar patternto the conditions of the cryopreservation procedure. Parallel conclu-sions were obtained by Lahnsteiner et al. (1997), indicating thatsemen of different Salmoniformes subfamilies, such as salmoninae andthymallinae can effectively be cryopreserved with the use of the sameprocedure. However, each species is characterized by specific parame-ters of spermatozoa, therefore, further studieswill include the examina-tion of the suitability of glucose–methanol extender for the spermcryopreservation of other families of Salmoniformes.

In conclusion, an efficient method for the cryopreservation ofEuropeanhuchen and grayling semenwith theuse ofmethanol–glucoseextender has been demonstrated. Thismethod seems to be universal forthe cryopreservation of semen of Salmonidae. In our opinion, it can be avery useful tool for ongoing efforts for conservation of European huchenand grayling in Central Europe, for aquaculture of those species, as well

151J. Nynca et al. / Aquaculture 435 (2015) 146–151

as this procedure can be implemented in hatchery conditions afterscaling-up.

Acknowledgments

Thisworkwas supported by Iuventus grant IP2011 0390 71 from thePolish Ministry of Higher Education, funds of the National ScienceCentre, Poland granted on research project nr 2011/01/D/NZ9/03738,and funds appropriated to the Institute of Animal Reproduction andFood Research, Polish Academy of Sciences.

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