Recent advances in cryopreservation od salmonid fish semen. Andrzej Ciereszko

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1 Recent dvnces in cryopreservtion od slmonid fish semen Andrzej Ciereszko Institute of Animl Reproduction nd Food Reserch, Polish Acdemy of Sciences in Olsztyn, Polnd

2 Justifiction for the studies Poor performnce of published protocols, low post-thw qulity of semen nd very short recommended time for fertiliztion (3 s) Promising preliminry results indicting good post-thw rinbow trout sperm qulity with the use of glucose-methnol extender.

3 Justifiction for the study Low qulity of cryopreserved semen.

4 Cryopreservtion of rinbow trout semen using four different extenders I.3 M glucose, 1% methnol; II.3 M glucose, 1% DMSO Methnol permeting cryoprotectnt Glucose nonpermeting cryoprotectnt

5 LIN ( ) ALH ( m) VAP ( m/s) VSL ( m/s) Motility (%) VCL ( m/s) Glucose concentrtion in the extender is importnt for the cryopreservtion of rinbow trout semen Fresh-diluted x * y * z * Cryopreserved Fresh-diluted x * y * y * Cryopreserved Dilution: 1: x * y * xy * 15 1 x * x * x * 5 5 Fresh-diluted Cryopreserved Fresh-diluted Cryopreserved x x * x 3 2 x * y xy * 2 1 Fresh-diluted Cryopreserved Fresh-diluted Cryopreserved.1 M glucose.2 M glucose.3 M glucose

6 LIN ( ) ALH ( m) VAP ( m/s) VSL ( m/s) Motility (%) VCL ( m/s) Effects of 1:3 nd 1:5 sperm-to-extender dilution rtios on sperm motility prmeters of fresh nd cryopreserved semen x * y * x x 2 5 Fresh-diluted Cryopreserved Fresh-diluted Cryopreserved 15 1 x * x * 8 6 x x * Fresh-diluted Cryopreserved Fresh-diluted Cryopreserved 6 4 x x x x * Fresh-diluted Cryopreserved Fresh-diluted Cryopreserved 15 min 1:3 15 min 1:5

7 Sperm motility chrcteristics nd fertiliztion rtes of fresh nd cryopreserved semen Eyed embryos (%) y * y x * Fresh-diluted Cryopreserved 1,:1 3,:1 6,:1 Post-thw motility 49.9 ± 6.8% Men sperm concentrtion nd osmollity of fresh undiluted semen were 1.87 ± 2.48 x 1 9 spermtozo nd 251 ± 39 mosm/kg, respectively.

8 Brown trout (Slmo trutt m. frio L.) - mjor source of freshwter fish resources in Europe becuse of its commercil vlue for quculture nd extreme importnce for ngling. - nturlly subdivided into lrge number of reproductively isolted nd geneticlly distinct popultions

9 The tiger trout (Slmo trutt X Slvelinus fontinlis) is sterile, intergeneric hybrid of the brown trout (Slmo trutt) nd the brook trout (Slvelinus fontinlis).

10 Brook trout (Slvelinus fontinlis; Mitchill) Importnt commercilly, recretionlly, nd ecologiclly in Europe. It is of interest in quculture becuse it is lmost completely resistnt to the virl hemorrhgic septicemi virus nd cn esily be subjected to genome mnipultion.

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12 Source of milt Brown trout (n=9); 3 yers of ge Brook trout (n=9; 2 yers of ge Cryopreservtion.2M glucose in 9% MeOH; Dilution 1:5 in.25 ml strws; 15 min equilibrtion, Thwing 4ºC, 5 s. Mterils nd Methods Fertiliztion Brown trout 1; 3; sperm/egg rtio Brook trout 3; sperm/egg rtio Fertiliztion rtes were mesured t the eyed nd htched stges. Mesurements of sperm motility nd concentrtion Sperm motility - mesured in fresh semen fter dilution nd in frozen smples. Sperm concentrtion - mesured using Nucleocounter SP-1.

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14 Sperm collection using ctheter

15 Collection of whitefish

16 Europen huchen

17 Reproductive system of sex-reversed femles of rinbow trout

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19 CASA nlysis of sperm motility

20 Extension of semen with glucose-methnol extender

21 Equilibrtion of strws filled with extended semen

22 Equilibrtion of strws filled with extended semen

23 Freezing

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26 Thwing 4 C, 5 s

27 LIN (%) ALH ( m) VSL ( m/s) VAP ( m/s) Motility (%) VCL ( m s -1 ) Effect of cryopreservtion on sperm motility prmeters Brown trout Brook trout b b Fresh Equilibrted Cryopreserved Fresh Equilibrted Cryopreserved Fresh Equilibrted Cryopreserved Fresh Equilibrted Cryopreserved 6 b bc c 2 1 Fresh Equilibrted Cryopreserved Fresh Equilibrted Cryopreserved

28 Fertiliztion rte (%) Fertiliztion rte (%) Effect of sperm-to-egg rtio on fertility of post-thw cryopreserved sperm Brown trout Brook trout 1 8 b b b b 6 5 b b :1 3 :1 6 :1 eyed stge (%) htched stge (%) 3 :1 6 :1 eyed stge (%) htched stge (%)

29 Effect of post-thw sperm storge on motility prmeters VCL ( m s -1 ) Motility (%) LIN (%) VAP ( m s -1 ) ALH ( m) VSL ( m s 1 ) Brown trout Brook trout min 6 min min 6 min b min 6 min min 6 min b 3 2 b min 6 min min 6 min

30 Effect of post-thw sperm storge on motility prmeters It hd been ssumed tht thwed semen must be used immeditely for fertiliztion within 3-second sperm storge fter thwing significntly reduces the fertiliztion rte. 3/5s = 6 strws 6 (min) * 6 s = 36 s/5s = 72 strws Prolonged hndling time of brook trout thwed semen could led to better orgniztion of htchery work becuse the thwing procedure of severl sperm smples for fertiliztion trils is time consuming.

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32 CryoSperm Bnk Implementtion Cryopreserved sperm Whitefish Arctic chrr Brook trout Fertiliztion Gynogenetic Hybrids Interspecific hybrids Lines of slmonid fish sprctic for ex. rinbow trout

33 Further experiments Different species Improvement of technology Addition of ntioxidnts (cystemine, glutthione, etc., ntioxidtive enzymes) Different sugrs (sucrose, trehlose, etc.) Anti-freeze proteins Potssium ions Buffers Higher volume of strws Higher sperm concentrtions in strws

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36 Preliminry results 16 October 215 Cryopreservtion of brown trout, cherry slmon nd white-spotted chr semen Andrzej Ciereszko Jonn Nync Mriol Dietrich Konrd Oclewicz Nne Fresh-Wter Sttion Director Etsuro Ymh

37 Brown trout Mle Fresh Equilbrted Cryopreserved Men SD

38 Cherry slmon Oncorhynchus msou

39 Cherry slmon Mle Fresh Equilibrted Cryopreserved Men SD Very thick semen, observed gglutintion of spermtoz fter ddition of extender to the fresh semen nd in the thwed semen. Fish were t the end of spwning seson.

40 White spotted chr Slvelinus leucomenis

41 White spotted chr Mle No. Fresh Equilibrted Cryopreserved Men SD Agglutintion of spermtoz fter ddition of extender nd fter thwing. Motility of the thwed sperm ws short sec. Fish were smpled one dy before experiment.

42 Effect of supplementtion of glucose-methnol extender with potssium ions on sperm motility of slmonid fish

43 Replcement of glucose with trehlose or sucrose in GM extender Slmon

44 Summry GM extender seems to be well suited for cryopreservtion of slmonid fish semen. Species specific modifiction my be necessry. For exmple trehlose for whitefish. The possibility of post-thw semen storge for the prolonged time (t lest 6 min) s well s the obtinment of high fertiliztion rte t low sperm-to-egg rtio cn led to the significnt improvement in implementtion of cryopreservtion in htchery prctice. Further studies should be focused on scling up this efficient cryopreservtion technique for ppliction in htchery conditions. This work ws supported by Iuventus grnt IP from Polish Ministry of Higher Eduction, funds of the Ntionl Science Centre grnted on reserch project nr 211/1/D/NZ9/3738, nd funds pproprited to the Institute of Animl Reproduction nd Food Reserch, Polish Acdemy of Sciences. This work ws lso prtilly funded by COST Office (Food nd Agriculture COST Action FA125: AQUAGAMETE).