Aquaculture Research. 1997. 28,471-479

Methanol as cryoprotectant and the suitability of1.2 ml and 5 ml straws for cryopreservation of semenfrom salmonid fishes

F LahnsteinerInstitute for Zoology. University of Salzburg. Salzburg. Austria

T WeismannBundesamt fur Wasserwirtschaft. institut fur Gewasserokoiogie. Fischereibiologie und Seenkunde. Mondsee. Austria

R A PatznerInstitute for Zoology. University of Salzburg. Salzburg. Austria.

Correspondence: Dr Franz lahnsteiner. Institute for Zoology. University of Salzburg. Hellbrunnerstr. 34. A-5O2(I Salzburg. Austria.

E-mail; Franz.Lahnsteiner@sbg.ac.at

Abstract

For salmonid semen, the cryoprotective action of10% methanol was compared with a 5% dimethylsulphoxide (DMSO). 1% glycerol mixture, until nowone of the most effective cryoprotectants. InOncorhynchus mykiss (Walbaum). Salmo trutta L. /.fario. Salmo trutta L. /. lacustris and Salvelinus alpinus(L.). semen crj'opreserved with hoth cr>'oprotectantsyielded post-thaw fertilization rates of 90-100% ofcontrol with untreated semen at sperm-to-egg ratiosof 1.8 X 10''-2.4 X 10^ spermatozoa per egg.However, at sperm-to-egg ratios of 0.9 X 10' '-1.2 X 10*' spermatozoa per egg. semen cryo-preserved with methanol had significantly higherfertilization rates than semen frozen with the DMSO/glycerol mixture. In other studies we obtained similardata for Coregonus sp.. Salvelimis fontinalis (Mitchill).Thymallus thymallus (L.) and Hucho hucho (L.).proving that methanol is the most effective andgenerally applicable cryoprotectant lor semen of thestudied salmonid species.

To facilitate the insemination of large egg batcheswe investigated the suitability of 1.2 ml and 5 mlstraws for deep freezing of semen of Oncorhynchusmykiss, Salmo trutta f. fario. Salmo trutla f. lacustris

and Salvelimis alpinus. With 1.2 ml straws thefertilization rates were similar to 0.5 ml strawswhen using lower freezing and higher thawingtemperatures. The 5 ml straws resulted in afertilization success of only about 40% of freshsemen control.

Introduction

Recently we presented a cr\'opreser\'ation methodapplicable for seven salmonid species - rainbowtrout. Oncorhynchus mykiss (Walbaum); brown trout,Salmo trutta L. / fario: lake trout. Sahno trutta L. /lacustris: brook trout. Salvelinus fontinalis (Mitchill);whitefish. Coretjonus sp.: Danube salmon. Huchohucho (L.); and grayling. Thymallus thymallus (L.) -hy which post-thaw fertility rates of 90-100% ofcontrols with untreated semen could be obtained(Lahnsteiner. Weismann & Patzner 1995. 1996a.b;Lahnsteiner. Berger. Weistnann & Patzner 1996c).

For semen cryopreservation of rainbow trout,brown trout. lake trout, brook trout and whitefish,a dimethyl sulphoxide/glycerol mixture is a veryeffective cryoprotectant (Lahnsteiner, Berger,Weismann & Patzner 1996d). Methanol has not

© 1997 Blackwell Science Lid. 471

Methanol as crj'oprotectanl (or salmonid semen F Lahnsteiner el al. Aquaculture Research. 1997. 28, 471-479

been tested for semen cryopreser\'ation in thesespecies, but in the Danube salmon and the graylingit is still superior to DMSO/glycerol (Lahnsteineretal. 1996a). Methanol has also been successfullyused for semen crj'opresen'ation in Clarias gariepinus(Burchell) (Steyn & Van Vuren 1987), Sarotlwrodonmossambictis (Peters) (Harvey 1983: Rana &McAndrew 1989) and iMtcs calcarifer (BJoch) (Leung1987). Therefore we investigated if methanolincreases the semen post-thaw quality of the rainbowtrout, brown trout, lake trout, and Arctic charr.Salvelimis alpimis (L.).

Straws with a volume of 0.5 ml have beensuccessfully applied to cryopreserve semen ofsalmonid fishes and to inseminate small egg batches(Lahnsteiner etal. 1995, 1996b). This is useful forgene banking, but insemination of large egg batchesis difficult because numerous straws must beprocessed at the same time (for 500 ml eggs, about40 straws according to a sperm density of 3 X ] 0**-4X10 '* spermatozoa ml"' and to a sperm-to-eggratio of 3 X 10'̂ spermatozoa per egg). Large-scaleinsemination with cr)'opreserved semen is importantin routine aquaculture when single catches ofspawners are frequent (e.g. in the lake trout) andfor selective breeding methods. Freezing the semenin pellets of 100-200 jil volume as described inother freezing protocols (review: Leung & Jamieson1991) has no advantages for large-scaleinsemination. Wheeler & Thorgaard (1991) testedthe suitability of larger straws with a volume of5 ml for freezing rainbow trout semen and obtaineda fertilization success of about 50%, of control. Toreduce the number of straws necessary forinsemination, we tested straws with a volume of1.2 ml and 5 ml for crj'opreservatlon of semen ofrainbow trout, lake trout and Arctic charr.

Material and methods

Collection of gametes

Mature rainbow trout, Oncorhynchus mykiss, autumnand winter strain (total length (TL) 3 5-50 cm), laketrout, Salmo truttaj. lacustris (TL 40-60 cm), browntrout, Salmo trutta f. fario {TL 45-65 cm) and Arcticcharr, Salvelinus alpinus (TL 20-30 cm) were usedas gamete donors. Rainbow trout, brook trout, laketrout and brown trout were cultivated at the fishfarm of Kreuzstein (Sankt Gilgen, Salzburg) in 5-m^basins supplied with ground water at a flow rate of0.5 1 s ' and at a temperature of 8.5 ± 0.2°C. Fish

were fed a pelleted diet consisting of 50% proteinand maximal stock density was 6 kg m"^. State ofmaturity was controlled from about 2 weeks beforethe expected date of spawning until they were in arunning stage. Charr were captured from spawningwild populations in Lake Grundlsee. The gameteswere collected by gentle abdominal massage at 5-8°C and care was taken to avoid contaminationwith blood, urine atid faeces. Sperm density wasdetermined bj' a spectrophotometric method(Ciereszko & Dabrowski 1992) and was 4.9 ± 0.6X 10^ cells ml"' for Oucorhynchtis wykiss (mean istandard deviation, n = 24), 2.3 ± 0.5 X l o ' "cells ml"' for Salmo trutta f. hwustris {n = 12), 7.9± 0.9 X lO'J cells ml"' for Salmo trutta f. fario (n =15) and 0.9 ± 0.2 X lO** cells ml"^ for Salvelinusalpinus {ii = 18). Semen batches with sperm motilityrates > 70% (evaluated by subjective estimation orcomputer-assisted cell motility analysis -Lahnsleiner etal. 1996c) were used for theexperiments. Semen and egg batches were storedon ice for not more than 30 min before the onset ofthe experiments.

General protocol for semen cryopreservation

Four to eight semen samples were pooled in equalratios in each species to volumes of 5-20 mlaccording to the required amount. Sperm density ofthe pools is reported together with the results.Following the freezing protocol of Lahnsteiner et al.(1995. 1996b). semen samples were diluted in 4°Ccold extender (NaCl, 103 mM: KCl, 40 mM: CaCl2,1 mM: MgS04. 0.8 mM: Hepes 20 mM pH 7.8:dimethyl sulphoxide (DMSO). 5%: glycerol, 1%:bovine serum albumin, 1.5%: egg yolk, 7%: sucrose,0.5%). The dilution ratio was 1:3 (semen:extender)for the rainbow trout and charr, 1:5 for the browntrout, and 1:7 for the lake trout). As equilibrationtimes up to 15 min did not affect the post-thawfertility of salmonid semen (Lahnsteiner et al1996b), 30-50 straws were filled with diluted semenin 5 min. Then they were frozen in the vapour ofliquid nitrogen on a horizontally mounted tray inan insulated box. After a freezing period of 10 min(0.5 ml and 1.2 ml straws) to 1 5 min (5 ml straws),straws were placed in liquid nitrogen and stored forabout 60 min. Short storage periods were necessaryto perform controls and experiments with the samesemen batches. For thawing, straws were immersedin a 25°C thermostat-regulated water bath for 30 s.

472 C 1997 Blackwell Science Ltd. Aquaculiure Research. 28 , 471-479

Aquaculture Research, 1997. 28. 471^79 Methanol as cryoprotectant for salmonid semen F Lahnsteiner et al.

Straws were removed, opened, and the thawedsemen was poured onto the eggs.

Fertilization assays

Egg pools, consisting of eggs from three tosix females, were used for the fertilizationexperiments. The ovarian fluid was poured off2-A min before fertilization, and replaced by afertilization solution at 4°C. modified according toScheerer & Thorgaard (1989) (NallCOj, 60 mM:glycine. 20 mM: theophylline, 5 mM: Tris, 50 mM:pH 9) (fertilization solution:eggs ratio = 1:2). Inthe experiment, the desired amounts of eggswere transferred into Petrl dishes containingfertilization solution, the straws were thawedand mixed with the eggs by gentle stirring.After 2-3 min. about 50 ml well water wasadded. Eggs were rinsed and incubated in flowincubators at Kreuzstein. Hatching was donein well water of 5.0-6.0°C (pH = 7.8,conductivity = 3]5nS at 20°C. O, = 11.35 mg1-1. Fe--^ = 0 mgl"'. NHi = 0 mg ]-\ NOi = 0mg r ' . NO3 = 1.42 mgl"'. total phosphate =0.002 mg rM. The experimental success wasdetermined by percentage eyed embrj'os 2 8 -30 days after fertilization in the rainbow troutand 3 5-40 days after fertilization in the lake trout,brown trout and charr. All experiments wererepeated at least three times.

as cryoprotectant and frozen in 0.5 ml (diameter3 mm. length 135 mm: made by Minitub. Germany).1.2 ml (diameter 4 mm. length 135 mm: made byBicef, France) or 5 ml (diameter 5 mm, length280 mm. made by Minitub) straws. Freezing andthawing conditions for 1.2 ml and 5 ml strawswere determined in preliminary investigations byevaluation of the motility rate as this parametercorrelates with the post-thaw fertility rate(Lahnsteiner etal. 1996c). Conditions yielding thehighest post-thaw motility rate were investigatedlor their post-thaw fertilization capacity. In theexperiments 1.2 ml straws filled with diluted semenwere frozen 0.5 cm (-150°C). 1cm (-130°C).1.5 cm (-110°C) and 2.0 cm (-100°C) above thelevel of liquid nitrogen for 10 min and thawed at25°C for 30 s and at 30°C for 25, 30 and 35 s.Semen placed in 5 ml straws was frozen 0.5 cmabove the level of liquid nitrogen for 15 min. Lowerfreezing levels could not be exactly adjusted in theopen system used. Straws were thawed at 35°C for40 s and at 40°C for 40 s. Control experiments wereperformed with semen frozen in 0.5 ml straws andwith untreated semen.

The amount of semen used for fertilization wasadjusted to obtain similar sperm-to-egg ratios in allexperiments. The 12.5 ml batches of eggs werefertilized viith the content of one or half a 0.5 mlstraw. 30 ml egg batches with the content of oneor half a 1.2 ml straw and 125 ml egg batches withthe content of one 5 ml straw.

Experiments

Methanp] as cryoprotectant for salmonid semen

Rainbow trout, lake trout, brook trout and charrsemen was frozen in extenders containing either a5% DMSO, 1 % glycerol mixture or 10% methanol ascryoprotectant. This concentration was determinedas the most effective in preliminary experiments.Semen was frozen in 0.5 ml straws 1.5 cm (-110°C)above the surface of liquid nitrogen for rainbow trout,lake trout and brown trout semen and 2.5 cm (-92°C)above the level of liquid nitrogen for charr and brooktrout semen, and 12.5 ml eggs were fertilized withthe content of either one or half a straw.

1.2 ml and 5 ml straws for deep freezing of salmonid

semen

Rainbow trout, lake trout, brook trout and charrsemen was diluted in extenders with lO'Xi methanol

Determination of freezing and thawing rates

Straws (0.5. 1.2 and 5 ml) were filled with semendiluted in extender with 10% methanol and athermoelectrode (Chessel M301) was inserted.Freezing and thawing was performed as describedand the changes in temperature were monitored.Each determination was repeated eight times.

Statistics

Fertilization rate was determined by the percentageof eyed-stage embryos in relation to the total numberof eggs. All experiments were repeated at least threetimes. Percentage data were subjected to angulartransformation, and two-way analysis of varianceand Student's f-test were used for data analysis ata probability level of P < 0.005.

© 1997 Blackwell Science Ltd. Aquacultun Research, 28 , 4 7 1 ^ 7 9 473

Methanol as cryoprotectant for salmonid semen F Lahnsteiner ei a\. Aquaculture Research. 1997. 28. 471-479

Results

Efflciency of methanol as cryoprotectant forsalmonid semen

In the rainbow trout, at sperm-to-egg ratios of1.8 X 10''-2.4 X 10^ spermatozoa per egg, semenfrozen with 10% methanol as well as with the 5%DMSO / 1% glycerol mixture yielded post-thawfertilization rates of 90-100% of control withuntreated semen (Table 1). At lower sperm-to-eggratios of 0.9 X lO**-!.! X 10^ spermatozoa per egg,semen frozen with methanol had a significantlyhigher (P < 0.005) fertilization capacity than semenfrozen with the DMSO / glycerol mixture.

In the brown trout, semen cryopreservation with10% methanol yielded higher post-thaw fertilizationrates than cryopreservation with the 5% DMSO. 1%

'bble 1 Cryoprotective efficiency of 10% methanol incomparison with a 5% dimethyl sulphoxide. 1% glycerolmixture for rainbow trout semen. Freezing: 0.5 ml straw.s.1.5 cm above the level of liquid nitrogen: thawing: 25°C.30 s. Values (mean ± SD. n = 3) belonging to the sameseries of experiments and superscripted by the same letterwere not significantly different. Three trials with differentsemen and egg pools were performed (trial I: spermdensity. 4.8 X 10^ spermatozoa ml"'; number of eggs perexperiment. 206 ± 11: trial II: sperm density, 4.3 x 10 'spermatozoa ml"': number of eggs. 175 ± 8: trial III:sperm density. 3.1 X 10 ' spermatozoa ml ': number ofeggs 190 ± 10)

Treatment Sperm/egg Absoluteratio fertilization (%)

glycerol mixture at all tested sperm-to-egg ratios (1,2X 10''-2.4 X lO*" spermatozoa per egg) (Table 2)( P < 0.005). In the lake trout, fertilization ratessimilar to control were obtained with methanol aswell as with the DMSO / glycerol mixture at sperm-to-egg ratios (P > 0,005) of 1.2 X 10* spermatozoapereggandof2.3 X 10^-2.4 X 10^ spermatozoa peregg (Table 2), indicating a very high quality of thesesemen batches.

Suitability of 1.2 ml and 5 ml straws forcryopreservation of salmonid semen

The optimal freezing conditions for 1.2 ml strawswere 1.0 cm (-130°C) above the level of liquidnitrogen for the rainbow trout, lake trout (Fig. 1,Table 3) and brown trout (data not shown) and1.5 cm (-110°C) above the level of liquid nitrogen forthe charr (Fig. 1, Table 3) and the brook trout (similarresults, not shown). When freezing 1.2 ml straws1.0 cm above the level of liquid nitrogen, the initialfreezing rates were similar to those when freezing 0.5ml straws 1.5 cm (-110°C) above the level of liquid

2 Cryoprotective efficiency of 10% methanol incomparison with a 5% dimethyl sulphoxide. 1% glycerolmixture for lake trout and brown trout semen. Values(mean ± SD. n = 3) belonging to the same series ofexperiments and superscripted by the same letter were notsignificantly different. For Salmo trutta f. fario. the spermdensity was 7.8 x l o ' spermatozoa ml"' and the numberof eggs per experiment was 185 ± 12: for Salmo trutta f.lacustris. the sperm density was 2.8 X lO '" spermatozoaml"' and the number of eggs per experiment was 168 ± 7

Triai 1Untreated semen

5% DMSO. 1% glycerolmixture

10% Methanol

Trial IIUntreated semen

5% DMSO. 1% glycerolmixture

10% Methanol

Triai IIIUntreated semen5% DMSO, 1% glycerol

mixture10% Methanoi : ' •- ••

2.4 X 10^

1.2 X 102.4 X 1061.2 X 10^2.4 X 106

1.2 X 10^

1.8 X 10^0.9 X 1061.8 X 10^

0.9 X 1061.8 X 10^0.9 X 106

1.0 X 1061.0 X 10^

1.0 X 106

84.4 ± 2.2 =QQ Q -•- Q 1 aOO.i? — O. 1

82.4 ± 2.0 "70.8 ± 1 . 8 "81.6 ± 1.0 ^77.5 ± 1.1 •=

94.3 ± 0.6 ^95.8 ±1 .8 =89.6 ± 1 . 8 "65.0 ± 1.9<=88.1 ± 1.2"75.3 ± 1 . 5 "

96.2 ± 0.4 "64.0 ± 5.4 "

89.2 ± 7.4 "

Treatment

Salmo trutta f. farioUntreated control

5% DMSO. 1% glycerolmixture

10% Methanol

Salmo trutta f. lacustrisUntreated semen

5% DMSO. 1% glyceroimixture

10% Methanoi

Sperm/eggratio

2.4 X 1061.2 X 10^2.4 X 1061.2 X 10^2.4 X 10^

1.2 X 10^

2.3 X 10^1.2 X 10^2.3 X 10^1.2 X 10^

2.3 X 10^1.2 X 106

Absolutefertilization (%)

95.3 ±1 .4 =90.4 ± 2.0 "85.3 ± 6.4 =71.5 ± 5.1 <̂95.7 ± 1.1 =90.2 = 1 . 7 "

97.2 ±1 .8 =97.8 ± 1 . 7 "95.1 ± 2.0°95.5 ± 0.9 =98.6 ± 0.3 =97.0 ± 0.7 '

474 C 1997 BlackweU Science Ltd, Aquaculture Research. 28, 471-479

Aquaculture Research, 1997, 28, 471-479 Methanol as cryoprotectant for salmonid semen F Lahnsteiner et al.

20

-140

80 100 120Time (sec)

140 160 180 200

Figure 1 Freezing curves for 0.5 ml. 1.2 ml and 5 ml straws in the vapour of liquid nltogen. Determination of each freezingcurve was repeated eight times and the standard deviation for each measuring point was < ± 3°C: (a) 5 ml straws, 0.5 cm(-150°C) above the surface of liquid nitogen;(b) 0.5 ml straws. 2.5 cm (-92°C) above the surface of liquid nitogen:(c) 1.2 mlstraws. 1.5 cm (-110°C) above the surface of liquid nitogen; (d) 0.5 ml straws. 1.5 cm (-110°C) above the surface of liquidnitogen; le) 1.2 ml straws. 1.0 cm (-1 30°C) above the surface of liquid nitogen. The initial freezing rates are similar for:0.5 ml straws at -92°C and 1.2 ml straws at -110°C, 0.5 ml straws at -110°C and 1.2 ml straws at -130°C.

-200 T

10— 1 —

15—r—

20

1

25

Time

1—

30

(8ec)

— 1 —35

— 1 —

401

45

' " 1 •

50—r

55 60

Figured Thawing curves for 5 ml. 1.2 ml and 0.5 ml straws: (a) the thawing curves are similar for 0.5 ml straws at25°C and for 1.2 ml straws at 30°C; (b) 5 ml straw 35°C, 40 s. Determination of each thawing curve was repeated eighttimes and the standard deviation for each meastiring point was < ± 3°C.

nitrogen. When freezing 1.2 ml straws 1.5 cm above

the level of liquid nitrogen, the initial freezing rates

were similar to those when freezing 0.5 ml straws 2.5

cm (-92°C) above the level of liquid nitrogen. Two-

factor analysis of variance revealed that freezing

conditions were the highly significant main efTect

(P < 0.005). The other experimental parameters,

which were strongly intercorrelated, were combined

to a single variable which did not significantly

influence the fertilization rate (P > 0.005).

© 1997 BlackweU Science Ltd, Aquaculture Research, 28 , 471-479 475

Methianol as cryoprotectant for salmonid semen F Lahnsteiner et al. Aquaculture Research. 1997. 28. 471- i79

Table 3 Standardization of freezing conditions for rainbow trout, lake trout and Arctic charr semen placed in 1.2 mlstraws. Values (mean ± SD. » = 3) belonging to the same series of experiments and superscripted by the same letterwere not significantly different. Freezing level is the distance of straws from the surface of liquid nitrogen. For Oncoriiymhusmykiss. the sperm density was 4.3 X lO'* spermatozoa mtK the number of eggs per experiment 206 ± 11, and thesperm-to-egg ratio 1.8 X lO*" spermatozoa per egg; for Salmo trutta f lacustris. the sperm density was 2.8 X

spermatozoa ml"', the number of eggs per experiment was 168 ± 7. and the sperm-to-egg ratio 2.3 X lO'' spermatozoaper egg: for Salvelinus alpinus. the sperm density was 2.8 X lO'" spermatozoa ml"', the number of eggs per experimentwas 200 ± 8. and the spenm-to-egg ratio 0.8 X 1 ()'' spermatozoa per egg

Species

Oncorhynchusmykiss

Salmo trutta1. lacustris

Salvelinusalpinus

Straw size

(ml)

Untreated semen0.51.21.21.2

Untreated semen0.51.21.2

Untreated semen0.51.21.21.2

Freezing

(cm)

1.50.51.01.5

-

1.50.51.0

_

2.51.01.52.0

level

(°C)

-110-150-130-110

_

-110-150-130

_

-92-130-110-100

Thawing

CC)

25303030

_

253030

_

25303030

(s)

30303030

_

303030

_

30303030

Absolute

fertilization (%)

94.3 ± 0.6 "88.1 ± 1.2 *=41.4 ± 5.2":86.1 ± 0.1 "58.7 i 6.9 "

97.2 ±1.8 =98.6 ± 0.3 ^90.3 d: 1.2 '•

97.7 ± 1.1 "

75.3 = 8.3='57 4 ± 4.7*'26.6 ± 2.4 =55.4 ± 2.2 "36.2 ± 5.6 "

Optimal thawing conditions yielding the highestpost-thaw fertilization rate were in a 30°C water bathfor 30 s for all investigated species (Fig. 2. Table 4).With this thawing procedure, the thawing rates weresimilar to those when thawing 0.5 ml straws at 2 5°Cfor 30 s. The two-way analysis of variancecharacterized the thawing conditions as highlysignificant main effect (P < 0.005). In this set ofexperiments, straw size and freezing conditions werethe parameters correlated with each other and theywere combined to a single variable. It had nosignificant effect (P > 0.005) on the fertilization rate.The insemination dose was similar for 1.2 ml strawsand 0.5 ml straws (Table 5).

The use of 5 ml straws yielded in a relatively lowpost-thaw fertilization of at most 39% of the freshsemen control (P < 0.005). These results wereobtained when freezing the 5 ml straws 0.5 cm (-150°C) above the level of liquid nitrogen (Fig. 1) andthawing them at 35°C for 40 s (Fig. 2, Table 6).

4 Standardization of the thawing conditions forrainbow trout semen frozen in ] .2 ml straws. Values (mean± SD. n = 3) belonging to the same series of experimentsand superscripted by the same letter were not significantlydifferent. Freezing level is the distance of the straws fromthe surface of liquid nitrogen. Sperm density of the eggpool was 4.8 X lO** spermatozoa ml"', sperm-to-egg ratiowas 2.4 X 10'' spermatozoa per egg. and the number ofeggs per experiment was 206 ± 1 1

Straw

size (ml)

Untreatedsemen0.51.21.21.21.2

Freezing level

(cm)

_

1.51.01.01.01.0

(°C)

_

-110-130-130-130-130

Thawing

(°C)

2530303025

(s)

3025303530

Absolute

fertilization (%)

84.4 ± 2.2 ^

81.6 ± 1.0^75.4 i 5.3 ^84.1 ± 3 . 8 "

72.1 ± 2 . 2 "

75.1 ± 3.4 "

476 © 1997 Blackwell Science Ltd. Aquaculture Researth. 28, 471-479

Aquaculture Research, 1997, 28, 471-479 Methanol as cryoprotectant for salmonid semen F Lahnsteiner et al.

Table 5 Standardization of the sperm-to-egg ratio for rainbow trout and lake trout semen cryopreserved in 1.2 ml straws.The freezing level was the distance of straws from the surface of liquid nitrogen. Values (mean ± SD, n = 3) belongingto the same series of experiments and superscripted by the same letter were not significantly different. For Oncorhynchusmykiss. the sperm density was 4.1 X lO** spermatozoa ml ' ' and the number of eggs per experiment was 156 ± 9: forSalmo truttaf lacustris, the sperm density was 2.8 x 10 ' " spermatozoa ml"' and the number of eggs per experiment was168 ± 7

Species

Oncorhynchusmykiss

Salmo truttaf. lacustris

Straw size

(ml)

Untreated semenUntreated semen0.50.51.21.2Untreated semenUntreated semen0.50.51.21.2

Freezing

(cm)

_

-

1.51.01.01.0--

1.51.51.01.0

level

(°C)

_

--110-130-130-130--

-110-110-130-130

Thawing

_

-25253030--

25253030

(s)

-30303030--

30303030

Sperm/egg

ratio

3.8 X 10^1.9 X 10^3.8 X 10^1.9 X 10^3.8 X 10^1.9 X 10^2.3 X 10^1.2 X 10^2.3 X 10^1.2 X 10^2.3 X 10^1.2 X 10''

Absolute

fertilization (%)

93.8 ± 3.0 '93.6 =: 3.0 "89.8 ± 2.4 «90.6 ± 2.2 "90.4 ± 1.9»89.8 ± 2.4 =97.2 ±1 .8 =97.8 ± 1.7''98.6 ± 0.3 "95.3 ± 2.0 =97.7 ± 1.1 "94.2 ± 3.2 ^

6 Post-thaiv fertilization rates for rainbow trout semen frozen in 5 ml straws. Sperm densitj' of the egg pool was4.9 X lO** spermatozoa m r ' , sperm-to-egg ratio 3.8 x lO*" spermatozoa per egg. Values (mean ± SD, ;i = 31 belongingto the same experiment and superscripted by the same letter were not significantly different. The freezing level was definedas the distance of straws from Ihe surface of liquid nitrogen

Straw size

(ml)

Untreated semen0.555

Freezing level

(cm)

150.50.5

( X )

-110-150-150

Thawing

(°C)

253540

(s)

304040

No. of eggs per

experiment

156 ± 9156 2: 9

1480 ± 161480 - 16

Absolutefertilization (%)

93.8 - 3.0"89.8 ± 2.4 "46.6 r 2.2 "32.6 i 5.8 "

Discussion

Methanol as cryoprotectant

Dimethyl sulphoxide (DMSO) (Legendre & Billard1980; Stoss & Holtz 1983: Baynes & Scott 1987;Holtz 1993), glycerol (Piironen 1993), DMSO/glycerol mixtures (Lahnsteiner et al 1995, 1996d),propanediol (Lahnsteiner etal 1996d), dimethylacetamide (McNiven, Gallant & Richardson 1993)and polyethylene glycol (Lahnsteiner & Berger,unpublished) have been applied for cryopreservationof salmonid semen. By sperm motility analysis.

vitality staining and measurements of intracellularlactate dehydrogenase liberation, a mixture of 5%DMSO and 1% glycerol (Lahnsteiner etal 1996d)has been determined as the most effectivecryoprotectant which was established by the highpost-thaw fertilization success (Lahnsteiner et al1995, 1996d). The present data demonstrate thatmethanol gives a further increase in frozen-thawedsemen quality of rainbow trout, lake trout, browntrout and Arctic charr, an effect which is mostsignificant at low sperm-to-egg ratios of 0.9 X lO' '-1.2 X 10*' spermatozoa per egg. Owing to this

® 1997 Biackweil Science Ltd. Aquaculture Research. 28 , 471-479 477

Methanol as cryoprotectant for salmonid semen F Lahnsteiner et al. Aquaculture Research. 1997. 28, 471-^79

quality improvement, the reliable averageinsemination doses to obtain a fertilization rate of90-100% of fresh semen control can be loweredfrom about 4 X 10^ spermatozoa per egg (calculatedfor the DMSO / glycerol mixture, Lahnsteiner et al.1996b) to 2.5 X lO*" spermatozoa per egg. Also inthe whitefish and brook trout (Lahnsteiner,unpublished data) and in the Danube salmon andthe grayling (Lahnsteiner etal 1996a), methanolsignificantly increased the quality of cryopreservedsemen. As only small semen quantities wereavailable, low sperm-to-egg ratios of 0.8 x 10*spermatozoa per egg had to be used in S. alpinus,which reduced the fertilization success withcryopreser\'ed semen in relation to fresh semencontrol.

In summary, the data prove that methanol is themost effective cryoprotectant for semen of salmonidfishes and that it is generally applicable for deepfreezing of semen of all salmonids investigated todate. Post-thaw motility rate, a sensitive indicatorfor frozen/thawed semen quality (Lahnsteiner et al1996c). is also increased by methanol by about 10%in comparison with the DMSO / glycerol mixture.Quality of semen for cryopreservation can becontrolled by the seminal plasma pH and osmolality,and by the post-thaw motility rate and swimmingvelocity (Lahnsteiner etal, 1996c). Using semen ofhigh quality, the insemination doses can be reducedto sperm-to-egg ratios of less than 2 X 10*spermatozoa per egg (Lahnsteiner and Weismann.unpublished data). For fresh semen, minimal sperm/egg ratios of 3 X 10 ' -6 X lO ' spermatozoa per eggare recommended (Billard 1988).

necessary for the fertilization of 500 ml eggs. Thisnumber of straws could not be handled at the sametime (Weismann & Lahnsteiner, unpublished data),limiting the utilization of 0.5 ml straws forfertilization of small egg batches. Only 15 strawswith a volume of 1.2 ml are necessary for fertilizationof 500 ml egg batches and therefore this methodgives the first possibility for the insemination of largeegg batches with frozen-thawed semen.

The 5 ml straws are not suited for cryo-preservation of salmonid semen as the fertilizationsuccess is only about 40% of the fresh semen control.Temperature measurements inside the strawsindicated that in 5 ml straws, both the freezing andthawing rates were insufficient. Further increase ofthe freezing rate was not possible in the opensystem used, and direct immersion in liquid nitrogendecreased the post-thaw motility rate to zero(Lahnsteiner and Weismann, unpublished data).Thawing temperatures higher than 40°C causeddenaturation of semen enzymes and proteins,especially in the peripheral portions of the strawswhich thawed more quickly than the central ones(Lahnsteiner and Weismann, unpublished data).

Acknowledgments

Studies were supported by the AustrianBundesminsterium fur Land- und Forstwirtschaft.We are grateful to the workers in the Kreuzsteinfish farm for providing material, help in fertilizationand hatching of fry and to the Bundesanstalt furkunstliche Befruchtung und Fortpflanzung vonHaustieren in Wels for technical advice.

Straw size

The present data demonstrate that 1.2 ml strawsare suited to cryopreservation of salmonid semen asthe fertilization capacity of semen frozen in 1.2 mlstraws is similar to that of semen frozen in 0.5 mlstraws. However, for 1.2 ml straws, lower freezingand higher thawing temperatures are necessary toobtain the same freezing and thawing rates as for0.5 ml straws. As already reported (Lahnsteiner et al1995. 1996a), the thawing of semen is a verysensitive process and slight alterations in time ortemperature decrease its quality. At a sperm-to-egg ratio of 2 x lO^-3> X 10* spermatozoa per egg(insemination doses for the methanol extender),about 35 straws with a volume of 0.5 ml are

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