Fish sperm cryopreservation in France: from laboratory studies
to application in selective breeding programs
by
Pierrick Haffray (1), Catherine Labbe (2), IMV Technologies (3) & Gérard Maisse (2)
AbsTract. - Application of sperm cryopreservation by the fish farming industry is still limited, despite great interest in
optimising genetic resource conservation; limiting risks associated with horizontal disease transmission; increasing genetic
progress; and accelerating genetic selection from prime specimens, as in other livestock production. Several factors are
listed. With the aim of developing coherent broodstock selection programmes, SYSAAF (Union of the French poultry and
aquaculture broodstock industry), INRA and IMV-Technologies, standardised semen cryopreservation procedures between
1994 and 2000. The first step was to develop multispecies (salmonids, turbot, sea bream, sea bass, gold fish, tilapia, European catfish) extenders to collect, induce maturation and conserve (> 7 days) fresh sperm or testicular spermatozoa
(StorFish™); to wash ova from faeces and blood (OvaFish™); to freeze sperm (Cryofish™); and to improve fertilisation
success (ActiFish™). The second step was to evaluate the practical limits of the technology and to test several potential
improvements (GnRH stimulation, anaesthetic, frequency of collection, cryopreservation devices, optimal egg/sperm volumes, etc.). The third step was to standardise sperm collection procedures, from the efferent duct or testicular spermatozoa;
to ensure absolute traceability by straw printing; to fill and seal a large number of straws by means of an automated filling
and sealing machine (MRS1); and to freeze sperm in liquid vapour in adapted container. The entire procedure, using optimal extenders and standardised procedures, was transferred to the industry. From 2000 to 2006, 50,080 0.5 ml straws from
1554 males, originating from 26 populations of rainbow trout, sea bream, turbot and sea bass, were frozen by breeding
companies. The development of a collective gene bank is in progress, with the aim of securing private gene banks (breeding companies, research organisations and Cryobanque Nationale).
Key words. - fish - Sperm cryopreservation - IMV- Gene bank.
Introduction
Technical feasibility of semen cryopreservation is published for most aquaculture farmed species today (Mounib,
1978; Stoss, 1983; Billard, 1984; Tiersch and Mazik, 2000).
90% fertilisation success is reported in marine fishes, as
compared to 50-70 % for salmonids where more variability
is observed mainly because of a shorter duration of motility
after thawing (3-4 s vs 60-90 s in marine species). French
fish farmers first initiated selective breeding programs at the
beginning of the nineties (with advice on genetic selection
provided by SYSAAF) (Haffray et al., 2002), leading on to
the development of an important project on fish sperm cryopreservation, in collaboration with INRA and IMV-Technologies. The objective was to develop extenders, protocols,
adapted devices, know-how and procedures to make this
technology accessible to fish broodstock companies.
Material and methods
The standardisation of the freezing procedure was performed following 3 successive steps :
Step 1. - Development of multispecies extenders (salmonids, turbot, sea bream, sea bass, gold fish, tilapia, European
catfish) and evaluation in fish farms before marketing by
IMV Technologies (Maisse et al., 1998).
Step 2. - Evaluation of practical limits and technical
improvement.
Step 3. - Adaptation and standardisation of procedures
for on-farm practices.
The freezing method of reference was based on the use
of a freezing extender (see below) with 10% DMSO (or
another cryoprotectant, depending on the species) and 10%
egg yolk. 0.5 ml IMV “bovine” straws were filled with 1 vol.
extender + 3 vol sperm. Straws were frozen in a liquid nitrogen vapour, 4 cm over the LN2 surface. Thawing was performed in a water bath for 10 s at 37°C. Fertilisation success
was estimated by duplicate trials, based on fertilisation of
10 g of eggs by 1 straw, plus 10 ml of salmonid activation
extender, or 10 ml seawater for marine fishes.
Results
Step 1
- The StorFish™ extender is well adapted to dilute and
enhance/induce maturation of spermatozoa from testicular
extracts of sex reversed rainbow trout. After 3 h of incubation (2°C), mean sperm motility (n = 45) increased from
13.6% ± 19.3 to 83.7% ± 12.9. StorFish can also be used as
an immobilising and storage extender. In the medium term
(up to 7 days), good spermatozoa motility is maintained for
most of the species (example Fig. 1) when 1 volume of testicular extract, or fresh sperm, is diluted in 9 volumes of
StorFish™ and stored in a static thin layer.
- The OvaFish™ extender was developed to rinse faeces,
blood and coelomic fluid from the salmonid egg surface, to
obtain the best environmental conditions for sperm movement. When eggs are rinsed, a significant increase in fertilisation success is observed (Fig. 2).
(1) SYSAAF, Campus de Beaulieu, 35000, Rennes, France. [[email protected]]
(2) INRA, SCRIBE, UR1037, IFR140, Génopole Ouest, Campus de Beaulieu, 35000, Rennes, France.
(3) IMV-Technologies, L’Aigle, France.
Cybium 2008, 32(2) suppl.: 127-129.
Development of fish semen cryopreservation in France Figure 1. - Fertility of testicular sperm from sex reversed female
rainbow trout, conserved at 2-4°C for 1 day or 6 days (m ± S.E.M.;
n = 10) in StorFish™ extender (ANOVA, p < 0.05).
Figure 2. - Effect of rinsing rainbow trout eggs prior to fertilisation
with OvaFish™ extender or MMLC (medium from Billard and Jalabert 1974), on fertilisation success with cryopreserved sperm (m ±
S.E.M. ; n = 10 ; ANOVA, p < 0.05).
Figure 3. - Effect of ActiFish™ activating extender on fertilisation
success with rainbow trout cryopreserved sperm (mean ± S.E.M. ;
n = 6 ; (ANOVA, p < 0.05).
- The ActFish™ is used as an activating medium for
improved fertilisation success with cryopreserved semen
(trout, Fig. 3), and to allow production of progeny from low
quality sperm.
- The CryoFish improved cryopreservation (Fig. 4) when
compared with the Mounib (1978) extender.
Step 2
- No adverse effect in trout of GnRH (Ovaprim) stimulation of spermiation on cryopreservation success.
- No adverse effect in trout of the frequency of collection
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Haffray et al.
Figure 4. - Effect of sperm freezing with CryoFish™ extender compared to Mounib (1978) extender on fertilisation success (paired
t-test p < 0.05).
(1 week and + 2, + 3 or + 4 weeks).
- No adverse effect in trout of anaesthetics (phenoxy ethanol or CO2).
- In trout, 0°C (40 or 60 min) is the best temperature for
fresh sperm conservation before freezing (e.g. 5, 10, 15, 20
or 25°C under O2).
- Microwave thawing is not suitable and provokes a risk
of straw explosion (5 ml straw).
- 0.25, 0.5, 1 or 5 ml IMV TBS or 0.5, 1 ml IMV CBS
straws or 20 ml pig bag are all suitable.
- No effect of the distance to LN2 (3 to 11 cm), and therefore of freezing speed e.g. programmable freezer
(KRYO10).
- Thawing temperature of 37°C/min is better than 49 or
55°C/min?
- 10 g to 80 g of eggs could be fertilised by 1straw;
(0.5 ml) / 10 g of eggs.
- No substitute egg yolk could be obtained (e.g. of tested
products: protein (BSA, PPCN, b-lactoglobulin) or lipid
(lecithin, liposomes)) .
- DMSO as a cryoprotectant allowed succesful fertilization for all species studied, except the European catfish
(DMA) and Nile tilapia (MeOH).
Step 3
- Collection of semen from the sperm duct was performed
by aspiration in an essay tube using a 10 ml poultry sperm
collector. Graduations allow for the collection of a definite
volume, or for collection in StorFish™ immobilizing solution at the right dilution ratio.
- Straw traceability was developed using automated
printing technology, in collaboration with an artificial insemination centre for bovines. Information on the broodstock
company, the species, the lineage, the individual, and the
year of collection are printed on the straw and stored in a
database.
- Filling and sealing of straws was automated using a
MRS1 machine for 0.5 ml bovine straws (TBS). This portaCybium 2008, 32(2) suppl.
Haffray et al.
Development of fish semen cryopreservation in France
breeding candidates and their Breeding Value. Today, 7 private cryobanks owned by fish breeders, INRA and IFREMER are in operation for rainbow trout, brown trout, sea bass,
sea bream and turbot. At the end of 2006, 50,080 0.5 ml
straws from 1554 males, originating from 26 different populations or generations, were frozen by broodstock companies.
Figure 5. - Speed of freezing depending 1) on freezing equipment :
polystyrene box (black curves) or stainless steel insulated freezer
box (grey curves) and 2) on the distance of straws from the liquid
nitrogen surface : 4 cm (thin curve), 7.1 cm (medium curve) or
10 cm (thick curve)
ble equipment (4000 straws/h capacity) can be disinfected.
Disposable accessories for the manipulation of diluted sperm
(semen receptacle, flexible tube, and nozzle) prevent contamination between semen samples.
- Semen freezing: As for bovine sperm, an adapted freezer was developed to freeze 2 racks of 52 straws, providing
for a greater storage capacity than with polystyrene boxes.
This insulated stainless steel box provides a temperature of
-80°C in less than 100 s when straws are placed 4 cm above
the LN2 surface. The low temperature vapour reduces the
duration of recrystallisation (Fig. 5).
Attracted by the reliable fertilization rates obtained, and
the accessible procedures developed, fish breeders are now
investing in sperm cryopreservation for 3 main reasons: to
secure their selective breeding programs from technical,
genetic or pathological problems; to optimise breeding
schemes; and to store genetic material to enable future evaluation of genetic selection progress. Assistance for on-farm
cryopreservation is provided by SYSAAF together with parentage assignment (fingerprints) to improve breeding
schemes according to the genealogical relationship between
Cybium 2008, 32(2) suppl.
Conclusion
The procedure described here allowed for the transfer of
sperm cryopreservation from the research domaine to the
fish farm. The high cost of maintenance of individual cryobanks and the high risk of accidental loss prompted the setting up of a cooperative cryobank, whose development will
associate aquaculture broodstock companies, research
organisations (INRA, Ifremer, etc.) and the “Cryobanque
Nationale”, which stores public livestock genetic resources
in France. For sanitary reasons, the use of high security
CBS™ straws is recommended for the future aquatic species
patrimonial depository in the “Cryobanque Nationale”.
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